12

Kidney and urinary tract

12

Kidney and urinary tract

12

Kidney and urinary tract

12

Kidney and urinary tract

12.1 Clinical laboratory diagnosis of kidney and the urinary tract diseases

Christian Thomas, Lothar Thomas

In patients with symptoms of renal diseases and urinary tract disorders, serum and urine biomarkers are important investigations for diagnosis, differentiation, and therapeutic monitoring:

  • Of acute renal failure (ARF)
  • Of chronic kidney disease (CKD)
  • Of congenital kidney disease
  • Of urinary tract infection
  • Of stone disease.

Screening tests (Tab. 12.1-1 – Tests for renal diseases) are indicated:

  • For asymptomatic patients
  • For the estimation of the renal glomerular and tubular function
  • For the diagnosis of renal and urinary tract infections.

An isolated elevated serum creatinine or a positive test strip result in urine should, initially, be seen as a screening result and be confirmed with repeated testing and more specific tests. In patients with suspected renal disease such results should be considered to be relevant, and they should be verified with further examinations and, particularly, by clinical findings.

For renal disease screening, reference intervals of biomarkers are definitive. More sensitive tests (renal ultrasound, albuminuria testing) and clinical investigations are needed for symptomatic patients with underlying illness that also effects renal function (diabetes, hypertension), and individuals with a relevant family history.

12.1.1 Acute kidney injury

Acute kidney injury (AKI), also termed acute renal failure (ARF), is defined as a rapid, acute onset (i.e., over hours to weeks) and usually reversible decline in glomerular filtration rate (GFR) /1/. ARF is generally characterized by an abrupt rise in serum creatinine, decreased urinary output or both. Chronic hypertension and heart failure are risk factors for acute kidney injury and can hamper recovery from kidney injury. ARF can occur either in the setting of previously normal renal function (classic ARF) or in a patient with pre-existing chronic renal failure (acute-on-chronic renal failure). Clinically, ARF is further characterized as being oliguric (urine volume < 500 mL/24 h), non oliguric (urine volume ≥ 500 mL/24 h), and on the basis of dialysis dependence. ARF is associated with a disturbed water, electrolyte and acid-base balance. Patients with suspected ARF should be diagnosed rapidly and appropriate clinical treatment should be initiated.

12.1.1.1 Classification of acute renal failure

Recommendations for the definition and classification of ARF were developed by nephrologists and intensive care specialists. They are based upon serum and urine creatinine determination, the urine output, and the GFR. These include the following (Tab. 12.1-2 – Definition of acute renal failure according to RIFLE and AKIN classification):

  • The RIFLE staging system /2/ of the Acute Dialysis Quality Initiative (ADQI) Working Group. The system includes three levels of progressive kidney dysfunction (risk, injury and failure) and two outcomes (loss of function and end stage renal disease). A value obtained within 7 days prior to the suspicion of ARF is taken as reference for assessing the creatinine rise.
  • The Acute Kidney Injury Network (AKIN) criteria preferring the term injury to failure (Tab. 12.1-2).

Since studies have shown that relatively small changes in creatinine are associated with a significant rise in mortality, the AKIN has proposed modifications to the RIFLE criteria that included a threshold of absolute change in serum creatinine of > 0.3 mg/dL (26 μmol/L) to fulfill the criteria within a 48 h period /3/.

In the opinion of many authors there is general agreement that the RIFLE criteria perform well, are a valid and robust tool and have clinical relevance for correlation with patient-centred outcomes /4/. For the diagnosis and classification of ARF a pre morbid serum creatinine baseline value must be known for application as a component of the RIFLE or AKIN criteria. Since a pre morbid creatinine value is often not available, the estimation of a baseline serum creatinine value by use of the Modification of Diet in Renal Disease (MDRD) equation is recommended assuming a lower limit of normal glomerular filtration rate of 75 [mL × min–1 × (1.73 m2)–1]. The use of the MDRD for determining RIFLE category results in false positive rates for AKI of 18% /4/.

The incidence of ARF has increased in recent years, while the mortality rate has remained constant. Up to 30% of intensive care patients develop ARF with mortality rates up to 50%. However, of the surviving patients, less than 5% require renal replacement therapy /2/.

Etiologically, ARF is classified into pre renal, renal and post renal ARF (Tab. 12.1-3 – Etiology of acute renal failure). The cause and laboratory findings in ARF are shown in Tab. 12.1-4 – Acute renal failure: clinical and laboratory findings.

12.1.1.2 Pre renal acute renal failure

Pre renal ARF is by far the most common cause of ARF /6/. The most systemic factor is a response to renal hypo perfusion caused by sepsis and septic shock. The integrity of the kidney tissue is maintained. Pre renal ARF can complicate any other underlying disease that causes a reduction in the effective blood volume. Vasoconstrictor mechanisms are activated by the hypovolemia, in order that the blood pressure be maintained. The kidneys normally respond to changes in renal perfusion pressure through an autoregulatory process which keeps the renal blood flow (RBF) and the glomerular filtration rate (GFR) constant. This occurs with a gradual expansion of the pre glomerular capillaries, mediated by angiotensin, prostaglandins and NO. A simultaneous vasoconstriction of the post glomerular capillaries, mediated by angiotensin II, preserves the glomerular capillary hydrostatic pressure. A tubulo-glomerular feedback mechanism stabilizes the GFR and the delivery of fluid to the distal tubule.

ARF is triggered by drugs that cause a disturbance in the autoregulation of RBF and the GFR, or by renal hypo perfusion following vomiting, diarrhea, bleeding, diuretic therapy or massive burns. Treatment with ACE-inhibitors triggers ARF in 6–23% and in up to 38% of patients with bilateral and unilateral renal artery stenosis, respectively. Angiotensin receptor blockers can also trigger ARF.

12.1.1.3 Acute renal failure

Acute renal failure (parenchymal renal failure), also known as acute kidney injury (AKI) /7/ is a syndrome in which the principal source of damage is within the kidney and typical structural changes can be seen on microscopy /2/.

The following disturbances due to tubular necrosis occur:

  • Decrease in the GFR
  • Reduction in the concentrating capability of the kidneys
  • Proteinuria and interstitial fibrosis
  • Reduced tubular Na+ reabsorption.

Two components are responsible for an acute decrease in the GFR:

  • A vascular component, which comprises pre-glomerular vasoconstriction with a decrease in the GFR, decreased blood flow in the external medulla and an activation of the tubuloglomerular feedback mechanism
  • A tubular component; it involves tubular obstruction, the trans tubular back flow of ultra filtrate, and interstitial inflammation.

Histologically, the tubular cells manifest vacuolization, the loss of brush border membrane and desquamation of the cells into the lumen. Interstitially, edema with mild leukocyte infiltration occurs. As the damage increases, the tubular cells desquamate from the basement membrane and aggregate in the tubular lumen.

In the kidney cortex many tubular cells are injured only to a sub-lethal extent, and may regenerate after restored re perfusion. Following the desquamation of necrotic and apoptotic cells and the accumulation of monocytes, quiescent kidney cells are activated and generate tubular cells. In the next phase poorly differentiated epithelial cells are activated, and they pre-differentiate to tubular cells. In the last phase, survived tubular cells proliferate and the regeneration of the tubule takes place along with the renewal of its regular functions.

12.1.1.4 Post renal acute renal failure

Obstruction to urine output causes the so-called post renal renal failure, which is the most common cause of acute functional impairment in ambulatory patients /7/. Post renal acute renal failure is often secondary to prostatic hypertrophy. Frequent cases are bladder neck obstruction from an enlarged prostate, ureteric obstruction from pelvic tumors or retroperitoneal fibrosis, papillary necrosis or large calculi. In addition, in their medical history these patients report alternating anuria, oliguria and polyuria as well as, occasionally, hematuria.

12.1.1.5 Laboratory findings in acute kidney injury

AKI is diagnosed utilizing with the aid biomarkers listed in Tab. 12.1-5 – Diagnostically significant laboratory tests in acute renal failure. The disturbances of renal tubular flow and epithelial transport result in hyperkalemia, metabolic acidosis, increased creatinine and urea, the formation of isotonic Na+-rich urine, and varying urine output; < 500 mL/day in oliguric ARF and > 500 mL/day in non-oliguric ARF (Tab. 12.1-6 – Urine findings in the etiological groups of acute renal failure).

12.1.2 Chronic kidney disease

Definition

Chronic kidney disease (CKD) is defined as abnormalities of kidney structure or function present for > 3 months, with implications for health. The prevalence of CKD is 10–16% and comprises /13/:

  • Decreased GFR below 60 [mL × min–1 × (1.73 m2)–1]. The GFR is considered to be the best marker of renal function and is decreased in a broad spectrum of kidney diseases. A GFR decrease is associated with a higher risk of CKD complications in comparison to individuals with a normal GFR. Patients with end stage renal disease have a GFR < 15 [mL × min–1 × (1.73 m2)–1]. The GFR < 60 [mL × min–1 × (1.73 m2)–1] for definition of CKD was chosen because it lies just below one half of the GFR value in young adults, namely 125 [mL × min–1 × (1.73 m2)–1].
  • Markers of kidney damage (one or more): albuminuria, urine sediment abnormalities, electrolyte and other abnormalities due to tubular disorders, abnormalities detected by histology, structural abnormalities detected by imaging, history of kidney transplantation.
  • The time frame of 3 months is a measure of the chronic disease. If the time frame cannot be documented, CKD is not confirmed.

The definition of CKD is also valid for newborns and for children up to 18 years of age. However, the following exceptions apply to children below 2 years:

  • The time frame of 3 months does not hold for newborns
  • For the criterion of GFR < 60 [mL × min–1 × (1.73 m2)–1] an age-appropriate value must be utilized
  • For the criterion of albuminuria ≥ 30 mg/24 h, an age-appropriate value must be utilized.

12.1.2.1 Staging of chronic kidney disease

CKD is classified according to the Kidney Disease Global Outcomes (KDIGO) based on cause (C), GFR category (G) and albuminuria category (A). The classification is designated as CGA /13/.

Cause

The cause of CKD is based on presence or absence of systemic disease and the location within the kidney of observed or presumed pathologic anatomic findings (Tab. 12.1-7 – Classification of CKD based on primary renal disease or systemic disease). The cause of disease is included in the classification of CKD because of its fundamental importance in predicting the outcome of CKD and choice of cause-specific treatment.

GFR category

The GFR category is assigned as shown in Tab. 12.1-8 – GFR categories in CKD.

Albumin category

Albuminuria is included as an additional expression of severity of disease not only because it is a marker of the severity of injury but also because albuminuria itself strongly associates with progression of kidney disease (Tab. 12.1-9 – Albuminuria categories in CKD).

12.1.2.2 Prognosis of CKD

In predicting the risk for outcome of CKD the cause of CKD, the GFR category, the albuminuria category, other risk factors and co morbid conditions have to be evaluated /13/. The relative strength of each of these factors for kidney disease endpoints will vary for each complication or outcome of interest. Risk for kidney disease and endpoints is individually driven by an individual patient’s diagnosis.

Independent of the cause of CKD, the GFR and albuminuria are risk multipliers of further complications of CKD, such as overall mortality and mortality due to cardiovascular disease (CVD). On their own, neither the GFR categories nor the albuminuria categories enable prognostic statements. Risk associations for the progression of CKD, CVD-associated mortality and kidney failure are, however, possible with the combination of GFR and albuminuria categories (see Fig. 12.9-1 – Assessment of the course of the glomerular filtration rate (GFR) and of albumin excretion in type 1 diabetes mellitus). The lower the GFR and the higher the degree of albuminuria at the time of the CKD diagnosis, the more marked is the progression of CKD.

12.1.2.3 Clinical symptomatology in CKD

Most renal diseases only manifest clinical symptoms or significant laboratory findings in the CKD stage. Irreversibility is usually already present and the therapy consists of retarding progression to kidney failure. In some diseases, however, the CKD is reversible, or a therapy (immunosuppressive therapy of glomerulonephritis) can achieve a partial improvement in the kidney injury and function.

The decrease in GFR leads to metabolic and endocrine complications such as metabolic acidosis, anemia, bone mineralization disturbances, increased cardiovascular and overall mortality. The final stage of CKD (end stage renal disease, ESRD) is the state of kidney failure that can only be treated with kidney replacement therapy (dialysis or kidney transplant). Proteinuria, hypertension, diabetes mellitus, race, and ethnicity are strong risk factors for progression of CKD to ESRD and the higher ESRD incidence among men than among women is most pronounced in elderly patients. Declines in renal function, measured by creatinine clearance, occur in two thirds of healthy older adults over time but progress to ESRD in only 1–2% of these individuals /14/.

The 5-year survival rates among dialysis patients are low (Europe 40.5%, USA 55.2%) and has remained constant during the past decade. CKD is a powerful risk factor for poor survival. The mortality rates are considerably higher among CKD patients than among the general population (17.7 versus 5.5/100 patient years) /15/.

Cardiovascular disease (CVD) is the single largest cause of mortality, responsible for 40–50% of deaths among CKD patients. CKD patients are more likely to die of CVD than to develop ESRD, with mortality rates estimated to be 10 to 30-fold higher in dialysis patients as compared to the healthy population /15/.

CKD patients have an elevated prevalence of traditional risk factors for cardiovascular mortality such as atrial fibrillation, hypertension, diabetes mellitus, obesity, hypercholesterolemia as well as of newer risk factors like low grade inflammation (CRP ≥ 3 mg/L), abnormal lipoproteins and markers of oxidative stress. However, some CKD patients do not have hypertension or dyslipidemia and nonetheless manifest elevated cardiovascular risk /15/.

Finally, mineral and bone disorders develop early in CKD patients and biochemical abnormalities are common (increased phosphate and parathyroid hormone, decline of vitamin D). Diseases such as insulin resistance and amenorrhea are also diagnosed in CKD. Also meaningful are neurological diseases that cause muscle weakness and sensory disturbances in the extremities and, in acute renal failure, are associated with altered mental status and encephalopathy.

Diabetes mellitus is the most frequent cause of CKD in industrial nations, while in developing countries inflammatory renal disease such as glomerulonephritis and interstitial nephritis are common.

The association of CKD with diseases is described in Tab. 12.1-10 – Chronic kidney disease in association with systemic disease.

12.1.2.4 Progression of CKD and acute kidney injury

Patients with CKD, particularly elderly individuals, are more likely to experience acute renal failure (ARF) and ARF is a risk factor for progression to ESRD /14/. In ischemia associated hypoxia developing ARF is characterized by a temporary decrease in GFR, reduced urine-concentrating ability, proteinuria, impaired Na+ processing and interstitial fibrosis. In elderly individuals, in the absence of other kidney insults glomerular capillary density and interstitial peri tubular vascular density decrease and are exacerbated by acute injury. The progression of CKD is not, apparently, a smoothly proceeding continuous event but rather one that occurs in phases.

In CKD a continuous decline in the number of nephrones and a compensatory hypertrophy of the remaining nephrones develops. The result is, initially, compensatory glomerular hyper filtration, which is contingent upon a rise in the capillary pressure and an increase in capillary flow. With time, however, and as a consequence of elevated capillary pressure, sclerosis of the nephrones along with a decrease in performance occurs.

12.1.2.5 Laboratory findings in CKD, and prediction of CKD progression

In CKD patients GFR and albuminuria should be assessed at least annually. Small fluctuations in GFR are common and are not necessarily indicative of progression. CKD progression is defined based on one of more of the following /13/:

  • Decline in GFR category (G1 ≥ 90; G2 60–89; G3a 45–59; G3b 30–44; G4 15–29; G5 < 15 [mL × min–1 × (1.73 m2)–1]. A certain drop in eGFR is defined as a drop in GFR category accompanied by a 25% or greater drop in eGFR from baseline.
  • Rapid progression is defined as a sustained decline in eGFR of more than 5 [mL × min–1 × (1.73 m2)–1] per year.

See Tab. 12.1-8 – GFR categories in CKD and Tab. 12.1-9 – Albuminuria categories in CKD.

12.1.2.6 Hemodialysis

The incidence of CKD is > 300 per 1 million individuals per year, and the prevalence of individuals with renal replacement therapy is 804 per million. The optimum timing of dialysis treatment is still a matter of debate, and mainly based on clinical judgement and clinical experience. K/DOQI guidelines suggest that the estimation of estimated GFR in conjunction with the evaluation of the nutritional and clinical status of the patient should guide decision making /31/. A problem is, however, that the initiation of hemodialysis at higher, as compared with lower, eGFR values is associated with increased mortality. In the literature widely differing eGFR values were published for the initiation of dialysis, e.g. ≤ 6–15 [mL × min–1 × (1.73 m2)–1]. Since, however, the eGFR is dependent not only on renal function, but also on nutritional status, muscular mass and fluid overload, the European Best Practice Guidelines do not recommend the eGFR as an exclusive marker for the optimal timing of dialysis but rather, the evaluation of the patient’s co morbidities. In addition, the ratio of the urea clearance to the creatinine clearance is recommended (see Section 12.6.5 – Clinical significance).

Inflammation, hyper hydration and CVD are determining mortality factors in dialysis patients. Therefore, the following are determined as risk markers:

  • For the monitoring of hyper hydration, surrogate markers such as inter dialytic weight gain, ultrafiltration rate and blood pressure
  • CRP as inflammation marker
  • NT-proBNP as a stress marker of the cardiac wall
  • Cardiac troponin for the detection of ischemic heart muscle injury.

12.1.3 Glomerulonephritis (GN)

The kidneys are perfused by some 25% of the cardiac output. Daily, 180 liters of primary urine are formed, and this is accomplished by filtration at the glomerular filtration barrier. The filtration barrier in the glomerulus (Fig. 12.1-1 – Structure of the renal filtration mechanism) which separates the blood and urine compartments, enables only the passage of a limited amount of plasma proteins. The filtration barrier is composed of the glomerular endothelium, the glomerular basement membrane (GBM) and the podocyte (glomerular-visceral epithelial cell). Podocytes extend long processes toward the GBM to which they affix by cell surface adhesion proteins (α3β1 integrin and dystroglycan). The foot processes of adjacent podocytes interdigitate and are separated by narrow spaces (30–40 nm) that are bridged by a porous membrane called the slit diaphragm. The diaphragm contains pores freely permeable to plasma proteins. The integrity of the slit diaphragm is one of the principal determinants of the perm-selective properties of the glomerular filtration barrier.

The NPHS1 genes code for the protein nephrin and the NPHS2 genes for podocin. Mutations of the NPHS1 genes are diagnosed in severe forms of the nephrotic syndrome and mutations of the NPHS2 genes in familial segmental glomerulosclerosis and the sporadic steroid refractory nephrotic syndrome in children.

GN is a disease with intraglomerular inflammation and cellular proliferation. It is associated with hematuria. Numerous disorders which, strictly speaking, are believed to be of inflammatory genesis, are concealed under the term GN. Because, however, the transition to non-inflammatory glomerulopathy is smooth, the terms GN and glomerulopathy are often used as synonyms. The distinction between primary and secondary GN is clinically more relevant, however. This is often possible only through the exclusion of a systemic disease or the investigation of a kidney biopsy. Primary and secondary GN with and without proliferative changes are distinguished /32/ (Tab. 12.1-11 – Types of glomerular diseases).

12.1.3.1 Primary glomerulonephritis

In primary GN, also often called idiopathic GN, the origin of the pathogenesis is in the kidneys and the systemic manifestations are the result of the renal dysfunction. The most common form of primary GN is IgA nephropathy /33/. Post-streptococcal GN is also regarded as primary GN, in spite of the fact that, in the strict sense of the term, it cannot be classified as idiopathic.

In IgA nephropathy, IgG antibodies are detected in the blood against only minimally galactosylated IgA subclass 1 antibodies. The concept is that these IgA1 molecules act as auto antigens, triggering the synthesis of IgG autoantibodies. The auto antigens tend toward self-aggregation and the formation of immune complexes with the autoantibodies. The immune complexes have a high affinity for the matrix components fibrinogen and type 4 collagen; they bind and activate mesangial cells and activate the complement system via the lectin pathway. An IgA nephropathy classification has been developed.

12.1.3.2 Secondary glomerulonephritis

Secondary GN results from renal involvement associated with systemic disease. Diabetic nephropathy and lupus nephritis are examples of multisystem disease-dependent GN. Renal lesions are considered diffuse if they affect more than 50% of the glomeruli, and as focal if the number is lower. Global lesions affect the entire nephron, while with segmental lesions only a part of the nephron is involved. Due to leukocyte infiltration, proliferative glomerulopathies have an increased number of glomerular cells. Crescent-shaped glomerular lesions are those in which the Bowman’s capsule is filled with proliferating epithelial cells and infiltrating monocytes.

The clinical course of GN is as follows:

  • Acute nephritic syndrome. This manifests clinically as edema and hypertension within a period of days to a few weeks and can develop into acute kidney failure. These classical clinical symptoms are evident in the proliferative forms of GN.
  • Rapid progressive GN (RPGN) associated with a rapid decline in renal function. RPGN can result in end -stage renal failure within a period of days to a few weeks /32/. The pathological hallmark of this disease is the presence of cellular crescents surrounding most glomeruli. RPGN is, like the acute nephritic syndrome, the consequence of immune-mediated proliferative GN, which can be either of primary or secondary nature occurring as a complication of systemic disease. From a laboratory diagnostic standpoint, the urinalysis is nephritic.
  • Primary chronic GN, a latent form of the disease that progresses slowly over a period of years. The indicators for this form are as follows: elevated creatinine, proteinuria or hematuria, and anemia.

12.1.3.3 Laboratory findings in glomerulonephritis

Increase in creatinine and nephritic urinalysis with macroscopic hematuria (frequent), dysmorphic erythrocytes, erythrocyte casts, sub nephrotic proteinuria (< 3 g/24 h). In non-proliferative forms of GN, such as focal segmental glomerulosclerosis, the clinical and laboratory findings are less pronounced.

In IgA nephropathy, the laboratory findings include mild hematuria, proteinuria of > 0.5 g/24 h and a GFR of > 30 [mL × min–1 × (1.73 m2)–1].

12.1.3.4 Nephrotic syndrome (NS)

The NPHS1 genes code for the protein nephrin, the glomerular slit diaphragm which, along with podocin, regulates signal transmission (Fig. 12.1-1 – Structure of the renal filtration mechanism). Mutations in NPHS1 are responsible for the development of a severe congenital nephrotic syndrome. The nephrotic syndrome is defined as protein excretion of > 3.5 [g × 24 h–1 × (1.73 m2)–1]. Severe proteinuria is present and the consequences are, independent of the underlying disease, uniform: Na+ retention with edema, hyperlipidemia, elevated risk of thrombosis and susceptibility to infections. Diabetic nephropathy is the most frequent cause of the nephrotic syndrome /34/.

The most important non-diabetic kidney diseases associated with a nephrotic syndrome are the primary forms of GN which include minimal change GN, focal segmental glomerulosclerosis, membranous GN, and vasculitis/necrotizing GN.

12.1.3.4.1 Laboratory findings in nephrotic syndrome

Proteinuria, serum albumin < 25 g/L, often only with protein excretion > 10 g/L, also a decrease in antithrombin < 75%, hyperlipidemia with either raised cholesterol only or combined elevated cholesterol and triglycerides. The VLDL, IDL und LDL fraction and Lp(a) are elevated, the HDL fraction is usually normal /36/.

12.1.3.5 Minimal change disease (MCD)

MCD is a podocytopathy and is the cause of the nephrotic syndrome in 76% of affected children and 20% of affected adults. The steroid-resistant form is caused by a mutation in the NPHS2 gene. Under light and fluorescence microscopy, the glomeruli appear to be normal. The clinical presentation is sudden with the nephrotic syndrome and, occasionally, with hematuria and hypertension. Secondary MCD occurs in non-Hodgkin lymphoma and is drug-induced. Medications that can cause MCD are listed in Tab. 12.1-12 – Drugs with tubulo-interstitial, glomerular and vascular toxic effects /35/. Some MCD cases convert into focal segmental glomerulosclerosis.

12.1.3.6 Focal segmental glomerulosclerosis (FSGS)

FSGS is, likewise, a podocytopathy. A few or many glomeruli manifest segmental sclerosis. With immunofluorescence, some of the glomeruli show segmental glomerular IgM and C3. FSGS can be primary in nature and is then based upon a mutation in the NPHS2 gene, or it may be secondary (e.g., drug-induced). FSGS is similar to MCD clinically, although its onset is less sudden, it is associated with nephrotic proteinuria, mild hematuria and, occasionally, hypertension. The frequency of FSGS of the nephrotic syndrome is 8% in children and 15% in adults. Medications can lead to immunological and non-immunological injury. Segmental capillary obliteration occurs due to the accumulation of foam cells, matrix accumulation and adhesion to the Bowman’s capsule.

12.1.3.7 Membranoproliferative glomerulonephritis (MPGN)

MPGN is a pathological entity, characterized by subepithelial immune complex depositions and corresponding changes in the glomerular basement membrane. Histologically, characteristic thickening of the capillary wall is seen. Patients are often in their 4th to 5th decade of life, but one third is below the age of 16 or older than 60. Clinically, the patients present with a nephrotic syndrome, micro hematuria, temporary hypertension and mild renal insufficiency. Membranous GN can also be linked to neoplasms. In elderly patients with neoplasms or a nephrotic syndrome this link should, therefore, be taken into consideration. In addition, in order to exclude multiple myeloma, the urine should be tested for monoclonal immune protein excretion /34/.

12.1.3.8 Vasculitis/necrotizing glomerulonephritis

An inflammation of the vessel wall, the capillaries or the glomeruli is present. Vasculitis of the small kidney vessels leads to necrotizing or crescentic glomerulonephritis (the histological criteria are capillary fibrinoid necrosis and crescents). Based upon immunohistology, this form is differentiated into the categories with antibodies to the glomerular basement membrane, the immune complex form, and pauci-immune vasculitis. Most cases of pauci-immune vasculitis are associated with the presence of anti neutrophil cytoplasmic antibodies (ANCA). ANCA vasculitis can occur in a renally isolated manner or within the context of Wegener’s granulomatosis or Churg-Strauss syndrome. The patients present with rapidly progressing GN with hematuria, erythrocyte casts, proteinuria and acute renal failure. Fever, joint pain and myalgia are often present. Most cases are idiopathic in nature, while some are drug-induced (Tab. 12.1-12 – Drugs with tubulo-interstitial, glomerular and vascular toxic effects).

12.1.4 Tubulo-interstitial disease

The renal tubulo-interstitial compartment is the space between the basement membrane of the glomeruli, the tubuli and the peri tubular capillaries. The compartment comprises 4–7% of the healthy renal cortex and consists of fibroblasts and dendritic cells. The exchange of substances between the blood and the primary urine in the tubular lumen includes the passage through the tubulointerstitium. Tubulointerstitial diseases are differentiated into acute tubular necrosis and tubulointerstitial nephritis. In the latter, acute, chronic and crystal-induced forms and primary and secondary forms are distinguished (Tab. 12.1-13 – Types of tubulointerstitial nephritis).

12.1.4.1 Acute tubular necrosis

Acute tubular necrosis is defined as acute kidney failure with tubular injury in the absence of significant or vascular renal pathology. A sudden rise in creatinine occurs, sometimes in combination with microscopic hematuria and mild proteinuria. The etiology is that of acute kidney failure. Medications that are utilized in cancer therapy play an important role as a cause of acute tubular necrosis.

12.1.4.2 Tubulointerstitial nephritis (TIN)

TIN is characterized by inflammation of the renal interstitium and the tubuli, resulting in interstitial edema, acute tubular injury or interstitial fibrosis with tubular atrophy /35/. Mechanisms that cause TIN can be attributed to direct cytotoxic reactions as well as systemic inflammatory or immunological reactions. Direct cytotoxic mechanisms, as in analgesic nephropathy and lead poisoning-related nephropathy, are dose and time-dependent. Of the functional renal disorders, 5–10% are based upon primary TIN. The largest proportion (85%) is drug-induced, while 10% are due to an infectious process (e.g., Epstein-Barr virus infection) and 5% are idiopathic /38/.

Acute TIN

Clinically, acute TIN is similar to acute tubular necrosis. Histologically, interstitial edema and tubular injury are present, along with a mixed infiltrate composed of mononuclear cells and also, in some cases, of eosinophilic granulocytes (if drug-induced). Clinically, symptoms of hypersensitivity as well as exanthema and eosinophilia are also often diagnosed; these are relatively specific attributes. The causes of acute TIN are medications (Tab. 12.1-12 – Drugs with tubulo-interstitial, glomerular and vascular toxic effects), infections and autoimmune diseases (M. Sjögren). In contrast to acute tubular necrosis, leukocyturia is present and not uncommonly in drug-induced TIN, eosinophiluria of > 5%. The incidence of TIN in patients with ESRD is 26–42% /37/.

Chronic TIN

The chronic TIN involves tubulointerstitial fibrosis, which is an integral feature of the structural changes in chronic progressive renal insufficiency /39/. The accumulation of extracellular matrix in the tubulointerstitial space is mediated by myofibroblasts. These are formed from localized fibroblasts, tubular epithelial cells, periadventitial cells and, probably, from mesenchymal stem cells and endothelial cells as well. The fibrosis usually precedes the tubulointerstitial infiltration with mononuclear inflammatory cells. Proteinuria is one of many triggering events of glomerular or vascular disease, which transfer the disease process to the interstitium. The elevated protein filtration is likely toxic at the level of the tubular cells and induces the activation of chemokines and cytokines and increased expression of adhesion molecules. All of this leads to an augmented influx of inflammatory cells into the interstitium. The result is tubular atrophy with a gradual decline in kidney function.

Crystal-induced TIN

It is not uncommon, since many drugs tend to precipitate as crystals within the tubules (Tab. 12.1-12 – Drugs with tubulointerstitial, glomerular and vascular toxic effects).

12.1.4.3 Laboratory findings in tubulo-interstitial disease

In acute TIN, early symptoms include decreased urine concentration, sterile leukocyturia, leukocyte casts, erythrocyturia, erythrocyte casts, and occasionally macro hematuria /37/. Neither serum creatinine nor the eGFR provide a hint with regard to etiogenesis, but they do allow an important prognostic statement to be made.

In chronic TIN, in addition to a gradual decrease in the GFR, glucosuria, phosphaturia and acidification disorders of urine due to disturbed NH4+ formation may be detectable (see Section 8.8.4 – Disturbances of chloride excretion).

A typical finding, regardless of the etiology of the TIN, is tubular proteinuria, caused by inadequate tubular reabsorption of low molecular weight proteins such as α1-microglobulin. Total protein excretion is < 2.5 g/24 h and, in the chronic form, usually < 1 g/24 h, and in random specimen < 1 g/g creatinine.

The diagnosis of tubular proteinuria can be made semi quantitatively using SDS polyacrylamide gel electrophoresis, and quantitatively with the fractional clearance of β2-microglobulin or α1-microglobulin, relative to the creatinine excretion (see also Section 12.7 – Cystatin C).

12.1.5 Hepatorenal syndrome (HRS)

HRS is a fully reversible impairment of renal function in patients with severe hepatic failure. The prevalence in patients with liver cirrhosis and ascites is 18% after 1 year and increases to 39% at 5 years. Renal failure is a serious complication in patients with advanced liver cirrhosis and occurs in about 20% of patients hospitalized with decompensated liver cirrhosis /40/. In about 70% impaired renal function is caused by pre renal failure due to gastrointestinal hemorrhage, bacterial infection and hypovolemia. In about 30% of cases renal failure is caused by intrarenal etiology. HRS is due to renal hypo perfusion causing reduced filtration fraction. Vasodilation in the splanchnic region is the main trigger of hypo perfusion /40/. The vasodilation is associated with increased formation of vasodilators and vascular hypo responsiveness to vasoconstrictors.

12.1.5.1 Laboratory findings in hepatorenal syndrome

Creatinine based formulae overestimate the true glomerular filtration rate, especially in patients under 50 years and in those with ascites /41/. Refer to Tab. 12.7-2 – Diagnostic significance of cystatin C in impaired renal function.

12.1.6 Acute pyelonephritis

Acute pyelonephritis denotes inflammation of the renal pelvis and kidney /42/. Pyelonephritis usually occurs when enteric bacteria enter the bladder and ascend to the kidneys. Clinical presentation manifests suddenly with signs and symptoms of systemic inflammation (fever, chills, and malaise) and bladder inflammation (urinary frequency, urgency, and dysuria). Approximately 10% of septicemia cases originate from pyelonephritis.

12.1.6.1 Laboratory findings in acute pyelonephritis

The confirmatory test is the urine culture, which yields 10,000/mL or more colony-forming units of a uropathogen, usually E. coli. The white blood cell count shows granulocytosis, urinalysis is positive for leukocyte esterase and nitrites. The estimated GFR is decreased.

References

1. Alscher MD, Erley C, Kuhlman MK. Acute renal failure of nosocomial origin. Dtsch Arztebl Int 2019; 116: 149–58.

2. Bellomo R, Ronco C, Kellum JA, et al. The Second International Consensus Conference of the Acute Dialysis Quality Initiative (ADQI) Group. Acute renal failure – definition, outcome measures, animal models, fluid therapy and information technology needs. Crit Care 2004; 8: R204–R212.

3. Levin A, Kellum JA, Mehta RL. Acute kidney injury: toward an integrated understanding through development of a research agenda. Acute Kidney Injury Network (AKIN). Clin J Am Soc Nephrol 2008; 3: 862–3.

4. Bagshaw SM, Uchino S, Cruz D, Bellomo R, Morimatsu H, Morgera S, et al. A comparison of obseved versus estimated baseline creatinine for determination of RIFLE class in patients with acute kidney injury. Nephrol Dial Transplant 2009; 24: 2739–44.

5. Murugan R, Karajala-Subramanyam V, Lee M, Yende S, Kong L, Carter M, et al. Acute kidney injury in non-severe pneumonia is associated with an increased immune response and lower survival. Kidney Int 2010; 77: 527–35.

6. Weiss R, Meersch M, Pavenstädt HJ, Zarbock A.Acute kidney injury– a frequently underestimated problem in perioperative medicine. Dtsch Arztebl Int 2019; 116: 833–42.

7. Legrand M, Rossignol P. Cardiovascular consequences of acute renal failure. N Engl J Med 2020; 382, 23: 2238–47.

8. Kaddourah A, Basu RK, Bagshaw SM, Goldstein SL. Epidemiology of acute kidney injury in critically ill children and young adults. N Engl J Med 2017; 376, 1: 11–20.

9. Uchino S, Kellum JA, Bellomo R, et al. Acute renal failure in critically ill patients: a multinational multicenter study. JAMA 2005; 294: 813–8.

10. Bosch X, Poch E, Grau JM. Rhabdomyolysis and acute kidney injury. N Engl J Med 2009; 361: 62–72.

11. Gines P, Schrier RW. Renal failure in cirrhosis. N Engl J Med 2009; 361: 1279–90.

12. Endre ZH, Pickering JW. Outcome definitions in non-dialysis intervention and prevention trials in acute kidney injury (AKI). Nephrol Dial Transplant 2010; 25: 107–18.

13. Kidney Disease: Improving Global Outcome (KDIGO) CKD Work Group. KDIGO clinical practice guideline for the evaluation and management of chronic kidney disease. Kidney Int Suppl 2013: 3: 1–150.

14. Anderson S, Halter JB, Hazzard WR, Himmelfarb J, McFarland Horne F, Kaysen GA, et al. Prediction, progression, and outcomes of chronic kidney disease in older adults. J Am Soc Nephrol 2009; 20: 1199–1209.

15. Bansall S, Zelnick L, Shlipak M, Anderson A, Christenson R, Doe R, et al. Cardiac stress biomarkers and chronic kidney disease progression: the CRIC study. Clin Chem 2019; 65, 11: 1448-57.

16. Cirillo P, Sautin YY, Kanellis Y, Kang DH, Gesualdo L, Nakagawa T, et al. Systemic inflammation, metabolic syndrome and progressive renal disease. Nephrol Dial Transplant 2009; 24: 1384–7.

17. Kurella M, Lo JC, Chertow GM. Metabolic syndrome and the risk for chronic kidney disease in US adults. J Am Soc Nephrol 2005; 16: 2134–40.

18. Adler A, et al. Development and progression of nephropathy in type 2 diabetes: the United Kingdom Prospective Diabetes Study (UKPDS 64). Kidney Int 2003; 63: 225–32.

19. Atkins RC, Zimmer P. Diabetic kidney disease: act now or pay later. Nephrol Dial Transplant 2010: 25: 331–3.

20. Groop PH, Thomas MC, Moran JL, et al. The presence and severity of chronic kidney disease predicts all-cause mortality in type 1 diabetes. Diabetes 2009; 58: 1651–8.

21. Hovind P, Rossing P, Tarnow L, et al. Serum uric acid as a predictor for development of diabetic nephropathy in type 1 diabetes: an inception cohort study. Diabetes 2009; 58: 1668–71.

22. Ekbohm P, Damm P, Feldt-Rasmussen PB, et al. Pregnancy outcome in type 1 diabetic women with microalbuminuria. Diabetes Care 2001; 24: 1739–44.

23. Krishnan AV, Kiernan M. Neurological complications of chronic kidney disease. Nat Rev Neurol 2009; 5: 542–51.

24. Matsushita K, Selvin E, Bash LD, Franceschini N, Astor BC, Coresh J. Change in estimated GFR associates with coronary heart disease and mortality. J Am Soc Nephrol 2009; 20: 2617–24.

25. Bansal S, Zelnick L, Shlipak MG, Anderson A, Christenson R, Deo R, et al. Cardiac stress biomarkers and chronic kidney disease progression: The CRIC study. Clin Chem 2019; 65 (11): 1448–57.

26. Munkhaugen J, Lydersen S, Romundstad PR, Wideroe TE, Vikse BE, Hallan S. Kidney function and future risk for adverse pregnancy outcomes: a population-based study from HUNT II, Norway. Nephrol Dial Transplant 2009; 24: 3744–50.

27. Woodward A, McCann S, Al-Jubori M. The relationship between estimated glomerular filtration rate and thyroid function: an observational study. Ann Clin Biochem 2008; 45: 515–7.

28. Boudville N, Salama M, Jeffrey GP, Ferrari P. The inaccuracy of cystatin C and creatinine-based equations in predicting GFR in orthoptic liver transplant recipients. Nephrol Dial Transplant 2009; 24: 2926–30.

29. Ferrari P, Xiao J, Ukich R, Irish A. Estimation of glomerular filtration rate: does haemoglobin discriminate between ageing and true CKD? Nephrol Dial Transplant 2009; 24: 1828–33.

30. Ware AE, Rees RC, Sarnaik SA, Iyer RV, Alvarez OA, Casella JF, et al. Renal function in infants with sickle cell anemia: baseline data from the BABY HUG trial. J Pedriatr 2010; 156: 66–70.

31. Hemodialysis Adequacy 2006 Working Group. Clinical practice guidelines for hemodialysis adequacy, update 2006. Am J Kidney Dis 2006; 48 (suppl 1): pg. 2 – pg. 90.

32. Hricik DE, Chung-Park M, Sedor JR. Glomerulonephritis. N Engl J Med 1998; 339: 888–99.

33. Wyatt RJ, Julian BA. IgA nephropathy. N Engl J Med 2013; 368: 2402–14.

34. Orth SR. Ritz E. The nephrotic syndrome. N Engl J Med 1998; 338: 1202–11.

35. John R, Herzenberg AM. Renal toxicity of therapeutic drugs. J Clin Pathol 2009; 62: 505–15.

36. Joven J, Villabona C, Villela F, et al. Abnormalities lipoprotein metabolism in patients with nephrotic syndrome. N Engl J Med 1990; 323: 579–84.

37. Rastegar A, Kashgarian M. The clinical spectrum of tubulo-interstitial nephritis. Kidney Int 1998; 54: 313–27.

38. Schwarz A, Krause PH, Kunzendorf U, Keller F, Distler A. The outcome of acute interstitial nephritis: risk factors for the transition from acute to chronic interstitial nephritis. Clin Nephrol 2000; 54: 179–90.

39. Strutz FM. EMT and proteinuria as progression factors. Kidney Int 2009; 75: 475–81.

40. Lenz K, Buder R, Kapun L, Voglmayr M. Treatment and management of ascites and hepatorenal syndrome: an update. Ther Adv Gsatroenterol 2015; 8: 83–100.

41. Francoz C, Prie D, AbdelRazek W, Moreau R, Mandot A, Belghitti J, et al. Inaccuracies of creatinine and creatinine based equations in candidates for liver transplantation with low creatinine: impact on the model for endstage liver disease score. Liver Transplant 2010; 16: 1169–77.

42. Johnson JR, Russo TA. Acute pyelonephritis in adults. N Engl J Med 2018; 378: 48–59.

12.2 Glomerular filtration rate (GFR)

Lothar Thomas

Kidney damage refers to a broad range of abnormalities observed during clinical assessment, which may be insensitive and non-specific for the cause of disease but may precede reduction in kidney function. Most kidney diseases do not have symptoms or findings until later in their course and are detected only when they are chronic /1/.

The kidney has excretory, endocrine and metabolic functions. The GFR is one component of the excretory function, but is widely accepted as the best overall index of kidney function because it is generally reduced after widespread structural damage and most other kidney functions decline in parallel with GFR in chronic kidney disease (CKD) /1/. Excretory, endocrine and metabolic functions decline together in most CKDs. This applies to:

  • Water and electrolyte metabolism
  • Acid-base homeostasis
  • The synthesis of 1,25 (OH)2D and erythropoietin
  • The metabolism of low molecular weight proteins and insulin
  • The excretion of metabolites like creatinine, urea, uric acid, and phosphate.

With reduction of the GFR due to CKD, all these functions are affected; a process that proceeds in parallel and, likely, in intervals rather than in a continuous manner /1/. It therefore makes sense to measure the GFR as the equivalent of renal function /1/. The KDIGO refers to a GFR < 60 [mL × min–1 × (1.73 m2)–1] as decreased GFR and a GFR < 15 [mL × min–1 × (1.73 m2)–1] as kidney failure. Physiologically, the GFR decreases with age. In individuals over 50 years, this occurs at the rate of 13 [mL × min–1 × (1.73 m2)–1] per decade of life. Thus, some elderly people have a GFR < 60 [mL × min–1 × (1.73 m2)–1] and a prognostic dilemma emerges in the range of 60–45 [mL × min–1 × (1.73 m2)–1].

12.2.1 Determination of GFR using exogenous filtration markers (measured GFR; mGFR)

GFR is measured indirectly through the concept of clearance, which is defined as the equivalent of plasma volume from which a filtration marker would have to be totally removed to account for its rate of excretion in urine per unit of time. Clearance is calculated by dividing the excretion rate of a filtration marker by its plasma concentration (Cx = UxV/Px) where Ux and Px are urine and plasma concentrations respectively, of substance X, and V is urine flow rate (Tab. 12.2-1 – Clearance formula). When the filtration marker is freely filtered by the kidney, then Cx = GFR. Exogenous filtration markers are inulin, iothalamate, iohexol, EDTA and DTPA (Tab. 12.2-2 – Determination of measured GFR using exogenous filtration markers (mGFR)). The gold standard for determining the mGFR is inulin clearance, but this is the most complex method. Due to their complexity, methods that use exogenous filtration markers are usually only employed as confirmatory tests.

12.2.1.1 Reference interval

Refer to Ref. /68 / and Tab. 12.2-3 – GFR reference intervals based on inulin clearance. The mGFR is associated with considerable inter individual and intraindividual variability. The day-to-day variability depends on the diurnal variation, protein intake and physical activity. Healthy young white males have an average mGFR of 130, while that of females is 120 [mL × min–1 × (1.73 m2)–1]. The lower limit for men is 90 and for women is 85 [mL × min–1 × (1.73 m2)–1].

With increasing age, the mGFR decreases, but there is no age group-specific reference interval for adults, particularly not for elderly and old people. The US National Kidney Foundation has, therefore, set the threshold for chronic kidney disease, independent of age, at < 60 [mL × min–1 × (1.73 m2)–1/7/.

The GFR is based on the body surface area of 1.73 m2 of a person weighing 75 kg and differentiated into six categories according to the KDIGO (Tab. 12.2-4 – GFR categories according to KDIGO).

12.2.2 Evaluation of GFR using endogenous filtration markers

GFR estimating equations are recommended for the evaluation of kidney function for routine clinical care.

Current clinical guidelines /1/ recommend:

12.2.2.1 Determination of eGFRcr in adults

KIDGO recommendations for clinical laboratories /1/:

  • Measure serum creatinine using a specific assay with calibration traceable to the international standard reference materials and minimal bias compared to isotope-dilution mass spectrometry reference methodology
  • Report eGFRcr in addition to the serum creatinine concentration in adults and specify the equation used whenever reporting eGFRcr
  • Report eGFRcr in adults using the CKD-EPI creatinine equation. An alternative creatinine-based GFR estimating equation is acceptable if it has been shown to improve accuracy of GFR estimates compared to the CKD-EPI creatinine equation.
  • Report serum creatinine concentration be rounded to the nearest whole number when expressed as standard international units (μmol/L) and rounded to the nearest 100th of a whole number when expressed as conventional units (mg/dL)
  • When reporting eGFRcr the result be rounded to the nearest whole number and relative to a body surface area of 1.73 m2 in adults using the units mL/min/1.73 m2
  • Understand clinical settings in which eGFRcr is less accurate
  • Values of eGFRcr < 60 [mL × min–1 × (1.73 m2)–1] should be reported as decreased.
12.2.2.1.1 Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) creatinine equation

The CKD-EPI creatinine equation is employed for adults and applies the variables of age, gender and ethnicity.

Equation: eGFR [mL × min–1 × (1.73 m2)–1] = 141 × min (SCr /k, 1)α × max (SCr /k, 1)–1,209 × 0.993age × 1.018 [if female] × 1.159 [if black]

Explanation: k is 0.7 for females and 0.9 for males, α is –0.329 for females and –0.411 for males; min indicates the minimum for SCr/k or 1; max indicates the maximum for SCr/k or 1.

CKD-EPI creatinine equations, specified for gender and creatinine value, are shown in Tab. 12.2-5 – CKD-EPI creatinine equations, specified for gender and creatinine concentration. Based on these equations, the GFR and the progression of renal impairment is differentiated into the G categories specified in Tab. 12.2-4 – GFR categories according to KDIGO.

12.2.2.2 Determination of eGFRcr in children

The criteria for CKD of GFR < 60 [mL × min–1 × (1.73 m2)–1] does not apply to children below 2 years of age in whom an age appropriate value should be applied. Since it is not until 2 years of age that one expects to see body surface area adjusted to GFR values comparable to seen in the adult the CKD-EPI equation can not be applied in children of lower age. The most robust pediatric eGFR equations, which have been derived from iohexol clearance, emerge from the CKDs study (Tab. 12.2-6 – Equations for estimation of GFR in children using serum creatinine/1/. Values of eGFRcr < 60 [mL × min–1 × (1.73 m2)–1] should be reported as decreased if evaluated with pediatric equations in children below the age of 2 years.

12.2.2.3 Determination of eGFRcys in adults

KDIGO suggests measuring cystatin C in adults with eGFRcr 45–59 [mL × min–1 × (1.73 m2)–1] who do not have markers of kidney damage in cases where confirmation is required. Use a GFR estimating equation to derive GFR from serum cystatin C (eGFRcys) rather than relying on the serum cystatin C concentration alone.

KDIGO recommendations for clinical laboratories /1/

  • Measure serum cystatin C using an assay with calibration traceable to the international standard reference material
  • Report eGFR from cystatin C in addition to the serum cystatin C level in adults and specify the equation used whenever reporting eGFRcys and eGFRcr-cys
  • Report eGFRcys and eGFRcr-cys in adults using the CKD-EPI cystatin C equations, respectively, or alternative cystatin C-based estimating equations if they have been shown to improve accuracy of GFR estimates compared to the CKD-EPI eGFRcys and CKD-EPI eGFRcr-cys
  • Report serum cystatin C concentration rounded to the nearest 100th whole number when expressed as conventional units (mg/dL)
  • When reporting eGFRcys and eGFRcr-cys the result be rounded to the nearest whole number and relative to a body surface area of 1.73 m2 in adults using the units mL/min./1.73 m2
  • Levels of eGFRcys and eGFRcr-cys < 60 [mL × min–1 × (1.73 m2)–1] should be reported as decreased.
12.2.2.3.1 Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) cystatin C equation

The equation is employed for adults and applies the variables of age, gender and ethnicity /1/.

Equation: eGFRCys [mL × min–1 × (1.73 m2)–1] = 133 × min (SCys /0.8, 1)–0.499 × max (SCys /0.8, 1)–1.328 × 0.996Age × 0.932 [if female].

Explanation: serum cystatin C (SCys) is expressed in mg/L; min indicates the minimum for SCys /0.8 or 1; max indicates the maximum for SCys /0.8 or 1.

Tab. 12.2-7 – CKD-EPI cystatin C equations for male and female persons shows the CKD-EPI cystatin C equation as a function of the cystatin C value.

12.2.2.4 Determination of the eGFRcys in children

The advantage of the cystatin C-based equation over the creatinine-based equation is that it is less subject to the effects of age, sex, and race. Cystatin C should be determined immunonephelometrically. The serum cystatin C value should be reported together with the eGFRcys. The eGFRcys is calculated according to the equation /9/:

eGFRcys [mL × min–1 × (1.73 m2)–1] = 70.69 × (Scys)0.931

The equation has good accuracy; 82.6% and 37.6% of the children fall within the 30% range and 10% range of the iohexol clearance. The majority of children with a GFR < 60 [mL × min–1 × (1.73 m2)–1] had either structural abnormalities of the kidneys or kidney damage, which become apparent with urine or serum analyses. In contrast to adults, an isolated GFR reduction occurs rarely.

12.2.2.5 Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) creatinine-cystatin C equation

The eGFRcr-cys is employed in adults; the variables are age, gender and ethnicity /10/. GFR estimates based on both filtration markers provides more precise GFR estimates, which may be useful as a confirmatory test for the diagnosis of CKD with a decreased eGFRcr

Equation: eGFR [mL × min–1 × (1.73 m2)–1] = 135 × min (SCr /k, 1)α × max (SCr /k, 1)–0.601 × min (SCys /0.8, 1)–0.375 × max (SCys /0.8, 1)–0.711 × 0.995Age × [0.969 in female] × [1.08 in male]

Explanation:

  • Serum creatinine is expressed in mg/dL
  • cystatin C in mg/L
  • k is 0.7 for females and 0.9 for males
  • α is –0.248 for females and –0.207 for males
  • min indicates the minimum for SCr/k or 1
  • max indicates the maximum for SCr/k or 1.

The eGFRcr-cys is a more powerful predictor of the course of CKD than is the eGFRcr. This relates particularly to the frequency of events and mortality in patients with cardiovascular disease and an eGFRcr of 45 to 60 [mL × min–1 × (1.73 m2)–1]. Equations, specified for gender, creatinine and cystatin C, are shown in Tab. 12.2-8 – CKD-EPI eGFRCr-Cys equations.

Data from the National Health and Nutrition surveys suggest that 3.6% of U.S. adults would be classified as having chronic kidney disease solely on the basis of a creatinine based GFR estimate of 45–59 [mL × min–1 × (1.73 m2)–1]. In a study /10/ participants whose eGFRcr was 45–74 [mL × min–1 × (1.73 m2)–1] , the eGFRcr-cys equation improved the classification of measured GFR as either 60 [mL × min–1 × (1.73 m2)–1] or greater than or equal to 60 [mL × min–1 × (1.73 m2)–1] and correctly classified 16.9% of those with an eGFRcr of 45 to 60 [mL × min–1 × (1.73 m2)–1] as having a GFR of ≥ 60 [mL × min–1 × (1.73 m2)–1].

Individuals with eGFR below 60 [mL × min–1 × (1.73 m2)–1] have markedly elevated risks for death, cardiovascular disease and end stage renal disease compared with individuals with eGFR > 60 [mL × min–1 × (1.73 m2)–1/1/. The consensus of the KDIGO Work Group was therefore that the large group of individuals with eGFRcr 45–59 [mL × min–1 × (1.73 m2)–1] without markers of kidney damage, but with eGFRcr-cys ≥ 60 [mL × min–1 × (1.73 m2)–1] could be considered not to have CKD.

12.2.3 Progression of CKD

In patients with CKD there is variability in the presence or rate of decline of kidney function /1/. The rate at which the decline occurs also varies based on the underlying population, cause of CKD, presence of albuminuria, co morbidities, age and kidney damage markers (Tab. 12.2-9 – Kidney damage markers).

12.2.3.1 Definition, identification, and prediction of CKD progression

The annual decline in GFR in various populations is shown in Tab. 12.2-10 – Annual decline of GFR in various populations.

Progression of CKD is based on one or more of the following /1/:

  • Decline in GFR category (Tab. 12.2-4 – GFR categories according to KDIGO). A certain decrease in eGFR is defined as a drop in GFR category accompanied by a 25% or greater decline in eGFR from baseline
  • Rapid progression is defined as a sustained decline in eGFR of more than 5 [mL × min–1 × (1.73 m2)–1] and year
  • The confidence in assessing progression is increased with increasing number of serum creatinine measurements and duration of the follow-up.

Factors associated with CKD progression:

  • Factors associated with CKD progression are: cause of CKD, level of GFR, level of albuminuria, age, sex, race/ethnicity, elevated blood pressure, hyperglycemia, dyslipidemia, smoking, obesity, history of cardiovascular disease, and ongoing exposure to nephrotic agents.

Evaluation of CKD progression:

  • Assess GFR and albuminuria at least annually in patients with CKD. Assess GFR and albuminuria more often for individuals at higher risk progression, and/or where measurement will impact therapeutic decisions.
  • Recognize that small fluctuations in GFR are common and are not necessarily indicative of progression.

12.2.4 Influence factor GFR decrease

With a decrease in GFR, reduced excretion and metabolism of endogenous and exogenous substances, as well as potentially toxic reactions, may occur. Examples are shown in Tab. 12.2-11 – Influencing variables in chronic kidney disease.

12.2.5 Referral to specialists

KDIGO recommends referral to specialist kidney services for people with CKD in the following circumstances /1/:

  • Acute renal failure or abrupt sustained fall in GFR
  • GFR below 30 [mL × min–1 × (1.73 m2)–1]
  • A consistent finding of significant albuminuria (albumin/creatinine ratio ≥ 300 mg/g (≥ 30 mg/mmol) or albumin excretion ≥ 300 mg/24 h
  • Progression of CKD
  • Urinary red cell casts, erythrocytes > 20 per high power field sustained and not readily explained
  • CKD and hypertension refractory to treatment with 4 or more antihypertensive agents
  • Persistent abnormalities of serum potassium
  • Recurrent extensive nephrolithiasis
  • Hereditary kidney disease.

12.2.6 Indexing GFR for body surface area

The GFR is expressed in [mL × min–1 × (1.73 m2)–1]. Indexing GFR for body surface area (BSA) is meaningful in individuals with a normal constitution, but not in obesity or cachexia /12/. Examples:

  • In obesity, a GFR indexed on BSA describes erroneously low clearance values. Thus, in a cohort of obese individuals, the measured GFR was 101 ± 24 mL/min., but only 76 ± 16 [mL × min–1 × (1.73 m2)–1], indexed on the BSA. The estimated GFR according to the MDRD equation was 90 ± 22 [mL × min–1 × (1.73 m2)–1].
  • In individuals with cachexia, a GFR indexed on BSA describes erroneously high clearance values. Thus, the measured GFR was 68 mL/min., but indexed on the BSA it was 80 [mL × min–1 × (1.73 m2)–1].

12.2.7 Pathophysiology

The mean number of nephrones is 860,000 ± 370,000 per kidney. The mean single-nephron GFR is 80 ± 40 nL per minute /13/. A higher single-nephron GFR is independently associated with larger nephrones and more glomerulosclerosis and arteriosclerosis than would be expected for age. The single-nephron GFR varies little according to age, sex and height (if ≤ 190 cm), despite substantial variation in the number of nephrones. The number of nephrons declines with age, owing to nephrosclerosis; but the single-nephron GFR in the remaining non sclerotic glomeruli does not increase with age.

Certain acquired risk factors for CKD (e.g., obesity) or inherent risk factors for CKD (e.g., family history for end-stage renal disease) are associated with a higher single-nephron GFR. Obesity is a risk factor for CKD and increases total GFR /13/. Nephrosclerosis exceeding that expected for age decreases the number of nephrons but is generally accompanied by a compensatory higher single-nephron GFR, which maintains the total GFR /13/.

CKD, which is defined as an eGFR below 60 [mL × min–1 × (1.73 m2)–1] and/or an albuminuria ≥ 30 mg/24 h, is associated with an increased cardiovascular disease mortality risk. Findings support the concept that eGFR and albuminuria are, over the entire range, associated with cardiac injury /14/.

References

1. Kidney Disease: Improving Global Outcome (KDIGO) CKD Work Group. KDIGO clinical practice guideline for the evaluation and management of chronic kidney disease. Kidney Int Suppl 2013: 3: 1–150.

2. Levey AS, Eckfeldt JH. Using glomerular filtration rate estimating equations: clinical and laboratory considerations. Clin Chem 2015; 61: 1226–9.

3. Davies DF, Shock NW. The variability of measurement of inulin and diodrast tests of kidney function. J Clin Invest 1950; 249: 491–5.

4. Stevens LA, Levey AS. Measured GFR as a confirmatory test for estimated GFR. J Am Soc Nephrol 2009; 20: 2305–13.

5. James T, Lewis AV, Tan GD, Altmann P, Taylor RP, Levy JC. Validity of simplified protocols to estimate glomerular filtration rate using iohexol clearance. Ann Clin Biochem 2007; 44: 369–76.

6. Piepsz A, Tondeur M, Ham H. Revisiting normal 51Crethylenediaminetetraacetic acid clearance values in children. Eur J Nucl Med Mol Imaging 2006; 33: 1477–82.

7. National Kidney Foundation-K/DOQ1. Clinical practice and guidelines for chronic kidney disease: evaluation, classification, and stratification. Am J Kidney Dis 2002; 39 (Suppl 1): pg. 1–266.

8. Schwartz GJ, Munoz A, Schneider MF, Mak RH, Kaskel F, Warady BA, et al. New equations to estimate GFR in children with CKD. J Am Soc Nephrol 2009; 20: 629–37.

9. Schwartz GJ, Schneider MF, Maier PS, Moxey-Mims M, Dharnidharka VM, Warady BA, et al. Improves equations estimating GFR in children with chronic kidney disease using an immunonephelometric determination of cystatin C. Kidney Int 2012; 82: 445–53.

10. Inker LA Schmidt CH, Tighiouart H, Eckfeld JH, Feldman HI, Kusek JW, et al. Estimating glomerular filtration rate from serum creatinine and cystatin C. N Engl J Med 2012; 367: 20–9.

11. Furth SL, Abraham AG, Jerry-Flunker J, Kaskel F, Make R, Schwartz G, et al. Metabolic abnormalities, cardiovascular disease risk factors, and GFR decline in children with chronic kidney disease. Clin J Am Soc Nephrol 2011; 6: 2132–6.

12. Delanaye P, Mariat C, Cavalier E, Krzesinski JM. Errors induced by indexing glomerular filtration rate for body surface area: reductio ad absurdum. Nephrol Dial Transplant 2009; 24: 3593–6.

13. Denic A, Mathew J, Lerman LO, Lieske JC, Larson JJ, Alexander MP, et al. Single-nepron glomerular filtration rate in healthy adults. N Engl J Med 2017; 376: 2349–57.

14. Martens RJH, Kimenai DM, Kooman JP, Stehouwer CDA, Tan FES, Bekers O, et al. Estimated glomerular filtration rate and albuminuria are associated with biomarkers of cardiac injury in a population-based cohort study: The Maastricht study. Clin Chem 2017; 63: 887–97.

12.3 Urinalysis

Christian Thomas, Lothar Thomas

In kidney and urinary tract patients, an effective diagnostic strategy in urine examinations should start from medical need followed by standard procedures for collection, transport, and examinations. Standardized procedures are needed for consistent reference intervals and interpretation of results /1/. Procedures for urine collection, transport and analysis are published in the European Analysis Guidelines /2/. The guidelines recommend a diagnostic algorithm for urinalysis (Fig. 12.3-1 – Algorithm for urinalysis in individuals with suspected renal and urinary tract disorders).

12.3.1 Indication

Medical indications for urinalysis according to the European Urinalysis Guidelines /2/:

  • Suspicion or clinical evidence of acute or chronic kidney disease, either primary or secondary to a systemic disease such as diabetes mellitus, metabolic syndrome, hypertension, gestosis of pregnancy, drug abuse, autoimmune disease, as well as the assessment of their course
  • Decrease in the glomerular filtration rate < 60 [mL × min–1 × (1.73 m2)–1]
  • Suspicion, clinical symptoms or situations suggestive of urinary tract infection as well as the assessment of its course
  • Suspicion of post renal disease and the assessment of its course (e.g., kidney stones, urinary tract obstruction)
  • Glucosuria in patient groups (e.g., pregnant women, children, emergency patients)
  • Assessment of the course in diabetics (e.g. children, in addition to blood glucose monitoring, for the detection of glucosuria and ketonuria)
  • Detection and assessment of the course of a metabolic disorder in patients with vomiting, diarrhea, acidosis/alkalosis, ketosis, or recurrent stone formers.

12.3.2 Specimen

Preparing the patient prior to specimen collection, selecting the appropriate type of urine specimen, using the best possible means to transport the sample, and starting the urinalysis within a reasonable amount of time are all important pre analytical requirements for obtaining accurate urinalysis results.

12.3.2.1 Patient preparation

Preparation must begin with the patient being instructed on the evening before the specimen is to be collected. The patient should be given the following information:

  • The reasons why the urine specimen needs to be collected
  • What type of urine specimen is to be collected
  • The procedures to be followed when collecting the urine specimen. The information should ideally be given to the patient both verbally and in writing. Detailed information on urine collection procedures can be found in reference /2/.

The information provided to the patient depends on the tests that are indicated.

  • For example /2/, if a sensitive urine screening is required, the urine must be highly concentrated with a urinary flow rate of no more than 20–50 mL/h. In this case, it is important that the patient fast for 8–12 hours before the specimen is collected in order to reduce diuresis.

12.3.2.2 Urine specimens

A first morning urine specimen is collected.

  • If the morphology of the particulate components (erythrocytes, leukocytes, or casts) is important, then urine that has incubated only for 1–2 hours in the bladder is preferred.

A second morning urine specimen suffices for the following purpose:

  • If bacterial infection is supposed, the bacteria should be given 4–8 hours of growth time in the bladder in order to ensure a high analytic sensitivity. First morning urine is therefore the preferred type of specimen. Antibiotics should be discontinued beforehand.

A 24 hour urine specimen is preferred if calcium excretion is to be measured, since calcium excretion while the patient is in bed is over twice as high as in the middle of the day.

In order to keep the urine sample as free as possible of contaminants, patients should refrain from sexual intercourse for 1 day before the urine specimen is collected. This is because contamination of the urine with vaginal or prostate secretions and semen cause increased cell counts and protein levels.

The following time-related urine specimens are used for urinalysis /2/:

  • First-void urine: this is the first urine of the morning after an overnight bed rest. The patient should not take fluids during the night. The urine collected must have remained in the bladder for at least 4 hours, even if the bladder was emptied during the night. The first morning void urine is the standard urine specimen used for urinalysis since it is concentrated and acidic. Compared with daytime urine samples, these specimens contain better preserved particulate components. Bacterial growth is also more significant due to the long time the urine remains in the bladder. First morning urine specimens are most often collected in clinics, but outpatient collection is also possible if the urine is brought in the laboratory quickly for testing.
  • Second-void urine: this is a single urine specimen collected 2–4 hours after voiding the first morning urine. The quality of second-void urine is affected by movement before the specimen is collected, as well as by eating and drinking. To improve the quality of the urine, it is recommended that a glass of about 200 mL of water be taken after 10:00 p.m., with the patient subsequently fasting until the specimen is collected. The second morning urine is used for testing on outpatients.
  • Timed urine collection: 24-hour urine collection is frequently used. Collection can begin at any time. The bladder must be emptied before the collection starts; all urine for the next 24 hours is then voided into the collection container.
  • Overnight urine collection: before the patient goes to bed, the bladder is emptied and the time recorded. All urine passed during the night is collected in the urine container and the time and total volume are recorded in the morning.
  • Random urine specimens: these spot urine specimens can be obtained at any time of the day, and no reference can therefore be made to the time of day, volume or patient preparation. This type of sample should only be used in acute situations, and caution should be exercised when evaluating the results since they are associated with an elevated incidence of false-positive or false-negative results.

12.3.2.3 Procedures for urine collection

When performing microbiology testing on urine, the procedures used to collect the urine specimens are important because the specimen must not be contaminated with commensal bacteria. A distinction is made between the following types of urine /2/:

  • Mid-stream urine: the first portion of the urine is discarded due to contamination from commensal bacteria in the urethra; then, 50–100 mL of urine is passed into the specimen cup. The remainder of the urine is also discarded. The area around the urethra on the glans penis or vaginal introitus is washed with water prior to urination. In this way, the number of false-positive bacterial cultures can be reduced by 20% /2/.
  • First-void urine: this type of urine is suitable for molecular biology assays for(e.g., Chlamydia trachomatis) but not for determining bacterial counts
  • Single catheter urine: is collected after the insertion of a bladder catheter. This method of collection is particularly suited for children with no bladder control.
  • Indwelling catheter urine: indwelling catheter urine is collected when a catheter is replaced or by puncturing an existing indwelling catheter. The collection bag must not be punctured.
  • Suprapubic aspiration urine: this type of urine can be used to confirm the presence of a urinary tract infection.
  • Bag urine: this method is used with children. The urine specimen should be collected within one hour of the bag being attached onto the patient.
  • Urostomy specimens (urine specimens from an ileal conduit): since chronic infection and bleeding are common in urostomies, it is recommended that the urine be collected after a sterile catheter has been used to clean the stoma.

12.3.2.4 Collection container, preservation of sample

Collection container

The collection container must have a volume of at least 50–100 mL, as well as an opening of at least 5 cm to allow both males and females to urinate directly into the container. For 24-hour total urine samples, the volume of the container must be 2–3 liters, and the contents must be protected from light.

Sample preservation

No stabilization is required for urine chemistry testing with test strips, as long as the test is performed within 24 hours and the urine is stored at 4–8 °C. Failure to refrigerate the urine frequently results in a false-positive nitrite reaction. For urine particle analysis, the urine must be refrigerated immediately if it cannot be tested within one hour of collection. However, the leukocyte count is questionable after 2–4 hours, even if the urine has been refrigerated /3/. For bacterial tests, the urine must not be more than 2 hours old. If testing cannot be performed within this time, the sample can be stored for up to 24 hours at 4–8 °C. Adding boric acid (1 g per liter of urine) makes it possible to stabilize the leukocyte and bacteria counts for 24 hours at + 20 °C. However, borate will inhibit the growth of Pseudomonas spp.

12.3.2.5 Visual inspection and odor of urine

Inspecting the color and turbidity of the urine can provide certain types of information about existing diseases and should not be neglected. Normal urine is yellow and has an aromatic odor. Tab. 12.3-1 – Characteristic appearances of urine shows the various colors of urine and their causes, while the characteristic odors associated with metabolic disorders are listed in Tab. 12.3-2 – Characteristic odors of urine in metabolic diseases.

12.3.3 Method of determination

The urine assays used to diagnose diseases of the kidney and the urinary tract include (Tab. 12.3-3 – Urine tests for evaluation of disorders of the kidney and the urinary tract):

  • Multiple test strips
  • Total protein excretion
  • Plasma protein excretion (e.g., albumin, IgG and α1-microglobulin)
  • Osmolality or relative density (specific gravity)
  • Microbiology urine examination
  • Creatinine determination to evaluate the excretion rates for proteins, hormones, or other analytes independently of the elimination of water.

12.3.3.1 Multiple test strips (dipsticks)

Rapid methods provide the user with a fast, reliable response to an individual patient. Because of easy handling of equipment rapid methods, like multiple test strips, are used to screen for renal and urinary tract disorders. The test strips can be read with the naked eye or automated spectrophotometric reading can be used for larger series.

When testing for hematuria and/or leukocyturia, the sample tested must contain urine that has not been centrifuged but is well mixed. Testing should be performed within two hours after collection of the sample.

It must be noted that the precision of the test strip assay is only moderate to good, and is poorer than is generally assumed /4/. Spectrophotometric reading is also no more precise than the eye of a good investigator /5/.

The test strips provide qualitative or semi-quantitative results. The detection limits are set with screening requirements in mind, and therefore provide a high degree of diagnostic specificity in order to exclude as many healthy individuals as possible.

For urology and nephrology purposes, a urine test strip should contain the following screening assays: protein, erythrocytes, leukocytes, pH, specific gravity, and nitrite. Other test fields, such as urobilinogen, bilirubin, glucose, and ketones are less suitable for screening as they lack diagnostic specificity.

When screening for renal and urinary tract disorders, the test strip fulfills the requirements for the preselection of individuals who are free of disease based on pH, leukocytes, and erythrocytes. The test strip is not sufficiently sensitive to detect mild proteinuria, and is totally unsuitable for detecting the excretion of free light chains. Overall, studies have found that test strip results are accurate in 71–92% of tests for those renal and urinary tract disorders that manifest themselves in the form of changes in the urine detectable by diagnostic laboratory testing /6/.

Reliability

Requirements for test strip assays need to be evaluated at the following two points of their measuring range /2/:

  • At the limit of detection (LD), which represents the analyte concentration for which the test field begins to show positive results
  • At the confirmation limit (LC), which is the analyte concentration for which almost all measurements are positive
  • There is a grey zone between LC and LD and the concentration ratio LC/LD should be 5
  • The following level of reliability can be expected: a false-positive rate of < 10% at the LD analyte concentration, and a false-negative rate of < 5% at the LC concentration.

12.3.3.2 Microscopy of urine

The microscopy of urine for clinically significant particles in urine sediment, or the standardized particle count in centrifuged or uncentrifuged urine, is diagnostically meaningful /7/:

  • In symptomatic patients
  • In patients with chronic diseases of the kidneys and the urinary tract
  • As a follow-up evaluation of a positive test strip finding in proteinuria and micro hematuria.

The morphological assessment of erythrocytes, casts and epithelia can provide clues as to whether, for example, the genesis of a disease is renal or post renal. For microscopic urine testing and standardized particle counting with urine chemistry analyzers, refer to Section 12.8 – Erythrocytes, leukocytes, casts in urine.

12.3.3.3 Biochemical urine examinations

The measurement of total protein in the urine is meaningful for the quantitative determination of proteinuria, since test strips are only semi quantitative and insensitive. By means of the follow-up determination of marker proteins, the etiogenesis of proteinuria can be differentiated into pre renal, renal and postrenal /8/. Renal proteinuria is differentiated into glomerular, tubular, and miscellaneous forms with the use of marker proteins. Marker proteins represent a characteristic size category for each form of proteinuria, and are present in the urine under normal conditions in concentrations that are easy to measure using immunonephelometric or immunoturbidimetric methods. Identifying marker protein levels makes it possible to classify proteinuria according to the various types. This testing also allows conclusions to be drawn regarding the location of any renal damage, but not regarding etiology. Refer also Section 12.9 – Urinary proteins.

12.3.3.4 Microbiological urine examinations

Urine from symptomatic patients with microscopic evidence of bacteriuria in uncentrifuged, native urine will usually have a microbe count of > 104/mL and should be cultured. This also applies to all urine samples with a leukocyte count of ≥ 10/μL in uncentrifuged urine /9/. The urinary tract infection prevalence in children 2 to 24 months of age with a fever without obvious source is 5%. The diagnostic sensitivity of leukocyturia (≥ 10 leukocytes/μL) for a urinary tract infection is about 95% and a specificity of 88%. In the absence of leukocyturia, the likelihood of a urinary tract infection decreases from 5% to 0.2–0.4% /10/.

Medical indications for microbiology investigation of urine include /2/:

  • Identifying the relevant pathogen
  • Locating the focus of infection
  • Optimizing the chemotherapeutic elimination of the pathogen.

Urine bacterial culture with identification of species and susceptibility testing

The culture methodology on plates is common in the clinical bacteriological laboratory /2/. A 1-μL disposable loop inoculum is used for routine practice. Quantitative culture is performed on a relatively nonselective agar plate, such as Cystine-Lactose Electrolyte Deficient (CLED) medium. In addition, culture on blood agar is performed as an optimum approach.

The following should be evaluated (Tab. 12.3-4 – Interpretation of microbiological urine findings):

  • Significant concentration of colony forming units
  • Whether the culture is a mono culture or a mixed culture
  • Species and type of the bacteria
  • Susceptibility testing of the bacteria.

Asymptomatic bacteriuria

The term describes a condition with pathogenic or facultative pathogenic bacteria in two consecutive urine samples and a bacterial count of ≥ 105/mL. At the same time, leukocyturia of ≥ 10 cells per view field in the urine sediment or ≥ 10/μL in the uncentrifuged urine are indicative of a local defense reaction. If the defense responses are disrupted, a urinary tract infection can develop /11/.

Asymptomatic bacteriuria occurs particularly frequently in diabetic patients, pregnant women, patients with organ transplants and elderly individuals. It should be taken into account that beyond the 85th year of life, asymptomatic bacteriuria is present in 10% of women and 5% of men; in individuals with an indwelling catheter this occurs quite regularly.

Mixed cultures

Most uncomplicated urinary tract infections result from one bacterial species /12/. The isolation of more than one organism from a single specimen of urine must always be interpreted in the light of features suggesting either true infection (presence of leukocytes) or contamination (presence of squamous epithelial cells). The demonstration of a maximum of two types of uropathogenic bacteria with ≥ 105 CFU/mL speaks in favor of an infection. The likelihood is augmented if, at the same time, leukocyturia is present or if the pathogen was already repeatedly isolated. In this case lower bacterial counts are also indicative of an infection.

12.3.4 Comments and problems

Proteinuria

The average coefficient of variation (CV) of one individual (CV within subject) is 40–50% at the upper reference interval value in health. In discrimination between health and disease (diagnostic testing) the average CV between-subject is about 25% for total protein and 75% for albumin in 24-hour output /2/.

Creatinine excretion

The average CV between-subject is about 20% giving an average CV for albumin/creatinine ratio of about 40%, and maximally 80%. This within-subject variation results in estimate of maximally allowable imprecision ≤ 20%. It would allow discrimination between twofold and threefold changes in excretion rates (analyte/creatinine ratios) in multiple monitoring of the same patient, by using the equation for critical difference DC = α × CV, where α = 2.77 at a statistical probability p < 0.05 /2/

In discrimination between health and disease (diagnostic testing) the average CV between-subject for creatinine in 24-hour excretion is about 25–30%.

Automated urinalysis systems

Some automated urinalysis systems have borderline trueness. In comparison to microscopy true classifications of leukocytes and erythrocytes were only above 85% and 72%, respectively. False negative casts were above 70% for all tested systems /16/.

References

1. Kouri TT, Gant VA, Fogazzi GB, Hofmann W, Hallander HO, Guder WG. Towards European urinalysis guidelines. Introduction of a project under European Confederation of Laboratory Medicine. Clin Chim Acta 2000; 297: 305–11.

2. European Urinalysis Guidelines: Summary. Scand J Clin Lab Invest 2000; 60: 1–96.

3. Roberts AP, Robinson IE, Beard RW. Some factors affecting bacterial colony counts in urinary infection. Br Med J 1967; I: 400–3.

4. Kierkegaard H, Feldt-Rasmussen U, Hoerder M, Andersen HJ, et al. Falsely negative urinary leukocyte counts due to delayed examination. Scand J Clin Lab Invest 1980; 40: 249–61.

5. Winkens RAG, Leffers P, Degenaar CP, Heuben AW. The reproducibility of urinalysis using multiple reagent test strips. Eur J Clin Chem Clin Biochem 1991; 29: 813–8.

6. Guder WG, Heidland A. Urinalysis. Report on a workshop conference. J Clin Chem Clin Biochem 1986; 24: 611–20.

7. Brody LH, Sallady JR. Urinalysis and urinary sediment. Med Clin North Am 1971; 55: 243–66.

8. Hofmann W, Regenbogen C, Edel H, Guder WG. Diagnostic strategies in urinalysis. Kidney Int 1994; 46 (Suppl 47): pg. 111– 4.

9. Graham JC, Galloway A. The laboratory diagnosis of urinary tract infection. J Clin Pathol 2001; 54: 911–9.

10. Finnell SME, Carroll AE, Downs SM. Technical report– Diagnosis and management of an initial UTI in febrile infants and young children. Pediatrics 2011; 128: e749–e770.

11. Fünfstück R, Stein G. Asymptomatische Bakterurie. Nieren- und Hochdruckkrankheiten 2007; 36: 269–76.

12. Pfister W. Klinische Relevanz mikrobiologischer Befunde. Nieren- und Hochdruckkrankheiten 2007; 36: 258–61.

13. The National Collaborating Center for Chronic Conditions (UK). Chronic Kidney Disease 2009. www.nice.org.uk/Guidance/CG73.

14. Collier G, Greenan MC, Brady JJ, Murray B, Cunningham SK. A study of the relationship between albuminuria, proteinuria and urinary reagent strips. Ann Clin Biochem 2009; 46: 247–9.

15. Hooton TM, Roberts PL, Cox ME, Stapleton AE. Voided midstream urine culture and acute cystitis in premonopausal women. N Engl J Med 2013; 369: 1883–91.

16. Bartosova K, Kubicek Z, Franekova J, Louzensky G, Larikova P, Jabor A. Analysis of four automated urinalysis systems compared to reference methods. Cin Lab 2016; 62: 2115–23.

12.4 Creatinine

Lothar Thomas

12.4.1 Indication

Assessment of renal function:

  • Symptomatic patients (proteinuria, leukocyturia, erythrocyturia, hypertension, diabetes mellitus, metabolic syndrome, hyperuricemia)
  • Clinical suspicion of acute renal failure or chronic kidney disease and its course
  • Extrarenal diseases with diarrhea, vomiting, profuse sweating
  • Acute disease conditions (e.g., preoperative and post-operative, in intensive care)
  • Sepsis, shock, poly trauma
  • Hemodialysis treatment
  • Pregnancy
  • Diseases with increased protein metabolism (e.g., chronic inflammation, multiple myeloma, acromegaly)
  • Therapy with renally excreted drugs that are potentially nephrotoxic if administered in too high a dosage.

12.4.2 Method of determination

12.4.2.1 Higher order reference materials and reference measurement procedure

Isotope dilution gas chromatography-mass spectrometry (IDGC-MS) is designated by the NCCLS and the Joint Committee on Traceability in Laboratory Medicine as a premium reference method for the determination of creatinine /1/. LC-MS is of equal value. The Laboratory Working Group of the National Kidney Disease Education Program (NKDEP) for improving serum creatinine measurements of NIST has developed the standard reference material SRM 967 for the calibration of the creatinine methods /2/. The certified creatinine concentrations of the two pools are 1 mg/dL (88.4 μmol/L) and 4 mg/dL (354 μmol/L).

12.4.2.2 Jaffé method

Principle: in alkaline medium creatinine reacts with alkaline picrate to form a red-orange solution which is measured spectrophotometrically. The absorption of the solution is, within a certain range, proportional to the creatinine concentration. The Jaffé method is implemented with different modifications and the kinetics of the reaction product that emerges is determined /3/. Undiluted serum and plasma is used in the assays. The reason for the many modifications is the low analytical specificity of the Jaffé method (i.e., about 50 pseudo creatinines, also referred as non-creatinine chromogens form a reaction product with alkaline picrate similar to that produced by creatinine). In comparison to true creatinine, some pseudo creatinines react faster while some others react more slowly with alkaline picrate. Following the addition of the sample, the reaction proceeds in three phases. In the first phase mainly the fast pseudo creatinines react; in the second phase, true creatinine; and subsequently, the slow pseudo creatinines. The change in absorption in the second phase is measured at 509 nm. It is proportional to the true creatinine concentration in the sample. Dependent upon the type of pseudo creatinines and the assay these lead to an intercept. To account for the sensitivity of alkaline picrate-based methods to non-creatinine chromogens, some manufacturers have adjusted the calibration to minimize the pseudo creatinine contribution (compensated methods), others did not (non-compensated methods) /4/.

  • Compensated methods: the pseudo creatinine contribution of plasma proteins is minimized by introducing a negative offset to compensate the positive intercept found in the correlation. Thus, for example, one manufacturer compensated Jaffé assay, 0.208 mg/dL (18 μmol/L) is automatically subtracted from each result /4/.
  • Non-compensated methods: the manufacturers do not compensate for pseudo creatinine but rather they attempt, with the reaction conditions, to capture a minimum of pseudo creatinines.

12.4.2.3 Enzymatic method

Enzymatic phenol-aminophenazone peroxidase method /5/

Principle: creatinine is hydrolyzed to creatine by creatinase. In a subsequent reaction step, both creatine produced from creatinine, as well as endogenous creatine, are degraded by creatinase to sarcosine and urea. The use of sarcosine oxidase results in the production of H2O2. In an indicator reaction with phenol amino phenazone (PAP reaction) H2O2 reacts by forming a red dye whose absorption at 546 (510) nm is proportional to the creatinine concentration (Fig. 12.4-1 – Principle of the creatinine PAP color test). Endogenous creatine must be compensated for by measuring a sample blank.

Creatinine determination in whole blood /6/

The determination is performed with blood gas analyzers or point of care instruments. The blood gas analyzer uses an amperometric biosensor (electrode) based on the enzymatic conversion of creatinine to creatine, then sarcosine, followed by a reaction that generates H2O2 from sarcosine.

Creatinine UV test /7/

Principle: creatinine imino hydrolase catalyzes creatinine degradation to N-methyl hydantoin and ammonia. The ammonia thus formed is determined in a subsequent enzymatically catalyzed reaction by measuring the absorption increase of NADH at 340 or 366 nm.

12.4.3 Specimen

Serum, plasma (heparin, EDTA, citrate): 1 ml

Whole blood (point of care)

12.4.4 Reference interval

The reference intervals for serum creatinine are dependent on the method of determination mentioned in Tab. 12.4-1 – Serum creatinine reference intervals.

12.4.5 Clinical significance

For the assessment of renal function, the KDIGO recommendations state that the serum creatinine value, along with the estimated GFR (eGFR), should be reported. The CKD-EPI equation is recommended for the calculation of the eGFR (see also Section 12.2 – Glomerular filtration rate (GFR)/11/. The creatinine value is of importance in the progression of chronic kidney disease due to the acceptable intraindividual variation. In addition the creatinine level contributes to the establishment of whether or not the renal function is in a steady state.

Intraindividual and inter individual variation

In healthy individuals the intraindividual variation in serum creatinine is minimal, while the inter individual variation is high. Therefore the reference interval is large. As a result, in an individual with low basal creatinine value and an impaired GFR, the creatinine level has to be increased by 13 times the intraindividual variation before the upper reference interval value is exceeded /12/.

The reasons for the high inter individual variation and the insensitivity to early changes in renal function are (Tab. 12.4-2 – Limitations of serum creatinine):

  • Differences in muscle mass and thus in creatinine formation. Given the same GFR, men have a higher creatinine level than women, muscular individuals have higher levels than less muscular individuals, and younger individuals higher levels than elderly /13/.
  • Varying intake of meat. One kilogram of meat contains 2–5 g of creatine, which is converted to creatinine during cooking. Food is the source of 15–30% of the daily renal creatinine excretion. Thus, for example, the creatinine value increases from 1.0 mg/dL (88 μmol/L) to 2.0 mg/dL (177 μmol/L) 3 hours following the ingestion of a large portion of goulash /14/.

The intraindividual variation of serum creatinine is lower than the MDRD value and the creatinine clearance (Tab. 12.4-3 – Variation in creatinine, cystatin C and MDRD values). Diseases and conditions with changes in serum creatinine levels are listed in Tab. 12.4-4 – Diseases and conditions that can cause a change in serum creatinine. The relationship between GFR and serum creatinine is shown in Fig. 12.4-2 – Relationship between glomerular filtration rate and serum creatinine.

12.4.6 Comments and problems

Reference interval

The upper and lower reference interval values depend on age, gender, body weight, and the method of determination. Over a long period of time, creatinine concentrations in an individual vary only to a small extent. Creatinine does not manifest a circadian rhythm.

Method of determination

Within the range of 0.2–2.0 mg/dL (18–176 μmol/L), the non-compensated procedure provides falsely high creatinine values. With the Jaffé method, the compensation of the non-creatinine chromogens is critical, particularly in children. Overcompensation occurs occasionally and incipient renal insufficiency is not identified. Therefore, it is generally recommended that exclusively enzymatic methods be used for the determination of creatinine in children.

Jaffé method: the Jaffé modifications are to a varying extent prone to interference and unspecificity. Kinetic methods require a high degree of stability with regard to temperature and pH. Generally, they measure pseudo creatinines more than the endpoint methods, because in these latter tests the serum proteins are removed beforehand by precipitation or dialysis. Falsely elevated values that are clinically relevant are determined in the range of below 2 mg/dL (177 μmol/L) are caused by ketone bodies, glucose, fructose, ascorbic acid, HbA (1.88 mmol/L) and HbF (2.03 mmol/L) /20/. Especially in samples from children with creatinine values of around 0.5 mg/dL (44 μmol/L) the values are raised by 8–27% /21/.

The following cephalosporins may interfere with the Jaffé method and thus cause elevated creatinine concentrations: cefoxitin, cephalothin, cefatril, cefazolin. Those that are believed not to interfere are moxalactam, cefoperazone, cefotaxime, ceftazidime, cephalexin, cefradine /22/. Flucytosine also causes increased creatinine levels.

Substances like acetoacetic acid react to some extent faster with alkaline picrate than creatinine while others, such as glucose, react slower. Depending on the modification employed, they are measured as creatinine and falsify the measured value. In the early reaction phase, fast-reacting substances contribute to the color reaction; during the middle phase it is almost exclusively creatinine that does so; in the late phase the reaction is influenced by slowly reacting non-creatinine chromogens.

The interference by bilirubin in the Jaffé method, varies according to the different analysis systems and can be mathematically corrected only to a limited degree. Up to a bilirubin concentration of 35 mg/dL (600 μmol/L), this interference can be eliminated by treatment of the icteric samples with peroxidase, bilirubin oxidase or ferrocyanide.

Enzymatic method: the PAP method shows excessively low creatinine levels in the presence of bilirubin concentrations > 7 mg/dL (120 μmol) /7/. The UV test is believed to measure falsely low creatinine in samples of bilirubin concentrations ≥ 12.1 mg/dL (207 μmol/L) /23/. Falsely low values are obtained with calcium dobesilate; the same is true, but only with high doses, of metamizole, ascorbic acid and α-methyldopa /7/.

With the UV creatinine test, the addition of a constant quantity to the creatinine concentration occurs due to high glucose values, amino acids and ammonia in the sample /16/. Dopamine and dobutamine lead to falsely low creatinine values by up to 67% if the creatinine to dopamine ratio in the sample is 4 : 1, expressed on a molar basis. Even a ratio of 50 :1 can lead to reductions of > 25%. The interference occurs only if the blood is collected via an indwelling intravenous tube /24/.

Separation of the cellular constituents

The erythrocytes should be separated from the serum within 16 hours from the time of the blood collection, otherwise an increase of 29% (21–63%) is measured after 48 hours with the Jaffé reaction /25/.

Biological variation

In a study /29/ the within-subject biological variation (CV) of creatinine and different eGFR equations in people with chronic kidney disease (CKD) and people without CKD was assessed. The CV creatinine was higher in people without CKD than in those with CKD (6.4% vs 2.5%) owing primarily to the more profound effect of meat consumption on creatinine variability in individuals with lower baseline creatinine concentrations.

Stability

In serum and plasma, no changes for up to 7 days at room temperature in closed containers /26/.

12.4.7 Biochemistry and physiology

Creatine, the precursor of creatinine, is produced by the liver and following its release is taken up by the muscles and other tissues. A man weighing 70 kg has a creatine pool of some 120 g, 98% of which is stored in the muscles. Creatine accounts for 20–30% while the remainder is phosphocreatine. The metabolism of creatine and creatinine is shown in Fig. 12.4-3 – Creatine and creatinine metabolism.

Phosphocreatine serves as an energy store for the muscles; during muscle contraction, chemical energy is transformed into mechanical energy by the cleavage of phosphocreatine /27/.

Creatinine is created by the non enzymatic dehydration of muscular creatine (Fig. 12.4-4 – Transformation of creatine into creatinine). In men, some 1.5% of the creatine pool is transformed daily into creatinine, representing a quantity of 1.8 g. The creatine and creatinine pools are increased by creatine that is ingested with food.

The size of the creatinine pool is determined by the muscular mass. As determining factors with regard to muscle mass, age and gender therefore have a marked influence on the creatinine pool. Further biological factors are dietary creatine intake and protein ingestion.

The intake of creatine and creatinine through meat consumption increases creatinine in the body. 1 g of beef contains 3.5 mg of creatine, of which 18–65% is transformed into creatinine during the process of cooking.

Limited protein intake diminishes the creatinine pool since arginine and glycine, as precursors of creatine synthesis, are not available (Fig. 12.4-3 – Creatine and creatinine metabolism/27/.

Because the muscle mass is diminished in myopathy, in cases of leg or arm amputation, and in intensive care patients, serum creatinine is low to normal and urinary creatinine excretion is reduced. Serum creatinine exceeds the upper reference value only when the GFR is markedly reduced.

Serum creatinine can only be used for the estimation of GFR if a steady state is present (i.e., the synthesis rate of creatinine equals its elimination rate). In order to check whether or not this situation is present, two determinations should be performed 24 hours apart. Differences of more than 10% indicate that a steady state is not present /28/.

In impaired renal function, GFR of 80–40 [mL × min–1 × (1.73 m2)–1], the GFR is overestimated with rising serum creatinine concentrations if creatinine clearance is measured. This is due to the fact that the elimination of creatinine is not only due to glomerular filtration is also excreted via the intestinal mucosa. In the intestines, creatinine is apparently metabolized as a result of the action of bacterial creatininase.

An approximately linear correlation between serum creatinine concentrations and the GFR exists only with an impairment of the GFR in the range of 40–20 [mL × min–1 × (1.73 m2)–1], which represents a serum creatinine range of some 2–4 mg/dL (177–354 μmol/L) in a person weighing 75 kg /15/.

References

1. Joint Committee on Traceability in Laboratory Medicine. Database of higher order reference materials and reference measurement procedure for laboratory medicine and in vitro diagnostics. JCTLM Reference measurement, 2006.

2. NIST. Certificate of analysis, standard reference material 967, creatinine in human serum. JCTLM Reference material, 2007.

3. Moss GA, Bondar RJ, Buzzelli DM. Kinetic enzymatic method for determining serum creatinine. Clin Chem 1975; 21: 1422–6.

4. Panteghini M on behalf of the IFCC Scientific Division. Enzymatic assays for creatinine: time for action. Clin Chem Lab Med 2008; 46: 567–72.

5. Guder WG, Hoffman GE. Multicenter evaluation of an enzymatic method for creatinine determination using a sensitive colour reagent. J Clin Chem Clin Biochem 1986; 24: 889–902.

6. Korpi-Steiner N, Williamson EE, Karon BS. Comparison of three whole blood creatinine methods for estimation of glomerular filtration rate before radiographic contrast administration. Am J Clin Pathol 2009; 132: 920–6.

7. Toffaletti J, Blosser N, Hall T, Smith S, Tompkins D. An automated dry-slide enzymatic method evaluated for automated enzymatic determination of plasma creatinine. Clin Chem 1983; 29: 684–7.

8. Ceriotti F, Boyd JC, Klein G, Henny J, Queralto J, Kairisto V, Parthegine M. Reference intervals for serum creatinine concentrations: assessment of available data for global application. Clin Chem 2008; 54: 559–65.

9. Pottel H, Vrydags N, Mahieu B, Vandewynckele E, Croes K, Martens F. Establishing age/sex related serum creatinine reference intervals from hospital laboratory data based on different statitical methods. Clin Chim Acta 2008; 396: 49–55.

10. Arzideh F, Wosniok W, Haeckel R. Reference limits of plasma and serum creatinine concentrations from intra-laboratory data bases of several German and Italian medical centres. Comparison between direct and indirect procedures. Clin Chim Acta 2010; 411: 215–21.

11. Kidney Disease: Improving Global Outcome (KDIGO) CKD Work Group. KDIGO clinical practice guideline for the evaluation and management of chronic kidney disease. Kidney Int Suppl 2013: 3: 1–150.

12. Perrone RD, Madias NE, Levey AS. Serum creatinine as an index of renal function: new insights into old concepts. Clin Chem 1992; 38: 1933–53.

13. Salive ME, Jones CA, Guralnik JM, et al. Serum creatinine levels in older adults: relationship with health status and medications. Age and Ageing 1995; 24: 142–50.

14. Jacobson FK, Christensen CK, Mogensen CE, Heilskov NSC. Evaluation of kidney function after meals. Lancet 1980; i: 319.

15. Filser D, Ritz E. Serum cystatin C concentration as a marker of renal dysfunction in the elderly. Am J Kidney Dis 2001; 37: 79–83.

16. Ferry N, Caillette A, Goudable J, Denicola C, Pozet N. Creatinine determination in peritoneal dialysis: what method should be used. Nephrol Dial Transplant 1996; 11: 2282–7.

17. Jungers P, Chauveau D. Pregnancy in renal disease. Kidney Int 1997; 52: 871–85.

18. Kierdorf HP, Seeliger S. Acute renal failure in multiple organ dysfunction syndrome. Kidney Blood Press Res 1997; 20: 164–6.

19. Andreev E, Koopman M, Arisz L. A rise in plasma creatinine that is not a sign of renal failure: which drugs can be responsible? J Internal Med 1999; 246: 247–52.

20. Soldin SJ, Henderson L, Hill JG. The effect of bilirubin and ketones on reaction rate methods for measurement of creatinine. Clin Biochem 1978; 11: 82–6.

21. Cobbaaert CM, Baadenhuisjen H, Weykamp CW. Prime time for enzymatic creatinine methods in pediatrics. Clin Chem 2009; 55: 549–58.

22. Kroll MH, Koch TR, Drusano GL, Warren JW. Paint of interference with creatinine assays by four cephalosporin-like antibiotics. Am J Clin Pathol 1984; 82: 214–6.

23. Spaett R, Gässeler N. Enzymatische Creatininbestimmung mit der Creatininiminohydrolase. Eine kritische Evaluation. Ärztl Lab 1990; 36: 136–44.

24. Saenger AK, Lockwood C, Snozek CL, Milz TC, Karon BS, Scott MC, et al. Catecholamine interference in enzymatic creatinine assays. Clin Chem 2009; 55: 1732–6.

25. Ford L, Berg J. Delay in separating blood samples affects creatinine measurement using the Roche kinetic Jaffe method. Ann Clin Biochem 2008; 45: 83–7.

26. Heins M, Heil W, Withold W. Storage of serum or whole blood samples? Effects of time and temperature on 22 serum analytes. Eur J Clin Chem Clin Biochem 1995; 33: 231–8.

27. Wyss M, Kaddurah-Daouk R. Creatine and creatinine metabolism. Physiological Reviews 2000; 80: 1107–1213.

28. Rutherford WE, Blondin J, Miller JP, et al. Chronic progressive renal disease: rate of change of serum creatinine concentration. Kidney Int 1977; 11: 62–70.

29. Hilderink JM, van der Linden N, Kimenai DM, Litjens EJR, Klinkenberg LJJ, Aref BM, et al. Biological variation of creatinine, cystatin C, and eGFR over 24 hours. Clin Chem 2018; 64: 851–60.

12.5 Creatinine clearance (ClCr)

Lothar Thomas

The ClCr is not a valid method for the assessment of the glomerular filtration rate (GFR). Only relative changes – and not absolute – in the GFR can be well assessed. It is, therefore, recommended that in symptomatic patients and in at risk patients with suspicion of impaired renal function, clinicians should order the measured GFR (mGFR). This method correlates closely with the real GFR (see Section 12.2 – Glomerular filtration rate (GFR)). If the GFR and the ClCr are known, the ClCr alone can, subsequently, be employed as a relative criterion for the assessment of the GFR course /1/.

In the following the ClCr will, in spite of the above-mentioned recommendation, be discussed in detail, because it is recommended in numerous drug studies in spite of a certain lack of reliability /2/.

12.5.1 Indication

  • Determination whether the GFR is almost normal, slightly or severely reduced
  • Assessment of a change in GFR under medication with potentially nephrotoxic substances
  • For the assessment of the necessity of dialysis in combination with urea clearance in patients with end stage renal failure (see Section 12.6 – Urea nitrogen.

12.5.2 Test protocol

1. Collection of 2 mL of blood for the determination of creatinine.

2. Collection of 24-hour urine (possibly 4-hour or 2-hour collections). The collection period for the 24-hour urine sample includes one daytime and one nighttime period. It starts at 7:00 or 8:00 a.m. At first the bladder is emptied. This urine is not included in the collection. As of this point in time until the very same time the following morning (any urine voided then is included as well), all urine is collected. A urinary flow rate of > 1 mL/min. should be maintained by an adequate fluid intake.

3. Determination of creatinine in the collected urine.

4. Calculation of the clearance value. Besides the volume of urine collection, the height and body weight of the patient need to be known as well.

Clearance results are related to 1.73 m2 body surface area of a person weighing 75 kg. The body surface area of the patient is derived from a nomogram using height and body weight. (Fig. 12.5-1 – Nomogram for determining the body surface area). The clearance is calculated according to the formula shown in Tab. 12.5-1 – Creatinine clearance formula.

Some laboratories employ the 4-hour urine collection for the calculation of the ClCr, especially under therapy with nephrotoxic medication. A clearance value is calculated on each of 3 successive days.

12.5.3 Specimen

  • Serum, obtained at the beginning and at the end of the collection period: 1 mL
  • Urine without additives (volume to be measured): 5 mL

12.5.4 Reference interval

Refer to Ref. /3456/ and Tab. 12.5-2 – Creatinine clearance reference intervals.

12.5.5 Clinical significance

The ClCr does not permit the measurement of the GFR; rather, it provides an approximation of it’s magnitude. For practical clinical concerns this is sufficient in order to clarify the following questions /7/:

  • Detection of a reduced ClCr and including an approximate but clearly reduced GFR, in particular for the recognition of the creatinine-blind range of the GFR of 80–40 [mL × min–1 × (1.73 m2)–1], that is to say when the serum creatinine value is likely still within the reference range
  • Assessment of the course of the GFR by serial measurements of the ClCr (e.g., under therapy with potentially nephrotoxic substances)
  • Establishment of when a patient with end stage renal failure will become dialysis-dependent, which can be the case as of a ClCr of less than 10–20 [mL × min–1 × (1.73 m2)–1].

In contrast to inulin clearance, which is the gold standard for the determination of GFR, ClCr provides values that are too high. The cause of this is that in contrast to inulin, in healthy individuals 10–14% of the creatinine that appears in the urine is actively secreted by the tubules. Therefore, even a normal GFR already produces falsely elevated values if ClCr is used. The overestimation of the GFR with ClCr increases with the impairment of the renal function. The absolute difference between CrCl and GFR (CrCl-GFR) is, however, greatest in the GFR range of 80–40 [mL × min–1 × (1.73 m2)–1] (Tab. 12.5-3 – Relationship between the clearances of creatinine and inulin depending on the GFR/8/.

According to a study /9/, 42% of patients with an impaired GFR of 70–61 mL had normal ClCr, while in those with impairment down to 60–51 mL this was still the case in 23% of the patients. Only with a ClCr above 115 [mL × min–1 × (1.73 m2)–1], a reduction in the GFR was no longer found /3/.

With serum creatinine values ≥ 3 mg/dL (265 μmol/L), ClCr is superfluous. In such cases the GFR is below 20 [mL × min–1 × (1.7 m2)–1], and the relative deviation of the ClCr to the GFR (ClCr/GFR) is double (Tab. 12.5-3). This is due to the increasing tubular secretion and the intestinal elimination of creatinine.

In the assessment of the ClCr, the age dependence of the GFR must be taken into consideration. In adults the following relationship exists between the GFR and age /10/:

GFR = 157 – (1.16 × age in years)

12.5.5.1 Urinary creatinine excretion

The completeness of a 24-hour urine collection can be estimated roughly by means of the quantity of excreted creatinine /11/. The excretion values for adults are listed in Tab. 12.5-4 – Creatinine excretion in adults; in children they are calculated according to the following formula /10/:

mg creatinine/kg body weight/24 h = 15.4 + (0.46 × age in years)

12.5.6 Comments and problems

Method of determination

The ClCr correlates better with the GFR in 24-hour urine collections than in shorter collection periods. In comparison with 24-hour samples, urine samples collected at daytime periods result in an increase in the ClCr/GFR ratio (Tab. 12.5-3 – Relationship between the clearances of creatinine and inulin depending on the GFR/10/.

For the determination of urinary creatinine concentrations, the specimen has to be diluted 1: 20 to 1 : 50 with water. In order to determine serum creatinine for ClCr, blood should be obtained before and after the 24-hour urine collection, and the serum should be analyzed in the same test run. The results are only used for averaging if the difference is less than 10% of the highest value. If the difference is greater, no clearance calculation should be performed.

The method for the determination of creatinine has a considerable influence on the ClCr. If the Jaffé method for serum and urine creatinine determination is employed, it must be noted that within the reference interval, with the use of a non-compensated method 20% of the measured value is attributed to non-creatinine chromogens. These are, however, so diluted in the urine that they do not interfere.

If serum and urinary creatinine is determined with the enzymatic method, only true creatinine is measured and the serum creatinine level is lower than that determined with the non-compensated Jaffé method. Therefore, a higher ClCr is determined with the insertion of the creatinine value into the clearance formula. Since the ClCr reference intervals were usually established with the Jaffé method, it is recommended that the enzymatically established creatinine value in the concentration range of up to 1.5 mg/dL be raised by 20% to achieve comparability with the reference ranges described in literature /12/.

Urine collection

The most critical errors made in determining the ClCr are insufficient bladder emptying and incomplete collection of the urine. Often, the first morning urine is already added to the collection container (instead of being discarded). Loss of urine also occurs if urine is spontaneously voided during defecation. Occasionally, the patient forgets that at the end of the collection period the bladder contents needs to be completely emptied into collection container. Precise instruction about the urine collection procedure is an indispensable prerequisite to the implementation of a ClCr.

Reference interval

The ClCr has high interindividual variability (Tab. 12.4-3 – Variation in creatinine, cystatin C and MDRD values).

References

1. Payne RB. Creatinine clearance and glomerular filtration rate. Ann Clin Biochem 2000; 37: 98–9.

2. Hasslacher C. Praxis-Leitlinien der Deutschen Diabetes-Gesellschaft. Diabetische Nephropathie. Diabetes und Stoffwechsel 2002; 11, Suppl 2: 17–9.

3. Schirmeister J, Willmann H, Kiefer H, Hallauer W. Für und wider die Brauchbarkeit der endogenen Creatininclearance in der funktionellen Nierendiagnostik. Dtsch Med Wschr 1964; 89: 1640–7.

4. Apple F. Creatinine clearance: enzymatic vs. Jaffé determinations of creatinine in plasma and urine. Clin Chem 1986; 32: 388–90.

5. Kampmann J, Siersbaek-Nielsen K, Kristensen M, Hansen JM. Rapid evaluation of creatinine clearance. Acta Med Scand 1974; 196: 517–20.

6. Schwartz GJ, Feld LG, Langford DJ. A simple estimate of glomerular filtration rate in full-term infants during the first year of life. J Pediatr 1984; 104: 849–54.

7. Schirmeister J. Standpunkte: Creatininclearance. Diagnostik 1984; 17: 9.

8. Shemesh O, Golbetz H, Kriss JP, Myers BD. Limitations of creatinine as a filtration marker in glomerulopathic patients. Kidney Int 1985; 28: 830–8.

9. Kim KE, Questi G, Ramirez O, Brest AN, Swartz C. Creatinine clearance in renal disease: a reappraisal. Brit Med J 1969; 4: 11–14.

10. Watkins DM, Shock NW. Agewise standard value for Cin, CPAH and TMPAH in adult males. J Clin Invest 1965; 34: 969–73.

11. Filler G, Browne R, Seikaly MG. Glomerular filtration rate as a putative »surrogate end-point« for renal transplant clinical trials in children. Pediatr Transplant 2003; 7: 18–24.

12. Kertai MD, Boersma E, Bax JJ, et al. Comparison between serum creatinine and creatinine clearance for the prediction of postoperative mortality in patients undergoing major vascular surgery. Clin Nephrol 2003; 59: 17–23.

12.6 Urea nitrogen (BUN)

Lothar Thomas

The terms urea and blood urea nitrogen (BUN) are used in medical diagnostics as synonyms. Urea is calculated from the BUN value by multiplication with the factor 2.14 or BUN is calculated from the urea value by multiplication with the factor 0.46.

Conditions with an elevated serum urea value are referred to as azotemia. Pre renal, renal and post renal azotemia are distinguished.

12.6.1 Indication

  • Differential diagnosis of acute renal failure (ARF) using the urea/creatinine ratio
  • Differential diagnosis of ARF using fractional urea clearance
  • Assessment of end stage renal failure, because the signs of uremic intoxication, particularly the gastrointestinal signs, correlate better with serum urea than with creatinine concentrations
  • Assessment of metabolic status in intensive care and dialysis patients, since urea concentrations are representative of protein metabolism
  • Assessment of the urea distribution volume in dialysis patients.

12.6.2 Method of determination

Urease-Berthelot reaction

Principle: urea is hydrolyzed to ammonium ions and CO2 with urease (urea amidohydrolase, EC 3.5.1.5). The ammonium ions are quantitated in a reaction with phenol and sodium hypochlorite, thus forming a blue dye which is measured spectrophotometrically between 530 and 570 nm /1/.

Diacetyl monoxime method

Principle: the condensation of diacetyl with urea forms the chromogen diazine. The specimen added to diacetyl monoxim forms a pink dye which is stabilized by the addition of thiosemicarbazide and iron(III) chloride. The pink dye is quantified spectrophotometrically /2/.

Urease UV method

Principle: urea is hydrolyzed to ammonium ions and CO2 with urease (urea amidohydrolase, EC 3.5.1.5). In the presence of large excess of 2-oxoglutarate, the ammonium ions arising from the hydrolysis of urea are stoichiometrically consumed by the GLD reaction. Thus, the decrease in NADH concentration, measured by the change in absorbance at 340 nm is proportional to the amount of urea in the specimen (Tab. 12.6-1 – Urease UV method/3/.

Additional methods

They include the hydrolysis of urea by urease. Either the change in pH or in conductivity, or the formation of ammonia, is measured with an NH4+ electrode.

12.6.3 Specimen

Serum, plasma (no ammonium heparin), urine: 1 mL

12.6.4 Reference interval

Refer to Ref. /45/ and Tab. 12.6-2 – Reference intervals for urea nitrogen (BUN).

12.6.5 Clinical significance

Essentially, three factors determine the serum urea concentration:

  • Renal perfusion and thus the quantity of excreted water. In diuresis, re diffusion of urea in the distal tubule is minimal, a lot of urea are excreted in the urine, serum urea levels are low. In anti diuresis (e.g., in the case of thirst, exsiccosis or oliguric heart failure) urea re diffuses in the tubules at increased rate and hence serum urea increases.
  • Urea synthesis rate. It depends on the daily protein intake and the amount of endogenously metabolized protein. Urea concentrations of up to 100 mg/dL (16.7 mmol/L) can occur, particularly in combination with thirst or febrile illnesses.
  • Value of the glomerular filtration rate (GFR). A persistently elevated serum urea concentration indicates a significantly impaired GFR. With normal protein intake of approximately 75 g/day and normal renal perfusion, concentrations higher than the reference interval are not found until the GFR declines to below 30 [mL × min–1 × (1.73 m2)–1].

Because of the dependence on renal perfusion and protein intake, serum urea is not suitable for the first-time diagnosis of incipient renal insufficiency but is useful for monitoring the course in cases associated with a more clearly reduced GFR.

12.6.5.1 Urea/creatinine ratio

In acute renal failure pre renal and post renal disorders can be distinguished from the renal cause by determining the urea/creatinine ratio /67/. With normal daily protein intake of some 1 g/kg body weight, the urea/creatinine ratio in healthy individuals is approximately:

  • 35, if both are expressed in mmol/L
  • 25, if both are expressed in mg/dL
  • 12, if urea is determined as BUN and if both are expressed in mg/dL.

The clinical interpretation of the urea/creatinine ratio is shown in Tab. 12.6-3 – Serum urea/creatinine ratios in various diseases and conditions.

12.6.5.2 Fractional excretion of urea nitrogen (FEUN)

In acute renal failure (ARF) fractional Na+ excretion (FENa) is used in the diagnosis of ARF to distinguish between the two main causes of ARF, pre renal state and acute tubular necrosis (ATN) (see Section 8.8.3 – Fractional sodium excretion (FENa)). However, many patients with pre renal disorders receive diuretics, which decrease Na+ reabsorption. In contrast, the FEUN is primarily dependent on passive forces and is therefore less influenced by diuretic therapy.

A spectrum of investigations such as the urea/creatinine ratio, urine Na+concentration, urine osmolality, the serum/urine creatinine ratio, and the FENa are frequently used markers.

The FEUN is a marker for the determination of the excreted fraction of glomerularly filtered urea-N. The FEUN is calculated according to the following formula:

FE UN (%) = UN (U) × creatinine (S) × 100 UN (S) × creatinine (U)

U: urine; S: serum; UN, urea nitrogen in mg/dL; creatinine in mg/dL

A low FEUrea (≤ 35%) was found to be a more sensitive and specific index than FENa in differentiating between ARF due to pre renal azotemia and that due to ATN, especially if antibiotics have been administered /8/.

12.6.5.3 Urea in advanced chronic kidney disease

In advanced chronic kidney disease, up to 60% of urine creatinine can be the result of tubular secretion, and the relationship between the factional excretions of creatinine and inulin may fluctuate between 1.2 and 2.1 in cases with GFR of 20–25 [mL × min –1 × (1.73 m2)–1/9/. For the calculation of the GFR in these patients, the average value of the urea and creatinine clearance is recommended. It is considered by some nephrologists to be a precise indicator of a GFR of < 15 [mL × min–1 × (1.73 m2)–1] and of the likelihood of a necessity of dialysis /10/.

GFR [mL × min–1 × (1.73 m2)–1] = (CCr + CUrea)/2

The urea clearance is performed like the creatinine clearance and, in each case, inserted into the above formula in [mL × min–1 × (1.73 m2)–1].

The behavior of urea in certain diseases and conditions is shown in Tab. 12.6-4 – Diseases and conditions that cause elevated serum urea.

12.6.5.4 Subnormal urea values

Subnormal urea concentrations are less meaningful diagnostically than elevated values, and can appear in cases of severe liver disease, in low-protein nutrition and in long-term treatment with hypotonic solutions. Physiologically, low urea values occur in children and in pregnant women.

12.6.6 Comments and problems

Reference interval

Within the reference interval, men have higher values than women. Low values are found in pregnant women, with low-protein nutrition, and following the intraoperative transfusion of plasma expanders. The circadian rhythm of the serum urea level is not higher than the methodological imprecision.

Possible methodological errors

The urease GLD method is specific; interferences occur due to contamination with ammonia. The diacetyl monoxime method is relatively nonspecific. Creatinine, allantoin, arginine, and some proteins are also determined.

Using the urease GLD method with serum start, urea values increase if the serum is stored (e.g., at 37 °C for 3 days). Some commercially produced control sera contain noteworthy quantities of ammonia (ammonium sulphate precipitation). Like the urease/Berthelot method, the serum start procedure provides higher urea concentrations than the method with a urease start.

Drugs

Ascorbic acid, sulfonylurea, guanethidine, thiazides, sulfonamides, chloramphenicol and dextran containing plasma expanders can produce artificially high values, mainly with the diacetyl monoxime method.

Stability

At room temperature for 2 days, at 4 °C for 1 week /14/.

12.6.7 Biochemistry and physiology

Urea is the final product of protein and amino acid metabolism; it is synthesized in the liver. During protein catabolism, the proteins are split into amino acids and de aminated. The ammonia thus produced is converted to urea inside the mitochondria via a series of reactions that are jointly referred to as urea cycle (see Fig. 5.1-1 – Organization and regulation of the hepatic and renal ammonia metabolism). On the average, dietary protein contains 16% nitrogen. Of this nitrogen, over 90% is not used for metabolic processes but is converted to urea. In adults, some 16 g of urea are formed daily /15/.

Most of urea is eliminated renally via glomerular filtration. Approximately 40–60% of the filtered urea is reabsorbed, independently of the tubular flow rate, in the proximal tubule. The re diffusion in the distal tubule is dependent upon the urine flow and is controlled by the anti-diuretic hormone. Thus, during diuresis approximately 40% of the urea that arrives at the distal tubule is reabsorbed and in anti diuresis this value increases to 70% /16/.

In pre renal and post renal ARF, in contrast to renal ARF the tubular urine flow is decreased. Due to the increased re diffusion of urea in the distal tubule and the increased creatinine secretion, a disproportionate rise in plasma urea relative to creatinine, as well as an increase in the urea/creatinine ratio, occurs.

In childhood protein catabolism is diminished, because of higher growth-related protein requirements. Average urea values are, therefore, generally lower than in adults. In pregnant women, the subnormal urea values are due to the increased protein requirement of the fetus and to renal hyper perfusion.

Because of the dependence on protein metabolism and the complex process of renal excretion the reference interval for serum urea is broad. Urea determination is, therefore, a less specific and less sensitive parameter for the assessment of incipient GFR impairment.

The upper reference interval value for serum is not exceeded until the GFR declines by 75%. With more marked impairment of the renal function, serum urea correlates better with the GFR. With a decrease of the GFR to 10% of normal, the serum urea concentrations increases by approximately 10 fold.

A correlation of serum urea with the GFR is, however, reliable only if the urea level is not influenced by extrarenal factors. For instance in chronic renal failure with polyuria or in the case of diarrhea, vomiting and hepatic insufficiency, the urea value may be less elevated than anticipated. In contrast, it is higher than expected if in addition to chronic renal failure, oliguria, excessive protein intake, heart failure or gastrointestinal hemorrhage are also present.

High protein intake leads to urea induced diuresis. This is associated with high urine osmolality of 700–900 mmol/kg. The urea induced diuresis can lead to a decrease in extracellular fluid volume combined with hypernatremia. For the clinician, this results in an apparently contradictory situation including hypernatremia together with a maximally concentrated urine at the same time /7/.

References

1. Fawcett JK, Scott JE. A rapid and precise method for the determination of urea. J Clin Pathol 1960; 13: 156–9.

2. Marsh WH, Fingerhut B, Miller H. Automated and manual direct methods for the determination of blood urea. Clin Chem 1965; 11: 624–7.

3. Kerscher L, Ziegenhorn J. Urea. In: Bergmeyer HU. Methods of enzymatic analysis. Weinheim: VCH, 1985: III, 444.

4. Rodger R, Laker M, Fletcher K, White TF, Heaton A, Ward MK, et al. Factors influencing normal reference intervals for creatinine, urea, and electrolytes in plasma as measured with a Beckman Astra 8 analyzer. Clin Chem 1985; 31: 292–5.

5. Soldin SJ, Hicks JM. Pediatric reference ranges. Washington: AACC-Press, 1995: 135.

6. Schuster VL, Seldin DW. Renal clearance. In: Seldin DW, Giebisch G (eds). The kidney. New York: Raven, 1992: 951–78.

7. Star RA. Pathogenesis of diabetes insipidus and other polyuric states. In: Seldin DW, Giebisch G (eds). Clinical disturbances of water metabolism. New York: Raven, 1995: 211–24.

8. Carvounis CP, Nisar S, Guro-Razuman S. Significance of fractional excretion of urea in the differential diagnosis of acute renal failure. Kidney International 2002; 62: 2223–9.

9. Bauer JH, Brooks CS, Burch RN. Renal function studies in man with advanced renal insufficiency. Am J Kidney Dis 1982; 3: 30–5.

10. Lubowitz H, Slatopolsky E, Shankel S, et al. Glomerular filtration rate: Determination in patients with chronic renal disease. JAMA 1969; 199: 100–4.

11. Harten J, Hay A, McMillan DC, McArdle CS, O’Reilly DStJ, Kinsella J. Postoperative serum urea is associated with 30-day mortality in patients undergoing emergency abdominal surgery. Ann Clin Biochem 2006; 43: 295–9.

12. Morgan DB, Carver ME, Payne RB. Plasma creatinine and urea creatinine ratio in patients with raised plasma urea. Brit Med J 1977; 2: 929–32.

13. Buchborn E. Erhöhte Harnstoffwerte im Blut. Monatskurse Ärztl Fortbild 1970: 20: 328–31.

14. Heins M, Heil W, Withold W. Storage of serum or whole blood samples? Effects of time and temperature on 22 serum analytes. Eur J Clin Chem Clin Biochem 1995; 33: 231–8.

15. Atkinson DE. Functional roles of urea synthesis in vertebrates. Physiological Zoology 1992; 65: 243–67.

16. Gillin AG, Sands JM. Urea transport in kidney. Semin Nephrol 1993; 13: 146–54.

12.7 Cystatin C

Lothar Thomas

Cystatin C is a non glycosylated basic protein (MW 13.36 kDa) and is found in a variety of biologic fluids. Cystatin C is endogenously produced at a constant rate, freely filtered in the glomerulus, neither reabsorbed nor secreted in the renal tubule, and not extra renally eliminated. All endogenous filtration markers are affected by factors other than GFR (non-GFR determinants), including generation, renal tubular reabsorption and secretion, and extrarenal elimination. In comparison with creatinine, cystatin C is less influenced by muscle mass and has often been proposed to be more accurate than creatinine for estimation of GFR in subgroups of the population /1/.

In addition, cystatin C is a novel risk factor for cardiovascular events and increasing concentrations are associated with higher mortality in patients with acute coronary syndromes /2/.

12.7.1 Indication

The 2012 Kidney Disease Improving Global Outcomes (KDIGO) guideline suggest measuring cystatin C in adults with eGFRCr 45–59 [mL × min–1 × (1.73 m2)–1] who do not have markers of kidney damage if confirmation of chronic kidney disease (CKD) is required /3/.

Further indications are:

  • Patients with assumed moderate impairment of the GFR, such as in hypertension, diabetes mellitus, metabolic syndrome, hyperuricemia, cardiovascular disease, liver disease, obstructive uropathy
  • Children and elderly individuals (≥ 70 years)
  • Suspicion of acute renal failure
  • Monitoring of renal function in transplant patients; using the eGFRCr–Cys
  • Monitoring of the renal function under cytostatic therapy, e.g., cisplatin, carboplatin.

12.7.2 Method of determination

Particle-enhanced homogeneous immunoassays using latex or polystyrene particles which are coated with specific antibodies to cystatin C. Two different versions of particle-enhanced assays are offered /4/:

  • Particle-Enhanced Immuno Turbidimetric Assay (PETIA)
  • Particle-Enhanced Nephelometric Assay (PENIA).

PETIA

Principle: polyclonal rabbit antibodies to cystatin C are covalently attached to carboxylate-modified uniform latex particles /4/. The increase in absorbance at 340 nm produced by the agglutination reaction is then measured after 240 seconds to give an endpoint value. Lyophilized recombinant human cystatin C is used as calibrator.

PENIA

Principle: serum cystatin C is measured by a nephelometric immunoassay based on rabbit mono specific anti-human cystatin C antiserum covalently coated with 80-nm diameter chloromethyl styrene particles (latex reagent) /5/. This refers to a fixed time method, in which the formation of agglutinates is measured following incubation of diluted samples with the latex reagent. These scatter the infrared light (840 nm) of a diode. Changes in the scattered light signal are transformed into mg/L using a calibration curve.

A certified reference material, ERM-DA471/IFCC, is available for cytatin C.

12.7.2.1 KDIGO recommendations for laboratories that measure cystatin C

Recommendations:

  • Measure cystatin C using an assay with calibration traceable to the international standard reference material
  • Report eGFRcys in addition to the serum cystatin C concentration in adults and specify the equation used whenever reporting eGFRcys and eGFRcreat-cys
  • Report eGFRcys and eGFRcreat-cys in adults using the 2012 CKD-EPI cystatin C and 2012 CKD-EPI creatinine-cystatin C equations respectively
  • eGFRcys and eGFRcreat-cys levels less than 60 [mL × min–1 × (1.73 m2)–1] should be reported as decreased.

For calculation and clinical assessment of eGFRcys and eGFRcreat-cys refer to Chapter 12.2 – Glomerular filtration rate.

12.7.3 Specimen

Serum, plasma (heparin, EDTA): 1 mL

12.7.4 Reference interval

Refer to Ref. /678, 91011/ and Tab. 12.7-1 – Reference intervals for cystatin C.

12.7.5 Clinical significance

Cystatin C is an additional marker for the estimation of renal function and also for prediction of cardiovascular risk.

12.7.5.1 Cystatin C and renal function

Serum cystatin C has been proposed as a better marker of chronic kidney disease (CKD) than serum creatinine. The most important reasons are /12/:

  • Cystatic C- based GFR-estimating equations do not require the use of vague terms like race and sex
  • Cystatic C- based GFR-estimating equations are useful for both children and adults, including the elderly
  • Cystatic C- based GFR-estimating equations are superior to creatinine-based equations in predicting end-stage renal disease, cardiovascular manifestations, hospitalization and death
  • Cystatin C is required to diagnose the new syndrome shrunken pore syndrome with its high mortality and morbidity, even in the absence of reduced GFR.

Different studies in patients with reduced measured GFR (mGFR) < 80 [mL × min–1 × (1.73 m2)–1] have found a better correlation of mGFR to cystatin C than to serum creatinine. In some cases it was possible to diagnose CKD as early as stage 2 /1314/. If the diagnostic sensitivity of cystatin C and creatinine was set, in each case, at 100% for the recognition of a GFR of < 80 [mL × min–1 × (1.73 m2)–1], the diagnostic specificity of cystatin C was 75%, while that of creatinine was 0% /4/. In a study, for the detection of mild impairment of GFR < 72 [mL × min–1 × (1.73 m2)–1], the diagnostic sensitivity of cystatin C was 71.4% and of creatinine it was only 52.4% /14/.

Cystatin C is more accurate than creatinine in subgroups of the population, including vegetarians, those with limb amputation, muscle wasting or chronic disease /1/.

The diagnostic sensitivity of cystatin C for the diagnosis of CKD is higher than for creatinine because of its lower inter individual variation. Thus, a creatinine value at the lower reference interval value must increase by 13 times the standard deviation of the intra individual variation to exceed the upper reference interval value; with cystatin C, only a 4-fold rise is necessary /15/.

The cystatin C-based eGFRcys is not more accurate for routine GFR estimation than eGFRCr. The eGFR based on the combination of creatinine and cystatin C (eGFRCr-Cys) is more accurate than either alone, reflecting the lesser influence of non-GFR determinants of either marker when both are used /16/.

The KIDGO recommends the determination of the eGFRcys or eGFRcr-cys in patients with eGFRcr in the range of 45–59 [mL × min–1 × (1.73 m2)–1/3/. Data from the CKD-EPI showed improved accuracy in GFR estimation using eGFRCr-Cys compared to either marker alone. In patients with eGFRCr 45–59 [mL × min–1 × (1.73 m2)–1], the combined equation correctly reclassified 16.8% of those with eGFRCr 45–59 [mL × min–1 × (1.73 m2)–1] to mGFR ≥ 60 [mL × min–1 × (1.73 m2)–1/17/. The consensus was therefore that individuals with eGFRCr 45–59 [mL × min–1 × (1.73 m2)–1] without markers of kidney damage, but with eGFRcys or eGFRcr-cys ≥ 60 [mL × min–1 × (1.73 m2)–1] could be considered not to have CKD.

Co morbidities such as solid-organ transplant strongly influence the relationship between mGFR, eGFRCr, eGFRcys or eGFRcr-cys. Among the 3 CKD-EPI equations, eGFRCr-Cys performed most consistently across potential kidney donors and transplant recipients /318/. The three CKD-EPI equations are depicted in Section 12.2 – Glomerular filtration rate (GFR).

Recently a simple cystatin C-based equation for estimation of GFR comprising only two variables, cystatin C concentration and age was introduced /19/:

eGFR = 130 × cystatin C–1.069 × age–0.117 –7

The new equation includes age, a single coefficient (exponent) for serum cystatin C (–1.069), and a value for extrarenal elimination of cystatin C [7 mL × min–1 × (1.73 m2)–1] across the range of GFR. By contrast to the CKD-EPI eGFRcys the new equation does not require specification of sex and takes into account extrarenal cystatin C elimination /19/.

The diagnostic value of cystatin C for the detection of impaired kidney function and for the assessment of their course is shown in Tab. 12.7-2 – Diagnostic significance of cystatin C in impaired renal function.

12.7.5.2 Cystatin C in children

Cystatin C concentrations in serum during the first weeks of life are approximately twice as high as in adults; they then fall continuously and reach adult values by the end of the first year of life /10/. At the age of < 4 years, serum creatinine is in the range of 0.2–0.4 mg/dL (17.7–35.4 μmol/L) and, because of insensitivity of the Jaffé method, slight changes are difficult to detect. These children benefit from the determination of cystatin C.

12.7.5.3 Cystatin C, age and metabolic disturbances

Because there is a constant relationship between the mGFR and the cystatin C serum level, the age-dependent decrease in the GFR leads to a rise in concentrations. However, metabolic abnormalities, which occur frequently in old age, also contribute to the rise in cystatin C. Thus, in the Third National Health and Nutrition Examination Survey /20/, individuals ≥ 60 years with a cystatin C level of > 1.0 mg/L (PENIA), but without stage 3 or 4 CKD, had a higher prevalence of elevated uric acid, phosphate, homocysteine, CRP and fibrinogen and more often a reduced hemoglobin level than those with a lower cystatin C value. Hence, elevated cystatin C may be an indicator of preclinical metabolic disease.

12.7.5.4 Discrepant results of serum creatinine and cystatin C

Factors that are not associated with the GFR may influence serum cystatin C concentrations.

Corticosteroids

High-dose corticoidsteroids stimulate cystatin C production. The eGFRcys indicates a false low clearance. Glucocorticoids affect the extrarenal metabolism of cystatin C. This was demonstrated in asthmatics and in organ transplant patients under corticosteroid therapy /21/.

Thyroid gland hormones

Hyperthyroidism is associated with a reversible increase in cystatin C production. The eGFRcys indicates a false low clearance. Untreated hypothyroidism leads to decreased cystatin C concentration. The eGFRcys indicates a false increased clearance. Treatment causes a 14% increase in cystatin C in hypothyroid patients and a 21% decrease in the hyperthyroid group, producing similar final concentrations /22/.

Other factors

In a study involving 3418 patients in whom the measured GFR was determined with 125I-iothalamate or 51Cr EDTA in comparison to cystatin C and creatinine the results were in summary /2123/:

  • After adjustment for GFR, cystatin C was 4,3% lower for every 20 years of age, 9,2% lower for female gender but only 1.9% lower in blacks
  • Diabetes was associated with 8.5% higher levels of cystatin C and 3.9% lower levels of creatinine
  • Higher C-reactive protein and white blood cell count and lower serum albumin were associated with higher levels of cystatin C and lower levels of creatinine. Adjustment for age, gender and race had a greater effect on the association of factors with creatinine than cystatin C.
  • Low creatinine levels in the presence of cystatin C concentrations at or above the reference interval are suggestive of muscle wasting, while the opposite may indicate very high muscle mass or excessive intake of cooked meat., fish or creatinine supplements (e.g., body builders) /23/.
  • Although cystatin C is predominantly excreted via the kidney, there is some hepatic elimination accounting for about 5% at normal. The relative contribution of extrarenal elimination increases with declining GFR and underlies the observation that cystatin C levels do not exceed 10 mg/L even in anuric patients. Thus, compared to creatinine, the rise in cystatin C is stronger with mildly to moderately impaired renal function (CKD stage 2-3), whereas the opposite applies to severe and end-stage renal failure (CKD stage 4-5) /23/.

12.7.6 Comments and problems

Specimen

The differences in cystatin C values in serum, heparin and EDTA plasma are slight and, therefore, all three specimens can be used. Estimated GFR based on capillary cystatin C concentrations is higher than the venous cystatin C based GFR. The limits of agreement between estimated GFR based on capillary and venous measurements exceed ± 20% at the upper reference concentration, which hampers the usefulness of capillary sampling /24/.

Method of determination

For PENIA one manufacturer provides the laboratory with instruments and reagents. For PETIA there are several manufacturer of reagents, but these are adapted to various analytical platforms. The calibrators are adjusted to the reference preparation ERM-DA471/IFCC with a concentration of 5.48 mg/L in the dissolved state /25/. The analytical imprecision of PENIA and PETIA and the upper reference interval values for individuals aged 70 and older were comparable /24/.

Although some manufacturers have clearly improved their calibration protocols relative to ERM-DA471, most of them failed to meet the criteria for acceptable cystatin C measurement /28/.

Reference interval

It is reported predominantly that in the age group of 1–49 years, the reference interval is stable. Newborns have significantly higher values which decrease during the first year in life. From the age of 50 onwards, a continuous rise in cystatin C occurs, due to reduction in the GFR /24/. A few publications hint at gender-specific differences, but the majority denies their existence /14/.

Biological variation

In a study 24-h profiles of cystatin C and its derived estimates of GFR were determined in people with and without CKD. The CV of cystatin C was in the same range for both people. Despite differences in the biological variation the reference change values of all eGFR equations were within 13 to 20% in both study groups /29/.

Interference factors

PENIA: hemolysis > 6–12 g Hb/L, bilirubin > 24.5 mg/dL (418 μmol/L), triglycerides > 800 mg/dL (10 mmol/L), rheumatoid factors > 2000 kIU/L /27/.

PETIA: hemolysis > 1 g Hb/L, bilirubin > 9–40 mg/dL (120–700 μmol/L), triglycerides > 700 mg/dL (8 mmol/L), rheumatoid factor > 300 kIU/L /26/.

Stability

7 days at room temperature, 1–2 months at –20 °C, at least 6 months at –80 °C /26/.

12.7.7 Biochemistry and physiology

Cystatin C, also termed γ-trace and post-γ-globulin, is a protease inhibitor belonging to the cystatin super family. Eleven proteins from this protease inhibitor family are known; clinically cystatin C is the most important of these. It is believed that cystatin C neutralizes proteases that are released from lysozymes or that originate from dying cells.

Cystatin C has a MW of 13,359 Da, is composed of 120 amino acids, is non-glycosylated, and contains two disulfide bonds. It is a basic protein with a pI of 9.3. Because it is synthesized as a pre protein, this is an indication of its extracellular function /27/. In bodily fluids, the highest concentrations are found in seminal plasma (41–62 mg/L) and in the cerebrospinal fluid (3.2–12.5 mg/L), the lowest concentrations are found in the urine (0.03–0.29 mg/L).

Cystatin C has been demonstrated in all organs and in all nucleated cells; it is synthesized by the cells at a relatively constant rate. There are, as yet, no concrete indications that infections, cancer, or exogenous substances may have a significant effect on cystatin C levels in serum. Only high concentrations of dexamethasone stimulate the formation of cystatin C in cell culture. In addition, the extreme doses that are used for immunosuppression lead to elevated values in transplant patients.

The higher reliability of cystatin C, in comparison with serum creatinine, for the assessment of the GFR is based on the following facts /27/:

  • As demonstrated in rat studies, 94% of cystatin C undergoes glomerular filtration as compared to 51Cr-EDTA clearance. There is only minimal and serum concentration-independent extrarenal clearance.
  • Following glomerular filtration, cystatin C is metabolized by the proximal tubular cells and is not reabsorbed in the intact form
  • In the presence of an intact tubular epithelium, there is no secretion of cystatin C from the renal vessels into the tubuli.

References

1. Levey AS, Fan L, Eckfeldt JH, Inker LA. Cystatin C for glomerular filtration rate estimation: coming of age. Clin Chem 2014; 60: 916–9.

2. Taglieri N, Koenig W, Kaski JC. Cystatin C and kardiovascular risk. Clin Chem 2009; 55: 1932–43.

3. Kidney Disease: Improving Global Outcome (KDIGO) CKD Work Group. KDIGO clinical practice guideline for the evaluation and management of chronic kidney disease. Kidney Int Suppl 2013: 3: 1–150.

4. Kyhse Andersen J, Schmidt C, Nordin G,Andersson B, Nilsson-Ehle P, Lindström V, Grubb A. Serum cystatin C, determined by a rapid, particle-enhanced turbidimetric method, is a better marker than serum creatinine for glomerular filtration rate. Clin Chem 1994; 40: 1921–6.

5. Mussap M, Ruzzante M, Varagnolo M, Plebani M. Quantitative automated particle-enhanced immunonephelometric assay for the routinary measurement of human cystatin C. Clin Chem Lab Med 1998; 36: 859–65.

6. Montini G, Cosmo L, Amici G, Mussap M, Zacchello G. Plasma cystatin C values and inulin clearances in premature neonates. Pediatr Nephrol 2001; 16: 463–4.

7. Bökenkamp A, Domanetzki M, Zinck R, Schumann G, Brodehl J. Reference values for cystatin C serum concentrations in children. Pediatr Nephrol 1998; 12: 125–9.

8. Norlund L, Fex G, Lanke J, von Schenck H, Nilsson JE, Leksell H, et al. Reference intervals for the glomerular filtration rate and cell proliferation markers: serum cystatin C and serum β2-microglobulin/cystatin C ratio. Scand J Clin Lab Invest 1997; 57: 463–70.

9. Bahar A, Yilmaz Y, Unver S, Gocmen I, Karademir F. Reference values of umbilical cord and third-day cystatin C levels for determining glomerular filtration rates in newborns. J Int Med Res 2003; 31: 231–5.

10. Fischbach M, Graff V, Terzic J, Bergere V, Oudet M, Hamel G. Impact of age on reference values for serum concentration of cystatin C in children. Pediatr Nephrol 2002; 17: 104–6.

11. Finney H, Newman DJ, Price CP. Adult reference ranges for serum cystatin C, creatinine and predicted creatinine clearance. Ann Clin Biochem 2000; 37: 49–59.

12. Grubb A. Cystatin C is indispensable for evaluation of kidney disease. eJIFCC 2017; 28: 268–76.

13. Newman DJ, Thakkar H, Edwards RG, et al. Serum cystatin C, measured by automated immunoassay: a more sensitive marker of changes in GFR than serum creatinine. Kidney Int 1995; 47: 312–8.

14. Keevil BG, Kilpatrick ES, Nichols SP, Maylor PW. Biological variation of cystatin C: implications for the assessment of glomerular filtration rate. Clin Chem 1988; 44: 1535–9.

15. Inker LA, Schmid CH, Tighioart H, Eckfeldt JH, Feldman HI, Greene T, et al. Estimating glomerular filtration rate from serum creatinine and cystatin C. N Engl J Med 2012; 367: 20–9.

16. Kidney Disease: Improving Global Outcome (KDIGO) CKD Work Group. KDIGO clinical practice guideline for the evaluation and management of chronic kidney disease. Kidney Int Suppl 2013: 3: 1–150.

17. Levey AS, Eckfeldt JH. Using glomerular filtration rate estimating equations: clinical and laboratory considerations. Clin Chem 2015; 61: 1226–9.

18. Oellerich M, Shipkova M, Assendorf T,Walson PD, Schauerte V, Mettenmeyer N, et al. Absolute quatification of donor derived cell free DNA as a marker of rejection and graft injury in kidney transplantation. Results from a prospective obsevational study. Am J Transplant 2019; 19 (11): 3087–99.

19. Grubb A, Horio M, Hansson LO, Björk J, Nyman U, Flodin M, et al. Generation of a new cystatin C-based estimating equation for glomerular filtration rate by use of 7 assays standardized to the international calibrator. Clin Chem 2014; 60: 974–86.

20. Muntner P, Vapputuri S, Coresh J, Uribarri J, Fox CS. Metabolic abnormalities are present in adults with elevated serum cystatin C. Kidney Int 2009; 76: 81–8.

21. Stevens LA, Schmid CH, Greene T, Li L, Beck GJ, Joffe MM, et al. Factors other than glomerular filtration rate affect serum cystatin C levels. Kidney Int 2009; 75: 652–60.

22. Jayacopal V, Keevil B G, Atkin SL, Jennings PE, Kilpatrick ES. Paradoxical changes in cystatin C and serum creatinine in patients with hypo- and hyperthyroidism. Clin Chem 2003; 49: 680–1.

23. van Roij KGE, van der Horst HJR, Hubeek I, van Wijk JAE, Bökenkamp A. Discrepant results of serum creatinine and cystatin C in a urological patient. Clin Chem 2017; 63: 812–5.

24. Schäfer E, Ebert N. Turbidimetrische versus nephelometrische cystatin C Analyse zur Einschätzung der Nierenfunktion bei Personen im Alter von 70 Jahren und älter. Klin Chem Mitteilungen 2016; 47. 18–28.

25. Grubb A, Blirup-Jensen S, Lindström V, Schmidt C, Althaus H, Zegers I. First certified reference material for cytatin C in human serum ERM-DA471/IFCC. Clin Chem Lab Med 2010; 48: 1619–21.

26. Newman DJ. Cystatin C. Ann Clin Biochem 2002; 39: 89–104.

27. Abrahamson M, Mason RW, Hansson H, Buttle DJ, Grubb A, Ohlsson K. Human cystatin C. Biochem J 1991; 273: 621–6.

28. Barnoux AS, Pieroni L, Cristol JP, Kuster N, Delanaye P, Carlier MC, et al. Multicenter evaluation of cystatin C measurement after assay standardization. Clin Chem 2017; 63: 833–41.

29. Hilderink JM, van der Linden N, Kimenai DM, Litjens EJR, Klinkenberg LJJ, Aref BM, et al. Biological variation of creatinine, cystatin C, and eGFR over 24 hours. Clin Chem 2018; 64: 851–60.

30. Shimizu-Tokiwa A, Kobata M, Io H, et al. Serum cystatin C is a more sensitive marker of glomerular function than serum creatinine. Nephron 2002; 92: 224–6.

31. Grubb A, Horio M, Hansson LO, Björk J, Nyman U, Flodin M, et al. Generation of a new cystatin C-based estimating equation for glomerular filtration rate by use of 7 assays standardized to the international calibrator. Clin Chem 2014; 60: 974–86.

32. Thomassen SA, Johannesen IL, Erlandsen EJ, Abrahamsen J, Randers E. Serum cystatin C as a marker of renal function in patients with spinal cord injury. Spinal Cord 2002; 40: 524–8.

33. Herget-Rosenthal S, Margraf G, Hüsing J, et al. Early detection of acute renal failure by serum cystatin C. Kidney Int 2004; 66: 1115–22.

34. Le Bricon T, Thervet E, Froissart M, Benlakehal M, Bousquet B, Legendre C et al. Plasma cystatin C is superior to 24-h creatinine clearance and plasma creatinine for estimation of glomerular filtration rate 3 months after kidney transplantation. Clin Chem 2000; 46: 1206–7.

35. Filler G. Cystatin C should be measured in pediatric renal transplant patients. Pediatr Transplantation 2002; 6: 357–60.

36. Boudville N, Salama M, Jeffrey GP, Ferrari P. The inaccuracy of cystatin C and creatinine-based equations in predicting GFR in orthoptic liver transplant recipients. Nephrol Dial Transplant 2009; 24: 2926–30.

37. Biancofiore G, Pucci L, Cerutti E, Penno G, Pardini E, Esposito M, et al. Cystatin C as a marker of renal function immediately after liver transplantation. Liver Transpl 2006; 12: 285–91.ann intern med

38. Ling Q, Xu X, Li J, Wu J, Chen J, Xie H, Zheng S. A new cystatin C based equation for assessing glomerular filtration rate in liver transplantation. Clin Chem Lab Med 2008; 46: 405–10.

39. Townsend DM, Deng M, Zhang L, Lapus MG, Hanigan MH. Metabolism of cisplatin to a nephrotoxin in proximal tubule cells. J Am Soc Nephrol 2003; 14: 1–10.

40. Weiss RB. Nephrotoxicity. In: deVita VT, Hellman S, Rosenberg SA (eds). Principles and practice of oncology. Philadelphia; Lippincott-Raven 1997: 2796–800.

41. Stabuc B, Vrhovec L, Stabuc-Silih M, Cizej TE. Improved prediction of decreased creatinine clearance by serum cystatin C: use in cancer patients before and during chemotherapy. Clin Chem 2000; 46: 193–7.

42. Mangge H, Liebmann P, Tanil H, et al. Cystatin C, an early indicator for incipient renal disease in rheumatoid arthritis. Clin Chim Acta 2000; 300: 195–202.

43. Calvo-Alen J, De Cos MA, Rodriguez-Valverde V, et al. Subclinical renal toxicity in rheumatic patients receiving longterm treatment with nonsteroidal antiinflammatory drugs. J Rheumatol 1994; 21: 1742–7.

44. Tan GD, Lewis AV, James TJ, Altman P, Taylor RP, Levy JC. Clinical usefulness of cystatin C for the estimation of glomerular filtration rate in type 1 diabetes. Diabetes Care 2002; 25: 2004–9.

45. Pucci C, Triscornia S, Lucchesi D, Fotino C, Pellegrini G, Pardinin E, et al. Cystatic C and estimates of renal function: searching for a better measure of kidney function in diabetic patients. Clin Chem 2007; 53: 480–8.

46. Stevens LA, Schmid CH, Zhang YL, Coresh J, Manzi J, Landis R, Bakoush O, et al. Development and validation of GFR-estimating equations using diabetes, transplant and weight. Nephrol Dial Transplant 2010; 25: 449–57.

47. Gerbes AL, Gülberg V, Bilzer M, Vogeser M. Evaluation of serum cystatin C concentration as a marker of renal function in patients with cirrhosis of the liver. Gut 2002; 50: 106–10.

48. Orlando R, Mussap M, Plebani M, Piccoli P, De Martin S, Floreani M, et al. Diagnostic value of cystatin C as a glomerular filtration marker in decompensated liver cirrhosis. Clin Chem 2002; 48: 850–8.

49. Randers E, Ivarsen P, Erlandsen EJ, et al. Plasma cystatin C as a marker of renal function in patients with liver cirrhosis. Scand J Clin Lab Invest 2002; 62: 129–34.

50. Woitas P, Stoffel-Wagner P, Flommersfeld S, Poege U, Schiedermaier P, Klehr HU et al. Correlation of serum concentrations of cystatin C and creatinine to inulin clearance in liver cirrhosis. Clin Chem 2000; 46: 712–5.

51. Bökenkamp A, Stoffel-Wagner B, Hasan C, Henne T, Offner G, Lentze MJ. The β-microglobulin/cystatin C ratio: a potential marker of post-transplant lymphoproliferative disease. Clin Nephrol 2001; 48: 417–22.

52. Parikh Ni, Hwang SJ, Yang Q, Larson MG, Guo Cy, Robins SJ, et al. Clinical correlates and heretability of cystatin C (from the Framingham Offspring Study). Am J Cardiol 2008; 102: 1194–8.

53. Kestenbaum B, Rudser KD, de BI Peralta CA, Fried LF, Shlipak MG, et al. Differences in kidney function and incident hypertension: the multiethnic study of atherosclerosis. Ann Intern Med 2008; 148: 501–8.

54. Shlipak MG, Sarnak MJ, Katz R, Fried LF, Seliger SL, Newman AB, et al. Cystatin C and the risk of death and cardiovascular events among elderly persons. N Engl J Med 2005; 352: 2049–60.

55. Sarnak MJ, Katz R, Stehman-Breen CO, Fried LF, Swords Jenny N, Psaty BM, et al. Cystatin C concentration as a risk factor for heart failure. Ann Intern Med 2005; 142: 497–505.

56. Akerbloom A, Wallentin L, Siegbahn A, Becker RC, Budaj A, Buck K, et al. Cystatin C and eGFR as predictors of adverse outcome in patients with ST-elevation and non-ST-elevation acute coronary syndromes: results from the platelet inhibition and patient outcomes study. Clin Chem 2012; 58: 190–9.

57. Akerbloom A, Wallentin L, Larsson A, Siegbahn A, Becker RC, Budaj A, et al. Cystatin C- and creatinine-based eatimates of renal function and their value for risk prediction in patients with acute coronary syndrome: results from the platelet inhibition and patient outcomes study. Clin Chem 2013; 59: 11369–75.

58 Chew-Harris JSC, Florkowski CM, George PM, Elmslie JL, Endre ZH. The relative effects of fat versus muscle mass on cystatin C and estimates of renal function in healthy young men. Ann Clin Biochem 2013; 50: 39–46.

59. Ncube V, Starkey B, Wang T. Effect of fenobibrate treatment for hyperlipidaemia on serum creatinine and cystatin C. Ann Clin Biochem 2012; 49: 491–3.

12.8 Erythrocytes, leukocytes, casts in urine

Christian Thomas, Lothar Thomas

The detection of hematuria, leukocyturia and proteinuria are early indicators of kidney and urinary tract disorders. Evidence of casts provides an indication of a pathological process in the kidney.

Screening for hematuria and leukocyturia includes the use of:

  • Test strips with test fields for erythrocytes/hemoglobin and leukocytes
  • Particle examination by means of microscopy or flow cytometry.

In asymptomatic patients the test strip screening precedes particle examination and this is only performed if the test yields a positive result either for erythrocytes, leukocytes, protein, and glucose. The diagnostic sensitivity of the test strips is adequate for the detection of clinically significant erythrocyturia and leukocyturia /1/.

Depending on the clinical concerns in the presence of proteinuria, hematuria or leukocyturia, the following further examinations may be useful for the location of the underlying injury:

  • Macroscopic inspection of the urine before and after centrifugation
  • Particle analysis
  • Investigation for the presence of dysmorphic erythrocytes
  • Urine cytology if hematuria is present, for the diagnosis of tumor cells.

12.8.1 Indication

Test strips

Screening during the first examination for the exploratory exclusion of a kidney and urinary tract disease.

Particle examination

  • Patients with symptoms of a kidney and urinary tract disease
  • If the test strip indicates a positive finding (e.g., proteinuria, erythrocyturia, leukocyturia, and glucosuria).

Dysmorphic erythrocytes

Differentiation between renal and extrarenal hematuria.

Urine cytology

Diagnosis and monitoring of bladder tumors.

12.8.2 Method of determination

For the detection of hematuria and/or leukocyturia, a sample of uncentrifuged, well-mixed urine is examined within 2 hours following collection.

12.8.2.1 Erythocyte examination

Principle of reagent strip test

The test detects pseudo-esterase activity of erythrocytes. The reagent pad contains an organic hydro peroxide, which oxidizes a highly sensitive chromogen to a blue dye through catalytic pseudo-esterase activity. The presence of erythrocytes is recorded as spots on the test field, while hemoglobin or myoglobin are recorded as a homogeneous appearance of color on the reagent pad. For erythrocyte count of 10 × 106/L, the diagnostic sensitivity of the test strip is 70–80% /2/. The specificity of the red blood cell detection is, in comparison with chamber counting, only seemingly diminished, since some of the cells in the urine lyse and the test strips provide a positive result, while chamber counting shows a negative outcome.

12.8.2.2 Leukocyte examination

Principle of reagent strip test

Neutrophil granulocytes and macrophages contain the enzyme indoxyl esterase. The reagent pad on the test strip contains an indoxyl ester. The ester is cleaved in the presence of leukocytes. The indoxyl produced is oxidized to indigo blue under the influence of atmospheric oxygen which results in a color change on the test pad from beige to blue /3/. The detection limit of the test strip is 20 × 106 leukocytes/L and the accuracy 80–90% when compared with microscopic leukocyte count. At a cell count of 100 × 106 leukocytes/L, the diagnostic sensitivity is 95%. At the limit of detection, the diagnostic specificity is 80–90% /4/.

12.8.2.3 Particle examination

Clinically significant particles in the urine include /4/:

  • Erythrocytes as indicators of hematuria. These can result from a general bleeding tendency, a renal disorder, a urinary tract disorder, or vaginal contamination. In cases of isolated hematuria, dysmorphic erythrocytes indicate that the patient requires further nephrological treatment.
  • Leukocytes, which can indicate a urinary tract infection, glomerulonephritis, or interstitial nephritis
  • Casts, which can indicate a renal disorder. The diagnostic sensitivity for casts as indicators of renal disorders is low.
  • Epithelial cells from the external genitals and the urethra. Except in pregnant individuals, these indicate a mistake in the urine specimen collection method used.
  • Cells from the transitional epithelium (urothelium), from the renal pelvis to the ureters. These are indicative of a urinary tract infection, but are also evident in noninfectious urological disorders.
  • Renal tubular epithelial cells. These are small, round epithelial cells and are present in disorders such as acute tubular necrosis, acute interstitial nephritis, or kidney transplant rejection.
  • Fatty droplets or lipid-laden tubular cells. They are a sign of damage to the glomerular basement membranes, through which lipids can permeate.
  • Bacteria which are only clinically significant in spontaneously voided urine at counts of ≥ 105/mL.

Particle counts should be performed within 1 hour of the sample being collected. If this is not possible, the sample can be kept refrigerated for 2–4 hours /4/.

In routine diagnostic testing, particle identification is performed microscopically and qualitatively using urine sediment under a coverslip. Quantitative testing is performed on uncentrifuged urine either using a counting chamber or by automated particle analysis.

12.8.2.4 Standardized urine sediment under a coverslip

Principle of urine sediment test

Urine collected no more than 2 hours previously is briefly shaken, and then 10 mL is decanted into a centrifuge vial. The specimen is then centrifuged for 5 minutes at 400 × g to obtain a 25-fold concentration. The test tube is tipped and emptied, taking care to allow excess material to drip off. The sediment is thoroughly homogenized with the remaining supernatant by drawing it in to and out of a rubber-bulbed Pasteur pipette five times. A small drop of the homogenate is placed on a slide and a cover slip is placed on top. Under the microscope, the distribution density of the cells is first evaluated with the 10 × ocular lens and 10 × objective. If the distribution is homogeneous, a count is performed using the 40 × objective. Microscopy should ideally be performed using a phase contrast microscope with a polarization system /45/.

Centrifugation methods such as the coverslip urine sediment method can never be used for the quantitative assessment of erythrocyte and leukoctyte counts. Variable amounts of cells (on the order of 20–80%) are lost during centrifugation /6/.

12.8.2.5 Chamber counting of uncentrifuged samples

Principle of chamber counting test

A sample from an uncentrifuged, well-mixed spot urine is placed in a counting chamber, and the number of erythrocytes and leukocytes per microliter are determined. If the counting chamber has,for example, a depth of 0.25 mm and squares with an area of 1 mm2, then counts are performed in 4 of the 1 mm2 squares and the cell count calculated as follows:

Cell count/μl = Counted cells Chamber depth (mm) × 4

One disadvantage of using the counting chamber method is that it is highly time-consuming. Without staining or phase contrast microscopy, only inadequate particle differentiation is possible.

12.8.2.6 Automated urinalysis systems

Urine chemistry analyzers determine chemical constituents of the urine and count and differentiate erythrocytes, leukocytes, epithelial cells and casts flow cytometrically /7/. For particle examination, the cellular DNA is stained with a fluorochrome, and particle size is determined following hydrodynamic focusing of the cells using forward and side scatter.

12.8.2.7 Erythrocyte morphology

Principle of erythrocyte morphology testing

Mid-stream morning urine is examined 1–2 hours following collection /8/. The test needs not to be performed immediately if, following collection, approximately 50 mg of the antiseptic compound thiomersal is added to about 10–20 mL of the urine sample. In urine conserved in this manner, the urinary corpuscular constituents remain stable for 3 to maximally 7 days /9/.

Procedure: 10 mL of urine are centrifuged for 5 minutes at 400 × g (swing bucket rotor), and 9.5 mL of supernatant are discarded. The sediment is resuspended in the remaining 0.5 mL of urine.

The erythrocyte morphology can be assessed as follows:

  • With the phase contrast microscope; 1 drop of sediment is placed on a slide, covered with a cover slip and immediately examined microscopically
  • With the bright field microscope, following staining of the urine sediment; 1 drop of sediment is put onto the middle of a cover slip, which is then placed on a dye-coated slide. The erythrocytes are examined following 10 minutes of staining time. Slides pre-colored with the dyes new methylene blue N and cresyl violet acetate are well suited and commercially available. The staining of the sediment on the slides, according to Papanicolaou, also supports the morphological assessment /9/.

The morphology of 100 erythrocytes is assessed, and the percentage of isomorphic and dysmorphic forms is determined.

12.8.2.8 Urine cytology

Mid-stream urine from the second voided morning urine is examined, and a sediment is prepared. For the differentiation of leukocytes, a drop of resuspended sediment is placed on a dye-coated slide (see above). For the assessment of the morphology of tubular cells or bladder epithelial cells, these cells are prepared, fixed, and stained according to May-Grünwald-Giemsa or Papanicolaou.

12.8.3 Specimen

Mid-stream morning urine should be collected for the microscopic assessment of cells and casts. It should not be older than 2 hours at the time of the microscopic examination. For the different urine specimens see Section 12.3 – Urinalysis.

The testing for dysmorphic erythrocytes is only considered to be meaningful if the test strips are positive for blood, or as of an erythrocyte count of (6–8) × 106/L of urine, and not in cases of macro hematuria.

12.8.4 Reference interval

Refer to Ref. /410111213/ and Tab. 12.8-1 – Reference intervals for erythrocytes, leukocytes and casts in urine.

12.8.5 Clinical significance

The use of test strips to analyze urine and the use of microscopy to analyze urine sediment enable the semi quantitative identification of erythrocytes and leukocytes; microscopy can also be used to test for casts and epithelial cells. Patients generally do not need to undergo microscopic urine sediment testing in cases with test strip results being negative for nitrite, normal protein, erythocytes and glucose (< 1 g/L) in the initial evaluation /13/.

For microscopic urine analysis, positivity criteria are defined as the detection of the cellular components erythocytes and/or leukocytes per ≥ 4 per high power field (10 × 40).

12.8.5.1 Hematuria

The microscopic examination is a sensitive way of detecting erythrocytes in urine. The test strip can detect (2.5–5) × 107 erythrocytes per liter (25–50 erythrocytes per μL) with high accuracy corresponding to 3 erythrocytes per high power field (10 × 40). The detection limit of microscopic examination is 1.2 × 107 erythrocytes per liter /14/. Definitions for micro hematuria vary and are ≥ 4 erythrocytes with the standardized urine sediment under cover slip, > 10 erythrocytes/μL, in chamber counting of uncentrifuged specimens and in particle counting instruments > 15/μL /15/. Macro hematuria (visible with the naked eye) and micro hematuria (only visible with the microscope) are distinguished.

Clinically, hematuria is classified into a glomerular and a non-glomerular form /15/.

In glomerular hematuria, the erythrocytes are often dysmorphic and casts are demonstrable in 30% of the cases. In the non-glomerular form, the erythrocytes are normal and casts are not present.

Non-glomerular hematuria can be of urological or non-urological genesis. The absence of proteinuria differentiates urological causes of non glomerular hematuria from other diseases that are associated with hematuria. Frequently causes of non-urological, non glomerular genesis are tubulo-interstitial diseases (vascular diseases, papillary necrosis).

Urological causes of hematuria are divided into painful (infectious disease, nephrolithiasis) and pain-free (urological cancers, benign prostatic hyperplasia). Frequent causes of hematuria are shown in Tab. 12.8-2 – Causes of hematuria, an algorithm for the location of source in Fig. 12.8-1 – Diagnostic procedure for investigating hematuria.

12.8.5.1.1 Prevalence of hematuria

Children

The prevalence of persistent, isolated hematuria in children is 0.4–4% /16/. In one study involving 12,000 schoolchildren, 6% had hematuria (> 5 erythrocytes per high power field) when first tested. Hematuria was more common in girls than in boys. The incidence of hematuria in a second set of samples taken within a week of the first was only half as high. The annual incidence of new cases in 6–12 year-old children was 0.4% /17/. In another study involving 9000 schoolchildren, 4.1% had hematuria, while only 0.5% had the condition for over 6 months. Macro hematuria is rare in childhood.

Adults

In one study /18/, 2.5% of males aged 28–57 had hematuria, while in another study /19/, 5.4% of males aged 18–54 had the condition. The prevalence in postmenopausal females is reported to be up to 13% /20/. Among urological patients, the frequency of hematuria is about 10%. According to the American Urological Association, up to 56% of hematuria cases are associated with a significant disease, while the prevalence of malignant urologic cases among this patient pool is up to 25.8% /14/. The prevalence of hematuria depends on age and sex, and is higher in older individuals and in males. Prospective studies with hematuria patients found the cause to be urinary tract cancer in 37% of cases, while 15% were due to nephrolithiasis, urinary tract obstruction, or chronic urinary retention /21/. Another study /14/ also found neoplasms to be the most common cause of hematuria (in 41.8% of cases). Primary carcinomas of the genitourinary system were found in 22% of cases, of which 9% were in the bladder, 6% in the kidneys, and 6% in the prostate. The benign causes included prostatic hypertrophy (19%), urinary tract infections (26%), nephrolithiasis (13.6%), congenital abnormalities (3.6%), trauma (2%), and unidentified etiology (12%) /22/. No increase in risk of microscopic hematuria with aspirin use of 75–325 mg per day is found by asymptomatic healthy people /23/.

12.8.5.1.2 Macro hematuria

Macro hematuria is present with > 1000 erythrocytes/μL. 1 mL of blood in 1 liter of urine is equivalent to some 25,000 erythrocytes/μL. Depending on the source of the blood, the concentration of blood in the urine, and other factors, the color of the urine can range from light red to pink to tea or cola-brown.

Tea or cola-colored urine tends to be indicative of a glomerular disorder, while red or pink-colored urine indicates bleeding in the urinary tract.

Blood clots indicate the ureters or bladder as the source of the blood. They do not occur with glomerular hematuria, since the glomerular filtrate contains urokinase and t-PA (tissue-type plasminogen activator).

Hematuria at the end of urination indicates the bladder as the source of the blood, hematuria at the start of urination indicates the urethra or prostate, and hematuria throughout urination points to the kidneys, ureters, or diffuse bleeding as the source.

Children

The prevalence of macro hematuria is reported to be 0.13%. The possible causes that need to be considered include renal stone disease, primary glomerulonephritis (GN), systemic disease with GN, hemolytic uremic syndrome, Alport syndrome, and a malignant tumor. The most common glomerulonephritic disorders associated with macro hematuria are IgA nephropathy, acute post infectious GN, and GN associated with Henoch-Schoenlein purpura /24/.

Adults

Macro hematuria is a common symptom of polycystic kidney disease, IgA nephropathy, kidney tumors, kidney stones, prostate disorders, bladder tumors, bladder tuberculosis, bladder stones, hemorrhagic cystitis, hemorrhagic diathesis, and hydronephrosis /25/.

12.8.5.1.3 Pseudo hematuria

Red urine comparable to macro hematuria can be passed following circumstances that include (see also Tab. 12.3-1 – Characteristic appearances of urine):

  • Eating foods such as rhubarb, red beets or substances containing aniline dyes
  • Use of medications such as phenothiazine, phenazopyridine, or phenindione
  • Porphyria, rhabdomyolysis, or hemolysis.

False-positive test strip results can be caused by substances that contain iodine and other oxidants, as well as by peroxidases in cases of bacteriuria /26/.

Hematuria can occur during treatment with the following medications /27/: phenytoin, rifampicin, anticoagulants (including aspirin), NSAIDs such as ibuprofen, and cytostatics such as cyclophosphamide, ifosfamide, and danazol.

12.8.5.1.4 Microhematuria

Microhematuria is a type of hematuria that can only be identified by a test strip assay, examination of the sediment under a cover slip, or by chamber counting. The prevalence of microhematuria among general medical clinics in outpatients is 5–13% in adults, 1–2% in infants and 4% in school children /28/. Any incidence of microhematuria in the absence of a urinary tract infection or menstrual bleeding should be further investigated. Often microhematuria is an accidental finding.

If the microhematuria was identified by urine test strip, the result must be confirmed by microscopy to rule out pseudo hematuria.

The discovery of micro hematuria without proteinuria is often an incidental finding. About 70% of cases cannot be explained even after a thorough examination of the upper urinary tract with diagnostic imaging.

The most common causes of micro hematuria in the upper urinary tract include:

  • In adults, the most common cause of glomerular micro hematuria is IgA nephropathy and thin basement membrane nephropathy (TBMN). Thirty percent of patients with isolated microscopic hematuria have mesangial immunoglobulin A glomerulonephritis and 20–40% of these patients will progress to renal failure without treatment /29/. TBMN is often diagnosed clinically when there is persistent dysmorphic or glomerular hematuria, but minimal proteinuria, normal kidney function, and no other obvious cause /30/.
  • In children, urinary tract infections, lithiasis, idiopathic hypercalcuria (in 22% of cases), or hyper uricosuria /31/
  • Other common etiologies are chronic glomerulonephritis and the after-effects of acute glomerulonephritis /24/. In addition to urolithiasis, the occurrence of hematuria after 2–3 years is a complication in about one-third of patients following a kidney transplant.

Micro hematuria has two types, and can be:

  • Transient in nature, for example due to extreme physical exertion (as seen in long-distance runners), infection, or trauma
  • Permanent or recurrent in nature, resulting from kidney stones, malignant tumors in the kidneys and urinary tract, and glomerular or interstitial nephritis.

In one study /32/, patients with clinically confirmed asymptomatic micro hematuria and an average age of 44.2 years were followed for an average of 3.7 years. All of the patients remained asymptomatic during this time. When the initial testing revealed > 9 erythrocytes per high power field, the hematuria was more constant in nature than when 3–9 erythrocytes were detected.

Refer to Tab. 12.8-4 – Diagnosis and definition of microhematuria.

12.8.5.1.5 Dysmorphic erythrocytes

The morphology of red blood cells is important for the differential diagnosis of isolated hematuria. Dysmorphic erythrocytes appear in the urine as a consequence of glomerulonephritis. These erythrocytes feature characteristic, microscopically recognizable changes in shape (Fig. 12.8-2 – Display of non-glomerular, osmotically modified erythrocytes and glomerular dysmorphic erythrocytes). The acanthocytes are particularly relevant. A fraction of > 10% of the microscopically counted erythrocytes is believed to be indicative of glomerulonephritis, with a diagnostic sensitivity and specificity of > 90%. Most of the dysmorphic erythrocytes shown in Fig. 12.8-2 can also be formed in vitro. This is not as often the case with acanthocytes. These cells have a ring-like form with protuberances. According to one study /33/ in 101 patients with histologically confirmed glomerulonephritis, the fraction of dysmorphic erythrocytes was 68 ± 24%; of these, 16.7 ± 16.3% were acanthocytes. The highest proportion of dysmorphic cells was observed in membranous and mesangio-proliferative glomerulonephritis, the lowest in minimal change nephropathy.

The determination of dysmorphic erythrocytes is of limited value as far as urologists are concerned. Since patients with post glomerular hematuria are more seen by such specialists the diagnostic specificity of dysmorphic erythrocytes is not high enough for glomerular hematuria, too many false positive cases of glomerular hematuria are diagnosed /34/.

12.8.5.1.6 Free hemoglobin, myoglobin

Hemoglobinuria and myoglobinuria can be mistaken for hematuria. The causes are acute intravasal hemolysis or severe muscular damage (e.g., due to trauma, electric shock, seizure).

12.8.5.2 Leukocyturia

If urine is collected according to the conditions described in Section 53.2.4 – Urine collection, a leukocyte count > 10 × 106 per liter (> 10/μl) in males and > 20 × 106 per liter (> 20/μl) in females is to be considered as pathological. Leukocyturia and bacteriuria are characteristic of a urogenital tract infection. The presence of granulocyte casts speaks for an involvement of the kidneys in the pathological process.

The clinical significance of isolated leukocyturia incidentally diagnosed in young men is unclear, while in women it often indicates contamination. A study of 1000 men with asymptomatic leukocyturia showed that all remained free of urogenital tract complaints after an average of 7.6 years /19/. Sterile isolated leukocyturia is seldom a sign of urogenital tuberculosis.

In combination with tubular proteinuria, sterile isolated leukocyturia is suggestive of analgesic nephropathy in adults, and of Kawasaki syndrome in children.

Granulocyturia

The most commonly detected leukocytes in urine are polymorphonuclear granulocytes. In most cases they are associated with an infection of the urogenital tract, but are also found in interstitial nephritis, glomerulonephritis, and aseptic cystitis.

Lymphocyturia

Lymphocyturia is a sign of a chronic inflammatory process in the urogenital tract and can be associated with viral infections and renal graft rejection.

Monocyturia

Appears in urinary tract infections.

Eosinophil granulocytes

They can be a sign of acute interstitial nephritis, but also occur in other renal diseases /4/.

12.8.5.3 Excretion of casts

Casts are formed in the distal tubules and collecting ducts. Cast formation requires favorable conditions in terms of osmolality, pH, electrolyte and protein concentrations. Under these conditions, the Tamm-Horsfall protein secreted by tubular epithelial cells can polymerize to fibrils. Fibrillar precipitates incorporate plasma proteins, lipids, various types of cells, microorganisms, hemoglobin, myoglobin, bilirubin, and crystals. The matrix for all casts is Tamm-Horsfall protein, which from the visual perspective has a very similar refractive index to urine. Hyaline casts, which consist almost entirely of Tamm-Horsfall protein, are therefore barely visible under a high power field microscopy, but are clearly visible using phase contrast microscopy.

Casts can be categorized as:

  • Non-cellular casts (e.g., hyaline casts, granulated casts, waxy casts, and fatty casts)
  • Cellular casts (e.g., epithelial, erythrocyte, leukocyte, and bacterial casts).

The detection of casts is a specific indicator for renal disorders, but is not very sensitive. While non-cellular casts are normally evident in the urine under circumstances such as strenuous exercise (e.g., long-distance running) this is not the case with cellular casts. The diagnostic significance of the individual cast types is shown in Tab. 12.8-3 – Diagnostic significance of urinary casts.

12.8.6 Comments and problems

Specimen collection

Morning mid-stream urine is recommended for the screening of asymptomatic patients. A first morning, mid-stream sample is recommended for hospital inpatients with nephrological and urological conditions, while a second morning sample is recommended for outpatients. The genitals must be washed prior to specimen collection.

Test strip for hematuria

Erythrocytes, free hemoglobin, myoglobin (≥ 0.5 mg/L), and erythrocyte casts will give a positive reaction.

False-positive results can be caused by oxidants such as cleaning agents and disinfectants that contain chlorine or iodine, as well as by microbial peroxidases.

False-negative results can arise due to reducing agents such as ascorbic acid, particularly when 1 g or more is taken daily. It should be noted that ascorbic acid is frequently used as a stabilizer for other drugs, such as tetracycline.

Test strip for leukocyte detection

The test strip detects leukocyte-bound esterase in the urine, dissolved leukocyte esterase, and the leukocyte esterase from leukocyte casts.

False-positive leukocyte reactions are caused by a high level of tubular esterase activity and by formaldehyde. When compared with quantitative cell counts and the examination of sediment under a cover slip, ostensibly false positive reactions are often seen after leukocytes have been lysed. Lysis can result from a pH > 6, low urine osmolality, and allowing the urine to stand for hours prior to analysis. In hypotonic urine, the leukocyte count will fall to about 25% of its original level within 4 hours.

False-negative leukocyte reactions are caused by a high urine protein concentration (> 5 g/L) which interferes with the reaction in the leukocyte test field. False-negative results have also been reported in patients taking doxycycline, gentamycin, cephalexin, and cephalothin, as well as due to excessive glucose excretion (> 20 g/L) and high concentrations of oxalic acid.

Quantitative cell count

At least 100 of a given type of cell must be counted in order for a reliable result to be obtained. If the cell count is low, the chamber should be filled several times.

Standardized urine sediment under a coverslip

A standardized test procedure is essential for obtaining accurate results. Well homogenized, resuspended sediment and homogeneous cell distribution in the specimen are important. The homogenization must be verified by an initial assessment using the 10 x objective. Should the aggregation of eythrocytes and leukocytes occur despite thorough homogenization, this indicates that the cells did not originate in the kidneys, and therefore points to the urinary tract or prostate /5/.

Detection of casts

Alkaline and hypotonic urine (< 260 mmol/kg) facilitate the rapid dissolution of casts and cells. This can largely be avoided if the patient is appropriately prepared (nothing to eat or drink for 8–10 hours before the first morning mid-stream urine is collected). Screening for casts should be performed using a phase contrast microscope at 100 x magnification, while cast identification should be performed at about 400 × magnification.

Stability

Testing for erythrocytes and leukocytes should be performed within 2 hours of the specimen being collected. Targeted testing for dysmorphic erythrocytes and casts should be performed immediately after the sample is collected, on urine that is no more than one hour old. Dysmorphic erythrocytes reportedly remain stable for at least 3 days in urine that has been preserved using thiomersal /9/.

References

1. Braun JS, Straube W. Die Diagnostik der Mikrohämaturie mit einem neuen Teststreifen. Ein Vergleich mit mikroskopischen Untersuchungsmethoden. Dtsch Med Wschr 1975; 100: 87–9.

2. Gambke B, Kouri T, Kutter D, Nagel D, et al. Multicenter evaluation of the urine analyser Miditron Junior. Scand J Clin Lab Invest 1997; 57: 605–11.

3. Kutter D, Figueiredo G, Klemmer L. Chemical detection of leukocytes in urine by means of a new multiple test strip. J Clin Chem Clin Biochem 1987; 25: 91–4.

4. European Urinalysis Guidelines: Summary. Scand J Clin Lab Invest 2000; 60: 1–9.

5. Thiel G. Urinuntersuchung in der Praxis. Schweiz Rdsch Med 1977; 66: 689–96.

6. Gadeholt H. Quantitative estimation of urinary sediment, with special regard to sources of error. Br Med J 1964; 1: 1547–9.

7. Hoffmann P, Hoffmann C, Ziebig R, Zimmermann M. Evaluation of the iChem velocity urine chemistry analyzer in a hospital routine laboratory. Clin Chem Lab Med 2011; 49: 509–13.

8. Birch DR, Fairly KF, Whitworth JA, et al. Urinary erythrocyte morphology in the diagnosis of glomerular haematuria. Clin Nephrol 1983; 20: 78–84.

9. Roth St, Renner E, Rathert P. Diagnostik der glomerulären Mikrohämaturie. Urologe B 1992; 32: 71–6.

10. Loh EH, Keng VW, Ward PB. Blood cells and red cell morphology in the urine of healthy children. Clin Nephrol 1990; 34: 185–7.

11. Fairley KF, Birch DF. Microscopic urinalysis in glomerulonephritis. Kidney Int 1993; 44 suppl 42: pg. 9 – pg. 12.

12. Thiel G, Bielmann D, Wegmann W, Brunner FP. Erythrozyten im Urin: Erkennung und Bedeutung. Schweiz Med Wschr 1986; 116: 790–7.

13. Chambliss AB, Mason HM, Van TT. Correlation of chemical urinalysis to microscopic urinanalysis and urine culture: implications for reflex urinalysis workflows. JALM 2020; July: 724–31.

14. Copley JB. Isolated asymptomatic hematuria in the adult. Amer J Med Sci 1986; 29: 102–11.

15. Cohen RA, Brown RS. Microscopic hematuria. N Engl J Med 2003; 348: 2330–8.

16. Diven SC, Travis LB. A practical primary care approach to hematuria in children. Pediatr Nephrol 2000; 14: 65–72

17. Dodge WF, West EF, Smith EH, Bruce H III. Proteinuria and hematuria in school children: epidemiology and early natural history. J Pediatr 1976; 88: 327–47.

18. Ritchie CD, Bevan EA, Collier SJ. Importance of occult hematuria found at screening. BMJ 1986; 292: 681–3.

19. Froom P, Gross M, Froom J, Caine Y, Margaliot S, Benbassat J. Factors associated with microhematuria in asymptomatic young men. Clin Chem 1986; 32: 2013–5.

20. Ahmed Z. Asymptomatic urinary abnormalities. Hematuria and proteinuria. Med Clin North Am 1997; 81: 641–52.

21. Bolenz C, Schröppel B, Eisenhardt A, Schmitz-Dräger DJ, Grimm MO. The investigation of hematuria. Dtsch Arztebl Int 2018; 115: 801–7.

22. Carter WC, Rous SN. Gross hematuria in 110 adult urologic hospital patients. Urology 1981; 18: 342–4.

23. Jeong CW, Lee S, Byun SS, Lee DH, Lee SE. No increase in risk of microscopic hematuria with aspirin use by asymptomatic healthy people. JAMA Intern Med 2013; 173: 1145–6.

24. Gillat DA, O’Reilly PH. Hematuria analyzed: a prospective study. J R Soc Med 1987; 80: 559–62.

25. Froom P, Ribak J, Benbassat J. Significance of macrohematuria in young adults. Br Med J 1984; 288: 20–22.

26. Lam MH. False “hematuria” due to bacteriuria. Arch Pathol Lab Med 1995; 119: 717–21.

27. Bryden AAG, Paul AB, Kyriakides C. Investigation of haematuria. Br J Hosp Med 1995; 54: 455–8.

28. Cohen RH,Brown RS. Microscopic hematuria. N Engl J Med 2003; 348: 2330–8.

29. Kincaid-Smith P, Fairley K. The investigation of hematuria. Semin Nephrol 2005; 25: 127–35.

30. Packham DK, Perkovic V, Savige J, Broome MRA. Hematuria in thin basement membrane nephropathy. Semin Nephrol 2005; 25: 146–8.

31. Stapleton FB. Hematuria associated wit hypercalciuria and hyperuricosuria: a practical approach. Pediatr Nephrol 1994; 8: 756–61.

32. Rüttimann S, Dreifuss M, Di Gallo A, Huser B, Dubach UC. Asymptomatische Hämaturie: Verlaufsbeobachtung bei 39 Patienten. Schweiz Med Wschr 1990; 120: 1461–5.

33. Fünfstück R, Klinzing S, Stein G, Schultz W, Risler T, Rösch R, et al Dysmorphe Erythrozyten im Urin bei unterschiedlichen Glomerulonephritisformen. Nieren- und Hochdruckkrankheiten 1995; 24: 554–8.

34. Offringa M, Benbassat J. The value of urinary red cell shape in the diagnosis of glomerular and post-glomerular haematuria. A metaanalysis. Postgrad Med J 1992; 68: 648–54.

35. Barocas D, Boorjian S, Alvarez RD, Downs T, Gross CP, Hamilton BD, Kobashi K, et al. Microhematuria: AUA/SUFU guideline. J Urol 2020; 204 (4): 778–86.

12.9 Urinary proteins

Lothar Thomas

12.9.1 Proteinuria

The distinction between primary kidney disease and systemic disease is based on the origin and locus of the disease process. In primary kidney disease the process arises and is confined to the kidney, whereas in systemic disease the kidney is compromised by a specific systemic process, for example diabetes mellitus and hypertension. In chronic kidney disease (CKD) proteinuria, especially albuminuria, is a marker of kidney damage /1/. CKD is not a diagnosis in and of itself and the assignment of cause is important for prognosis and treatment. The cause of CKD has been traditionally assigned based on presence or absence of underlying systemic diseases and location of presumed pathologic-anatomic abnormalities. The location of pathologic-anatomic findings is based on the magnitude of proteinuria, findings from the urine sediment examination, imaging and renal pathology. Proteinuria and albuminuria express the severity of kidney disease not only because they are indicators of the severity of injury but also because albuminuria itself strongly associates with progression of kidney disease /1/.

CKD is classified based on cause, GFR category, and albuminuria category. The extent of kidney damage and glomerular permeability can be assessed using quantitative measurement of albumin or protein excretion. The pattern of the excreted plasma proteins allows differentiation into selective glomerular, selective tubular or non-selective proteinuria. Urinary albumin measurements may be useful in early risk profiling and prevention of cardiovascular disease in the population at large. A 24-hour urine collection is no longer necessary for the quantitative assessment of proteinuria, and test strip methods are insufficiently sensitive and specific for its detection /23/.

Proteinuria is defined as a loss of:

  • Albumin /2/; generally proteinuria reflects albuminuria. Albuminuria is the earliest marker of glomerular disease, including diabetic glomerulosclerosis, where it generally appears before the reduction in GFR. Albumin is the predominant protein in the majority of proteinuric kidney diseases.
  • Total protein /3/; in pathological states, total urinary protein is made up of low levels of physiological proteins (e.g., uromodulin), albumin (the predominant protein in most disease states), non-albumin proteins (comprising a wide range of proteins, of varying molecular weights) and specific pathologic proteins (e.g., immunoglobulin light chains).

The 24-hour urine collection is the gold standard for the quantification of proteinuria, but is associated with considerable collection errors and is burdensome for the patients. Therefore, the spot measurement is recommended, which has a good overall correlation with the 24-hour total proteinuria. In the spot urine sample the total protein/creatinine ratio or the albumin/creatinine ratio should be measured. If the excretion of total protein and albumin in the 24-hour urine is compared with the total protein/creatinine ratio, and the albumin/creatinine ratio, significant proteinuria can be excluded with acceptable degree of reliability. Further proteinurias with important diagnostic thresholds (> 0,5 g total protein/day or > 1 g/day) are correctly identified /45/. Proteinuria > 1 g/day is frequently an indication for renal biopsy.

12.9.1.1 Diagnostic approach

The KDIGO /1/ recommends using the following measurements for initial testing of proteinuria (in descending order of preference; in all cases an early morning urine sample is preferred):

  • Albumin/creatinine ratio (ACR)
  • Total protein/creatinine ratio (PCR)
  • Reagent strip analysis for total protein with automated reading
  • Reagent strip analysis for total protein with manual reading.

Further recommendations are:

  • Confirm reagent strip positive albuminuria and proteinuria by quantitative laboratory measurement and express as a ratio to creatinine whenever possible
  • Confirm ACR of ≥ 30 mg/g (≥ 3 mg/mmol) on a random untimed urine with a subsequent early morning urine sample
  • If a more accurate estimate of albuminuria or total proteinuria is required, measure albumin excretion rate or total protein excretion rate in a timed urine sample
  • If significant non-albumin proteinuria is suspected, use assays for specific urine proteins (e.g., α1-microglobulin, monoclonal heavy or light chains). Specific marker proteins may help to localize the damage, or the protein pattern in urine may be analyzed with sodium dodecylsulfate (SDS) polyacrylamide gel electrophoresis (SDS-PAGE).

In order to decide how proteinuria should be detected and measured, the following must be kept in mind /6/:

  • Albumin represents only a small fraction of the total protein excreted physiologically. As proteinuria develops, the albumin fraction increases more and more; roughly calculated, twice the albumin value is equivalent to the total protein concentration, if that concentration is ≤ 1.0 g/L
  • For the diagnosis of renal disease, in primary and secondary prevention of cardiovascular events and in hypertensive patients, the ACR and not the PCR is the diagnostic criterion
  • Mild tubulo-interstitial proteinuria and the excretion of free light chains are only diagnosed with the total protein determination.

12.9.1.2 Classification of proteinuria

Pre renal proteinuria

Increased amounts of systemically produced low-molecular weight proteins such as free immunoglobulin light chains, hemoglobin, or myoglobin enter the primary urine as a result of systemic disease processes which do not initially cause any renal damage. If the tubular reabsorption capacity is exceeded, the proteins are excreted in the final urine, resulting in so-called overflow proteinuria.

Renal proteinuria

Renal proteinurias can be glomerular, tubular, or mixed (glomerular/tubular). Glomerular proteinurias are divided into selective and nonselective forms. In the former, medium-sized proteins (41–100 kDa) are excreted while in the latter, high-molecular weight proteins are also excreted (41–400 kDa). The mixed glomerular/tubular proteinurias are caused by:

  • A combination of glomerular and tubular damage
  • Glomerular damage only and, in consequence, overload of the tubular reabsorption capacity for low molecular weight proteins.

Post renal proteinuria

In this form of proteinuria, proteins of all sizes are evident, but there is a significant proportion of very large proteins with a molecular weight over 400 kDa that are unable to pass through even a severely damaged glomerular basement membrane. These proteins enter the urine via bleeding or exudates in the lower urinary tract after passage through the tubules.

12.9.1.3 Differentiation of proteinuria

The composition of the excreted proteins depends on whether the proteinuria is renal, pre renal, or post renal, and depending on the type and location of any renal damage. Therefore, based on the pattern of urine protein excretion, it is possible within certain limits to deduce the type and location of renal proteinuria, or to judge whether a disorder is pre renal or post renal. The amount of characteristic marker proteins that is excreted correlates with the activity of the disease; the exception to this is minimal change nephropathy.

Certain marker proteins are characteristic of the following localizations of kidney damage:

  • Isolated proteinuria of medium molecular weight proteins (albumin, transferrin) filtered by the glomeruli is indicative of glomerular injury
  • Isolated proteinuria of the low molecular weight proteins (α1-microglobulin and β2-microglobulin), which are normally reabsorbed by the tubuli, points to tubulo-interstitial disease
  • The presence of high molecular weight proteins such as IgG is diagnostically relevant in renal diseases with severe glomerular filtration impairment
  • The presence of α2-macroglobulin, which is admixed from the urinary tract into the urine, is suggestive of post renal proteinuria
  • Elevated excretion of neutrophil gelatinase-associated lipocalin (NGAL) is indicative of acute kidney damage.

The excreted quantity of typical marker proteins correlates with the disease activity.

12.9.2 Indication

Test strips

  • Screening of asymptomatic patients for proteinuria
  • Patient self-monitoring for proteinuria.

Albumin

Diagnosis and management of chronic kidney disease (e.g., in patients with):

  • Diabetes mellitus types 1 and 2
  • Metabolic syndrome
  • Hypertension, edema, obesity
  • Cardiovascular disease.

Total protein

  • Quantification and monitoring of known proteinuria
  • Classification of nephropathy by the type and severity of disease
  • In comparison to test strip analysis: detection of Bence-Jones proteinuria
  • Plausibility control regarding the individual immunochemical protein determinations: ruling out erroneous measurements due to antigen excess or due to alterations in immunoreactivity.

Determination of the protein excretion pattern

Information for location of renal pathologic-anatomic abnormalities (e.g., detection and differentiation between pre renal, renal (glomerular, tubular and mixed forms) and post renal proteinuria). The examination is performed either with SDS-polyacrylamide gel electrophoresis or a pattern of the marker proteins is determined:

  • Low molecular weight proteins (α1-microglobulin, β2-microglobulin, free light chains)
  • Medium molecular weight proteins (albumin, transferrin)
  • Proteins of high molecular weight (IgG, α2-macroglobulin).

Individual proteins with regard to specific questions

12.9.3 Method of determination

12.9.3.1 Test strips for the semi quantitative determination of total protein

Principle: the protein error of certain pH indicators is used for a colorimetric detection of proteins. The acid-buffered reaction zone on the test strip contains the indicator tetra bromphenol blue, which can release hydrogen ions. If the reagent strip is immersed in protein-free solutions, no hydrogen ions are released and the zone remains yellow. In protein-containing solutions, the indicator donates hydrogen ions to the proteins, and the color changes to greenish-blue. Of the diagnostically relevant urine proteins, only albumin and transferrin are good hydrogen ion acceptors. The test strips are therefore primarily sensitive to these proteins. Test strips have a diagnostic sensitivity of 90–95% for albuminuria of > 200 mg/L. This is lower for immunoglobulins, mucoproteins and low molecular weight proteins, and is very low for free light chains.

12.9.3.2 Test strips for the estimation of the albumin/creatinine ration (ACR)

The following tests are commercially available /7/:

Test 1: the test measures the albumin and creatinine in the sample. In the albumin test field, the albumin binds to a sulfonaphthyl dye and causes a color reaction. In a separate test field, the creatinine forms a copper-creatinine complex. This complex has peroxidase activity and catalyzes a reaction between diisopropylbenzene dihydroperoxide and 3,3’, 5,5’-tetramethylbenzidine. The reactions in both test fields result in colors that can be measured using a reflectometer. The test gives the following results:

  • Albumin in concentrations of 10, 30, 80 and 150 mg/L
  • Creatinine in concentrations of 10, 50, 100 and 200 mg/dL (0.9; 4.4; 8.8; 17.7 and 26.5 mmol/L)
  • The albumin/creatinine ratio (< 30, 30–300, and > 300 mg/g).

Test 2: this test only measures albumin. Albumin in the sample passes over a guide pad onto a conjugate pad. On the conjugate pad, the albumin binds with specific gold-labeled antibodies then flows into the detection field. Here, a chemical reaction takes place and the color is produced. The color intensity is compared with standard color blocks representing albumin concentrations of 0, 20, 50 and 100 mg/L.

12.9.3.3 Quantitative determination of total protein

The Biuret assay after protein precipitation and the turbidimetric measurement following precipitation of the proteins with benzethonium chloride or trichloroacetic acid are the most frequently used assays. All of the tests are more or less equally effective at detecting albumin, IgG, and hemoglobin. However, the turbidimetric methods are less sensitive when used to detect glycoproteins such as α1-microglobulin and Tamm-Horsfall protein than is the biuret assay.

12.9.3.4 Selective determination of urinary proteins

The determination of albumin, α1-microglobulin, transferrin, β2-microglobulin, IgG and α2-macroglobulin is performed with immunonephelometric and immunoturbidimetric methodology (see Chapter 52 – Selected analytical laboratory techniques).

Quantitative determination of albumin, transferrin, α1-microglobulin, and IgG

Immunonephelometric and immunoturbidimetric assays are used. The CRM 470 reference material is used as reference to calibrate the assays.

Principle of the immunonephelometric assay: the protein in the sample binds to a specific antibody, triggering an antigen-antibody complex. The increase in light scattering is measured with a nephelometer and converted into a concentration.

Principle of the immunoturbidimetric assay: the protein in the sample and the same protein bound to latex particles compete for a specific monoclonal antibody, which aggregates the latex particles. The resultant number of aggregates is measured spectrophotometrically and is inversely proportional to the concentration of the protein in the sample.

12.9.3.5 Determination of the protein excretion pattern

The sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (SDS-PAGE) is used /8/.

Principle: the urine is treated with SDS. The binding of the SDS to the proteins in the urine generates mixed micelles with a molecular radius that is proportional to the logarithm of the molecular mass, and the proteins acquire an excess negative charge.

Electrophoretic separation takes place in polyacrylamide gradient gels in an SDS containing alkaline buffer medium whereby the protein zones are compressed on the front of a discontinuous system with running and following buffers. The proteins can therefore migrate as sharp bands toward the anode and are slowed and separated according to molecular weight by the molecular sieving effect of the polyacrylamide gel. The distance migrated is inversely proportional to the logarithm of the molecular weight. The polyacrylamide gradient is selected to ensure that albumin will migrate about halfway across the gel plate.

The separated proteins will then be located as follows:

  • On the anodic side of albumin will be the low-molecular weight (tubular) urine proteins
  • On the cathodic side will be the high-molecular weight (glomerular) proteins.

The proteins are fixed and stained with Coomassie Blue, and the relative ratios between albumin and the high- and low-molecular weight proteins are then determined.

There are characteristic protein patterns that are indicative of location of pathologic-anatomic abnormalities in the kidney. In addition the test indicates pre renal and post renal proteins such as free light chains and hemoglobin monomers. All diagnostically significant urine proteins are detected qualitatively using SDS-PAGE and are classified according to molecular weight.

12.9.4 Specimen

First spontaneous voided morning urine. Determined are the albumin/creatinine ratio (ACR) or the total protein/creatinine ratio (PCR).

The relation to creatinine is a reference to the elimination of the volume rate of the urine excretion. Conversion: 1 g of creatinine represents 8.85 mmol.

The initial determination of the ACR in a urine sample collected during the course of the day (random urine), and, in the case of a pathological finding, additionally in a first morning urine sample, is proposed /9/.

A 24-hour urine collection (no additive), entire quantity to be brought to the laboratory. If this is not possible, remove 10 mL following repeated swiveling of the collection container and send it to the laboratory.

12.9.5 Reference interval

Total protein (adults)

Test strips /6/: Negative reaction at:

  • < 300 mg/L
  • < 500 mg/24 h (urine volume ~ 1.5 L)
  • < 50 mg/mmol creatinine (443 mg/g creatinine).

Biuret method /10/:

  • < 100 mg/L
  • < 150 mg/24 h (urine volume ~ 1.5 L)
  • < 25 mg/mmol creatinine (220 mg/g creatinine).

Turbidimetric test /11/:

  • < 50 mg/L
  • < 75 mg/24 (urine volume ~ 1.5 L)
  • < 12.5 mg/mmol creatinine (110 mg/g creatinine).

Method-independent /12/:

  • Below 23 mg/mmol creatinine (200 mg/g creatinine)
  • Pregnant women: below 300 mg/L /13/.

Total protein excretion (children) /141516/

Age

mg/24 h

mg/m2/24 h

Premature infants

14–60

88–377

Full-term neonates

15–68

68–309

2–12 months

17–85

48–244

13 months – 3 yrs

20–121

37–233

4–9 yrs

26–194

31–234

10–16 yrs

29–238

22–181

Values are the 2.5th and 97.5th percentiles.

Total protein in children (random urine)

  • 6 months to 2 years /17/: Below 56 mg/mmol creatinine (500 mg/g creatinine)
  • Over 2 years: ≤ 23 mg/mmol creatinine (200 mg/g creatinine) after rest and ≤ 28 mg/g creatinine (250 mg/g creatinine) after orthostasis /18/.

Albumin

Test strips (semi-quantitative):

  • Test strips for total protein: Negative reaction with albumin excretion of ≤ 200 mg/L /19/
  • Albumin-specific test strips: Negative reaction with albumin concentrations < 10 mg/L /7/.

Quantitative according to the Kidney Disease Improving Global Outcomes (KDIGO) classification of chronic kidney disease /1/:

  • ACR ≤ 10 mg albumin/g creatinine (1.0 mg albumin/mmol creatinine); young adults
  • Normal to mildly elevated < 30 mg albumin/24 h.

The reference values of the American Diabetes Association and the National Institute for Health and Clinical Excellence (NICE) are shown in Tab. 12.9-1 – Definition of albuminuria according to the American Diabetes Association and NICE /2021/.

Transferrin /22/: 0.2–1.2 mg/L

Immunoglobulin G (IgG)

  • First morning urine: ≤ 0.7 mg IgG/mmol creatinine (6 mg IgG/g creatinine) /23/
  • Second morning specimen (random urine): ≤ 1.0 mg IgG /mmol creatinine (9 mg IgG/g creatinine) /24/.

Values are the 95th percentiles.

α1-microglobulin (α1-M)

  • First morning specimen ≤ 1.75 mg α1-M/mmol creatinine (14 mg α1-M/g creatinine) /24/
  • Second morning specimen (random urine): ≤ 2.0 mg α1-M/mmol creatinine (17 mg α1-M/g creatinine) /25/.

Values are the 95th percentiles.

β2-microglobulin (β2-M) /25/

  • ≤ 0.2 mg/L
  • ≤ 0.023 mg β2-M/mmol creatinine (0.2 mg β2-M/g creatinine)
  • Clearance: 0.03–0.12 mL/min.

α2-macroglobulin (α2-M) /26/

≤ 0.79 mg α2-M/mmol creatinine (7.0 mg α2-M/g creatinine)

Neutrophil gelatinase-associated lipocalin (NGAL)

Adults ≤ 150 μg/L, children ≤ 135 μg/L /27/

12.9.6 Clinical significance

Total protein excretion in healthy individuals is ≤ 150 mg/24 h. The total protein is composed of filtered plasma proteins (60%) and non-albumin proteins (40%) mainly tubular Tamm-Horsfall protein. Albumin is the major filtered plasma protein, making up 20% of the total protein excreted; this is, as a rule, up to 20–30 mg/24 h or 14–21 μg/min. in healthy individuals. Glomerular proteinuria is the most common form and involved with a prevalence of 90% in elevated protein excretion.

12.9.6.1 Definition of albuminuria

  • The National Institute for Health and Clinical Excellence (NICE) CKD Guideline /21/ defines albuminuria as ≥ 30 mg/mmol creatinine (266 mg/g creatinine), corresponding to an excretion of ≥ 300 mg/24 h
  • According to the KDIGO classification of chronic kidney disease /1/, a value ≥ 3.4 mg/mmol creatinine (30 mg/g creatinine) is a marker of kidney damage
  • According to the American Diabetes Association /20/ and the National Institute for Health and Clinical Excellence CKD Guidelines /21/, albuminuria is classified as per the criteria listed in Tab. 12.9-1 – Definition of albuminuria according to the American Diabetes Association and NICE.

12.9.6.2 Definition of proteinuria

The definition of proteinuria varies:

  • Depending upon the laboratory, pathological proteinuria starts with the excretion of 150–300 mg/24 h
  • According to the UK CKD guidelines /28/, proteinuria is defined as a protein/creatinine ratio ≥ 45 mg/mmol creatinine (400 mg/g creatinine), but if hematuria is not present this should not be a reason to take more extensive measures, unless the value of 100 mg/mmol creatinine (885 mg/g creatinine) is exceeded
  • The National Institute for Health and Clinical Excellence CKD Guideline /21/ defines proteinuria ≥ 50 mg/mmol creatinine (443 mg/g creatinine)
  • According to the National Kidney Foundation /12/ proteinuria is defined as a protein/creatinine ratio ≥ 23 mg/mmol creatinine (200 mg/g creatinine).

In preeclampsia, proteinuria is ≥ 300 mg/24 h /13/.

In adults, proteinuria ≥ 3 g/24 h is defined to be nephrotic. In children, the following evaluations apply regarding 12-hour or 24-hour urine:

  • < 4 mg/m2/h = normal
  • 4–40 mg/m2/h = abnormal
  • > 40 mg/m2/h = nephrotic.

12.9.6.3 Epidemiology of proteinuria

Proteinuria is a frequently occurring symptom, particularly in adolescents. When Japanese schoolchildren aged 6–15 years were screened using test strips for total protein and albumin, 4.3% showed elevated levels of total protein excretion, while increased albumin excretion was evident in 2.1% /17/.

However, most proteinurias are functional in nature and are only transient or intermittent (Tab. 12.9-2 – Functional, transient and isolated proteinuria). Studies involving test strip assays performed on children in the U.S. showed that up to 10% of the children tested had proteinuria when tested once. The prevalence fell to about 2.5% for cases where two of four urine samples collected at set time intervals were positive, while only 1 in 1000 children exhibited proteinuria in all four samples /29/. In only 10% of the children with proteinuria did the condition persist for 6–12 months /30/. The prevalence of proteinuria is age-dependent, gradually increasing with age, and reaching a maximum in adolescence.

12.9.6.4 Significance and treatment of proteinuria

If proteinuria is present, examinations to answer the following questions are necessary /31/:

  • Is the proteinuria evident consistently?
  • How much albumin or total protein is excreted?
  • Is the protein pattern glomerular, tubular, mixed, pre renal or post renal?
  • Can the cause of the renal injury be recognized or localized?
  • If glomerular damage is present, is it progressive?

12.9.6.5 Etiology of proteinuria

From the pathophysiological point of view, proteinuria is an important factor in the genesis of kidney disease. The type and severity of the existing kidney disease is often unrelated to the clinical symptoms. Commonly, patients with proteinuria are also asymptomatic. A classification and the etiology of proteinuria is shown in Tab. 12.9-3 – Etiology of proteinuria.

In patients with total protein of < 1 g/24 h, without pathological laboratory findings or clinical symptoms (isolated proteinuria), separate daytime and night time urine collections should be performed to rule out orthostatic proteinuria.

Nephrotic proteinuria [albumin > 2,200 mg/24 h, ACR > 2,200 mg/g creatinine (> 220 mg/mmol creatinine)] or [total protein > 3,500 mg/24 h, PCR > 3,500 mg/g creatinine (> 350 mg/mmol creatinine)] are caused by glomerulonephritis and indicate a high progression rate in chronic kidney disease /3/.

12.9.6.6 Albuminuria

The National Kidney Foundation of the USA recommends the indications mentioned in Tab. 12.9-4 – Recommendations of the National Kidney Foundation for albuminuria as a clinical marker of kidney damage for the determination of urinary albumin /32/.

In kidney disease there is a positive relationship between decreasing GFR, increasing proteinuria, and the consequences. Risks which must be prognostically estimated are: progression of a chronic kidney disease (CKD), end stage renal failure, acute kidney injury, cardiovascular mortality, and overall mortality. Because of increasing evidence demonstrating the importance of proteinuria as a predictor of adverse outcomes albumin excretion, measured as ACR, was integrated by KDIGO into the staging of CKD in addition to the GFR /1/. Three albumin (A) categories were created (Tab. 12.9-5 – Categories of albumin excretion in chronic kidney disease).

The relationship of albumin excretion (abscissa) to the GFR (ordinate) is shown in a diagram (Fig. 12.9-1 – Assessment of the course of the GFR and of albumin excretion in type 1 diabetes mellitus). The risk increases progressively in both directions, downwards the GFR categories and from left to right the albumin categories.

For patients with reduced GFR and albuminuria, the prognosis regarding cardiovascular events and overall mortality is more worse than for those with GFR reduction without albuminuria. The GFR and albuminuria are independent risk factors for cardiovascular disease and mortality /33/. Cardiovascular disease is 10–20-fold more frequent in patients with end stage renal failure than in age-matched control groups. Albuminuria usually reflects injury to the vascular endothelium and associated diseases.

It is generally accepted that incipient glomerular disease is recognized years before GFR impairment by detection with albumin tests with a sensitivity of ≤ 5 mg/L. Tab. 12.9-6 – Albuminuria as a marker of nephropathy and systemic diseases shows albuminuria-associated diseases.

Albumin excretion is an important criterion for the . The heritability of albuminuria ranges from 0.17 to 0.20 in the general population, 0.20 in families with diabetes, and 0.12 to 0.49 in families with hypertension, depending on ethnicity. Genom wide scans for albuminuria have mostly yielded regions with suggestive evidence for linkage. Evidence of linkage for urine albumin/creatinine ratio to 20q12 was isolated. It has been suggested that the genes controlling urinary albumin excretion are the same in relatives both with and without diabetes.

Refer to the following figure and section:

Albuminuria, defined as urine albumin/creatinine ratio (ACR) ≥ 30 mg/g creatinine is a diagnostic component of CKD. In a study /9/, overall, 43.5% of the adults had increased ACR (≥ 30 mg/g creatinine) in a first morning urine. This proportion was higher among individuals ≥ 50 years old (48.9%), males (53.3%), participants with diagnosed diabetes (56.3%), and hypertension (51.5%), and eGFR < 60 [mL × min–1 × (1.72 m2)–1] (56,9%).

12.9.6.7 Proteinuria and secondary prevention of renal disorders

In CKD, the severity of total protein excretion is related to the degree of renal function loss, and there is also a connection between proteinuria and the progression of nephropathy. In secondary prevention, the reduction of proteinuria is used as a measure of the effectiveness of nephroprotective therapy. The following nephroprotective therapies have been indicated:

  • In patients with nondiabetic nephropathy and proteinuria ≥ 3 g/24 h who are under treatment with drugs that inhibit the renin-angiotensin system, proteinuria can be reduced and the rate of renal failure slowed down (Renal Efficiency in Nephrology Study /34/).
  • The use of ACE inhibitors or angiotensin II type 1 receptor antagonists to block the renin-angiotensin system leads to a reduction of proteinuria in chronic nephropathies. However, this therapy will only benefit those patients in whom the proteinuria declines by a significant amount (30–60% /35/) within the first 8–12 weeks after the treatment was initiated. The antiproteinuric and nephroprotective effects of the ACE inhibitors stem not only from the fact that these drugs reduce blood pressure, but also from their reduction of the proteinuria itself, which in turn also exerts a nephroprotective effect /35/.
  • The relationship between proteinuria and the progression of nephropathy also applies to patients with diabetes mellitus. Baseline protein excretion is also a strong predictor of renal failure in patients with type 2 diabetes mellitus. Thus, it was shown in a study /36/ that a two-fold increase in proteinuria also doubles the risk of renal failure. The risk of renal failure was reduced by 50% by administering the appropriate therapy; thus halving the level of proteinuria within 12 months. Treatment with the angiotensin receptor blocker irbesartan allowed proteinuria to be reduced by 41% within one year and had a nephroprotective effect of 36%.
  • In patients with focal segmental glomerular sclerosis (FSGS), specifically with primary FSGS, which most frequently results in end-stage renal failure. These patients had a proteinuria of 4.7 (0.2–98.3) g/24 h /37/. Under immunosuppresive therapy, the reduction in proteinuria was a valuable surrogate marker both for the prediction of future renal failure and for the rate of relapse from complete to partial remission in FSGS patients. Therapy was at its most effective when the proteinuria had declined by over 50% to a low-point of less than 3.5 g/24 h /37/.

In addition to the level of proteinuria, the underlying renal disorder and the proteinuria pattern are also important for secondary prevention. For example, patients with minimal change glomuleronephritis have severe proteinuria without any resultant chronic deterioration of renal function. This is because not every protein molecule causes the same degree of damage to the tubule cells. In interstitial nephritis, the selectivity index is a predictive factor.

12.9.6.8 Marker proteins

Marker proteins represent a characteristic size category for each form of proteinuria, and are present in the urine under normal conditions in concentrations that are easy to measure using immunonephelometric or immunoturbidimetric methods. Identifying marker protein levels makes it possible to classify proteinuria according to the various types. This testing also allows conclusions to be drawn regarding the location of any renal damage, but not regarding etiology.

Marker proteins can be determined qualitatively by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) or quantitatively with immunoassays. In SDS-PAGE assays, the marker proteins are identified by determining their relative mobility compared with a standard. This results in characteristic protein patterns that make it possible to classify the proteinuria according to the various possible types, and to draw conclusions regarding the location of the renal damage (Tab. 12.9-7 – Proteinuria types of SDS-PAGE).

Based upon the typical molecular weight category of the respective protein form, the following are differentiated:

  • Non-selective versus selective proteinuria, by the quantitative determination of IgG and albumin or transferrin, or the implementation of SDS-PAGE. Increased excretion of albumin, transferrin and IgG is a sign of glomerular damage. The increase in the excretion of these proteins is consistent with the hypothesis that glomerular selectivity decreases progressively with the increase in albuminuria.
  • Tubular proteinuria through the quantitative determination of α1-microglobulin, β2-microglobulin or the implementation of SDS-PAGE
  • Pre renal proteinuria by determining the free light chains κ and λ, myoglobin and hemoglobin, or the implementation of SDS-PAGE
  • Post renal proteinuria through the determination of α2-macroglobulin and albumin or the implementation of SDS-PAGE. Renal and post renal forms of hematuria can be differentiated based upon the concentration relationships of both of these proteins.
12.9.6.8.1 Concentration ratios of the urinary proteins

The concentration ratios of the urinary proteins provide an indication as to the mechanism of the proteinuria (Tab. 12.9-8 – Concentration ratios of urinary proteins and their predictive value). It must, however, be noted that common pathological mechanisms can underlie chronic nephropathies with highly diverse etiologies and can cause their progression, irrespective of the original etiology. Thus many etiologies lead to the same pathogenesis. This often has the following course: systemic hypertension, augmented glomerular filtration pressure, increased protein ultrafiltration, glomerular and tubular protein overload, chronic inflammation and, finally, structural kidney damage. In many of these cases the proteinuria pattern is similar and provides no information regarding the etiology of the disease /38/.

12.9.6.8.2 Selectivity index

The concept of proteinuria selectivity is useful for predicting the success of steroid therapy for nephrotic syndrome. The selectivity index (SI) is determined:

IgG (urine) × transferrin (serum)IgG (serum) × transferrin (urine) SI =

A proteinuria is considered highly selective with an SI < 0.10, moderately selective for an SI > 0.11 to ≤ 0.20 and nonselective for an SI > 0.21 /39/.

12.9.6.8.3 α1-microglobulinuria

α1-microglobulin (α1-M) is produced in the liver and by lymphocytes, and has a molecular weight of 30–33 kDa. It is a glycoprotein that is present in the free form in serum to the extent of 50%, up to 40% is bound to IgA, and up to under 10% is bound to albumin. The serum concentration is 10–20 mg/L. Only the free form is filtered and 99.8% is reabsorbed in the proximal tubule. Under normal glomerular filtration conditions and with normal serum creatinine levels, urine concentrations > 10 mg/L or an elevated α1-M/creatinine ratio indicate proximal tubular dysfunction. In the case of glomerular disorders and elevated serum creatinine, this is not necessarily the case. This is because under these conditions, the serum level of α1-M increases as a result of a reduced GFR. The load on the remaining intact nephrones from α1-M increases, resulting in an elevated α1-M concentration in the ultra filtrate. This results in a saturation of α1-M reabsorption capacity of proximal tubular renal cells, leading to an increase in α1-M concentration in the end urine without there being any tubular dysfunction . It is therefore important to distinguish these overflow proteinuria from that due to tubular dysfunction. This can be accomplished using the urine α1-M/albumin ratio (which is measured in mg/mg). A value < 0.1 is indicative of glomerular proteinuria, while a value ≥ 0.1 indicates a mixed proteinuria /40/.Mixed proteinuria is the result of a combination of glomerular and tubular damage. The diseases and factors that can cause tubular proteinuria are listed in Tab. 12.9-9 – Glomerular and tubulo-interstitial proteinuria.

The various forms of proteinuria can be distinguished from one another by determining the excretion of α1-M in relation to the excretion of albumin using a diagnostic diagram /41/ (Fig. 12.9-3 – Differentiation of proteinuria forms). This makes it possible to distinguish between primary and secondary glomerulopathy, as well as tubulo-interstitial nephropathy.

12.9.6.8.4 IgG excretion

The glomerular filtration system has a charge-sensitive permeability which is further enhanced by pores used for mechanical size selectivity. In this way, the passage of proteins through the glomerular barrier depends on charge and pore size.

A reduced anionic charge in the filtration system results in an increased excretion of negatively-charged proteins such as albumin without macromolecular proteins such as IgG appearing in the urine, since their molecular weight prevents them from being filtered out. A prime example of impaired charge selectivity is minimal change glomerulopathy. If the pore size in the filtration system is pathologically enlarged, larger molecules can also enter the urine.

By determining the urine albumin and IgG levels, selective (charge-dependent) proteinuria, characterized by a high proportion of albumin, can be distinguished from nonselective proteinuria, which is characterized by pathological changes in charge and pore size, as well as by a high proportion of IgG. The IgG/albumin ratio (in mg/mg) can be used as a measure of selectivity. A ratio ≤ 0.03 is indicative of selective proteinuria, while a ratio > 0.03 indicates a nonselective proteinuria /40/.

In glomerular disorders, not only increased amounts of albumin and IgG are excreted, but also low-molecular weight proteins such as α1-M, resulting in the development of a mixed proteinuria pattern. However, neither an α1-M/albumin ratio > 1 nor an α1-M excretion rate > 100 mg/g of creatinine are ever observed in cases of glomerular proteinuria /42/. Diseases associated with glomerular proteinuria are listed in Tab. 12.9-9 – Glomerular and tubulo-interstitial proteinuria.

12.9.6.8.5 Neutrophil gelatinase-associated lipocalin (NGAL)

Human NGAL, also known as lipocalin 2, is expressed in a variety of tissues. NGAL consists of a single disulfide-bridged polypeptide with molecular weight of 25 kDa. The majority of the NGAL molecules exist as monomers, dimers and trimers, as well as in a complex with neutrophil gelatinase. The monomeric NGAL form is secreted by injured kidney tubule epithelial cells, whereas the dimeric form is the predominant form secreted by neutrophils. Siderophores are the major ligands for NGAL. Iron containing NGAL binds to surface receptors such as megalin, is internalized and releases its iron to the labile iron pool of the cell. Plasma NGAL originates from injured kidneys and from extrarenal organs. The urine NGAL is derived predominantly from epithelial cells of the distal nephron, although a fraction may come from the systemic pool escaping reabsorption due proximal tubular damage /43/.

NGAL is a biomarker for acute renal failure (ARF) and is elevated in serum and random urine samples within 2–6 hours of the onset of ARF. Following cardiac surgery and renal transplantation, and in the critically ill, values > 150 μg/L are to be considered as high risk predictors of ARF (area under curve 0.88). The level of NGAL correlates with the severity of the ARF. Values of ≤ 100 μg/L rule out ARF with high accuracy. In the range of 100–150 μg/L, the risk of ARF is moderate, patients with ARF risk factors (older individuals, chronic kidney disease, cardiovascular disease) and with values in this range should be considered to be at high risk for ARF.

In a systematic review /44/ the utility of NGAL to predict the occurence of AKI in septic patients was investigated. For plasma NGAL the pooled sensitivity and specificity were 83% and 57%, respectively. The pooled positive and negative likelihood ratios were 3.10 and 0.24, respectively. The pooled diagnostic odds ratio was 14.72 (95% CI: 6.55–33.10).

12.9.6.8.6 Marker proteins in pre renal proteinuria

Pre renal proteinurias involve increased amounts of systemic, low-molecular weight proteins entering the primary urine. These proteins come from the systemic pool escaping due to exceeding the tubular reabsorption capacity or due to proximal tubular damage, and the proteins are excreted in the final urine. Clinically important pre renal proteinurias are free light chain excretion (associated with multiple myeloma), myoglobinuria (associated with rhabdomyolysis), or hemoglobinuria (associated with a transfusion-related condition). Hemoglobin and myoglobin are detected using a test strip, while light chain excretion is identified by quantitative immunochemistry assays for free κ light chains and free λ light chains in the urine (refer to Section 22.3.4 – Immunosubtraction and capillary zone electrophoresis (CZE)).

12.9.6.8.7 Marker proteins in renal proteinuria

Renal proteinuria occurs as glomerular, mixed glomerular-tubular, and tubular forms. The determination of the marker proteins albumin, IgG and α1-microglobulin or the SDS-PAGE permit the differentiation of the three types of proteinuria. Glomerular proteinuria is subdivided into selective (disturbances of anion filtration) and non-selective (structural damage to the molecular sieve) forms (Tab. 12.9-10 – Clinical assessment of urine protein marker analysis).

12.9.6.8.8 Marker proteins in post renal proteinuria

It is possible to distinguish between renal and post renal hematuria by testing for urine marker proteins. In the case of post renal (urological) bleeding, the protein pattern in the urine is comparable to that in serum, while in the case of renal (glomerular) hematuria, a typical glomerular protein pattern (over 80% albumin) is evident. The marker proteins for post renal proteinuria is albumin and α2-macroglobulin (α2-M). α2-M is a high-molecular weight protein that is not filtered through the glomeruli, even if the glomerular filtration system is injured. Renal hematuria can be distinguished from post renal hematuria by evaluating the excretion rate of α2-M in relation to the excretion of albumin by means of the urine α2-M/albumin ratio (measured in mg/mg). A ratio < 0.02 is indicative of renal hematuria, while a ratio ≥ 0.02 indicates post renal hematuria. However, it is not possible to differentiate between renal and post renal hematuria if the urine albumin concentration is less than 100 mg/L /42/. The causes of post renal proteinuria are listed in Tab. 12.9-11 – Post-renal proteinuria.

12.9.6.8.9 Medication-dependent proteinuria

Pharmaceuticals, environmental pollutants and radio-opaque substances that are excreted via the kidneys can cause tubulopathy and/or glomerulopathy. Nephrotoxic drugs and X-ray contrast media are frequent causes of iatrogenic nephropathy.

Tubular and glomerular proteinuria are early signs of toxic nephropathy and can serve as indicators for dose reduction or for nephroprotective measures (Tab. 12.9-12 – Toxic nephropathies: urine protein findings and clinical evaluation).

12.9.6.8.10 Marker proteins in unilateral kidney

Individuals with only a single kidney, a normal GFR and normal blood pressure have, in comparison with healthy individuals, higher 24-hour total protein excretion, but no selective proteinuria.

12.9.7 Comments and problems

Specimen

24-hour urine collection /40/: the volume of urine excreted in 24 hours varies considerably both in individual patients and between patients. One problem is the procedure used to collect the urine, which is rarely reliable. On average, the amount of urine excreted in 24 hours is ~ 1.5 liters, while the creatinine excretion for an individual weighing 70 kg is about 1.5–2.5 g. This means that the creatinine concentration in a 24 hour urine sample is on average about 100 mg/dL (8.84 mmol/L). Creatinine concentrations < 30 mg/dL (2.65 mmol/L) are indicative of polyuria, artificial dilution of the urine, or an individual with low body weight.

First morning urine /40/: first morning urine provides relatively reliable sample material as it is collected after the patient has been inactive for about 8 hours. The urine is concentrated, and the urine volume rate is 20 –50 mL/h as long as no liquids have been taken after 10:00 p.m. on the evening before.

Second morning urine /40/: the sample should be collected 2–4 hours after the first morning void. The quality of this type of sample will be better if one 200 mL glass of water is taken after 10:00 p.m. Early morning physical exercise is one of the major interfering factors. This causes dehydration while at the same time inducing benign exertional proteinuria.

Protein/creatinine ratio /40/: the ratio has gained general acceptance for correction of diuresis. The excretion of creatinine over time is relatively constant and distorting effects caused by diuresis and anti diuresis can be eliminated by reference to a specifically defined amount of excreted creatinine. The amount of creatinine excreted by a healthy individual is about 30 mg/kg of body weight per 24 hours. The ratio is higher in females than in males, and can frequently decline with age.

Factors affecting proteinuria results: stress, physical exertion, and fever before sample collection can cause proteinuria. Therefore, pathological protein results from spot urine samples should be confirmed by more than two independent consecutive tests.

Method of determination of total protein

Test strip: due to the high degree of affinity between the indicators and amino groups, test strips exhibit a high degree of sensitivity for albumin and transferrin when compared with other urine proteins. Other plasma proteins and mucoproteins from the urinary tract will only cause a positive result if they are present in high concentrations. Bence-Jones protein will be detected only occasionally or not at all. False-negative results occur at pH levels of < 4 and > 8. False-positive results may be caused by drugs, as well as by disinfectants and cleaners that contain quaternary ammonium bases.

Biuret reaction: this is an acceptable method if the proteins are first precipitated using trichloroacetic acid and then dissolved in the biuret reagent. A better precipitant is Tsuchiya’s reagent: 6 mol/L HCl and 1.7% (w/v) of tungstophosphoric acid in 80% (v/v) ethanol. Results within the reference interval are not precise. Protein fragments and free light chains are also detected.

Turbidimetric total protein assays: the concentrations of protein fragments, tubular proteins, glycoproteins, and Tamm-Horsfall protein are too low or are not detected. Contrast media, uric acid, detergents, and polyethylene glycol can mimic excessively high results.

Method of determination of albumin

A fresh midstream collection for urine albumin measurement is preferred.

Albumin-specific test strip assays: test strips do not meet the requirements for the early detection of diabetic nephropathy or of albuminuria as a cardiovascular risk factor if the albuminuria is still at the lower end of the established range, since their diagnostic specificity is only 70–80% /44/.

Quantitative albumin measurement: urine albumin is routinely measured by immunonephelometry or immunoturbidimetry, as well as by test strip assay. However, only immunoreactive albumin is detected. Measurement of urine albumin is not standardized due to the lack of a reference system, which include both a reference measurement procedure and certified reference materials /45/. In a study that evaluated the state of agreement among quantitative immunoassay measurement procedures from in-vitro diagnostic manufacturers results from urine albumin samples had total coefficients of variations of 5.2% to 8.1% and the effect of sample-specific influences were below 10% for most measurement procedures. The median difference range for routine measurement procedures versus comparator LC-MS/MS procedure was approximately 40%. Mean biases ranged from –35% to +34% for concentrations near 15 mg/L and –15% to +18% for concentrations near 30 mg/L /46/. The results of this study demonstrate that fixed decision thresholds cannot be effectively utilized due to lack of agreement among routine measurement procedures /45/.

Albumin levels in urine can vary by 20–30%, and can vary even more in diabetics /47/. Diagnostic conclusions should therefore not be based on only one test result.

Albumin-to- creatine ratio (ACR): the ACR of a random urine sample can be used in patients with kidney disease to rule in or rule out abnormal 24 h losses of albumin. An ACR cutoff of 30 mg/mmol showed a similar good performance in being able to rule in or rule out significant albuminuria (≥ 300 mg/day) /85/. Conversion: 1 mg albumin/g creatinine = 0.113 mg/mmol.

Stability

Total protein: urine can be kept after preservation with 0.1% sodium azide for up to 7 days at room temperature and for up to 30 days in a refrigerator, but must not be frozen /48/.

SDS-PAGE: urine can be kept after the addition of sodium azide (to a final concentration of 0.1% in the urine) for up to 45 days at room temperature and for more than 6 months at 4 °C. Specimens can be frozen after the addition of 50 g/L saccharose /49/.

Marker proteins: albumin, α1-M and IgG do not exhibit any significant reduction in concentration in untreated urine kept at room temperature and at 4 °C for 7 days or less /48/. Freezing at –20 °C for 6 months leads to a median decrease of 47% in the concentration of IgG, of 14% in the level of α1-M, and 5% in the albumin concentration /48/.

Albumin: Albumin can remain stable in urine for up to 8 weeks when stored under refrigerated conditions at 4 °C /45/. According to another study, the albumin concentration declines after one year of frozen storage at –20 °C by 9–28%, depending on the initial concentration. The greatest changes are observed at concentrations ranging from 10–20 mg/L, while the smallest changes are seen at concentrations of 200–500 mg/L /50/.

References

1. Kidney Disease: Improving Global Outcome (KDIGO) CKD Work Group. KDIGO clinical practice guideline for the evaluation and management of chronic kidney disease. Kidney Int Suppl 2013: 3: 1–150.

2. Lamb EJ, McTaggart MP, Stevens PE. Why albumin to creatinin ratio should replace protein to creatinine ratio: it is not just about nephrologists. Ann Clin Biochem 2013; 50: 301–5.

3. Methven S, MacGregor MS. Empiricism or rationalism: how should we measure proteinuria? Ann Clin Biochem 2013; 50: 296–300.

4. Price CP, Newall RG, Boyd JC. Use of protein: creatinin ratio measurements on random urine samples for prediction of significant proteinuria: a systematic review. Clin Chem 2005; 51: 1577–86.

5. Gansevoort RT, Verhave JC, Hillege HL, Burgerhof JG, Bagger SJ, de Zeeuw D, et al. The validity of screening based on spot morning urine samples to detect subjects with microalbuminuria in the general population. Kidney Int Suppl 2005; S28-S35.

6. Lamb EJ, MacKenzie F, Stevens PE. How should proteinuria be detected and measured ? Ann Clin Biochem 2009; 46: 205–17.

7. Graziani MS, Gambaro G, Mantovani L, Sorio A, Yabarek T, Abaterusso C, et al. Diagnostic accuracy of a reagent strip for assessing urinary albumin excretion in general population. Nephrol Dial Transplant 2009; 24: 1490–4.

8. Boesken W, Marmier A. Molekulargewichtsbezogene Urinprotein-Elektrophorese in der Diagnostik von Nierenkrankheiten. Lab Med 1985; 9: 285–90.

9. Saydah SH, Pavkov ME, Zhang C, Lacher DA, Eberhardrdt MS, Burrows NR, et al. Albuminuria prevalence in first morning void compared with previous random urine from adults in the National Health and Nutrition Examination Survey, 2009–2010. Clin Chem 2013; 59: 675–83.

10. Weichselbaum TE. An accurate and rapid method for the determination of proteins in small amounts of blood serum and plasma. Am J Clin Pathol 1946; 13: 40–9.

11. Henry RJ, Sobel C, Seglove M. Turbidimetric determination of proteins with sulphosalicylic and trichloroacetic acids. Proc Soc Exp Biol Med 1956; 92: 748–51.

12. National Kidney Foundation. Clinical practice guidelines for chronic kidney disease: evaluation, classification and stratification. Am J Kidney Dis 2002; 39: S1–S266

13. Davey DA, MacGillivray I. The classification and definition of the hypertensive disorders of pregnancy. Am J Obstet Gynecol 1988; 158: 892–8.

14. Ettenger RB. The evaluation of the child with proteinuria. Pediatric Annals 1994; 23: 486–91.

15. Miltenyi M. Urinary protein excretion in healthy children. Clin Nephrol 1979; 12: 216–21.

16. Vehaskari VM, Robson AM. Proteinuria. In: Edelman CM Jr (ed). Pediatric kidney disease. Boston 1992; Little, Brown: 531–51.

17. Houser MT. Assessment of proteinuria using random urine samples. J Pediatr 1984; 104: 845–8.

18. Houser MT, John MF, Kobayoshi A, Walburn J. Assessment of urinary protein excretion in the adolescent: effect of body position and exercise. J Pediatr 1986; 109: 556–61.

19. Pugia MJ, Lott JA, Kajima J, Saambe T, Sasaki M, Kuromoto K, et al. Screening school children for albuminuria, proteinuria and occult blood with dipsticks. Clin Chem Lab Med 1999; 37: 149–57.

20. American Diabetes Association. Diabetic nephropathy. Diabetes Care 2000; 23 (suppl 1): S69–S72.

21. National Institute for Health and Clinical Excellence. Chronic kidney disease: National clinical guideline for early identification and management in adults in primary and secondary care. Clinical Guide 73. 2008. www.nice.org.uk/Guidance/CG73 (last accessed 9 January 2009).

22. Verwiebe R, Wieneke U, Weber M. Immunnephelometrische Harnbestimmung von Albumin, Transferrin und Immunglobulin G (IgG). Nieren- und Hochdruckkrankheiten 1990; 19: 164–8.

23. Hoffmann W, Guder W. A diagnostic program for quantitative analysis of proteinuria. J Clin Chem Clin Biochem 1989; 27. 589–96.

24. Tencer J, Thysell H, Grubb A. Analysis of proteinuria: reference limits for urine excretion of albumin, protein HC, immunoglobulin G, κ- and λ-immunoreactivity, orosomucoid and α1-antitrypsin. Scand J Clin Lab Invest 1996; 56: 691–700.

25. Schardijn J, v Eps L, Swaak A. Urinary β2-microglobulin in upper and lower urinary tract infections. Lancet 1979: 805–12.

26. Hofmann W, Schmidt D, Guder W, Edel H. Differentiation of hematuria by quantitative determination of urinary marker proteins. Klin Wochenschr 1991; 69: 68–75.

27. Haase M, Bellomo R, Devarayan P, Schlattmann P, Haase-Fielitz A. Accuracy of neutrophil gelatinase-associated lipocalin (NGAL) in diagnosis and prognosis in acute kidney injury: a systemic review and metaanalysis. Am J Kidney Dis 2009; 54: 1012–24.

28. Joint Speciality Committee on Renal Medicine of the Royal College of Physicians and the Renal Association and the Royal College of General Practitioners. Chronic Kidney Disease in Adults. (Last accessed 9 January 2009).

29. Vehaskari VM, Rapolda J. Isolated proteinuria: analysis of a school-age population. J Pediatr 1982; 101: 661–8.

30. Randolph MF, Greenfield M. Proteinuria: a 6 year study of normal infants preschool, and school-aged population previously screened for urinary tract disease. Am J Dis Child 1967; 114: 631–8.

31. Mallick NP. The significance and management of proteinuria. Nephrol Dial Transplant 1990; Suppl 1: 35–6.

32. Eknoyan G, Hostetter T, Bakris GL, Hebert L, Levey AS, Parving HH, et al. Proteinuria and other markers of chronic kidney disease: a position statement of the National Kidney Foundation (NKF) and the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK). Am J Kidney Dis 2003; 42: 617–22.

33. Ninomiya T, Perkovic V, de Gaalen BE, Zoungas S, Pillai A, Parvin HH, et al. Albuminuria and kidney function independently predict cardiovascular and renal outcomes in diabetes. J Am Soc Nephrol 2009; 20: 1813–21.

34. Gruppo Italiano di Studi Epidemioloci in Nefrologia (GISEN). Randomized placebo controlled trial of effect of ramipril on decline in glomerular filtration rate and risk of terminal renal failure in proteinuric, non-diabetic nephropathy. Lancet 1997; 349: 1857–63.

35. Wapstra FH, Navis G, de Jong PE, de Zeeuw D. Prognostic value of the short-term antiproteinuric response to ACE inhibition for prediction of GFR decline in patients with nondiabetic disease. Exp Nephrol 1996; 4, Suppl 1: S47–S52.

36. Atkins RC, Briganti EM, Lewis JB, Hunsicker LG, Braden G, Champion de Crespigny PJ, et al. Proteinuria reduction and progression to renal failure patients with type 2 diabetes mellitus and overt nephropathy. Am J Kidney Dis 2005; 45: 281–7.

37. Troyanov S, Wall Ca, Miller JA, Scholey JW, Cattran DC. Focal and segmental glomerulosclerosis. J Am Soc Nephrol 2005; 16: 1061–8.

38. Remuzzi G, Chiurchiu C, Ruggenenti P. Proteinuria predicting outcome in renal disease: nondiabetic nephropathies (REIN). Kidney Int 2004; 66 (suppl 92): 90–96.

39. Bazzi C, Petrini C, Rizza V, Arrigo G, D’Amico G. A modern approach to selectivity of proteinuria and tubulointerstitial damage in nephrotic syndrome. Kidney Int 2000; 58: 1732–41.

40. European Urineanalysis Guidelines. Summary. Scand J Clin Invest 2000; 60: 1–96.

41. Hofmann W, Edel HE, Guder WG, Ivandic M, Scherberich JE. Harnuntersuchungen zur differenzierten Diagnostik einer Proteinurie. Dtsch Ärztebl 2001; 98: B637–44.

42. Guder W, Hofmann W. Differentiation of proteinuria and hematuria by single protein analysis in urine. Clin Biochem 1993; 26: 277–82.

43. Zhang A, Cai Y, Wang PF, Qu JN, Luo ZC, Chen XD, et al. Diagnosis and prognosis of neutrophil gelatinase-associated lipocalin for acute kidney injury with sepsis: a systemic review and meta-analysis.Critical Care 2016; 20: https://doi.org/10.1186/s13054-016-1212-x.

44. Zhang A, Cai Y, Wang PF, Qu JN, Luo ZC, Chen XD, et al. Diagnosis and prognosis of neutrophil gelatinase-associated lipocalin for acute kidney injury with sepsis: a systematic review. Critical Care 2016; 20: 41. https://doi.org/10.1186/s13054-016-1212-x.

45. Seegmiller JC, Miller WG, Bachmann LM. Moving toward standardization of urine albumin measurements. eJIFCC 2017, 28: 258–67.

46. Bachmann LM, Nilsson G, Bruns DE, McQueen MJ, Lieske JC. State of the art for measurement of urine albumin: comparison of routine measurement procedures to isotope dilution tandem mass spectrometry. Clin Chem 2014, 60: 471–80.

47. Miller WG, Bruns DE, Hortin GL, Sandberg S, Aakre KM, McQueen MJ, et al. Current issues in measurement and reporting of urinary albumin excretion. Clin Chem 2009; 55: 24–38.

48. Tencer J, Thysell H, Andersson K, Grubb A. Stability of albumin, protein HC, immunoglobulin G, kappa- and lambda-chain immunoreactivity, orosomucoid and alpha1-antitrypsin in urine stored at various conditions. Scand J Clin Lab Invest 1994; 54: 199–206.

49. Töpfer G, Pfisterer J, Schäfer P. Lagerungsbedingungen für Urin zur SDS-Polyacrylamidgel-Elektrophorese. Klin Lab 1993; 39: 205–6.

50. Brinkman JW, de Zeeuw D, Duker JJ, Gaansevoort RT, Kema IP, Hillege HL, et al. Falsely low urinary albumin concentrations after prolonged frozen storage of urine samples. Clin Chem 2005; 51: 2181–3.

51. Marks M, McLaine PN, Drummond KN. Proteinuria in children with febrile illness. Arch Dis Child 1970; 45: 250–3.

52. Poortmans JR. Postexercise proteinuria in humans. Facts and mechanisms. JAMA 1985; 253: 236–40.

53. Robertshaw M, Cheung CK, Fairly I, Swaminathan R. Protein excretion after prolonged exercise. Ann Clin Biochem 1993; 30: 34–7.

54. Urizar RE, Tinglof BO, Smith FG, McIntosh Jr RM. Persistent asyptomatic proteinuria in children. Am J Clin Pathol 1974; 62: 461.

55. Yoshikawa N, Kitagowa K, Ohta K, Tanaka R, Nakamura H. Asymptomatic constant isolated proteinuria in children. J Pediatr 1991; 110: 375–9.

56. Mogensen CE. Prediction of clinical diabetic nephropathy in IDDM patients. Alternatives to microalbuminuria. Diabetes 1990; 39: 761–7.

57. Hogg RJ, Portman RJ, Millner D, Lemley KV, Eddy A, Ingelfinger J. Evaluation and management of proteinuria and nephrotic syndrome in children: Recommendations from a pediatric nephrology panel established at the National Kidney Foundation Conference on Proteinuria, Risk, Assessment, Detection, Elimination (PARADE). Pediatrics 2000; 105: 1242–9.

58. Hillege HL, Janssen WMT, Bak AAA, Diercks GFH, Grobbee DE, Crijns HJGM, et al. Microalbuminuria is common, also in nondiabetic, nonhypertensive population, and an independent indicator of cardiovascular risk factors and cardiovascular morbidity. J Intern Med 2001; 249: 519–26.

59. Hillege HL, Fidler V, Diercks GFH, van Gielst WH. de Zeeuw D, van Veldhuisen DJ, et al. Urinary albumin excretion predicts cardiovascular and noncardiovascular mortality in general population. Circulation 2002; 106: 1777–82.

60. Kramer H, Jacobs DR, Jr, Bild D, Post W, Saad MF, Detrano R. Urine albumin excretion and subclinical cardiovascular disease. The Multi-Ethnic Study of Atherosclerosis. Hypertension 2005; 46: 38–43.

61. Van der Velde M, Halbesma N, de Charro FT, Bakker SJL, de Zeeuw D, de Jong PE, et al. Screening for albuminuria identifies individuals at increased renal risk. J Am Soc Nephrol 2009; 20: 852–62.

62. Ärnlöv J, Evans JC, Meigs JB, Wang TJ, Fox CS, Levy D, et al. Low grade albuminuria and incidence of cardiovascular disease events in nonhypertensive and nondiabetic individuals. The Framingham Heart Study. Circulation 2005; 112: 969–75.

63. Forman JP, Fisher ND, Schopick EL, et al. Higher levels of albuminuria within the normal range predict incident hypertension. J Am Soc Nephrol 2009; 19: 1983–8.

64. Schopick EL, Fisher ND, Lin J, Forman JP, Curhan GC. Post-menopausal hormone use and albuminuria. Nephrol Dial Transplant 2009; 24: 3739–44.

65. Sharma K. The link between obesity and albuminuria: adiponectin and podocyte dysfunction. Kidney International 2009; 76: 145–8.

66. Rifkin DE, Katz R, Chonchol M, Fried LF, Cao J, de Boer IH, et al. Albuminuria, impaired kidney function and cardiovascular outcomes or mortality in the elderly. Nephrol Dial Transplant 2010; 25: 1560–7.

67. De Jong PE, Brenner BM. From secondary to primary prevention of progressive renal disease: The case for screening albuminuria. Kidney Int 2004; 66: 2109–18.

68. Perkins BA, Ficociello LH, Roshan B, Warram JH, Krolewski AS. In patients with type 1 diabetes and new-onset microalbuminuria the development of advanced chronic kidney disease may not require progression to proteinuria. Kidney International 2010; 77: 57–64.

69. Ritz E, Viberti C, Ruilope LM, Rabelink AJ, Izzo Jr JL, Katayama S, et al. Determinants of urinary albumin excretion within the normal range in patients with type 2 diabetes: the randomized olmesartan and diabetes microalbuminuria prevention (ROADMAP) study. Diabetologia 2010; 53: 49–57.

70. Mottl AK, Vapputuri S, Cole SA, Almasy L, Göring HHH, Diego VP, Laston S, et al. Linkage analysis of albuminuria. J Am Soc Nephrol 2009; 20: 1597–1606.

71. The National High Blood Pressure Education Program Working Group. National High Blood Pressure Education Program Working Group report on hypertension and diabetes. Hypertension 1994; 23: 145–58.

72. Forman JP, Brenner BM. Hypertension and microalbuminuria: the bell tolls for three. Kidney International 2006; 22–8.

73. Palatini P, Graniero GR, Mormino P, et al. Prevalence and clinical correlates of microalbuminuria in stage I hypertension. Results from the Hypertension and Ambulatory Recording Venetia Study (HARVEST Study). Am J Hypertens 1996; 9: 334–41.

74. Agarwal R, Light R. GFR, proteinuria and circadian blood pressure. Nephrol Dial Transplant 2009; 24: 2400–6.

75. Hricik D, Chung-Park M, Sedor JR. Glomerulonephritis. N Engl J Med 1998; 339: 888–99.

76. Orth SR, Ritz E. The nephrotic syndrome. N Engl J Med 1998; 338: 1202–11

77. Boege F. Harnproteine. In: Thomas L (ed). Labor & Diagnose. Frankfurt; TH-Books 2005: 560–77.

78. Hofmann W, Schmidt D, Guder W, Edel H. Differentiation of hematuria by quantitative determination of urinary marker proteins. Klin Wochenschr 1991; 69: 68–75.

79. Reinhart HH, Spencer JR, Zaki NF, Sobel JD. Quantitation of urinary Tamm-Horsfall protein in children with urinary tract infection. Eur Urol 1992; 22: 194–9.

80. Kallerhoff M, Müller-Siegel K, Verwiebe R, et al. Lokalisation und Ausmaß einer Gewebeschädigung durch extrakorporale Lithotrypsie (ESWL). Urologe (A) 1991; 30: 85–8.

81. Hofmann W, Rossmüller B, Guder WG, Edel H. A new strategy for characterizing proteinuria and haematuria from single pattern of defined proteins in urine. Eur J Clin Chem Clin Biochem 1992; 30: 707–12.

82. Jung K. Urinary enzymes and low molecular weight proteins as markers of tubular dysfunction. Kidney Int 1994; 46, (suppl 47): 29–33.

83. Chlud K. Nierenschädigung unter parenteraler Goldtherapie. Aktuel Rheumatol 1988; 13: 220–3.

84. de Silva R, Eastmond C. Management of proteinuria secondary to penicillamine therapy in rheumatoid arthritis. Clin Rheumatol 1992; 11: 216–9.

85. Guy M, Borzomato JK, Newall RG, Kalra PA, Price CP. Protein and albumin-to-creatinine ratios in random urines accurately predict 24 h protein and albumin loss in patients with kidney disease. Ann Clin Biochem 2009; 46: 468–76.

12.10 Renal stones

Christian Thomas, Lothar Thomas

12.10.1 Urolithiasis

Renal stones occur in the renal pelvis, the ureter and the bladder. The prevalence of upper renal stone disease in the Western world ranges between 5% and 12%. It is estimated that 12% of men and 5% of women will develop stone disease by the age of 70 years. The recurrence rate without medical treatment is 10% at 1 year, 35% at 5 years and 50% at 10 years /1/. The highest risk for a first renal colic is in the age of 25–50 years. In the USA, the estimated annual incidences for men range from 100 to 300 per 100,000 /2/. Some 50% of the renal stones pass spontaneously, while one third of the patients require clinical treatment. The 10-year recurrence rate is 25% following the first renal colic and 75% with repeated colics /3/. The prevalence of urolithiasis is low in children; in the USA it is, depending upon the region, of the order of 1 in 1000 to 1 in 7600 hospital admissions.

In the Western world 75–80% of stones are composed of calcium oxalate, 5% of uric acid and 5% of calcium phosphate. In developing countries, the urinary stones are composed mainly of phosphates and urates. Life style factors have an influence on urolithiasis (Tab. 12.10-1 – Factors that increase the risk of renal stones). Renal stone diathesis increases with the prevalence of obesity, metabolic syndrome and diabetes mellitus. Apart from these patients, who are included in the group of idiopathic stone formers, other diseases such as hyperparathyroidism, cystinuria, hyper oxaluria and hypo citraturia are unequivocally associated with stone diathesis. Around 80% of stone formers have an idiopathic genesis and 20% have a family history of stones, probably indicative of a genetic predisposition.

The presence of stones can lead to renal tract obstruction with macro hematuria, infection, and loss of renal function.

In patients with renal stones the identification of risk factors and stone analysis for successful prophylactic management of stone disease are important.

Lithogenous substances are calcium, oxalate, uric acid, phosphate and cystine. The lithogenous effect is increased by acid urine, and a reduced urine volume.

Antilithogenous substances are citrate and magnesium.

12.10.1.1 Intratubular nephrocalcinosis

Crystal formation and crystal retention are the two key mechanisms in stone formers which determine the development of intratubular nephrocalcinosis. Calcium oxalate and calcium phosphate crystals frequently form in the tubular fluid as a result of supersaturation. Crystal formation in the distal nephron can be considered a renal mechanism to excrete an increased amount of waste per unit of volume. To ensure safe tubular crystal passage, the healthy kidney /4/:

  • Presents a non-crystal binding epithelium
  • Is able to keep crystal nucleation, growth and aggregation under control via urinary micro- and macromolecular constituents such as citrate, magnesium and proteins
  • Triggers a reduction in antidiuretic hormone-stimulated water permeability at high intracellular calcium concentrations. The increase in calcium level in the distal tubule leads to a decrease in arginine vasopressin secretion via the calcium-sensitive receptors and thus to an increased urine volume, which counteracts the over saturation.
  • The rate and extent of crystal formation and aggregation is a cell biological process in which a particular tubular epithelial phenotype is responsible for firm crystal adhesion. Under stress conditions the luminal surfaces of dedifferentiated or regenerating cells express multiple crystal-binding molecules, such as sialic acid containing proteins, phospholipids, hyaluran, phosphatidylserin, nucleolin-related protein, annexin II and osteopontin. The molecules are not present at the luminal membrane of intact tubular epithelia. Nucleation and crystal formation can, however, also primarily take place in the interstitium. The presence of crystals in the interstitium is termed interstitial nephrocalcinosis.

12.10.1.2 Clinical symptoms and diagnosis

The passage of a stone causes renal colic which is associated with pain, nausea and vomiting. The pain begins suddenly and can last from a few minutes to many hours. Hematuria is also often present. In children, the dramatic appearance of flank pain, as in adults, is rather unusual; hematuria occurs in up to 90% of the cases and pre-school children often have, in addition, a urinary tract infection /5/.

A preliminary rough clinical-radiological identification, if at all possible, of the stone or its fragments should be confirmed /6/. Important clues as to the etiology can be derived from the stone analysis. However, laboratory stone analysis is requested less frequently because of the advent of endoscopic stone removal and extra corporal shock-wave lithotripsy. Material that is worthy of being analyzed is not always obtained /7/. An overview of the composition and frequency of different kidney stones in the industrialized Western countries is shown in Tab. 12.10-2 – Composition and frequency of renal stones.

The stone analysis does not replace the identification of risk factors that are associated with the formation of renal stones. Diagnostic laboratory examinations for the assessment of the metabolic status of the stone former include /7/:

  • To recognize, with serum and urine tests, risk factors that favor the development of renal stones
  • To verify the effectiveness of the therapy.

Not uncommonly, conclusions concerning the stone formers metabolic disorders can be drawn from the composition of the renal stones:

  • With the demonstration of a cystine stone
  • Calcium phosphate stones can be attributable to hypercalcuria or renal tubular acidosis, owing to primary hyperparathyroidism
  • Calcium oxalate stones are indicative of primary hyper oxaluria, elevated dietary intake of oxalate precursors, or gastrointestinal disorders
  • Pure ammonium urate stones are rare in Western countries, but they are common in children in developing nations. Since the stones form in acidic urine, they occur in Western countries more frequently in the bladder in patients with laxative abuse. Due to enhanced intestinal loss of K+, laxative abuse causes increased renal excretion of NH3+ (refer to Section 8.8.4 – Disturbances of chloride excretion).

12.10.2 Examination of stones, lithogenic and antilithogenic substances

In patients with complaints of urolithiasis, the following investigations should be performed at initial presentation /78/:

  • An intravenous urogram to detect any anatomical abnormality of the renal tract, and any intercurrent urinary obstruction. Radiological examinations can give some hint regarding the presence and type of renal stone (Tab. 12.10-3 – X-ray appearance of renal stones).
  • Serum creatinine, uric acid, calcium, total protein or albumin and electrolytes
  • Urine status with regard to hematuria, leukocyturia, proteinuria, pH, osmolality or specific gravity and bacteriuria, possibly urine culture.

In patients with urolithiasis, the examinations listed in Ref. /9/ and Tab. 12.10-4 – Further investigation in addition to the basic program in renal stone formers are recommended in addition to the basic diagnostics (Tab. 12.10-4 – Further investigation in addition to the basic program in renal stone formers). The reference intervals of the urine tests are provided in Tab. 12.10-5 – Reference intervals of the solutes in the urine.

In principle, the examination of the stone patient on his usual diet is meaningful since it corresponds to the metabolic situation in which the urolithiasis has developed. Following the diagnosis of urolithiasis, the majority of patients maintain a certain diet which, however, is often insufficient for the prevention of the further development of stones. A dietary history is, in any event, important for the assessment of the urine findings.

A further step in patients with recurrent renal stones or who are at elevated risk (family history) is the clinical examination for metabolic diseases such as metabolic syndrome, diabetes mellitus, gout, osteopathy, inflammatory intestinal disease, chronic urinary tract infection and nephrocalcinosis /10/.

12.10.2.1 Analysis of stones

The chemical analyses of stones can provide information on the stone composition. The technique is facilitated by the use of test kits, however, they suffer from false-positive and false-negative results. This method is therefore best replaced by infrared spectroscopy, thermoanalysis and especially X-ray diffraction, which offer semi quantitative results /12/. As an alternative microscopy can be performed on thin sections of the concretion, which allows localization of stone components into the centre or periphery of the stone.

12.10.2.2 Analysis of lithogenic and antilithogenic substances

Urinary oxalic acid

Principle: the determination is performed with a broad spectrum of assays, all of which provide different values. Enzymatic methods, gas chromatographic procedures, ion chromatography and HPLC supported methods are employed.

Urinary citric acid

Principle: by the use of citrate lyase, citrate is transformed to oxalo acetate and acetate. In the presence of malate dehydrogenase and lactate dehydrogenase, oxalo acetate and its decarboxylation product pyruvate, are transformed by NADH2 to malate and lactate. The quantity of NADH2 consumed is measured spectrophotometrically /11/.

Qualitative test for urinary cystine

Principle: the reduction of cystine to cysteine by the use of cyanide; demonstration of SH-groups after adding sodium nitoprusside. Confirmation of a positive result by chromatography /12/.

Uric acid in urine refer to section 5.4, phosphate in urine refer to Section 6.3.

Estimation of additional covariates

The following covariates may significantly influence the excretion of lithogenic substances in stone formers /13/

  • Urinary sodium, as a proxy of salt intake
  • Urinary ammonium and citrate; are related to dietary acid load
  • Urinary sulfate, as a proxy of animal protein intake influencing calcium excretion independently of acid load
  • Urinary pH
  • Urinary volume
  • Estimated GFR
  • Family history

12.10.3 Specimen

Serum: 5 mL

24-hour urine collection (2.5–3 liter container):

  • No additives for urate excretion
  • No additives for electrolyte excretion
  • For the excretion of calcium, oxalate, magnesium, citrate, cystine, creatinine; requirement: 60 mL of HCl, 2 mol/L.

Random urine: 10 mL

12.10.4 Reference intervals

Reference intervals and abnormal excretions associated with nephrolithiasis are shown in Tab. 12.10-5 – Reference intervals and abnormal excretions associated with nephrolithiasis.

12.10.5 Clinical significance

Stone formation is the result of an imbalance between the solubility of the soluta and their crystallization. The urinary concentration of soluta is, therefore, the decisive factor. Studies in serum are only complementary and serve to clarify an underlying disease. Some stone formers do not have concentrations of lithogenic substances that represent a risk, but they do have low 24-hour urine volumes or significant pH changes.

At initial presentation /37/:

  • Patients with no previous stone history who show no abnormality of lithogenic substances are not investigated further
  • Patients with recurrent nephrolithiasis and those who have a renal stone for the first time but, based upon their medical history, are at risk for recurrent stones need long-term patient management.

Urinary supersaturation is a state that the concentration of solutes (e.g., lithogenic substances) in urine exceeds the saturation point, causing spontaneous precipitation of solutes, with the inadequacy of stone inhibitors (e.g., antilithogenic substances). The crystals will aggregate and adhere to the renal tubular epithelium with the presence of tubular adhesion molecules. In addition urinary proteins such as osteocalcin promote crystal aggregation. Some glycoproteins such as prothrombin fragment-1 and osteopontin exert stone-inhibiting property /10/.

12.10.5.1 Stone types

The most common type of urinary stone found in humans worldwide is calcium oxalate (Tab. 12.10-6 – Renal stones).

Calcium containing stones

These stones constitute the largest portion of renal stones and often contain oxalate. In serum the determination of calcium, phosphate and parathyroid hormone is important, in 24-hour urine the determination of calcium and oxalic acid.

Uric acid stones

For the less common pure uric acid stones, the determination of uric acid in the 24-hour urine collection is recommended.

Infection stones

So-called infection stones contain magnesium-ammonium phosphate. Patients complain about bouts of fever with flank pain. With imaging techniques large, low density stones are seen which require immediate removal and intensive antibiotic treatment.

Cystine stones

To rule out cystinuria, a qualitative test should be performed in every case of nephrolithiasis.

Drug-induced stones

With primary, difficult-to-identify stones, medication-induced cause must be considered. Stones may occur following treatment with triamterene, indinavir, salicylate, antibiotics, or due to oxypurinol, a metabolic product of allopurinol.

12.10.5.2 Hypercalcuria

Hypercalcuria is the most frequent pathological finding in patients with renal stones and can contribute to the formation of calcium oxalate and calcium phosphate stones (see also Section 6.2.2 – Urinary calcium excretion).

The mechanisms by which hypercalcuria may contribute to stone formation are /14/:

  • High urinary calcium concentration promotes crystallization of stone-forming calcium salts by increasing the ionic activity of calcium and in turn the saturation of calcium salts
  • Hypercalcuria attenuates urinary inhibitor activity against calcium salt nucleation and growth. Many of the known inhibitors are negatively charged (citrate and glycosaminoglycans), calcium can bind these inhibitors and inactivate their inhibitory activity in urine.

There are three important pathogenic mechanisms for the formation of calcium containing renal stones (Fig. 12.10-1 – Mechanisms that cause hypercalcuria):

  • Absorptive hypercalciuria
  • Renal hypercalciuria
  • Resorptive hypercalciuria.
12.10.5.2.1 Absorptive hypercalcuria

For a given calcium load, patients with absorptive hypercalcuria absorb greater proportions of calcium than normal individuals /1314/. The calcium concentration in blood increases slightly, raises the glomerularly filtered load of calcium and suppresses parathyroid hormone (PTH) secretion. Thus, the combination of increased filtered calcium load and the decreased tubular reabsorption of calcium from PTH suppression contributes to hypercalcuria. Absorptive hypercalcuria is the cause of calcium containing renal stones in up to 50% of cases, but it rarely appears in a pure form. Hypercalcuria is frequently accompanied by other metabolic derangements including bone disease, fasting hypercalcuria, and chronic acidosis These individuals often have a hypercalcuria of > 250 mg/24 h (6.2 mmol/24 h), which results from augmented intestinal Ca2+ absorption and PTH suppression.

A genetic and an acquired form of absorptive hypercalcuria are distinguished.

Genetic form of absorptive hypercalcuria

The major hormonal determinant of intestinal calcium absorption is vitamin D status. It is supposed that end organ sensitivity for 1,25(OH)2D is elevated due to a genetically determined increase in the number of vitamin D receptors in the small intestine /15/. The results, leading to hypercalcuria are: an increase in intestinal vitamin D receptor number, increased 1,25(OH)2D3 binding and an elevation in vitamin D dependent calcium-binding protein, resulting in enhanced intestinal calcium absorption /14/.

Acquired form of absorptive hypercalcuria

It is assumed that excessive dietary intake of animal protein leads to an increased membrane arachidonic acid content and an increase in the synthesis of prostaglandins. These alterations promote changes at the kidney, bone, and intestine that translate into increased urinary calcium excretion /1416/.

12.10.5.2.2 Renal hypercalcuria

There is a defect in the renal tubular reabsorptive mechanism for calcium /14/. Due to the continuous loss of calcium in the urine, secondary hyperparathyroidism enhances formation of 1,25(OH)2D and an increase in intestinal calcium absorption. Thiazide therapy reduces the renal tubule calcium leak and in turn corrects the fasting hypercalcuria, suppresses PTH secretion, and reduces calcitriol and intestinal calcium absorption /14/.

12.10.5.2.3 Resorptive hypercalcuria

Resorptive hypercalcuria is the result of an adenoma of the parathyroid glands with the development of primary hyperparathyroidism /14/. The excessive PTH synthesis leads not only to increased bone resorption of calcium, but also to renal synthesis of 1,25(OH)2D3. Thereby, elevated intestinal calcium absorption also occurs.

12.10.5.2.4 Hypercalcuria and hypernatruria

Hypercalcuria can be attributable to increased salt intake /17/. If the sodium excretion is > 100 mmol/24 h, then every further rise by 100 mmol leads to an increase in calcium excretion by 1.25 mmol/24 h (50 mg/24 h). Sodium excretion is therefore an important indicator for the assessment of the dietary influence on calcium excretion, since sodium restriction reduces the hypercalcuria.

12.10.5.2.5 Diagnostic interpretation of hypercalcuria

Resorptive hypercalcuria

If hypercalciuria is accompanied by hypercalcemia or by a serum calcium level that is near the upper reference interval value along with hypophosphatemia, primary hyperparathyroidism is suspected /18/. See also Section 6.4 – Parathyroid hormone. Elevated PTH values confirm the tentative diagnosis. If elevated serum calcium or hypernatruria cannot explain the hypercalcuria, absorptive or renal hypercalcuria must be taken into consideration.

Absorptive hypercalcuria

The findings are fasting hypercalcuria, plasma calcium near the upper reference interval, PTH low-normal, positive calcium absorption test (Tab. 12.10-7 – Calcium/creatinine ratio in healthy individuals and patients with absorptive and resorptive hypercalcuria in the Ca absorption test).

Renal hypercalcuria

The findings are fasting hypercalcuria, plasma calcium low-normal, PTH elevated. The administration of thiazide diuretics corrects fasting hypercalcuria and suppresses PTH secretion.

12.10.5.3 Hyperoxaluria

Oxalate, dicarbolic acid (HOOC-COOH) is a highly insoluble end product of metabolism in humans. It is excreted, particularly in the forms of its calcium salt, almost entirely by the kidneys. Oxalate has the tendency to crystallize in the renal tubules and the earliest symptoms among those affected are Randal’s plaques.They act as an anchor for calcium oxalate crystals and are considered to be a predisposing factor of renal stone formation and nephrocalcinosis. Progressive renal damage is caused by a combination of tubular toxicity from oxalate, intratubular and interstitial deposits of calcium oxalate, obstruction by renal stones and superimposed infection /19/. Hyperoxaluria is subdivided into primary and secondary forms.

12.10.5.3.1 Primary hyperoxaluria (PH)

PH has a prevalence of 1–3 cases per 1 million in the general population. In Europe, the incidence is 1 case per 120,000 live births per year and is involved in 1–2% of the end stage cases of chronic renal disease in children. The PH types I and II are autosomal recessive inherited diseases. Each type is caused by an enzyme defect and affects different cellular organelles /1920/.

The PH type 1 (OMIM 259900) is a rare autosomal recessive disorder of glyoxalate metabolism. The pyridoxal 5’-phosphate-dependent enzyme catalyzes the transamination of glyoxalate to glycine (Fig. 12.10-2 – Metabolic pathways of oxalate formation in the liver). The enzyme deficiency results in the accumulation of glyoxalate and excessive production of oxalate and glycolate. Mutations in AGXT, the gene encoding AGT, result in PH type 1. At least 178 mutations have been described. Gly170Arg and c.33dupC occur across populations at a frequency of 30% and 11%, respectively.

PH 2 (OMIM 260000) is attributable to a deficiency of glyoxylate reductase/hydroxy pyruvate reductase (GRHPR). The enzyme is localized mainly in the cytosol of hepatocytes and catalyzes the reduction of glyoxalate to glycolate and hydroxy pyruvate to D-glycerate (Fig. 12.10-2). In GRHPR deficiency, lactate dehydrogenase metabolizes accumulated glyoxalate to oxalic acid. A total of 30 mutations have been identified in the GRHPR gene, in which the c.103delG and c.403404+2delAAGT mutations are relatively common.

PH type 3 (OMIM 613616) results from a defect in the liver-specific enzyme 4-hydroxy-2-oxoglutarate aldolase (HOGA), which plays a role in the metabolism of hydroxyproline. A minimum of 19 mutations have been described in HOGA1.

Clinical symptoms

Clinical symptoms of PH can occur at any age, the median age is 5.5 years. The spectrum of the primary symptoms ranges from childhood nephrocalcinosis to nephrolithiasis in adulthood /19/. At the time of diagnosis, 20–50% of the patients have advanced chronic renal disease. Oxalate crystals are deposited preferentially in the kidneys, in the blood vessels, and in bone. The most common subtype is PH type 1, particularly in childhood.

Laboratory findings

Kidney stones consist of more than 95% calcium oxalate monohydrate (whewellite) have a non homogenous appearance and are of pale color. Children have elevated creatinine and metabolic acidosis. The following excretions are considered to be pathologic in PH type 1 /2021/:

  • Oxalic acid of ≥ 0.56 mmol × 24 h–1 or ≥ 50 mg × 24 h–1. Oxalic acid tends to be higher in PH type 1 with 2.14 ± 1.29 mmol × 24 h–1 than in PH type 2 with 1.46 ± 0.49 mmol × 24 h–1
  • Glycolate of ≥ 0.93 mmol × 24 h–1 or ≥ 70 mg × 24 h–1
  • An elevated glycolate value in PH type 1 is helpful in the differentiation of PH type 1 and PH type 2.

Oxalate in renal insufficiency: plasma concentrations in end stage renal disease (ESRD) are 20–60 μmol/L (1.8–5.4 mg/L), while in PH patients with ESRD they are ≥ 60–100 μmol/L (5.4–9.0 mg/L) /19/.

Calcium excretion: low in PH type 1 and PH type 2, variable in PH type 3. In uncategorized cases liver biopsy maybe performed to test the activities of AGT and GRHPR.

Definitive diagnosis of PH: requires genetic testing. Some 80% of patients have the PH type 1. If no AGT or GRHPR deficiencies are found on liver biopsy, PH 1 and PH 2 can be excluded.

12.10.5.3.2 Secondary hyperoxaluria

Secondary hyperoxaluria is the most common cause of renal stone formation and can be due to:

  • Elevated oxalic acid load
  • Increased intestinal absorption of oxalic acid
  • Chronic renal disease
  • Exaggerated vitamin C supplementation.

Elevated oxalic acid load

The elevated intake of oxalic acid-rich foods such as rhubarb, beet root, spinach, almonds, tofu, tea, and chocolate can lead to mild hyperoxaluria of up to 60 mg × 24 h–1 or 0.67 mmol × 24 h–1. The absorption of oxalic acid is dependent upon the calcium content of the food. If oxalate is ingested simultaneously with foods rich in calcium then much of oxalate will be complexed to calcium in the gut and intestinal absorption is reduced. Precursors of oxalate metabolism such as ascorbic acid, fructose, xylose and hydroxyproline can also lead to augmented synthesis of oxalic acid. Plasma oxalate levels are in the range of 1–5 μmol/L (90–450 μmol/L) and are supersaturated at concentrations of ≥ 30 μmol/L (2.7 mg/L) /2122/.

Enteric hyper oxaluria

The daily dietary oxalate intake is 50–200 mg, but may range to 1000 mg. Intestinal absorption ranges between 5% to 15%. Oxalate is absorbed throughout the length of the small intestine, and the colon also participates in its absorption. The major portion of the oxalate that is ingested with food is absorbed within 4–8 hours /22/.

Oxalate degrading bacteria play a role in the control of the transport of oxalate. In this regard, the colon is colonized with the anaerobic Oxalobacter formigenes. It is believed that deficient colonization with this bacterium may be a cause of elevated oxalate absorption and thereby a risk factor for renal stones.

Moderate to severe enteric hyper oxaluria occurs in bowel disease. The hyper oxaluria in bowel disease is not from increased endogenous production but rather intestinal over absorption. In the setting of malabsorption with steatorrhea, dietary calcium is bound by free fatty acids in the intestinal lumen. There is less calcium available to bind oxalate, resulting in increased amounts of oxalate free for absorption. In contrast to normal physiology, the colon appears to be the major site of oxalate absorption in enteric hyper oxaluria. Overall, enteric hyper oxaluria refers to a state in which disease or resection of the small bowel leads to malabsorption of fat and bile acids. It is most frequently seen in Crohn’s disease or patients with jejunal-ileal bypass /22/.

Renal oxaluria

Oxalate is not protein-bound, so it is freely filtered at the glomerulus. Net tubular secretion of oxalate with fractional excretions as high as 200–300% have been described. Serum oxalate levels are in the range of 1–5 μmol/L, but in urine the concentration is about 100-fold higher, mainly due to water reabsorption by the nephron. Urine is generally supersaturated with respect to calcium oxalate and the risk of crystallization is only in the urinary tract unless serum levels rise above 30 μmol/L. At the physiological pH of 7.0, oxalate forms insoluble crystals with calcium because in aqueous solution oxalic acid is only soluble up to a concentration of 56 μmol/L (5 mg/L). Generally, however, 0.44 mmol (40 mg) are excreted with 1.5 liters of urine in 24 hours /2122/.

In supersaturation, the calcium oxalate crystals adhere to the tubular cells. Some of these migrate into the interstitium and cause inflammation with subsequent interstitial fibrosis, which can progressively lead to nephrocalcinosis and the formation of renal stones /20/. Secondary complications are the obstruction of tubuli and collecting ducts, as well as urinary tract infection.

With impaired kidney function and a decline in the GFR below 30–40 [mL × min–1 × (1.73 m2)–1], a rise in serum oxalate concentration, which can slightly exceed the supersaturation concentration (30 mmol/L), occurs. The result is systemic deposition of calcium oxalate crystals in the kidneys and in extrarenal tissue such as myocardium, vessel walls, bone, the central nervous system and the eyes.

Supplementation with ascorbic acid : vitamin C is excreted in urine both in its unmetabolized form and as oxalate. Ascorbic acid intake of > 1 g daily increases the risk of kidney stones with a relative risk (RR) of 2.23 (1.28–3.88). With ingestion of less than 7 times weekly, the RR is 1.66 (0.99–2.79) /23/.

Laboratory findings

In all patients with recurrent calcium oxalate stone disease, a 24 h urine oxalate excretion should be determined. If the excretion is greater than 1.0 mmol × 24 h–1 glycolate and L-glyceric acid should be determined. These marker metabolites would distinguish PH type 1 from PH type II, but if negative, cannot exclude a diagnosis of pH /20/.

In oxalate stone formers with secondary hyper oxaluria the 24 h urine oxalate excretion is 0.55–0.8 mmol × 24 h–1 or 50–72 mg × 24 h–1; upper reference interval ≤ 0.50 mmol × 24 h–1 or 45 mg × 24 h–1.

12.10.5.4 Hyper uricosuria

Uric acid urolithiasis can present as pure uric acid stones or mixed uric acid/calcium stones. The incidence of uric acid stones shows wide regional differences. In Great Britain and the USA the incidence is 5–10%, in Germany it is 25%, and in the Middle East it is 30% in both adults and children /24/. Some 5% of kidney stone carriers have mixed uric acid/calcium stones /25/. A small proportion of patients with uric acid containing stones has hyper uricosuria, but the majority have normo uricosuria. The common element in these patients is low urinary pH promoting uric acid precipitation /24/. For hyper uricosuria, see also Section 5.4 – Uric acid.

Uric acid (C5H4N4O3) promotes the formation of salt crystals.

Factors related to uric acid crystallization are persistent acid urine, hyper uricosuria and small urine volume /24/:

  • Persistent acidic urine is the principal determinant of uric acid crystallization. Uric acid is a weak acid with a current urine pK of 5.35. At this pH, half of the uric acid is in its sparingly soluble non-dissociated, form. With a solubility of undissociated uric acid of 97 mg/L, to exceed solubility at urinary pH’s of 4.5, 5.5, and 6.5 would require total uric acid (undissociated and urate) concentrations of approximately 110, 200, and 1100 mg/L respectively /24/. This shows that increased solubilization of uric acid in urine is only possible with an increase in urinary pH. In the majority of patients with uric acid stones the urine pH is abnormally low because of increased renal H+ secretion, decreased NH4+ excretion, or a combination of both.
  • Hyper uricosuria is due to elevated synthesis, increased renal excretion or a combination of both. Only a small proportion of patients with uric acid stones has increased uric acid excretion. If hyper uricosuria is defined as the excretion of > 750 mg/24 h (4.5 mmol/24 h) in women and > 800 mg/24 h (4.8 mmol/24 h) in men, then approximately one-third of normal patients and one-third of calcium oxalate stone formers have hyper uricosuria. However, only 15% of the patients excrete more than 1000 mg/24 h (5.9 mmol/24 h) of uric acid. Of all stone formers undergoing a metabolic evaluation, hyper uricosuria was the only metabolic abnormality in 10% and was found in combination with other metabolic abnormalities in 40% of patients /25/.
  • Small urine volume. In dry regions, oliguria is an independent risk factor for urate nephrolithiasis /24/.
12.10.5.4.1 Etiology of uric acid stone diathesis

The etiological mechanisms of uric acid stone formation are various; they include congenital, acquired and idiopathic causes. The latter are the most common. Idiopathic genesis refers to etiology that is neither congenital, nor can it be ascribed to secondary causes. Uric acid stones occur in an augmented manner in the metabolic syndrome, in type 2 diabetes mellitus and in obesity /2425/.

Congenital causes

Monogenic disorders of uric acid metabolism: a defect in the enzyme hypoxanthine guanine phospho ribosyl transferase (Lesh-Nyhan syndrome), phospho ribosyl pyrophosphate synthetase over activity, and glucose-6-phosphatase deficiency lead to serum uric acid levels of > 10 mg/dL (595 μmol/L) and urinary uric acid excretion greater than 1000 mg/24 h (5.9 mmol/24 h).

Primary gout: the incidence of uric acid stones is directly proportional to the extent of hyper uricosuria. Primary gout is associated with a low urinary pH and with reduced fractional excretion of urate.

Adenine phospho ribosyl transferase deficiency: about 1 out of 1000 stone carriers has a deficiency of the enzyme adenine phospho ribosyl transferase, which plays a role in purine metabolism (see Section 5.4 – Uric acid). Adenine is oxidized exclusively to 2,8-dihydroxyadenine, excreted in the urine, where it leads to the formation of stones. The diagnosis of the autosomal recessive disease is made with the finding of the enzyme deficiency in red blood cells /7/.

Secondary causes

Diarrheal states: chronic diarrheal diseases, such as ulcerous colitis, M. Crohn, and ileostomy favor the formation of urate stones due to small urine volumes and the formation of acidic urine. The acidic urine is due the loss of HCO3 in the stool coupled with defects in urinary NH4+ excretion (see Section 8.8.4 – Disturbances of chloride excretion/24/.

Oliguria

Excessive sweating and exercise, particularly in arid regions, are important causes of the development of urate stones in the Near East /24/.

High animal protein diet

The excessive intake of animal protein raises the organism’s uric acid load, lowers the pH and reduces urinary citrate concentrations. The cause of hyper uricosuria is elevated purine intake in 70% of the patients. In most patients, however, urinary pH is higher than 5.5 due to adaptive increases in NH4+ excretion /24/.

Purine overproduction

Myeloproliferative diseases, hemolytic anemia and strong diet restriction lead to elevated uric acid production. The cause of hyper uricosuria is raised endogenous production in 30% of the patients.

Uricosuric drugs

Uricosuric drugs can lead to transient hyper uricosuria (e.g., probenecid, high dose salicylic acid, radio contrast agents).

12.10.5.4.2 Idiopathic uric acid nephrolithiasis

The two essential factors in the diathesis of idiopathic uric acid nephrolithiasis is persistent low urinary pH and the fractional excretion of urate is reduced. The causes of inappropriately low pH, are defective NH4+ formation and elevated net acid excretion. The acid-base balance and the effective buffering of the H+ in the kidneys is normally maintained by the H2CO3–HCO3 and the NH3 –NH4+ buffer systems. The latter system, with a pKa of 9.2, has a high buffering capacity for H+ (see also Fig. 8.8-4 – H+ secretion into the cortical collecting tubule). Remaining H+ are buffered by titratable acids. In this way, the urinary pH is held constant within a certain range, especially under acid load. If the NH4+ is low in urine, the buffering under acid load must be carried out exclusively by the titratable acids.

An inappropriately acidic urine, attributable to decreased NH4+ formation, is seen in obesity, metabolic syndrome and insulin resistance /21/. In these patients, inappropriately acidic urine is formed, e.g. due to a meat-rich diet. The intake of sulfur-containing amino acids affects the tubular secretion of H2SO4. The H+ that is released is titrated by HCO3. However, at a urinary pH of ≤ 6, no more HCO3 is available for titration and the titration must be carried out exclusively by NH4+.

12.10.5.5 Hypocitraturia

Urinary citrate inhibits calcium stone formation by complexing calcium in a soluble form and by effects on urinary crystals to prevent growth to stones. Citrate is a tricarboxylic acid with pKa of 2.3, 4.3 and 5.6. In urine citrate exists predominantly in its trivalent form. Since the last pKa is 5.6 the abundance of divalent species increases as the pH falls. Hypocitraturia contributes to calcium nephrolithiasis in 20–60% of stone formers. However, hypocitraturia is only infrequently the sole cause of calcium stones, except in certain unusual causes with renal tubular acidosis /26/. Causes of the high prevalence of hypocitraturia in Thai people are believed to be a low citrate intake and an increased urinary citrate reabsorption from chronic metabolic acidosis, which maybe caused by endemic incomplete renal tubular acidosis /27/.

Citrate is filtered freely in the kidney and 65–90% is reabsorbed as a divalent anion mainly in the proximal tubule. The remainder is found in the final urine.

Changes in acid-base homeostasis are the predominant determinant of proximal tubule reabsorption and urinary excretion of citrate (Fig. 12.10-3 – Transport of citrate in the proximal tubule of the kidney/26/.

Factors are:

  • Tubular reabsorption of citrate increases in intracellular tubular acidosis and hypocitraturia can ensue
  • Decrease in tubular pH cause increase in the proportion of divalent citrate and tubular citrate reabsorption will increase. For example the divalent citrate increases 3-fold as the luminal pH decreases from pH 7.4 to pH of 6.9 /26/.

Causes of hypocitraturia because of acidosis are /17/:

  • Conditions that lead to increased reabsorption of citrate (chronic systemic acidosis, increased tubular acid load). This is the case in chronic diarrhea, in infections or with high animal protein intake. Causes of metabolic acidosis with severe hypocitraturia (< 100 mg/24 h; 0.52 mmol/24 h) are chronic diarrhea, gastrectomy, and ulcerative colitis.
  • Hypokalemia and potassium deficiency. In potassium deficiency the intracellular pH declines and in spite of systemic alkalosis renal tubular H+ secretion increases. Potassium deficiency enhances citrate transport in the proximal tubule.
  • Type 1 renal tubular acidosis (see Tab. 8.8-5 – Renal-tubular acidoses).

Causes of hypocitraturia in the absence of acidosis are /17/:

  • Thiazide medication; thiazides lower the excretion of citrate, especially in potassium deficiency
  • Excessive physical work, chronic kidney disease and high salt intake.

Potassium citrate is administered in order to raise urinary citrate concentrations. This is metabolized to HCO3; the urinary alkaline load is thereby increased and tubular citrate reabsorption is reduced.

12.10.5.6 Hypomagnesuria

Urinary magnesium is an inhibitor of calcium salt formation. Low urinary concentrations promote the formation of calcium stones.

12.10.5.7 Low urinary pH

The excessive intake of acid with the food, due to an animal protein diet, can lead to high uric acid excretion, citrate and sulfate and can lower the pH of the 24-hour urine from 5.5 to 5.0. Constant acidic pH values below 6.0 in the daily urinary pH profile are an indicator of persistently acidic urine and they promote the formation of uric acid and calcium oxalate stones, and the crystallization of both /17/.

12.10.5.8 High urinary pH and hyper calcuria

Hyper phosphaturia could be caused by several etiologies, including phosphate intake, gastrointestinal absorption and the rate of phosphate reabsorption which is dependent on the abundance of phosphate co transporters, hormone regulation and dietary factors /28/. The prevalence of calcium phosphate stones is less common than calcium oxalate stones and seem preferentially likely in women /29/.

The following are distinguished based on the mineral form:

  • Carbonate apatite Ca10(PO4CO3OH)6(OH)2. It crystallizes at a pH of ≥ 6.8 and occurs particularly in association with urinary tract infections and as a constituent of calcium oxalate stones.
  • Brushite (CaHPO4 × 2 H2O), which is formed at the narrow urinary pH range of 6.5–6.8 with hypercalcuria of > 8.0 mmol/24h and phosphate excretions of > 35 mmol/24 h.

Calcium phosphate stone formation plug the inner medullary collecting ducts with apatite crystals. Formation of initial calcium phosphate deposits can lead to a vicious cycle of inner medullary collecting ducts cell injury, rising pH and more calcium phosphate deposition /28/. The patients have high calcium phosphate supersaturation due to high urine pH coupled with hyper calcuria. Hypercalcuria is genetic in nature or it is due to primary hyperparathyroidism. The elevated urinary pH is believed to be genetic in nature or to be based upon distal renal tubular acidosis (type 1 RTA). The latter is congenital or acquired. See also Section 8.8 – Renal electrolyte excretion).

It is believed that along with supersaturation of the urine, the fragmentation of calcium oxalate stones with shock wave lithotripsy is also responsible for the increase in calcium phosphate stones. It is possible that this favors the formation of calcium phosphate stones through renal injury /28/.

12.10.5.9 Small urine volume

The 24-hour urine volume should be 1.5–2 L. Since the insensible water loss in adults amounts to approximately 1 liter per day, daily fluid intake must be 2–3 liters. If less than 2 liters are ingested daily the urine volume will be below 1 liter and supersaturation of the urine with solutes can occur with the danger of crystal formation /17/.

12.10.5.10 Cystinuria

The worldwide incidence of cystinuria is variable. In Great Britain, the screening of newborns estimated an incidence of 1 : 2000; in Australia of 1 : 4000 and in the USA of 1 : 15,000 /18/. Cystinuria is characterized by a renal and intestinal transport defect in dibasic amino acids (cystine, lysine, ornithine and arginine). The physiological fractional excretion of cystine (C3H6O2NS) is 0.4%. The tubular reabsorption of cystine occurs to an extent of 80% via a low affinity, high capacity Na+-independent transport system, while the remaining 20% is effected through a high affinity system which also transports other dibasic amino acids /12/.

Three types of cystinuria are distinguished /31/:

  • The homozygous type with excretion of cystine and high levels of aminoaciduria. Because of the absence of the neutral and basic amino acid transport protein rBAT, a transport activator or co-transporter of cystine, renal tubular reabsorption is decreased. The homozygous type is clinically symptomatic (i.e., a stone carrier) in 90% of the cases.
  • Heterozygotes of type I show normal aminoaciduria, whereas heterozygotes of type II and type III show high and moderate levels of cystine and other dibasic amino acid excretion, respectively.

Cystine stones account for 1–2% of all kidney stones in adults with nephrolithiasis and 6–8% in children. The peak age of onset of stone disease is during the third decade of life. Patients present with symptoms related to urolithiasis. Cystine stones should be suspected in patients with /31/:

  • History of renal stones in childhood
  • Recurrent episodes of nephrolithiasis
  • Strong family history of stone disease
  • Amber colored kidney stones.

Cystine is almost insoluble at pH 5–7. Normal cystine excretion is below 20 mg/24 h (85 μmol/24 h). Due to its high pK value, the upper limit of solubility of cystine at physiological pH of the urine is about 300 mg/L (1,250 μmol/L) or 400 mg (1,660 μmol/24 h) /1230/. Supersaturation of the urine leads to the formation of renal stones. The solubility of cystine increases with rising pH; at a urinary pH of 8 it is almost 3 fold higher than at the physiological pH. The therapeutic objective is the alkalization of the urine.

By definition a urinary cystine excretion above 250 mg/24 h (1,000 μmol/24 h) in adults and above 75 mg/24 h (312 μmol/24 h) in children is abnormal. The excretion of > 250 mg (1,000 μmol) cystine/g creatinine or > 400 mg (1,660 μmol/24 h) is indicative of homozygous cystinuria. The final diagnosis is made based upon the analysis of the stone. Patients with homozygous cystinuria may have stones with calcium oxalate as the major component and cystine as the minor component. Heterozygous individuals, who excrete less cystine than homozygous patients, do not necessarily have a lower incidence of stone formation /30/.

Laboratory findings: on microscopic analysis of fresh urine, hexagonal cystine crystals are typical of cystinuria. These crystals are seen in only a minority of cystinuric patients /31/. The following tests are used for the chemical detection of cystinuria:

  • The cyanide nitroprusside method, a quick test for qualitative detection
  • The nickel/dithionite test for qualitative and semi-quantitative detection /30/.

12.10.6 Comments and problems

24-hour urine collection

For the determination of calcium, oxalate, magnesium, citrate, electrolytes, and cystine, the urine has to be acidified by HCl so that the pH is below 2. For the determination of urate the collection is to be made without additives /7/.

Excretion of calcium and oxalate

The urinary pH must be below pH 6.5 during the collection because otherwise calcium oxalate crystals that are not readily soluble will precipitate /7/.

Uric acid excretion

Uric acid precipitates in acidic urine. The urine collection should, therefore, not be performed with the addition of acid /7/. Before the uric acid determination, the urine has to be alkalized (pH 8–9) in the laboratory with the addition of NaOH to re-dissolve any uric acid that had precipitated in acid urine or due to low temperature.

Cystine excretion

The urine collection should be at a pH above 7.5 to improve the solubility and avoid false low concentrations due to precipitation at physiological pH. 2 g of sodium bicarbonate are added to the container before starting the collection to alkalize the urine /7/.

Cystine rapid test

False positive reactions with the sodium nitroprusside test in acetonuria and homocystinuria /32/.

pH value

The determination has to be performed on fresh fasting morning urine /17/.

Influence factors

Diuretics alter the excretion of calcium. Thiazides lead to reduced excretion and loop diuretics lead to increased excretion /3334/. Magnesium excretion reacts in the contrary manner. The excretion of uric acid decreases, as does that of oxalic acid, under thiazide treatment /35/.

The oral administration of phosphate salts leads to increased urinary excretion of phosphate /36/. The result is an increase in the excretion of pyrophosphates as suppressors of the growth of calcium oxalate crystals /35/ and a decrease in hyper calcuria /37/.

Vitamin C enhances the formation of oxalic acid and its excretion with a urinary pH of above 7 /38/.

References

1. Daudon M. Epidemiology of nephrolithiasis in France. Ann Urol 2005; 39: 209–31.

2. Coe FL, Evan A, Worcester E. Kidney stone disease. J Clin Invest 2005; 115: 2598–608.

3. Walker V, Stansbridge EM, Griffin DG. Demography and biochemistry of 2800 patients from renal stones clinic. Ann Clin Biochem 2013; 50: 127–39.

4. Vervaet B, Verhulst A, D’Haese PC, De Broe ME. Nephrol Dial Transplant 2009; 24: 2030–5.

5. Stapelton FB. Childhood stones. Endocrinol Metab Clin N Am 2002; 31: 1001–15.

6. Kok DJ. Clinical implications of physicochemistry of stone formation. Endocrinol Metab Clin N Am 2002; 31: 855–67.

7. Wilkinson H. Clinical investigation and management of patients with renal stones. Ann Clin Biochem 2001; 38: 180–7.

8. Leman J, Worcester EM. Nephrolithiasis. In: Massry SG, Glassock RJ (eds). Textbook of nephrology. 2nd ed. Baltimore: William & Wilkins, 1989: 920–41.

9. Knoll T. Leitlinien zur Diagnostik, Therapie und Metaphylaxe der Urolithiasis. AWMF Leitlinienregister 043/025. Febr. 2009.

10. Ticinesi A, Guerra A, Allegri F, Nouvenne A, Cervellin G, Maggio M, et al. Determination of calcium and oxalate excretion in subjects with calcium nephrolithiasis: the role of metabolic syndrome. J Nephrol 2018; 31: 395–403.

11. Welshmann SG, McChambridge H. The estimation of citrate in serum and urine using a citrate lyase technique. Clin Chim Acta 1973; 46: 243–6.

12. Shekarriz B, Stoller ML. Cystinuria and other noncalcareous calculi. Endocrinol Metab Clin N Am 2002: 31: 951–7.

13. Pak CYC, Sakhaee K, Moe OW, et al. Defining hypercalciuria in nephrolithiasis. Kidney Int 2011; 80: 777–82.

14. Zerwekh JE, Reed-Gitomer BY, Pak CYC. Pathogenesis of hypercalciuric nephrolithiasis. Endocrinol Metab Clin N Am 2002; 31: 869–84.

15. Bushinsky DA, Favus MJ. Mechanism of hypercalciuria in genetic hypercalciuric rats: inherited defect of intestinal calcium transport. J Clin Invest 1988; 82: 1585–91.

16. Baggio B, Gambaro G. Abnormal arachidonic acid content of membrane phospholipids – the unifying hypothesis for the genesis of hypercalciuria and hyperoxaluria in idiopathic calcium nephrolithiasis. Nephrol Dial Transplant 1999; 14: 553–5.

17. Pak CYC, Resnick MI. Medical therapy and new approaches to management of urolithiasis. Urol Clin N Am 2000; 27; 243–53.

18. Pak CYC, Kaplan R, Bone H, Townsend JT, Waters O. A simple test for the diagnosis of the absorptive, resorptive and renal hypercalciurias. N Engl J Med 1975; 292: 407–500.

19. Cochat P, Rumsby G. Primary Hyperoxaluria. N Engl J Med 2013; 369: 649–58.

20. Hoppe B, Beck BB, Milliner DS. The primary hyperoxalurias. Kidney Int 2009; 75: 1264–71.

21. Sakhaee K. Recent advances in the pathophysiology of nephrolithiasis. Kidney Int 2009; 75: 585–95.

22. Asplin JR. Hyperoxaluric calcium nephrolithiasis. Endocrinol Metab Clin N Am 2002; 31: 927–49.

23. Thomas LDK, Elinder CG, Tiselius HG, Wolk A, Akesson A. Ascorbic acid supplements and kidney stone incidence among men: a prospective study. JAMA Intern Med 2013; 173: 386–8.

24. Moe OW, Abate N, Sakhaee K. Pathophysiology of uric acid nephrolithiasis. Endocrinol Metab Clin N Am 2002; 31: 895–914.

25. Sorensen CM, Chandhoke PS. Hyperuricosuric calcium nephrolithiasis. Endocrinol Metab Clin N Am 2002; 31: 915–25.

26. Hamm LL, Hering-Smith KS. Pathophysiology of hypocitraturic nephrolithiasis. Endocrinol Metab Clin N Am 2002; 31: 885–93.

27. Dissayabutra T, Kalpongkul N, Rattanaphan J, Boonia C, Srisa-art M, Ungjaroenwathana W, et al. Urinary stone risk factors in the descendants of patients with kidney stone disease. Pediatric nephrology 2018; 33: 1173–81.

28. Hamm L, Alpers R. Regulation of acid-base balance, citrate, and urin pH. In: Coe F, Favus M, Pak C, Parks J, Preminger G (eds). Kidney stones: medical and surgical management. Philadelphia; Lippincott-Raven 1996: 289–302.

29. Parks JH, Coe FL, Evan AP, Worcester EM. Urine pH in renal calcium stone formers who do not increase stone phosphate content with time. Nephrol Dial Transplant 2009; 24: 130–6.

30. Knoll T, Janitzky V, Michel MS, Alken P, Köhrmann KU. Zystinurie – Zystinsteinleiden: Aktuelle Empfehlungen zur Diagnostik, Therapie und Nachsorge. Aktuel Urol 2003; 34: 97–101.

31. Berg W, Janitzky V, Schubert J. Einfacher Schnelltest zur Bestimmung von Zystin im Urin. Urologe B 1998; 38: 151–3.

32. Shekarriz B, Stoller ML. Cystinuria and other noncalcareous calculi. Endocrinol Metab Clin N Am 2002: 31: 951–7.

33. Martinez-Maldonado M, Eknoyan G, Suki WN. Diuretics in nonedematous states: physiological basis for the clinical use. Arch Int Med 1973; 131: 797–808.

34. Massry SG, Fiedler R, Coburn J. Excretion of phosphate and calcium. Physiology of their renal handling and relation to clinical medicine. Arch Int Med 1973; 131: 828–59.

35. Cohanim M, Yendt ER. Reduction of urine oxalate during longterm thiazide therapy in patients with calcium urolithiasis. Invest Urology 1980; 18: 170–3.

36. Fleisch H, Bisaz S, Care AD. Effect of orthophosphate on urinary pyrophosphate excretion and the prevention of urolithiasis. Lancet 1964; 1: 1065–7.

37. Pak CY, Cox JW, Powell E, Bartter FC. Effect of oral administration of ammonium chloride, sodium phosphate, cellulose phosphate and parathyroid extract on the activity product of brushite in urine. Am J Med 1971; 50: 67–76.

38. Chalmers AH, Cowley DM, McWinney BC. Stability of ascorbate in urine: relevance to analyses for ascorbate and oxalate. Clin Chem 1985; 31: 1703–5.

39. Hesse A, Tiselius HG, Jahnen A. Urinary stones. Diagnosis, treatment and prevention recurrence. Basel: Karger, 2002.

40. Carbone A, Al Salhi Y, Tasca A, Palleschi G, Fuschi A, De Nunzio C, et al. Obesity and kidney stone disease: a systematic review. Minerva Urol Nefrol 2018; https://doi.org/10.23736/s0393-2249.18.03113-2.

41. Sritippayawan S, Borvompadungkitti S, Paemanee A, Predanon C, Susaengrat W, Chuawattana D, et al. Evidence suggesting a genetic contribution to kidney stone in northeastern Thai population. Urol Res 2009; 37: 141–6.

Table 12.1-1 Tests for renal diseases

Screening

Symptomatic patient

Estimated GFR (eGFR)

Urine test strip (assay for protein, erythrocytes, leukocytes, nitrite)

Screening examinations

eGFRcr-cys (confirmatory test in cases with eGFR of 40–60 [mL × min–1 × (1.73 m2)–1])

Albumin/creatinine ratio in spontaneous voided morning urine

Urinary marker proteins (α1-microglobulin, IgG) or SDS polyacrylamide gel electrophoresis

Urine sediment for erythrocytes, leukocytes, casts, dysmorphic erythrocytes

Urine culture

Table 12.1-2 Definition of acute renal failure according to RIFLE and AKIN classification /5/

Criteria

Serum creatinine (Scr) criteria

Urinary output

RIFLE classification

Risk

Scr ≥ 1.5 times from baseline or a decrease in the GFR of ≥ 25%

< 0.5 mL/kg BW/h and ≥ 6 h

Injury

Scr ≥ 2.0 times from baseline or a decrease in the GFR of ≥ 50%

< 0.5 mL/kg BW/h and ≥ 12 h

Failure

Scr ≥ 3.0 times from baseline or a decrease in the GFR of ≥ 75% or an acute rise ≥ 0.5 mg/dL (44 μmol/L) from baseline

Scr ≥ 4 mg/dL (354 μmol/L)

< 0.3 mL/kg BW/h and ≥ 24 h or anuria ≥ 12 h

Loss of function

Complete loss of kidney function > 4 weeks

End-stage

Endstage renal disease > 3 months

AKIN classification

Stage 1

Scr ≥ 1.5 times from baseline or Scr increase ≥ 0.3 mg/dL (26 μmol/L) from baseline

< 0.5 mL/kg BW/h and ≥ 6 h

Stage 2

Scr increase ≥ 2.0 times from baseline

< 0.5 mL/kg BW/h and ≥ 12 h

Stage 3

Scr increase ≥ 3.0 times from baseline or an acute Scr rise ≥ 0.5 mg/dL (44 μmol/L) from baseline

Scr ≥ 4.0 mg/dL (354 μmol/L) or initiated on renal replacement therapy (irrespective at time of initiation)

< 0.3 mL/kg BW/h and ≥ 24 h or anuria ≥ 12 h

Only 1 criterion (serum creatinine or urine output has to be fulfilled to qualify for a specific category). BW, body weight

Table 12.1-3 Etiology of acute renal failure /67/

Pre renal kidney failure

The glomerular and renal tubular function are largely retained. If the cause is successfully corrected, the renal dysfunctions are immediately reversible. Etiology:

  • Insufficient renal perfusion (e.g., triggered by a reduction of the intravasal volume, drop in the arterial blood pressure or systemic vasodilation)
  • Systemic Inflammatory Response Syndrome (SIRS)
  • Medications such as non-steroidal anti-inflammatory drugs (NSAID), angiotensin-converting enzyme inhibitors (ACE inhibitors) and diuretics.

Renal failure

Macro vascular disease

This involves acute disorders affecting the arterial perfusion of the kidneys due to:

  • Thromboses, embolies, vasculitides, diseases of the aorta.

Micro vascular disease

They cause acute glomerulonephritis. The detection of dysmorphic erythrocytes or erythrocyte casts in the urine sediment is important for diagnosis

  • Rapid Progressive Glomerulonephritis (RPGN) with or without evidence of glomerular basement membrane antibodies (anti-GBM)
  • Goodpasture syndrome (RPGN with anti-GBM and pulmonary involvement)
  • Systemic diseases with autoimmune vasculitis such as Wegner’s granulomatosis or panarteritis nodosa
  • Thrombotic micro angiopathies such as hemolytic-uremic syndrome or thrombotic-thrombocytopenic purpura.

Tubulo-interstitial disease

Acute interstitial nephritis is characterized by inflammation of the tubules and the renal interstitium, together with the infiltration of mononuclear cells and eosinophils

  • Acute interstitial nephritis of allergic genesis due to medications (see Tab. 12.1-11 – Types of glomerular diseases)
  • Ischemic acute tubulus necrosis. This condition is distinct from pre renal acute renal failure in that once renal perfusion has been restored, renal function does not resume immediately, but rather requires several more weeks
  • Toxic acute tubulus necrosis. Due to the toxic effect (directly or immune-mediated), damage to the tubulus epithel occurs and urinary obstruction, either by means of renal vasoconstruction or directly. See also medications in Tab. 12.1-12 – Drugs with tubulo-interstitial, glomerular and vascular toxic effects.

Post renal kidney failure

The cause is obstructive uropathy. The condition is rare, and only occurs when the outflow of both kidneys is disrupted.

Table 12.1-4 Acute renal failure (ARF): clinical and laboratory findings

Clinical and laboratory findings

Community-acquired acute renal failure (ARF) /1/

Community acquired ARF in the USA has been reported to account for 1% of hospital admissions. Pre renal ARF and acute-on-chronic renal failure were associated with dehydration, use of drugs such as angiotensin-converting-enzyme inhibitors and angiotensin-receptor blockers in high risk patients, and heart failure. After exclusion of those with chronic renal insufficiency, ARF (defined as a serum creatinine concentration over 5.6 mg/dL [495 μmol/L]), developed in 172 adults per million people (pmp) per year. Incidence ranged between 17 pmp per year and 949 pmp per year for adults less than 50 years of age and those aged between 80 and 89 years, respectively. Acute dialysis was administered to 22 pmp per year. The etiologies of ARF are listed in Tab. 12.1-3 – Etiology of acute renal failure. A major further cause of ARF is community acquired pneumonia (CAP). In a study /5/, one third of these patients developed sepsis and ARF. They were, for the most part, elderly and had co morbidities.

In children, hemolytic uremic syndrome secondary to infection with E. coli and Shigella or post-streptococcal glomerulonephritis is a common cause of ARF. In tropical areas, diarrheal diseases, hemolysis, snake bites and non-tropical infections are still common causes of ARF.

Hospital-acquired ARF

The prevalence of hospital-acquired ARF is 0.15–7.2% which exceeds that of community-acquired ARF by 5–10 times. Between 5% and 20% of critically ill patients experience an episode of ARF, often accompanied by multiple organ dysfunction syndrome. Age also plays an important role. In a prospective 2-year population study, individuals over 70 years of age accounted for more than 70% of all cases of ARF. Crystalluria-induced ARF is common in AIDS patients under therapy, and 10–15% have ARF episodes. The incidence of post-operative ARF has dropped to 9.1%, but it is still high with a prevalence of up to 30% in cardiovascular surgery and in transplantation surgery (approximately 30%). About 6% of intensive care patients experience ARF at some point during their stay /8/. Sepsis and septic shock are the causes of over 50% of the ARF cases in critically ill patients in intensive care /9/.

Sepsis

Sepsis is a frequent cause of ARF which occurs in 19% of patients with moderate sepsis, in 23% of those with severe sepsis, and in 51% of patients with septic shock /6/. The hemodynamical event of sepsis is a generalized vasodilation, which is associated with a fall in systemic vascular resistance and arterial under filling. Endotoxinemia stimulates inducible NO synthetase, which leads to NO mediated vasodilation. The resulting arterial under filling is sensed by the baroreceptors and results in compensatory excretion of catecholamines, arginine vasopressin, and activation of the renin-angiotensin-aldosterone system. This leads to vasoconstriction of the vas afferens of the glomeruli and a decrease in the GFR, with Na+ and water retention. Renal ischemia leads to the loss of the brush border and tubular necrosis. In severe cases, only the basal membrane of the tubular cells remains. In the further course of events, the proximal tubular cells regenerate following the injury and proliferate; this can lead to a restitutio ad integrum weeks or months later /6/.

Special forms of ARF – Generalized

Special, but not uncommon forms of ARF are associated with acute rhabdomyolysis, liver cirrhosis and the hepatorenal syndrome.

– Acute rhabdomyolysis /10/

ARF is a potential complication of acute rhabdomyolysis and is the result of trauma or other causes. Acute rhabdomyolysis occasionally develops in patients with structural myopathies when they perform strenuous exercise, are under anesthesia, have taken drugs that are toxic to muscles or have viral infections. Exogenous agents hat can be toxic to muscles are alcohol, illicit drugs, and lipid lowering agents. In the USA 7–10% of all ARF cases are associated with rhabdomyolysis. Among patients in the intensive care unit the mortality rate is 59% when rhabdomyolysis and ARF are present and 22% in the absence.

Laboratory findings: CK reaches activities of 15,000–20,000 U/L; values of up to 5000 U/L are generally not associated with ARF. The rise in creatinine occurs more rapid than in other forms of ARF. Relatively marked is the rise in creatinine in relation to urea. The fractional excretion of Na+ (FENa) is, in contrast to the other forms of ARF, below 1%. The electrolyte abnormalities include hyperkalemia, hyperuricemia, hyper magnesemia, hyper phosphatemia and hypocalcemia; high anion gap, metabolic acidosis. Myoglobin is detected in the urine if the serum levels exceed 50–120 mg/L, and is visible with the naked eye in the urine if the serum values are greater than 1 g/L.

The renal tubular damage caused by myoglobin is the result of its Fe2+ which is necessary for binding oxygen. However, molecular oxygen can promote the oxidation of Fe2+ to Fe3+, thus generating a hydroxyl radical. This oxidative potential is normally counteracted by effective antioxidant molecules. However, cellular release of myoglobin leads to uncontrolled leakage of reactive oxygen species, and free radicals cause cellular injury (see also Section 19.2 – Oxidative stress).

Liver cirrhosis /11/

In liver cirrhosis, ARF is believed to be related to disturbances in circulatory function, mainly a reduction in systemic vascular resistance due to primary arterial vasodilation in the splanchnic circulation, triggered by portal hypertension. In advanced cirrhosis arterial pressure is maintained through activation of the vasoconstrictor systems (sympathetic nervous system, renin-angiotensin system, antidiuretic hormone). These mechanisms help maintain a relatively normal arterial pressure but have important effects on kidney function, particularly Na+ and solute-free water retention, that may lead to ascites and edema and to renal failure causing intra renal vosoconstriction and hypo perfusion.

Laboratory findings: the determination of serum creatinine is considered to be the most reliable biomarker for the diagnosis of ARF. Cirrhosis patients have low creatinine values due to reduced muscle mass, resulting in a falsely high estimation of the GFR. Neither the MDRD nor the Cockroft-Gault formula for GFR estimation should be employed. A creatinine value > 1.5 mg/dL (133 μmol/L) is considered to be an indicator of ARF. Thus the current definition of renal failure in cirrhosis identifies only those patients with severely reduced GFR below 30 [mL × min–1 × (1.73 m2)–1].

– Hepatorenal syndrome /11/

The hepatorenal syndrome is a frequent cause of ARF in liver cirrhosis and is characterized by functional renal vasoconstriction that leads to severe reduction in GFR with minimal renal histologic abnormalities. Hepatorenal syndrome is classified into two types with different clinical and prognostic characteristics.

Laboratory findings: type 1 is characterized by a doubling of the serum creatinine level above 2.5 mg/dL (221 μmol/L) within 2 weeks and is associated with severe multiple organ dysfunction. Type 2 follows a less progressive course than type 1 and is mainly characterized by refractory ascites. The FENa is below 1% and indicates tubular reabsorptive integrity.

Table 12.1-5 Diagnostically significant laboratory tests in acute renal failure

Investigation

Clinical and laboratory findings

Serum creatinine /12/

The serum creatinine level provides an assessment of glomerular filtration rate (GFR). To estimate GFR, it is essential that patients have a normal muscle mass and are in a steady state in terms of renal function. Creatinine is a weak marker in acute renal failure (ARF) because a steady state is often not achieved and interventions such as fluid loading can alter the concentration without reflecting the renal state. If creatinine clearance is determined, the GFR is over-estimated due to tubular creatinine secretion. The time required for serum creatinine to rise is dependent upon the half-life time of creatinine excretion. For a man weighing 75 kg with a GFR of 90 [mL × min–1 × (1.73 m2)–1], this value is 5.5 hours; it is twice that if the GFR is reduced by a factor of 2. Four half-life times (22 hours) are required for 94% of the equilibrium concentration to be reached, and it takes some 132 hours until the GFR is decreased to approximately 15 [mL × min–1 × (1.73 m2)–1]. In patients with acute kidney damage (earthquake victims), who had previously normal creatinine values, it may take 48 hours or longer until a significant rise in creatinine up to the pathological range occurs.

Urea

In end stage renal disease, creatinine concentration can not be used to assess renal function, but this can be obtained by measurement of urea. Its concentrations correlate with the severity of the clinical intoxication symptomatology, particularly the gastrointestinal symptoms. Azotemia is frequently defined as urea concentrations of above 84 mg/dL (14 mmol/L). A marked rise in urea relative to creatinine is an indication of sepsis, gastrointestinal bleeding or steroid hormone treatment. A relatively marked rise in creatinine in relation to urea suggests, rather, rhabdomyolysis dependent ARF.

Urine volume, osmolality

In acute pre renal renal failure small urine volumes (400–800 mL/day) of concentrated urine (osmolality of > 500 mmol/kg) are excreted. The urine contains low concentrations of Na+ (below 20 mmol/L), while in ARF (parenchymal renal failure) it is almost isotonic (> 40 mmol/L). See Tab. 12.1-2 – Definition of acute renal failure according to RIFLE and AKIN classification.

Fractional
sodium
excretion
(FENa)

FeNa < 1% indicates tubular re absorptive integrity, while higher values, especially above 2%, indicate ARF (see also Section 8.8.3 – Fractional sodium excretion (FENa)). The administration of diuretics must be ruled out. In the hepatorenal syndrome, FENa is < 1%; with other causes of ARF it is usually > 2%.

Urine sediment

In ARF, granular casts and tubular cells are detected in the urine sediment.

Potassium

Hyperkalemia is almost always present in ARF, while in chronic renal failure hyperkalemia occurs only if the GFR falls below 15 [mL × min –1 × (1.73 m2)–1] (see Section 8.7 – Potassium).

Sodium

See Section 8.2 – Sodium for the behavior of sodium in renal disease. Hyponatremia is suggestive of a hepatorenal syndrome, water intake despite existing oliguria, or massive use of diuretics.

Phosphate

Hyper phosphatemia occurs with a reduction in the GFR below 30 [mL × min –1 × (1.73 m2)–1]. High phosphate concentrations lead to the binding of calcium and the deposition of calcium phosphate complexes in the soft tissues. This is accompanied by the development of hypocalcemia.

Blood gas analysis

Metabolic acidosis occurs frequently in acute kidney injury, especially in the catabolic state (sepsis, infection, post-operatively).

Neutrophil gelatinase-associated lipocalin

Plasma and urinary NGAL concentrations are elevated in acute tubular necrosis, and they correlate with the duration and severity of the ARF.

Table 12.1-6 Urine findings in the etiological groups of acute renal failure

Examination

Pre renal

Post renal

Renal

Urine osmolality

> 500 mmol/kg

< 350 mmol/kg

< 350 mmol/kg

Urine/serum creatinine ratio

> 40

< 20

< 20

FENa

< 1%

> 1%

> 2%

FEUrea

≤ 35%

> 35%

> 35%

Urinary sodium

< 10 mmol/L

> 20 mmol/L

> 40 mmol/L

Specific gravity

> 1.018

≈ 1.010

≈ 1.010

Proteinuria

None

Approx.

2 g/24 h

None

Urine sediment

Possibly hyaline casts

Possibly hematuria

Dysmorphic erythrocytes

Erythrocyte casts, rough granulocyte casts

Urine/serum osmolality

> 1.5

≈ 1.0

≈ 1.0

FENa, fractional excretion of sodium, see also Section 8.8.2 – Disturbances of sodium excretion; FEUrea, fractional excretion of BUN, see also Section 12.6 – Urea nitrogen (BUN).

Table 12.1-7 Classification of CKD based on primary renal disease or systemic disease /13/

Classifi-

cation

Primary renal disease

Systemic disease

Glomerular disorder

Diffuse, focal or crescentic proliferative glomerulonephritis, focal segmental and glomerulosclerosis, membranous nephropathy, minimal change disease

Diabetes mellitus, systemic autoimmune diseases, systemic infections, drugs, neoplasia (including amyloidosis)

Tubulo-interstitial disorder

Obstruction, urinary tract infections, renal stones

Systemic infection, auto immune disease, sarcoidosis, drugs, urate, environmental toxins (lead, aristolochic acid), neoplasia (amyloidosis)

Vascular disorder

ANCA associated renal vasculitis, fibromuscular dysplasia

Atherosclerosis, hypertension, ischemia, systemic vasculitis, systemic sclerosis, thrombotic micro angiopathy, cholesterol emboli

Cystic and congenital diseases

Renal dysplasia, medullary cystic disease, podocytopathies

Polycystic kidney disease, Alport syndrome, Fabry disease

Table 12.1-8 GFR categories in CKD /13/

Category

GFR1)

Clinical significance

G1

≥ 90

Normal or high

G2

60–89

Mildly decreased

G3a

45–59

Mildly to moderately decreased

G3b

30–44

Moderately to severely decreased

G4

15–29

Severely decreased

G5

< 15

Kidney failure

1) Data in [mL × min–1 × (1.73 m2)–1]

Table 12.1-9 Albuminuria categories in CKD /13/

Cate-
gory

AER
(mg/24 h)

ACR
(mg/mmol)

ACR
(mg/g)

Increase

A1

< 30

< 3

< 30

Normal to mildly increased

A2

30–300

3–30

30–300

Moderately increased

A3

> 300

> 30

> 300

Severely increased

AER, albumin excretion in 24- hour urine; ACR, albumin/creatinine ratio

Table 12.1-10 Chronic kidney disease (CKD) in association with systemic disease

Clinical and laboratory findings

Systemic inflammation, metabolic syndrome and progressive CKD /16/

Systemic inflammation is an important criterion of the metabolic syndrome and cardiovascular disease, and is associated with elevated CRP values. Increased CRP levels also represent a risk for progression of CKD. In the metabolic syndrome, some of the patients have micro albuminuria in addition to the 5 classic characteristics insulin resistance, central obesity, decreased glucose tolerance, hypertension and dyslipidemia (hypertriglyceridemia, low HDL-cholesterol) /17/. It is suspected that high fructose consummation and low-grade inflammation are the main pathogenic reasons of the metabolic syndrome. Excessive fructose in diet leads to a depletion of ATP and to increased uric acid formation. Both are believed to induce the expression leukocyte adhesion molecules (ICAM-1) and chemokines (MCP-1), which, in combination with oxidative stress, cause endothelial dysfunction with renal lesion.

Diabetes mellitus type 2 and CKD /19/

Type 2 diabetes is, at 20–40%, the most important cause of end stage renal disease. In the United Kingdom Prospective Diabetes Study (UKPDS) /18/, the progression rate for newly diagnosed cases of type 2 diabetes within the stages of normo-, micro- and macro albuminuria and kidney failure was 2–3% per year. Within 15 years, 15% and 30% developed micro albuminuria and a decreased GFR, respectively, but only half of these developed micro albuminuria. Only 9% of type 2 diabetics with CKD are aware of their renal disease. For the diagnosis of CKD in diabetes type 2:

  • The Guidelines of the National Kidney Foundation Kidney Disease Outcome Quality Initiative (NKF-K/DOQI) recommends performing renal screening in persons at increased risk for kidney disease, including those with diabetes mellitus and hypertension
  • The American Diabetes Association recommends a yearly determination of serum creatinine in all diabetics, independent of the presence of albuminuria.

Diabetes mellitus type 1 and CKD

Mortality in type 1 diabetes patients is 3–5 times higher than in the general population. Investigations in the Finnish Diabetic Nephropathy (Finn Diane) study /20/ showed that CKD is the major reason for mortality. During the study, which lasted a median of 7 years, patients with normo albuminuria did not manifest increased mortality. In comparison with non-diabetic individuals, the presence of micro albuminuria, macro albuminuria and end stage renal disease was associated with mortality rates by factors of 2.8, 9.2 and 18.3, respectively. The FinnDiane study also showed that in patients with type 1 diabetes, the eGFR is an independent risk factor for mortality. Independent of the presence of albuminuria, diabetic patients with an eGFR of < 60 [mL × min–1 × (1.73 m2)–1] manifested, during the assessment of the course, a doubling of the mortality in comparison to non-diabetic individuals. Diabetics with a GFR of > 120 [mL × min–1 × (1.73 m2)–1] also had elevated mortality.

A relatively high uric acid level following the diagnosis of type 1 diabetes is associated with the subsequent emergence of diabetic nephropathy. During a median course of 18.1 years, 9% of the diabetics developed macro albuminuria (> 300 mg/24 h). In patients with uric acid levels in the highest quartile (values of > 4.1 mg/dL; 244 μmol/L) the cumulative incidence was 22.3% compared to 9.5% for lower values /21/.

Diabetic nephropathy is associated with a risk of hypertension during pregnancy, of pre-eclampsia and of premature birth, particularly if micro albuminuria is present. Accordingly, the premature birth rate is 20% with normo albuminuria and 71% with micro albuminuria /22/.

Mineral metabolism and CKD

CKD is associated with disturbances of mineral balance, especially with changes in phosphate, calcium and parathyroid hormone (PTH). In a systematic review /15/, relationships between these parameters, overall mortality, cardiovascular mortality and cardiovascular events were investigated in non-dialysis-dependent patients with ACD.

The threshold associated with a significant all-cause mortality risk varied:

  • For high phosphorus from 3.5 mg/dL (1.13 mmol/L) to 7.8 mg/dL (2.52 mmol/L)
  • For low phosphorus from < 3 mg/dL (0.97 mmol/L) to < 5 mg/dL (1.61 mmol/L)
  • For high Ca from 9.7 mg/dL (2.42 mmol/L) to > 10.5 mg/dL (2.62 mmol/L)
  • For low Ca from 8.8 mg/dL (2.20 mmol/L) to > 9.0 mg/dL (2.25 mmol/L)
  • For PTH > 300 ng/L to > 480 ng/L.

Thresholds at which cardiovascular mortality risk significantly increased were:

  • For phosphorus > 5.5–6.5 mg/dL (1.78–2.10 mmol/L)
  • For PTH > 476.1 ng/L (reference < 476.1 ng/L).

For cardiovascular events no significant threshold values emerged.

Neurological complications of CKD /23/

From a neurological perspective, clinical features of CKD include weakness, and length-dependent sensory impairment, which leads to functional disability, and, in patients with end stage renal disease, an altered mental state due to encephalopathy. Neurological complications occur in patients with severe renal insufficiency (GFR < 29 [mL × min–1 × (1.73 m2)–1]; stage 4). They can potentially affect all levels of the nervous system, from the brain to the peripheral nervous systems. Approximately 90% of CKD patients have peripheral neuropathy (weakness and sensory loss in the peripheral sections of the limbs); 60% have an autonomous neuropathy (impotence, postural hypotension); 15–20% suffer from restless legs syndrome, and 5–30% have carpal tunnel syndrome. In addition, 30–40% of dialysis patients have cognitive disorders.

Cardiovascular disease (CVD)

CKD is associated with an elevated prevalence of CVD and overall mortality, independently of the traditional risk factors. In the Atherosclerosis Risk in Communities (ARIC) study, the 3 and 9-year changes in the estimated GFR (eGFR) were correlated with the risk of CVD and overall mortality. Participants with an annual decrease in eGFR of ≥ 5.65% had, relative to those with a reduction between 0.33 and 0.47%, a significantly higher risk for CVD (hazard ratio 1.30) and overall mortality (hazard ratio 1.22) /24/. Increases in cardiac stress biomarkers may be associated with loss of kidney function through shared mechanisms involving cardiac and kidney injury. Increases with GDF-15, NT-proBNP, and hsTnT are associated with greater risk for CKD progression /25/.

Pregnancy and renal disease /26/

Pregnancy imposes significant stress on the kidneys, resulting in increased risk for maternal as well as fetal complications in individuals with established moderate-to-serious CKD. Pregnancies with CKD are complicated with preeclampsia and small for gestational age (SGA) offspring or pre term birth. The principal findings of the second Nord-Trøndelag Health Study (HUNT II) were that the risk for preeclampsia or SGA offspring was not increased in women with a reduced kidney function graded as eGFR of 60–89 [mL × min–1 × (1.73 m2)–1] with or without micro albuminuria. The risk of pre term birth was, however, increased two to three times. There was a strong and graded risk increase for preeclampsia, SGA or pre term birth when women with eGFR 60–89 [mL × min–1 × (1.73 m2)–1] were also hypertensive.

Thyroid function

Thyroid dysfunction leads to changes in the GFR. According to a study /27/ the eGFR is, on the average, 18% lower in hypothyroidism and 39% higher in hyperthyroidism than in the euthyroid state. In hypothyroidism reduced cardiac output, attributable to decreased heart rate and stroke volume, is assumed. In hyperthyroidism a decrease in vascular resistance is expected, with an increase in blood volume and renal blood flow, due to the relaxation effects of the thyroid hormones on vascular muscle.

Orthotopic liver transplantation (OLT)

Graft survival rates following OLT are, at 1, 5 and 8 years, 85%, 70% and 62%, respectively. The incidence of CKD following OLT is 18% after 5 years and 26% after 10 years. The etiology of CKD is multifactorial, where the nephrotoxicity of calcineurin inhibitors is believed to play an important role. It is difficult to control the GFR in these patients. If the GFR is determined as eGFR using the MDRD equation or the cystatin dependent Le Bricon equation (Tab. 12.2-6 – Equations for estimation of GFR in children using serum creatinine), then the eGFR is found to be within the ± 10% limits of the measured GFR only to a 22% or 27% accuracy /28/.

Anemia /29/

Anemia is relatively common in the elderly, as is an eGFR below 60 [mL × min–1 × (1.73 m2)–1]. Only at an eGFR below 45 [mL × min–1 × (1.73 m2)–1 is there an increased prevalence (i.e., a decrease of the Hb level below 120 g/L in women and below 130 g/L in men). For Hb levels below 100 g/L the corresponding odds ratios in individuals above the age of 65 for the eGFR ranges of 45–49, 30–44, 15–29 and below 15 [mL × min–1 × (1.73 m2)–1] are, respectively, 1.2; 1.9; 5.6 and 8.9.

Sickle cell anemia (SCA) /30/

Impaired urine concentrating ability, defects in urine acidification and electrolyte regulation, and high-normal proximal tubular function are commonly recognized in young patients with SCA. Glomerular enlargement with an increased GFR is perhaps the earliest renal abnormality in SCA. The elevated GFR is caused by a potentially reversible increase in renal plasma flow. Proteinuria, occurs later, often in the second decade of life. Proteinuria is usually asymptomatic with micro albuminuria, but 10% to 20% of young adults can develop macro albuminuria with nephrotic range protein loss. In adulthood, about one third of patients with SCA will develop chronic kidney failure, a major cause of death in this population. In the Baby HUG Trial it was shown that early estimation of the GFR is important, since this can reveal changes early. In children of average age 13.7 ± 2.6 months, the measured GFR was 125.2 ± 34.4 [mL × min–1 × (1.73 m2)–1], normal 91.5 ± 17.8 [mL × min–1 × (1.73 m2)–1]. The 10–90% range for the SCA group was 60–120 [mL × min–1 × (1.73 m2)–1. It is recommended that the GFR be measured with DTPA clearance, rather than estimating the eGFR by means like the Schwartz equation. Baseline GFR measurements suggest that renal dysfunction in SCA, evidenced by glomerular hyperfiltation, begins during infancy.

Table 12.1-11 Types of glomerular diseases /32/

Primary glomerulonephritis (GN)

Proliferative form

IgA nephropathy

IgM nephropathy

Other mesangioproliferative GN

Crescentic GN

  • Immune deposits
  • Pauci-immune membranoproliferative GN

Non-proliferative form

Focal segmental glomerulosclerosis

Membranous glomerulopathy

Minimal change disease

Thin basement membrane disease

Secondary glomerulonephritis (GN)

Proliferative form

Lupus nephritis

Post-infectious GN

GN attributable to hepatitis B or C

Systemic vasculitis

Wegener’s granulomatosis

  • Polyarteritis nodosa
  • Henoch-Schönlein purpura
  • Idiopathic

Non-proliferative form

Diabetic nephropathy

Amyloidosis

HIV-associated nephropathy

Alport’s syndrome

Medication-induced glomerulopathies

Table 12.1-12 Drugs with tubulo-interstitial, glomerular and vascular toxic effects /35/

Association with acute tubular necrosis

Non-steroidal anti-inflammatory drugs

Radiological contrast media

Aminoglycosides

Cephalosporins

Amphotericin

Anesthetic agents

Antiviral agents

Thiazides

Calcineurin inhibitors

Herbal medications

Association with interstitial nephritis

Acute interstitial nephritis

  • Antibiotics (cephalosporins, sulfonamides, penicillin, fluoroquinolones, rifampicin)
  • Non-steroidal anti-inflammatory drugs
  • Thiazides
  • Proton-pump inhibitors
  • Protease inhibitors
  • Phenytoin
  • 5-Aminosalicylates
  • Allopurinol

Association with glomerulopathy

Minimal change disease and focal segmental glomerulosclerosis

  • Non-steroidal anti-inflammatory drugs
  • Lithium
  • Interferon α and β
  • Pamidronate
  • Sirolimus

Membranous glomerulonephritis

  • Gold
  • Penicillamine
  • Bucillamine
  • Non-steroidal anti-inflammatory drugs
  • Captopril

Chronic interstitial nephritis

  • Lithium
  • Phenacetin
  • Non-steroidal anti-inflammatory drugs

Granulomatous interstitial nephritis

  • Penicillin
  • Cephalosporins
  • Phenytoin
  • Carbamazepine
  • Non-steroidal anti-inflammatory drugs
  • Allopurinol

Association with vascular pathology

Vasculitis, necrotizing glomerulonephritis

  • Propylthiouracil
  • Hydralazine
  • Methimazole
  • Sulfasalazine
  • Phenytoin
  • Minocycline
  • Penicillamine

Thrombotic microangiopathy

  • Quinine
  • Cyclosporin/tacrolimus
  • Clopidogrel/ticlopidine
  • Mitomycin, gemcitabine, sequential high-dosage chemotherapy
  • Anti-vascular endothelial growth factor antibody

Hyaline arteriolosclerosis

  • Calcineurin inhibitors

Table 12.1-13 Types of tubulo-interstitial nephritis /37/

Primary genesis

  • Infection (bacterial pyelonephritis, Hantavirus infection, leptospirosis)
  • Immune-mediated (Sjögren’s syndrome, anti-tubular basement membrane disease)
  • Drug-induced (antibiotics, analgesics, lithium, cyclosporine, Chinese herbs)
  • Toxic (lead)
  • Metabolic disease (gout nephropathy, hypocalcemic immune nephritis, hypokalemia)
  • Hematological diseases (light chain myeloma, sickle cell disease, amyloidosis)
  • Various (Balkan nephropathy)

Secondary genesis

  • Glomerulonephritis/glomerulopathy
  • Vascular disease
  • Structural kidney diseases (cystic kidneys, obstructive diseases, reflux)

Table 12.2-1 Clearance formula

C x = U x × V Px

Cx, clearance of the substance x; V, urinary flow rate; Px, Ux, plasma and urine concentrations of substance x

Table 12.2-2 Determination of GFR using exogenous filtration markers (mGFR)

Marker

Comment

Inulin

Inulin, MW 5.2 kDa, is an inert, neutral poly fructose molecule and an optimal filtration marker. The classical method for determination of the inulin clearance requires an intravenous priming dose of inulin, followed by a constant infusion to establish a steady-state inulin plasma concentration. After an equilibrium of 45 min., serial serum samples are collected every 10–20 min. and urine samples every 20–30 min. /2/. High urine flow is maintained throughout the test by providing an initial oral water load of 500–800 mL/m2. Inulin in plasma and urine is determined. Measured on 2 days, the intraindividual variation is 4.9–9 [mL × min–1 × (1.73 m2)–1] for individuals with a GFR of approximately 90 [mL × min–1 × (1.73 m2)–1]; according to other data the coefficient of variation is 11.3% /3/.

Iothalamate

Iothalamate is usually employed as a 125Iodine-labeled substance; in the nonradioactive form it is determined with HPLC. The marker is administered as an intravenous bolus or subcutaneously, and its excretion is measured 30 min. later. In order to prevent iodine uptake by the thyroid gland, cold iodine is administered along with the radioactive dose. In comparison to inulin clearance, iothalamate clearance shows slightly elevated values (likely positive bias due to low tubular secretion) /4/.

Iohexol

Iohexol is a nonradioactive iodine-containing contrast medium. The determination is carried out with HPLC. A 5 mL bolus (647 mg/mL of iohexol, corresponding to 300 mg of iodine/mL) is administered, followed by 10 mL of saline. Blood sampling for the determination of iohexol ensues 60, 90, 120, 150, 180 and 240 minutes later /5/. Iohexol- and iothalamate clearance correlate to a high degree /4/.

51Cr-EDTA

The clearance of 51Cr-labeled ethylenediamine tetra acetic acid is measured 30 min. following the intravenous administration of a bolus of 51Cr-EDTA /6/. Relative to inulin clearance, 51Cr-EDTA clearance generates GFR values that are 5–15% too low /4/.

99mTc-DTPA

Diethylene triamine penta acetic acid (DTPA) is filtered freely and has a half-life time of only 6 hours. Its excretion is measured 30 min. following bolus intravenous administration. Somewhat lower values are measured in comparison with inulin clearance, since 99mTc detaches from DTPA and binds to plasma proteins /4/.

Table 12.2-3 GFR reference intervals based on inulin clearance

Age

GFR
[mL × min–1 × (1.73 m2)–1]

Pre-term delivery /8/

  • 1–3 days

14.0 ± 5

  • 1–7 days

18.7 ± 5.5

  • 4–8 days

44.3 ± 9.3

  • 3–13 days

47.8 ± 10.7

  • 8–14 days

35.4 ± 13.4

  • 1.5–4 months

67.4 ± 16.6

Full-term delivery /8/

  • 1–3 days

20.8 ± 5

  • 3–4 days

39.0 ± 15.1

  • 4–14 days

36.8 ± 7.2

  • 6–14 days

54.6 ± 7.6

  • 15–19 days

46.9 ± 12.5

  • 1–3 months

85.3 ± 35.1

  • 0–3 months

60.4 ± 17.4

  • 4–6 months

87.4 ± 22.3

  • 7–12 months

96.2 ± 12.2

  • 1–2 years

105.2 ± 17.3

Children /8/

  • 3–4 yrs

111.2 ± 18.5

  • 5–6 yrs

114.1 ± 18.6

  • 7–8 yrs

111.3 ± 18.3

  • 9–10 yrs

110.0 ± 21.6

  • 11–12 yrs

116.4 ± 18.9

  • 13–15 yrs

117.2 ± 16.1

  • 2.7–11.6 yrs

127.1 ± 13.5

  • 9–12 yrs

116.6 ± 18.1

Adults /68/

  • 16.2–34 yrs

112 ± 13

  • 38 ± 1.8 yrs

118 ± 6

  • 28 ± 6.1 yrs

107 ± 11

  • 19–40 yrs

103 ± 16 51Cr EDTA clearance

  • 21–62 yrs

70–152 Iothalamat clearance

Values expressed as x ± s

Table 12.2-4 GFR categories according to KDIGO

Category

GFR1)

Clinical significance

G1

≥ 90

Normal or high

G2

60–89

Mildly decreased

G3a

45–59

Mildly to moderately decreased

G3b

30–44

Moderately to severely decreased

G4

15–29

Severely decreased

G5

< 15

Kidney failure

1) Data expressed in [mL × min–1 × (1.73 m2)–1]

Table 12.2-5 CKD-EPI creatinine equations, specified for gender and creatinine concentration /1/

Race,
gender

Serum creatinine

GFR equation
[ml × min–1 × (1.73 m2)–1]

Women

< 0.7 (62)

144 × (SCr/0.7)–0.329 × (0.993)Age

> 0.7 (62)

144 × (SCr/0.7)–1.209 × (0.993)Age

Men

< 0.9 (80)

141 × (SCr/0.9)–0.411 × (0.993)Age

> 0.9 (80)

141 × (SCr/0.9)–1.209 × (0.993)Age

Serum creatinine expressed in mg/dL (μmol/L); ×1.159 [if black]

Table 12.2-6 Equations for estimation of GFR in children using serum creatinine /1/

eGFR [mL × min–1 × (1.73 m2)–1] = 41.3 × height (m) × SCr (mg × dL–1)–1

Updated Schwartz equation for chronic kidney disease recommended by the 2012 KDIGO guideline; IDMS-traceable Jaffe method

eGFR [mL × min–1 × (1.73 m2)–1] = 40.7 × (height/SCr)0.64 × (30/BUN)0.202

Updated bedside Schwartz equation or 1B equation for chronic kidney disease recommended by the 2012 KDIGO guideline; IDMS-traceable Jaffe method

SCr and BUN in mg/dL, height in meters

Table 12.2-7 CKD-EPI cystatin C equations for male (M) and female (F) persons

Serum
cystatin C

GFR equation [ml × min–1 × (1.73 m2)–1]

F < 0.8

133 × (SCys/0.8)–0.499 × (0.996)Age

F* < 0.8

133 × (SCys/0.8)–0.499 × (0.996)Age × 0.932

M > 0.8

133 × (SCys/0.8)–1.328 × (0.996)Age

M* > 0.8

133 × (SCys/0.8)–1.328 × (0.996)Age × 0.932

* If black

Table 12.2-8 CKD-EPI eGFRCr-Cys equations /1/

Race,
gender

Creatinine

Cystatin C

GFR calculation [mL × min–1 × (1.73 m2)–1]

Women

< 0.7 (62)

< 0.8

130 × (SCr/0.7)–0.248 × (SCys/0.8)–0.375 × (0.995)Age × 1.08 [if black]

< 0.7 (62)

> 0.8

130 × (SCr/0.7)–0.248 × (SCys/0.8)–0.711 × (0.995)Age × 1.08 [if black]

Men

< 0.9 (80)

< 0.8

135 × (SCr/0.9)–0.207 × (SCys/0.8)–0.375 × (0.995)Age × 1.08 [if black]

< 0.9 (80)

> 0.8

135 × (SCr/0.9)–0.207 × (SCys/0.8)–0.711 × (0.995)Age × 1.08 [if black]

Women

> 0.7 (62)

< 0.8

130 × (SCr/0.7)–0.601 × (SCys/0.8)–0.375 × (0.995)Age × 1.08 [if black]

> 0.7 (62)

> 0.8

130 × (SCr/0.7)–0.601 × (SCys/0.8)–0.711 × (0.995)Age × 1.08 [if black]

Men

> 0.9 (80)

< 0.8

135 × (SCr/0.9)–0.601 × (SCys/0.8)–0.375 × (0.995)Age × 1.08 [if black]

> 0.9 (80)

> 0.8

135 × (SCr/0.9)–0.601 × (SCys/0.8)–0.711 × (0.995)Age × 1.08 [if black]

Whites do not multiply by the factor 1.08. Creatinine in serum in mg/dL (μmol/L). Cystatin C in serum in mg/L.

Table 12.2-9 Kidney damage markers /1/

Marker

Comment

Albuminuria

Excretion ≥ 30 mg/24 h, albumin/creatinine ratio ≥ 30 mg/g (3 mg/mmol)

Urine sediment

Micro hematuria, erythrocyte casts, granulocyte casts, granular casts (see Section 12.8 – Erythrocytes, leukocytes, casts in urine).

Renal tubular damage

Tubular proteinuria, renal tubular acidosis, nephrogenic diabetes insipidus, renal loss of K+, Mg2+, Fanconi syndrome, cystinuria

Histology

Imaging procedures

  • Glomerular diseases (autoimmune, diabetes, drugs, neoplasms)
  • Vascular diseases (atherosclerosis, hypertension, vasculitis, ischemia, thrombotic micro angiopathy)
  • Tubulo-interstitial diseases (urinary tract infections, kidney stones, urinary tract obstruction, drug-induced toxic)
  • Cystic and congenital diseases

Table 12.2-10 Annual decline of GFR in various populations /1/

Population

GFR1)

Annual decline1)

Healthy individuals

Approximately 0.5

Individuals with comorbidities2)

1–1.5

Diabetics > 65 yrs

45–59

1.5–2

Adults with CKD

51–58

1.0

Adults with CKD

< 60

2.7

Adults with CKD

25–55

(3.5)

Adults with CKD

13–45

(4.0)

Children

(4.3)

1) Data in [ml × min–1 × (1.73 m2)–1] 2) Example: hypertension, urinary tract infection: () approximate values

Table 12.2-11 Influencing variables in chronic kidney disease /1/

GFR*

Clinical and laboratory findings

< 60

In suspicion of cardiac disease, troponin and BNP/NT-proBNP should be cautiously assessed.

Potentially nephrotoxic drugs that are excreted renally (RAAS blockers, diuretics, NSAIDs, metformin, lithium, digoxin) may cause acute kidney failure in patients with severe intercurrent disease and should therefore be discontinued

In elective examinations, the intravenous administration of iodinated contrast media should be performed in agreement with the KDIGO Clinical Practice Guideline for Acute Kidney Injury

Phosphate-containing solutions for cleaning of the colon should not be administered orally.

30–44

The administration of metformin must be reconsidered.

< 30

In patients who require gadolinium-containing contrast agents, macro cyclic chelating substances should be employed with priority.

< 15

The use of gadolinium-containing contrast agents is not recommended, except if there is no alternative examination.

*[mL × min–1 × (1.73 m2)–1]

Table 12.3-1 Characteristic appearances of urine, modified according to Ref. /2/

Color

Cause

Colorless

Polyuria, dilute urine

Turbid, cloudy

Bicarbonate, phosphate, urate, leukocytes, bacteria, fungi, crystals, radiographic dye

Milky

Pyuria (infection), chyluria (lymphatic obstruction)

Blue-green

Biliverdin, pseudomonas infection, medications (arbutin, chlorophyll, creosote, indican, guaiacol, flavin, methylene blue)

Yellow

Flavines (vitamin B ingestion)

Yellow-orange

Concentrated urine, urobilin, bilirubin, rhubarb, senna, medications (phenacetin, pyridine derivatives, sulfasalazine, salazosulfapyridine)

Yellow-green

Bilirubin, biliverdin, riboflavin

Yellow brown

Bilirubin, biliverdin, nitrofurantoin

Red or brown

Hemoglobin, myoglobin, methemoglobin, bilifuscin, urobilin, porphyrin, beets, rhubarb, carotene, fuchsin, aniline derivatives, medications (amino phenazone, aminopyrine, antipyrine, bromsulphthalein, cascara, quinine, chloroquine, chrysarobin, hydro quinone, L-Dopa, naphthols, phenytoin, metronidazole, nitrite, nitrofurantoin, phenacetin, phenolphthalein, senna, phenothiazine, salazosulfapyridine, thymol)

Red-pink

Urate

Red-orange

Rifampicin

Red-purple

Porphyrins

Brown-black

Methemoglobin (blood in acidic urine), homogentisic acid (alcaptonuria), melanin

Darkening upon standing

Porphyrins, homogentisic acid, melanogen, serotonin, medications (cascara, chlorpromazine, methyl-dopa, metronidazole, phenacetin, imipenem)

Table 12.3-2 Characteristic odors of urine in metabolic diseases /2/

Odor

Metabolic disorder

Sweety feet

Isovaleric acidemia, glutaric acidemia

Maple syrup

Maple syrup urine disease

Cabbage, hops

Methionine malabsorption

Mousy

Phenylketonuria

Rotting fish

Trimethylaminuria

Rancid

Tyrosinemia

Table 12.3-3 Urine tests for evaluation of disorders of the kidney and the urinary tract

Clinical and laboratory findings

Test strips – Erythrocytes, hemoglobin, myoglobin

The test strip cannot provide a basic distinction between erythrocyturia, hemoglobinuria, and myoglobinuria /2/. The detection limit corresponds to the upper limit of the reference interval (10 erythrocytes/μL of urine), so that micro hematuria can also be detected. There is a distinction between micro hematuria (only visible through a microscope) and macro hematuria (visible to the naked eye).

Further investigation is important in the event of hematuria to determine whether it is renal or post renal. Proteinuria indicates a glomerular cause, as does the detection of erythrocyte casts or hemoglobin casts in the urine. It is possible to distinguish between renal and post renal hematuria based on the morphology of the erythrocytes present in the urine.

– Leukocytes

The esterase activity in neutrophils and histiocytes is measured, not the activity in lymphocytes, spermatozoa, or bacteria. Esterase released following the lysis of granulocytes can be measured even if no granulocytes are visible under the microscope. If first morning urine is tested, the analytical sensitivity is retained to the upper limit of the reference interval (10 leukocytes/μL of urine) /2/. Since leukocyturia is not always associated with a urinary tract infection and might also occur due to such factors as physical exertion and fever, a urine culture is always necessary. See also Section 12.8 – Erythrocytes, leukocytes, casts in urine.

– Protein

According to the UK Chronic Kidney Disease Guidelines /13/, proteinuria is present in non-diabetics:

  • With total protein excretion of ≥ 442 mg/g creatinine (50 mg/mmol creatinine)
  • With albumin excretion of ≥ 265 mg/g creatinine (30 mg/mmol creatinine) .

According to a study /14/, the test strips from a well-known manufacturer showed a reaction from 1+ with a median total protein excretion of 462 mg/g creatinine (52.3 mg/mmol creatinine) and an albumin excretion of 306 mg/g creatinine (34.6 mg/mmol creatinine).

Test strips are less sensitive for mucoproteins and low molecular weight proteins and are insensitive for free light chains /2/. However, the detection limit for albumin is too low for albumin excretion above 20 mg/L (micro albuminuria), as the early sign of incipient glomerular injury, in diabetic patients. Sensitive immunochemical test strips for albumin with a detection limit of 20 mg/L, are available. See also Section 12.9 – Urinary proteins.

– pH

Urine pH can range from 5–9, and reflects the intake of acids and bases in the diet. The concentrated morning urine is acidic. Meats in the diet cause an acid urine, plant-based foods are associated with an alkaline urine. Children’s urine is often alkaline. After lunch, urine is slightly alkaline, while after midnight it is acidic. Urea-cleaving bacteria cause alkaline urine via the formation of ammonia. The stability of leukocytes and casts is severely reduced at alkaline pH levels. Measuring urine pH can be an important tool in the investigation of acid-base balance disorders such as tubular acidosis, lithiasis, or of hypokalemic alkalosis with periodic aciduria /2/.

See also Section 8.8 – Renal electrolyte excretion.

– Nitrites

Many Gram-negative uropathogenic bacteria contain the enzyme nitrate reductase, and reduce nitrate to nitrite. Urine is normally free of nitrite. The detection of nitrite in freshly voided urine is evidence of a bacterial infection. Analytical sensitivity is 20–80% compared with the culture method. Diagnostic specificity is > 90% /2/. The nitrite test cannot replace a culture and microbe count, and constitutes a false negative result in the following circumstances:

  • In the absence of nitrate excretion, as is the case with premature infants, neonates, and individuals who do not excrete nitrate due to a lack of vegetables in their diet
  • Less than 105 CFU of bacteria per mL of urine
  • A very high bacterial count – in this case, nitrite is reduced to elemental nitrogen
  • An infection with bacteria that do not form nitrite from nitrate, such as staphylococci and enterococci.

Specific gravity

Specific gravity, also known as relative density or relative volume, depends on the concentration of electrolytes, glucose, phosphate, carbonate, and sometimes iodine-containing contrast media. In healthy individuals, specific gravity is often comparable with osmolality. This is not the case under pathological conditions.

Further information can be found in Section 8.5 – Osmolality.

Urine sediment

Urine sediment testing is used to detect particulate, mostly organic components such as erythrocytes, leukocytes, casts, and epithelial cells, but also substances such as hemosiderin. Testing must be performed on fresh, acidic, hypertonic morning urine, since it is in these samples that the spontaneous breakdown of the particulate components is most limited. See also Section 12.8 – Erythrocytes, leukocytes, casts in urine.

Total protein

The quantitative chemical determination of total protein is needed to diagnose proteinuria and to check the plausibility of urine protein results obtained by immunochemistry testing.

Albumin

The quantitative determination of urinary albumin in the form of the albumin/creatinine ratio or 24-hour urinary albumin excretion is, according to KDIGO, a criterion of chronic kidney disease and is recommended for the diagnosis of incipient kidney disease in diabetic and hypertensive patients. Albuminuria is often present years before the appearance of clinical symptoms or a fall in the glomerular filtration rate. Further information can be found in Section 12.9 – Urinary proteins.

Osmolality

Urine osmolality is dependent on the concentration of electrolytes, urea, and ammonia. The osmolality is an indicator for the diagnosis of impaired renal concentration ability, or if urine parameters have to be related to variable water excretion levels /2/.

Table 12.3-4 Interpretation of microbiological urine findings /12/

Sample type

CFU (mL)

Reporting/evaluation

Mid-stream urine

Below 103

In the absence of leukocyturia no urinary tract infection (UTI)

103 to 104

UTI in children and adults possible (if no mixed cultures are present, especially with typical uropathogenic bacteria)

≥ 104

Acute pyelonephritis in adults, particularly in the presence of leukocyturia as evidence of infection

104 to 105

Children: urinary tract infection (boys ≥ 104 CFU/mL, girls ≥ 105 CFU/mL)

≥ 105

Measurement of ≥ 105 CFU/mL in two consecutive samples in patients without clinical symptoms is suggestive of asymptomatic bacteriuria

Bladder puncture

Each CFU

All pathogenic bacteria should be considered as pathogens, irrespective of their number

Single use catheter urine

≥ 104

This bacterial count is considered to be proof of an infection

103 to 104

Suspicion of infection or contamination, repetition of the sampling

Indwelling catheter urine

≥ 104

If clinical symptoms are present, CFU ≥ 104/mL is considered as indication of an infection. Often mixed cultures are present, therefore careful interpretation and monitoring following change of catheter

In healthy premenopausal women with acute uncomplicated cystitis E. coli is the most pathogen but not Enterococcus fecalis or Group B streptococci, which are often isolated with E. coli /15/.

Table 12.4-1 Serum creatinine reference intervals

Children

IDGC-MS traceable method (values expressed as 2.5th and 97.5th percentiles) /8/

Umbilical cord blood

0.52–0.97 (46–86)

Premature 0–21 days

0.32–0.98 (28–87)

Neonate 0–14 days

0.30–1.00 (27–88)

2 months to < 1 yr

0.16–0.39 (16–39)

1 to < 3 yrs

0.17–0.35 (15–31)

3 to < 5 yrs

0.26–0.42 (23–37)

5 to < 7 yrs

0.29–0.48 (25–42)

7 to < 9 yrs

0.34–0.55 (30–48)

9 to < 11 yrs

0.32–0.64 (28–57)

11 to < 13 yrs

0.42–0.71 (37–63)

13 to < 15 yrs

0.46–0.81 (40–72)

Calculation via equations for children up to 14 years (girls and boys) determined using the enzymatic method /9/:

  • Lower limit: Cr (mg/dL) = 0.0199 × age (years) + 0.1504
  • Upper limit: Cr (mg/dL) = 0.0343 × age (years) + 0.3272

Adults

Jaffé method, non-compensated /10/

Women

≥ 18 yrs

0.61–1.12 (54–99)

18–49 yrs

0.58–1.05 (51–93)

50–79 yrs

0.61–1.12 (54–99)

Men

≥ 18 yrs

0.70–1.27 (62–112)

18–49 yrs

0.70–1.20 (62–106)

50–79 yrs

0.71–1.30 (63–115)

Jaffé method, compensated /10/

Women

≥ 18 yrs

0.45–1.00 (40–88)

18–49 yrs

0.40–0.94 (35–83)

50–79 yrs

0.51–1.00 (45–88)

Men

≥ 18 yrs

0.64–1.17 (57–103)

18–49 yrs

0.67–1.13 (59–100)

50–79 yrs

0.64–1.19 (57–105)

Enzymatic method /10/

Women

≥ 18 yrs

0.46–1.00 (41–88)

18–49 yrs

0.45–0.90 (40–80)

50–79 yrs

0.48–1.01 (42–89)

Men

≥ 18 yrs

0.57–1.18 (50–104)

18–49 yrs

0.57–1.11 (50–98)

50–79 yrs

0.58–1.23 (51–109)

Data expressed in mg/dL (μmol/L)

Conversion: mg/dL × 88.4 = μmol/L; mg/dL × 0.0884 = mmol/L

Table 12.4-2 Limitations of serum creatinine

A moderate impairment of the kidney function, GFR 40–80 [ml × min–1 × (1.73 m2)–1], is not detected (creatinine-blind range)

Interferences:

  • The serum concentrations depend on muscular mass, age, gender and meat consumption
  • The serum value decreases with malnutrition, liver cirrhosis and leg amputation
  • The serum concentration shows diurnal fluctuations (highest values in the early evening hours, lowest values in the morning hours)
  • The most frequently used determination method (alkaline picrate method) is subject to interfering factors (glucose, uric acid, ketone bodies, plasma proteins, cephalosporins)
  • The alkaline picrate method measures higher values than the enzymatic method.

Table 12.4-3 Variation in creatinine, cystatin C and MDRD values /11/

Parameter and value

Intra individual
variation (%)

Inter individual
variation (%)

Serum creatinine, 0.92 mg/dL (81 μmol/L)

5.8

211

Cystatin C (0.69 mg/L)

5.4

185

Creatinine clearance, 116 [mL × min–1× (1.73 m2)–1]

18.7

279

eGFR (MDRD) 82.4 [mL × min–1× (1.73 m2)–1]

6.7

202

Table 12.4-4 Diseases and conditions that can cause a change in serum creatinine

Clinical and laboratory findings

Age of The Individual

In spite of a decrease in GFR from, on the average 120 [mL × min–1 × (1.73 m2)–1] in young individuals, to 60–80 [mL × min–1 × (1.73 m2)–1] in the elderly, serum creatinine concentrations in elderly individuals do not increase, or increase only slightly, since the clearance and the formation of creatinine also decrease. The average decrease in GFR, from 50 years of age, is 5–10 [mL × min–1 × (1.73 m2)–1] every 10 years. In 80-year old individuals, the lower GFR reference interval value is approximately 50 mL. Because renal plasma flow falls as well at a similar rate, the filtration fraction remains about the same. An identical creatinine value in old and young individuals does not, therefore, signify the same GFR. In the elderly, serum creatinine is a poor indicator of GFR. Thus, in one study /15/, individuals of 67 ± 6 years had a GFR (inulin clearance) of 104 ± 12 [mL × min–1 × (1.73 m2)–1] and a creatinine clearance, estimated according to Cockroft and Gault, of 74 ± 18 mL/min. Individuals of the age of 25 ± 2 years had an inulin clearance of 119 ± 11 [mL × min–1 × (1.73 m2)–1] and a creatinine clearance, estimated according to Cockroft and Gault, of 114 ± 22 mL/min. If the lower reference interval of the GFR in young individuals was taken as the threshold value of normal kidney function, then only one out of 41 elderly individuals had a serum creatinine level above 1.15 mg/dL (104 μmol/L), but 12 had elevated serum cystatin C (i.e., a reduced GFR).

If, with the medication of renally excreted drugs, the GFR decreases and serum creatinine rises, the dose has to be reduced to a greater extent in elderly, as compared with young individuals, because the GFR is lower in the former group. For recommendations for dose adaptation, see Ref. /16/.

Pregnancy

In healthy pregnant women renal plasma flow at the end of the first trimester, measured using paraaminohippuric acid clearance, is increased by 80% and the GFR, measured using inulin clearance, is elevated by 50%. These values are maintained during pregnancy and normalize following delivery. Serum creatinine levels decrease by 10% and 30% during the first and third trimesters, respectively. Overall, and on the average, serum creatinine levels fall during the course of pregnancy, from prior to conception through the first and the third trimesters, as follows: 0.82 mg/dL (73 μmol/L), 0.73 mg/dL (65 μmol/L), 0.58 mg/dL (51 μmol/L), 0.53 mg/dL (47 μmol/L). A concentration of above 0.85 mg/dL (75 μmol/L) is considered to be an indication of incipient renal insufficiency. Formulas such as those of Cockroft and Gault or of the CKD-EPI should not be used for the estimation of GFR. A creatinine clearance test is also obsolete /17/.

Acute renal failure (ARF)

See also Section 12.1 – Clinical laboratory diagnosis of kidney and the urinary tract diseases. In ARF with a marked fall in GFR, a certain amount of time is required until the creatinine plateau, the level of which is a measure of the difference between creatinine formation and renal plus extrarenal elimination, is reached. Since in many acute diseases nutritional intake is low, and if elderly patients or patients with muscular weakness are involved, it is possible that in spite of significant impairment of the GFR, creatinine serum levels are increased only to a mild to moderate extent. The time required for serum creatinine to rise is dependent upon the half-life time of creatinine excretion. For a man weighing 75 kg with a GFR of 90 [mL × min–1 × (1.73 m2)–1] this value is 5.5 hours; it is twice that if the GFR is reduced by a factor of 2. Four half-life times are required (22 hours) for 94% of the equilibrium concentration to be reached, and it takes some 132 hours until the GFR is decreased to approximately 15 [mL × min–1 × (1.73 m2)–1]. In patients with acute damage (earthquake victims), who had previously normal creatinine values, it may take 48 hours or longer until a significant rise in creatinine up to the pathological range occurs.

While in ARF a rapid rise in creatinine, up to 2–3 mg/dL (177–265 μmol/L) daily, can occur as a consequence of rhabdomyolysis or the crush syndrome caused by other factors, the creatinine increase associated with renal insufficiency (e.g., due to shock) is usually slower. In renal insufficiency caused by multiple organ failure, a condition requiring dialysis is reached, on the average 9 ± 7 days following the acute event /18/. The transition from the oliguric to the poly uric phase of renal insufficiency does not signify a fall in serum creatinine; generally a further increase occurs, or the plateau value is maintained.

Chronic kidney disease (CKD)

Due to the hyperbolic relationship between GFR and serum creatinine level (Fig. 12.4-2 – Relationship between GFR and serum creatinine level), creatinine only becomes a useful indicator of a decreased GFR when it is markedly reduced. An elevation in creatinine is measured with a reduction in the GFR to 60–40 [mL × min–1 × (1.73 m2)–1]. A GFR impairment down to this value is therefore designated as the creatinine-blind range. Equilibrium conditions are a prerequisite for the diagnosis of CKD. The daily formation of creatinine, which is relatively constant and dependent to a considerable degree on the muscle mass, and its excretion must be in equilibrium. This is the case in individuals with normal renal function who are on a normal diet. If the GFR decreases, the renal elimination of creatinine also falls due to reduced creatinine filtration. If the formation of creatinine remains constant, this leads to a rise in serum creatinine. Ultimately, a new equilibrium is established, with reduced urinary creatinine excretion but with augmented serum value /12/. For the diagnosis of CKD, the creatinine concentration is a relatively weak indicator in comparison with the MDRD and the CKD-EPI equations, since in the equations those variables that influence serum creatinine level, such as age, gender and race, are taken into consideration. In impaired renal function there exists no direct inverse relationship between GFR and serum creatinine. This is attributable to:

  • The more marked renal tubular secretion of creatinine, particularly with a decrease in the GFR to 80–40 [mL × min–1 × (1.73 m2)–1]. In consequence, serum creatinine usually does not yet show an increase exceeding the reference interval, and creatinine clearance indicates a falsely high GFR. Conversely, in the recovery phase of ARF, an improvement in GFR due to decrease of creatinine secretion is reflected only insufficiently by serum creatinine. An improvement in the GFR of some 15 [mL × min–1 × (1.73 m2)–1] leads to a reduction in serum creatinine of some 0.2 mg/dL (18 μmol/L).
  • Extra-renal creatinine excretion which can be ignored if the GFR is normal. In CKD, however, up to 60% of the creatinine can be eliminated via extra-renal routes, particularly via the gastrointestinal tract.
  • A spontaneous reduction in protein intake if, in CKD, the GFR decreases into the range of 50–25 [mL × min–1 × (1.73 m2)–1]. Furthermore, the muscle mass decreases and serum creatinine is lower than what would be expected based upon the GFR impairment.

Chronic progressive renal insufficiency

The cause of GFR decrease is the reduction in the number of glomeruli, in renal blood flow, or in the clearance of the individual glomerulus. As a result, capillary blood flow and the perfusion of functional glomeruli are increased. In the GFR range of 80–40 [mL × min–1 × (1.73 m2)–1], serum creatinine hardly rises due to augmented renal tubular creatinine secretion. With a progressing decrease, serum creatinine is elevated but the absolute value is highly dependent upon individual factors such as muscle mass, reduced dietary meat, anorexia, or stimulation of the renin-angiotensin-aldosterone system /14/. All in all, in progressive CKD, serum creatinine levels do not accurately reflect the course of the progression. A rise is usually the sign of further impairment of the GFR, but a value that remains constant does not necessarily mean that kidney structure and function have stabilized.

Diabetes mellitus

The primary, noticeable morphological change in diabetic nephropathy is the increase in GFR and in kidney volume. In this early phase the structure of the hypertrophic glomeruli is normal. After several years a thickening of the basal membrane and an increase in the glomerular mesangial matrix develop. The result of the increase in glomerular matrix proteins is a reduction in basement membrane-associated heparan sulfate proteoglycan. This constitutes the negatively charged barrier of the glomerular basal membrane. Its reduction is associated with increased permeability to albumin /18/.

Micro albuminuria is the earliest indication of diabetic nephropathy. In this phase the GFR is normal or even elevated by 20–50% due to the renal hypertrophy and hyper perfusion. The serum creatinine levels in diabetic patients can vary over a wide range. Furthermore, marked hyperglycemia, osmotic diuresis, and the reduction in extracellular volume can cause the GFR to rise. In addition, falsely elevated creatinine values are determined in ketoacidosis with the Jaffe reaction, while falsely low values are obtained with the enzymatic creatinine imino hydrolase method /15/. During the course of micro albuminuria and its progression to clinical proteinuria, advanced chronic renal disease develops, with impaired GFR and high blood pressure. The determination of creatinine is inadequate for the timely diagnosis of this transition. More appropriate is the determination of cystatin C (see Section 12.7 – Cystatin C).

Drugs

Drugs can increase serum creatinine levels by causing a reduction in the GFR or an inhibition of renal tubular creatinine secretion. Drugs which inhibit tubular secretion usually do not have to be discontinued, because often the functional impairment is reversible.

Cimetidine: with normal kidney function, creatinine levels are increased by 15%, on the average, following oral therapy with 1.6 g/day, and by 20–30% a few hours following the intravenous administration of 0.8 g. In patients with chronic renal failure, a rise in creatinine of 22% occurs following 6–7 days of normal oral dosing /19/. The cause of the increase is cimetidine induced inhibition of tubular creatinine secretion. It is believed that cimetidine possesses a higher affinity than creatinine for the proximal tubule luminal cell membrane carrier. Ranitidine and famotidine, whose pharmacokinetic properties are similar to those of cimetidine, do not alter creatinine elimination.

Trimethoprim: on therapy with 160 mg of trimethoprim as monotherapy or in combination with 800 mg of sulphamethoxazol, 4 hours after the intake a reversible rise in serum creatinine of, on the average, 0.2 mg/dL (18 μmol/L), can occur. Trimethoprim competitively inhibits tubular creatinine secretion /19/.

Pyrimethamine: an increase in serum creatinine occurs in 20% of healthy individuals and in 27% of HIV-infected patients during mono therapy or combination therapy with dapsone. Tubular creatinine secretion is inhibited /19/.

Salicylates: they lead to an elevation in serum creatinine and to a reduction in creatinine clearance, with no decrease in the GFR. Thus aloxiprin, at a dose equivalent to 4 g of salicylate, leads to a 38% increase in serum creatinine and a 25% decrease in creatinine clearance. The inhibition of tubular creatinine secretion or altered protein binding of creatinine due to salicylate is assumed /19/.

Phenamacide: this anticonvulsive causes a reversible increase in serum creatinine.

Corticosteroids: increase in serum creatinine of some 10% in spite of an elevation of the GFR /19/.

Calcitriol, alfacalcidol: increase in serum creatinine and reduction in creatinine clearance.

Angiotensin converting enzyme (ACE) inhibitors: patients with pre-existing CKD and creatinine concentrations above 1.4 mg/dL (124 μmol/L) have a 55–75% reduction in disease progression risk in comparison to individuals with normal renal function. Following therapy initiation an acute decline in the GFR, along with a rise in creatinine concentrations of up to 30%, occurs /19/.

Table 12.5-1 Creatinine clearance

Clearance formula:

U × U vol × 1.73 S × t × A C [ml × min –1 × (1.73 m 2 ) –1 ] =

Cl = Clearance in mL per min.

U = Urinary creatinine concentration

S = Serum creatinine concentration, mean value from samples at the beginning and end of the collection period

Uvol = Quantity of urine per collection period

t = Collection period in min.

A = Body surface area of the patient, determined from nomogram or calculated

1.73 = Mean body surface area of persons weighing 75 kg in m2; the reference intervals are relative to this value.

Calculation of the body surface area (A):

A = 0.007184 (m/kg) × height (m) × 0.725 × weight (kg)0.425

Table 12.5-2 Creatinine clearance reference intervals

Adults, regardless of their age

  • Jaffé method /3/

95–160

98–156

  • Enzymatic /4/

+

Over 110*

Adults: Jaffé method, values are x ± s /5/

Years

20–29

95 ± 20

110 ± 21

30–39

103 ± 26

97 ± 36

40–49

81 ± 28

88 ± 20

50–59

74 ± 24

81 ± 19

60–69

63 ± 25

72 ± 21

70–79

54 ± 13

64 ± 15

80–89

46 ± 15

47 ± 15

90–99

39 ± 9

34 ± 9

Children: Jaffé reaction, values expressed as x ± 2s /6/

 

5–7 days

38–62

1–2 months

54–76

3–12 months

64–108

3–13 years

120–145

Data expressed in [mL × min–1 × (1.73 m2)–1]. * Examination only in a small population

Table 12.5-3 Relationship between the clearances of creatinine and inulin depending on the GFR /8/

GFR range

> 80 mL

80–40 mL

< 40 mL

ClInulin (x ± 2s)

113 ± 32

60 ± 7

22 ± 9

ClCr (x ± 2s)

134 ± 45

94 ± 23

42 ± 18

Difference
ClCr – ClInulin

21

34

20

Ratio
ClCr/ClInulin

1.16

1.57

1.92

ClInulin, glomerular filtration rate, determined with inulin clearance in [mL × min–1 × (1.73 m2)–1]; CCr, creatinine clearance; ClCr – ClInulin, erroneous rise in the GFR with creatinine clearance.

Table 12.5-4 Creatinine excretion in adults

Age (yrs)

Urinary creatinine
(mg/kg/24 h)

20–29

23.8 ± 2.3

30–39

21.9 ± 1.5

40–49

19.7 ± 3.2

50–59

19.3 ± 2.9

60–69

16.9 ± 2.9

70–79

14.2 ± 3.0

80–89

11.7 ± 4.0

90–99

9.4 ± 3.2

Values expressed as x ± s

Table 12.6-1 Urease UV method

Urea + H 2 O Urease 2 NH 3 + CO 2 2.2 oxoglutarate + 2 NADH + 2 NH 4 + GLDH 2 L-glutamate + 2 NAD + + 2 H 2 O

Table 12.6-2 Reference intervals for urea and (urea-N = BUN)

Adults /4/

Generally

17–43 (2.8–7.2)

< 50 yrs

15–40 (2.6–6.7)

> 50 yrs

21–43 (3.5–7.2)

< 50 yrs

19–44 (3.2–7.3)

> 50 yrs

18–55 (3.0–9.2)

Children /5/

1–3 yrs

11–36 (1.8–6.0)

4–13 yrs

15–36 (2.5–6.0)

14–19yrs

18–45 (2.9–7.5)

Data expressed in mg/dL (mmol/L)

Urea (mg/dL)/2.14 = BUN (mg/dL)

Conversion:

– Urea: mg/dL × 0.1665 = mmol/L; mmol/L × 6.006 = mg/dL

– BUN: mg/dL × 0.3561 = mmol/L; mmol/L × 2.808 = mg/dL

Table 12.6-3 Serum urea/creatinine ratios observed in various diseases and conditions /12/

Urea/creatinine ratio

Clinical and laboratory findings

25–401)

20–352)

10–163)

Normal urea/creatinine ratios with normal diets and no significant decline in the GFR.

< 251)

< 202)

< 103)

Decrease in the urea/creatinine ratio due to a decline in urea because of:

  • Reduced protein catabolism (e.g., low protein intake, undernourishment, cachexia, liver cirrhosis)
  • Decreased tubular rediffusion of urea (e.g., in acute tubular necrosis, diuresis).

Pre renal azotemia

> 401)

> 352)

> 163)

Increase in the urea/creatinine ratio due to an increase in urea because of:

  • Increased protein catabolism (e.g., high protein intake, tissue necrosis, burns, fever, high-dose glucocorticoid therapy, starvation, gastro-intestinal bleeding)
  • Reduced tubular perfusion and, in consequence, increased rediffusion of urea (e.g., in hypovolemia, severe heart failure, exsiccosis)
  • In pre renal azotemia serum creatinine is normal.

Post renal azotemia

Increase in both urea and creatinine, but the increase in urea is more pronounced, hence the increase in the urea/creatinine ratio. The cause is obstruction of the ureter, the bladder or the urethra. The more marked increase in urea is due to the tubular rediffusion of urea, as a consequence of the back pressure of urine.

1) Urea and creatinine in mmol/L; 2) Urea and creatinine in mg/dl; 3) BUN and creatinine in mg/dL

Table 12.6-4 Diseases and conditions that cause elevated serum urea /13/

Clinical and laboratory findings

Acute renal insufficiency, chronic kidney disease

Like creatinine, the serum urea concentration is also reversely related to the GFR. However, with normal protein intake and normal renal perfusion, values above the reference interval are only found until the GFR declines to approximately 30 [mL × min–1 × (1.73 m2)–1]. In chronic kidney disease and a GFR below 30 [mL × min–1 × (1.73 m2)–1] the urea is an important biomarker for monitoring the success of a protein-reduced diet (this also applies to the urinary excretion of urea).

Pre renal azotemia

In the case of hemorrhage, frequent vomiting, diarrhea, burns or insufficient fluid intake, the rise in urea is caused by the reduced extracellular fluid volume. As a result of this, renal perfusion is diminished and the tubular rediffusion of urea is increased because of underlying hypovolemia combined with electrolyte deficiency.

Post renal azotemia

The post renal azotemia is caused by obstruction of the ureters, the bladder or the urethra (e.g., due to prostatitis, urolithiasis or tumors). The urea in serum increases more than creatinine.

High intake of a protein-rich diet

With a high intake of protein (> 200 g/day), the serum urea can rise to values up to 80 mg/dL (13.3 mmol/L). This is especially the case if insufficient fluid intake, profuse sweating or alcohol-related polyuria are also present. Under such circumstances it can be useful to determine the creatinine clearance, if no other clinical method is successful.

Indicator of postoperative mortality /11/

The pre- and postoperative levels of serum urea are indicators of mortality within the postoperative 30 days for patients in abdominal surgery. Thus, the odds ratio for mortality in patients with pre- and postoperative urea values ≥ 48 mg/dL (8.0 mmol/L) was 2.29 and 4.79 respectively, in comparison to those with lower concentrations. The cause of the increased mortality is assumed to be an occult hypovolemia with renal hypo perfusion and reduced plasma clearance.

Table 12.7-1 Reference intervals for cystatin C

PETIA

Pre-term
delivery* /6/

1.8 (1.2–2.1)
only 14 participants

Children

1–18 yrs /7/

0.70–1.38

Adults

< 50 yrs /8/

0.79–1.05

≥ 50 yrs /8/

0.88–1.34

< 50 yrs /8/

0.75–0.99

≥ 50 yrs /8/

0.85–1.35

Data expressed in mg/L. Values are 2.5th and 97.5th percentiles.

PENIA

Children

3rd day /9/

0.72–1.98

1–18 months /10/

0.70–1.18

18 mos – 18 yrs /10/

0.44–0.94

Adults

< 50 yrs /11/

0.53–0.92

≥ 50 yrs /11/

0.58–1.02

Data expressed in mg/L. Values are 2.5th and 97.5 th percentiles. * Only 14 individuals were investigated.

Table 12.7-2 Diagnostic significance of cystatin C in impaired renal function

Clinical and laboratory findings

Chronic kidney disease (CKD)

In patients with CKD, there is a good correlation between the CKD stage and the increase in cystatin C, particularly in children and in elderly patients. The rise in cystatin C occurs earlier than that of creatinine. Thus in moderate impairment of eGFRcreat to 70–51 [mL × min–1 × (1.73 m2)–1] cystatin C values, but not those of serum creatinine, were generally increased /2730/.

In paraplegic and tetraplegic patients in whom, because of the reduced muscle mass, neither serum creatinine nor the CKD-EPI equation are indicators of renal function, cystatin C, in comparison with 51Cr-EDTA clearance proves to be an acceptable marker for the assessment of the GFR. The correlation coefficient between 51Cr-EDTA clearance and 1/cystatin C was 0.72 and that between 51Cr-EDTA clearance and 1/creatinine was only 0.26 /32/.

Acute renal failure (ARF) (acute kidney injury, AKI)

In patients who develop ARF according to RIFLE criteria (see Section 12.1 – Clinical laboratory diagnosis of kidney and the urinary tract diseases), the increase in cystatin C occurs 1–2 days earlier than does that of creatinine /33/.

Follow-up of kidney transplantation

Following kidney transplantation, there exists the risk of rejection episodes or of toxic transplant damage due to immunosuppressive agents. In the evaluation of GFR in the postoperative follow-up, cystatin C decreases more quickly than serum creatinine; this is believed to be due to tubular reabsorption of creatinine. Over time the creatinine/cystatin C ratio declines and stabilizes if complications do not occur. If acute impairment of kidney function develops, however, the ratio increases quickly and significantly. In kidney transplant recipients serum creatinine is 30% and creatinine clearance is 40% too high in comparison with mGFR; cystatin C is 14–25% too low /34/.

Of all serum markers cystatin C is the most reliable to estimate or measure GFR in immunosuppressive regimens in children after kidney transplantation. In children aged 2.0–17.1 years, the correlation between mGFR (125I-iothalamate clearance) and 1/cystatin C was 0.85, between mGFR and the Schwartz formula it was 0.66 and between mGFR and 1/creatinine it was only 0.49 /35/. Transplant recipients who receive high doses of glucocorticoids (500 mg methylprednisolone) have higher cystatin C levels than those on glucocorticoid-free immunosuppression. Dose-dependent glucocorticoid-induced cystatin C production is attributed to a promoter-mediated increase in the transcription of the cystatin C gene.

Absolute quantification of donor derived cell free DNA (dd-cfDNA) is a marker of rejection and graft injury in kidney transplantation /18/.

Liver transplant recipients

After 5 and 10 years 28% and 26%, respectively, of patients with liver transplantation develop CKD. This is because renal function is substantially affected by continuous immunosuppressive therapy.

In patients with mGFR of < 60 [mL× min–1 × (1.73 m2)–1, the eGFRcys and the MDRD equation show a satisfactory correlation with the mGFR /36/. According to other authors, the determination of serum creatinine is too insensitive for the assessment of changes in the GFR during the medication with cyclosporine and FK506. Liver transplant recipients often have reduced muscle mass. Thus, post-operative patients with an iohexol clearance of 60–80 [mL × min–1 × (1.73 m2)–1] had elevated cystatin C, but no elevated serum creatinine /37/. Cystatin C concentrations measured with PENIA of 1.4, 1.7 and 2.2 mg/L were predictive of GFR values of, respectively, 80, 60 and 40 [mL × min–1 × (1.73 m2)–1]. In comparison with serum creatinine and creatinine clearance, cystatin C was the more reliable marker of renal function.

For determining eGFR in recently transplanted liver recipients, a cystatin C based equation was developed, since the evaluation of kidney function using serum creatinine or eGFRcr is not reliable. There was a good correlation with mGFR, using 99mTc-DTPA /38/. The equation is as follows:

eGFR [mL × min–1 × (1.73 m2)–1 = 19.12 + 96.21 × 1/cystatin C (mg/L)

Chemotherapy of Malignant Disease

During treatment with chemotherapeutic agents oncologists must monitor renal function, because alteration in GFR may lead to impaired metabolism and accumulation of chemotherapeutic agents and their metabolites. Some chemotherapeutic agents are nephrotoxic and may cause dose-related tubular cell necrosis.

Cisplatin therapy: cisplatin is a potent chemotherapeutic agent which is used in the treatment of germ cell tumors, lung carcinoma, cervical carcinoma and head and neck tumors. The nephrotoxicity of cisplatin is dose-related. High doses of cisplatin cause necrosis of entire cell layers of the proximal tubule, while lower dosing leads to apoptosis. In order for its nephrotoxicity to be expressed, cisplatin must be converted to a toxic metabolite in the kidneys. The activation begins with the conjugation of cisplatin and glutathione. The glutathione conjugate is then metabolized to a cysteinyl-glycine conjugate and transformed, via a cysteine-conjugate, to a reactive thiol. The formation of the cisplatin-glutathione conjugate is the essential step in the formation of nephrotoxins /39/. With cisplatin therapy, creatinine clearance should not fall to below 60 [mL × min–1 × (1.73 m2)–1]. If this occurs, the dosage must be halved /40/. A decrease in the GFR with cisplatin therapy can be better detected by means of cystatin C, rather than serum creatinine determination. The determination of creatinine clearance is not necessary as long as the concentration of cystatin C is ≤ 1.33 mg/L /41/. According to our own investigations, the dosage should only be halved if the cystatin C concentration, as measured with PENIA (upper limit of the reference range 1.0 mg/L), is above 1.7 mg/L.

Rheumatoid arthritis (RA)

NSAIDs, which are usually used in long-term treatment of RA, cause a decrease in renal blood flow, prostaglandin production, glomerular filtration, and Na+ excretion, and they have been considered to induce interstitial nephritis. In a study /42/ in patients with RA affected for more than 5 years and treated with NSAIDs for more than 50 months, 57% showed a reduction in creatinine clearance, 60% had elevated cystatin C, but only 5% manifested increased serum creatinine. Due to the ingestion of NSAIDs, patients with prolonged RA represent a risk group for subclinical renal dysfunction, consistent with the early stages of analgesic nephropathy /43/.

Diabetes mellitus

Diabetes mellitus type 1: renal failure develops in ≤ 30% of patients with type 1 diabetes, however, the ability to assess renal function is poor, when active management is important. Serum creatinine level, the most commonly used surrogate measure of GFR does not increase until renal function decreases to about 50% of its normal value. In a study /44/, cystatin C proved to be more reliable than creatinine clearance for the detection of functional impairment. For the calculation of the GFR in these patients by means of cystatin C, the following equation is recommended:

GFR [mL × min–1 × (1.73 m2)–1] = 87.1 × 1/cystatin C – 6.9. (determined with PENIA)

Diabetes mellitus type 2: an increasing number of these diabetics lives longer and can thereby suffer from diabetic nephropathy or even end-stage renal failure. One reason is the improved treatment of diabetes and its sequelae such as hypertension and cardiovascular disease. The efficiency of the treatment is assessed based on urinary albumin excretion and the GFR (see also Section 12.2 – Glomerular filtration rate). In a study /45/ in diabetic patients with a 51Cr-EDTA clearance of 120–20 [mL × min–1 × (1.73 m2)–1], the diagnostic values of cystatin C, serum creatinine and the Cockroft-Gault formula were compared. With areas under the curve (AUC) of 0.954 (cystatin C), 0.812 (serum creatinine) and 0.873 (Cockroft-Gault), cystatin C proved to be the better marker for the identification of renal functional impairment. For the assessment of kidney function in diabetics, the CKD-EPI equation for the estimation of eGFR is more suitable than the MDRD equation, since a GFR above 60 [mL × min–1 × (1.73 m2)–1] is detected more reliably /46/.

Liver cirrhosis

Cirrhosis of the liver is often accompanied by functional renal failure. The reasons are hemodynamic alterations, mainly a peripheral vasodilation followed by activation of vasoconstrictive hormones and neurohumoral systems such as renin-aldosterone, vasopressin, endothelin and increased activity of the sympathetic nervous system. These alterations induce renal retention of Na+ and water and a decrease in GFR. The renal impairments are functional in nature and are not accompanied by morphological changes in the early stages and can be reversed by medical intervention /47/. The extreme stage of this renal failure however, the hepatorenal syndrome, is rarely reversible. Liver cirrhosis patients with renal function impairment are particular sensitive to decreases in plasma volume, which in turn further impairs the GFR. The early detection of renal impairment is important to counteract a further decline in GFR by means of appropriate measures (e.g., volume expansion). For the early detection of a reduced GFR /48/:

  • The determination of serum creatinine using the conventional upper reference interval values which, depending upon methodology and gender, are 0.9–1.1 mg/dL (80–97 μmol/L), is inappropriate. This is because the serum creatinine level is low due to reduced creatine metabolism. In addition, many of these patients have a reduced muscular mass.

The CKD-EPI equation is inapplicable since, because of the low serum creatinine and if ascites and edema are present, the GFR determination will provide falsely high values.

Various studies provided the following evaluations of cystatin C as a marker of CKD in liver cirrhosis patients:

  • Cystatin C was better suited than serum creatinine for the diagnosis of a decrease in GFR. With upper reference values of 1.0 mg/L (cystatin C) and 0.9 mg/dL (80 μmol/L) (creatinine), the diagnostic sensitivities were 69% and 45% and in women even 78% and 39% respectively /47/.
  • Based upon an inulin clearance of < 72 [mL × min–1 × (1.73 m2)–1] as indicator of impaired renal function, cystatin C showed a diagnostic sensitivity of 81%, serum creatinine of 23%, and the Cockroft-Gault equation of 53%. In spite of a falsely high GFR determination, creatinine clearance was considered to be equivalent to cystatin C /48/.
  • In relation to a creatinine clearance < 80 [mL × min–1 × (1.73 m2)–1], cystatin C showed a higher diagnostic accuracy than serum creatinine for the detection of functional renal impairment /49/. If an inulin clearance ≥ 90 [mL × min–1 × (1.73 m2)–1] was selected as normal, 42 out of 44 liver cirrhosis patients had a reduced GFR. The diagnostic sensitivity of cystatin C was 86%, while that of serum creatinine was 29%. Cystatin C was significantly higher in patients with Child B and Child C cirrhosis, while there were no significant differences between Child A and Child B /50/.

Post-transplantation lymphoproliferative disease (PTLD)

PTLD is the result of a monoclonal lymphoproliferative disorder and occurs in the post-organ transplant course. The incidence rates following kidney and lung transplantation are 1% and 8%, respectively. The incidence is directly proportional to the cumulative intensity of the immunosuppression. Many cases are caused by an Epstein-Barr virus infection. Since the mononuclear cells have a high production rate of β2-microglobulin (β2-M), the determination of β2-M is well suited to the diagnosis and therapeutic assessment of lymphoproliferative diseases. In order to evaluate the lymphoproliferation independently of the GFR, a correction via cystatin C using the ratio (β2-M/cystatin C) ratio was recommended /6/. Up to a GFR ≥ 40 [mL × min–1 × (1.73 m2)–1], which corresponds to a cystatin C value of 2.2 mg/L (PENIA upper reference interval value 1.0 mg/L), there is a close correlation between cystatin C and β2-M /51/. The ratio β2-M/cystatin C in the reference population was constant at 1.2–2.4. In all PTLD patients, apart from one, the values were in the range of 2.7–3.7. The ratio β2-M/cystatin C is recommended as a diagnostic marker for lymphoproliferative disorders in patients with mild GFR impairment /51/.

Cardiovascular disease (CVD) risk and mortality /1/

The eGFR is an independent risk factor for CVD and a predictor of myocardial infarction and cardiovascular death. The eGFR can be determined by means of creatinine and cystatin C.

Cystatin C and cardiovascular risk

  • The Framingham Offspring Prospective Cohort Study showed that in individuals with an eGFR < 60 [mL × min–1 × (1.73 m2)–1] a cystatin C level > 1.07 mg/L was associated with CVD, independently of risk factors such as age, female gender, body mass index, low HDL-cholesterol, and smoking. Patients with elevated cystatin C had a higher prevalence of obesity and hypertension than those with CKD and normal cystatin C /52/.
  • These results were confirmed in the Third National Health and Nutrition Examination Survey. The results also showed that in the absence of CKD stages 3 and 4, cystatin C levels > 1.00 mg/L (PENIA) were associated with an increased prevalence of serum uric acid, phosphate, homocysteine and CRP compared to a reference population with cystatin C levels ≤ 1.00 mg/L /3/.
  • The Multi-Ethnic Study of Atherosclerosis illustrated the association between cystatin C and developing hypertension /53/. For each 0.2 mg/L rise in cystatin C, the incidence of hypertension increased by 15%, even in individuals with an eGFR ≥ 90 [mL × min–1 × (1.73 m2)–1].
  • In the Cardiovascular Health Study /54/ the risk of cardiovascular events and of mortality as a function of the cystatin C value was investigated in individuals over the age of 65. The cystatin C (PENIA) quintiles in mg/L were: ≤ 0.89; 0.90–0.99; 1.00–1.10; 1.11–1.28, and ≥ 1.29. The hazard ratios for overall mortality were: 1.00; 1.08; 1.23; 1.34, and 2.18. The hazard ratios for cystatin C values ≥ 1.29 were 1.48 for acute myocardial infarction, 1.47 for stroke, and 2.27 for cardiovascular mortality of all causes.
  • In the Cardiovascular Health Study, the risk factors for development and progression of CVD were investigated in individuals over the age of 65 with and without previous heart failure /55/. The development of cardiac events within 8.3 years increased step-by-step as a function of the cystatin C level. With PENIA values of 1.00–1.09 mg/L, 1.10–1.25 mg/L and 1.26–6.75 mg/L, the hazard ratios were 1.44, 1.58 and 2.16. Serum creatinine did not exhibit such a behavior, indicating that elevated cystatin C is a marker for cardiovascular events.

Cystatin C in acute coronary syndrome (ACS)

Cystatin C independently predicts cardiovascular death or myocardial infarction. In the PLATO study the hazard ratio with cystatin C values ≥ 1.01 mg/L was 1.66. A better predictor was an eGFR ≤ 60.3 [mL × min–1 × (1.73 m2)–1] as determined with the creatinine-based CKD-EPI equation (eGFRcr-cys/56/. In the PLATO study, the eGFRcr-cys was also investigated in patients with acute coronary syndrome. The equation did not, however, improve the risk prognosis in comparison with basal cystatin C. The eGFRcr-cys showed the best predictive value for cardiac mortality /57/.

Obesity /1/

Obese individuals have higher cystatin C values than persons of normal body weight. Important determinants of the cystatin C value are body fat (> 30%) and the GFR.

Fenofibrate /59/

Fenofibrate treatment for hyperlipidemia increases cystatin C and creatinine concentrations by 9.9% and 15.1%, respectively.

Table 12.8-1 Reference intervals for erythrocytes, leukocytes and casts in urine

Test strips

Practical sensitivity

Erythrocytes /4/

LD: 10 × 106/L

LC: 50 × 106/L

Free hemoglobin

150–300 μg/L

Myoglobin

500 μg/L

Leukocytes /4/

LD: 20 × 106/L

LC: 100 × 106/L

Quantitative cell count

Counting chamber method*

  • Erythrocytes

≤ 13 × 106/L
(2–16 yrs) /10/

≤ 8 × 106/L (Adults) /11/

  • Leukocytes

≤ 4 × 106/L
(2–16 yrs) /10/

≤ 8 × 106/L (Adults) /11/

Standardized urine sediment under cover slip**

  • Erythrocytes

≤ 4 (5)***/view field

  • Dysmorphic erythrocytes

≤ 2/view field

  • Leukocytes

≤ 4 (10)***/view field

  • Cast

Only hyaline

Dysmorphic erythrocytes

  • Phase contrast

≤ 30% /12/

  • Immunocytochemistry

≤ 60% /13/

  • Acanthocytes

≤ 5% /4/

* Values are the 95th percentiles.

** Empirical values at a 400-fold magnification.

*** Some investigators.

LD, limit of detection

LC, positive result (sensitivity > 90%), see also Section 12.3.3.1 – Multiple test strips (dipsticks)

Table 12.8-2 Causes of hematuria /17/

Glomerular

  • Primary glomerulonephritis (GN): IgA nephropathy, post-infectious GN, membranoproliferative GN, progressive GN, focal glomerulosclerosis
  • Secondary glomerulonephritis: systemic lupus nephritis, vasculitis, essential cyroglobulinemia, hemolytic uremic syndrome, thrombotic thrombocytopenic purpura
  • Familial: thin basement membrane disease, Alport’s syndrome, Fabry disease, Nail-patella syndrome

Non-glomerular

  • Renal parenchymal: renal tumors, vascular (malignant hypertension, sickle cell disease, flank pain hematuria syndrome, arteriovenous malformation), metabolic (hyper calcuria, hyperuricemia), familial (polycystic kidney disease, sponge kidney), infections (pyelonephritis, tuberculosis)
  • Extrarenal: tumors (renal pelvis, ureter, bladder, prostate), benign prostatic hyperplasia, kidney stones, infections (cystitis, prostatitis, schistosoma, tuberculosis)
  • Miscellaneous: medications (heparin, warfarin, acetylsalicylic acid, ticlopidine, cyclophosphamide), systemic bleeding disorders, trauma (boxing, football, long distance running), fever, dehydration

Table 12.8-3 Diagnostic significance of urinary casts

Diagnostic significance

Hyaline casts

Hyaline casts are found in healthy individuals and in renal parenchymatous diseases. However, it is normal for the numbers of hyaline casts being excreted by healthy individuals to increase during physical exertion, during fever, and in the event of heart failure.

Granular casts

Granular casts are excreted by healthy individuals and renal disease patients, particularly in the event of proteinuria. Hyaline casts are about three times more common in healthy individuals than are granular casts. Granular casts are found in all patients with renal disease and urinary casts. The granularity of these casts is the result of the degeneration and lysis of epithelial and blood cells, as well as of proteins.

Waxy casts

The surface of these casts consists of amorphous material. Waxy casts are wide, transparent, and have sharp contours. They are present during chronic renal failure and in the poly uric phase of acute renal failure.

Fatty casts

Fatty casts are probably formed from degenerated tubular cells. They are excreted in the event of nephrotic syndrome and in severe cases of proteinuria.

Epithelial cell casts

Epithelial casts come from the epithelial cells lining the nephron. These adhere to a hyaline cast matrix. They indicate an increased elimination of tubular epithelial cells, which occurs under circumstances such as acute tubular necrosis, acute interstitial nephritis, renal transplant rejection, or the reparation phase of acute renal failure.

Erythrocyte casts

In these casts, erythrocytes are not only embedded in the matrix, but also adhere to the surface of hyaline casts. Erythrocyte casts are a sure sign of a renal parenchymatous disorder, and are usually indicative of glomerulopathy. Erythrocyte casts are evident in only about 40% of glomerulonephritides.

Hemoglobin casts

Hemoglobin casts are brownish in color and have a granular surface. They can either originate from erythrocyte casts (in which case they are an indicator of renal bleeding), or they are the result of hemoglobinuria associated with intravascular hemolysis.

Granulocyte casts

The matrix of these casts is hyaline in nature, with granulocytes and lymphocytes attaching to the surface via fibrillar fibers. Granulocyte casts are found in inflammatory renal disorders. These can be bacterial in nature such as in pyelonephritis, or non-bacterial such as in interstitial nephritis and proliferative glomerulonephritis.

Bacterial casts

These casts occur either as a mixture of leukocyte and bacterial casts, or in rare cases as purely bacterial casts. They are frequently wrongly classified as granular casts. Bacterial casts can be excreted in cases of pyelonephritis.

Table 12.8-4 Diagnosis and definition of microhematuria

Guideline statements /35/:

  • Clinicians should define microhematuria as ≥ 3 red blood cells per high power field on microscopic evaluation of a single urine specimen.
  • Clinicians should not define microhematuria by positive dipstick testing alone. A positive urine dipstick test (trace blood or greater) should prompt formal microscopic evaluation of the urine.
  • In patients with microhematuria, clinicians should perform a history and physical examination to assess risk factors for genitourinary malignancy, medical renal disease, gynecologic and non-malignant genitourinary causes of microhematuria.
  • Clinicians should perform the same evaluation of patients with microhematuria who are taking antiplatelet agents.
  • In patients with findings suggestive of a gynecologic or non-malignant urologic etiology clinicians should evaluate patients with appropriate physical examination techniques and tests to identify such an etiology.
  • In patients diagnosed with gynecologic or non-malignant genitourinary sources of microhematuria, clinicians should repeat urinalysis following resolution of the gynecologic or non-malignant cause.
  • In patients with hematuria attributed to a urinary tract infection clinicians should obtain a urinalysis with microscopic evaluation following treatment to ensure resolution of the hematuria.
  • Clinicians should refer patients with microhematuria for nephrologic evaluation if medical renal disease is suspected.
  • In low risk patients with microhematuria repeating urinanalysis within six months is recommended.
  • In patients with intermediate risk clinicians should perform cystoscopy and renal ultrasound in patients with microhematuria.
  • Follow up in patients with a negative hematuria evaluation: clinicians may obtain repeat urinalysis within 12 months.
  • For patients with a prior negative hematuria evaluation who develop gross hematuria, significant increase in degree of microhematuria, or new urologic symptoms, clinicians should initiate further evaluation. Women with hematuria have been especially prone to delays in evaluation, often due to practitioners ascribing hematuria to a urinary tract infection or gynecologic source, resulting in inadequate evaluation and delay in cancer diagnostics.

Table 12.9-1 Definition of albuminuria according to the American Diabetes Association /20/ and NICE /21/

Albuminuria

μg/min1)

mg/24 h2)

mg/L3)

mg/g
creatinine4)

mg/mmol
creatinine

Normal

< 20

< 30

< 30

< 30

≤ 2.5
≤ 3.5

Micro albuminuria

20–200

30–300

30–300

30–300

> 2.5
> 3.5

Macro albuminuria

> 200

> 300

> 300

> 300

≥ 30
≥ 30

1) Timed urine collection (e.g. overnight or from 8–10 a.m.); 2) 24-hour urine collection; 3) second morning urine, not recommended at present; 4) first morning urine

Table 12.9-2 Functional, transient and isolated proteinuria

Clinical and laboratory findings

Functional/transient proteinuria

Functional proteinuria is typically transient in nature and disappears if the cause abates or disappears. The degree of the proteinuria rarely exceeds a 2+ reaction on the test strip, while total protein excretion is less than 1 g/24 h. Transient proteinuria associated with fever, congestive heart failure, and physical exertion is the result of hemodynamic circulatory changes. These cause increased permeability in the glomerular basement membrane.

Fever: febrile proteinuria appears when the fever develops and generally lasts 10–14 days, even if the fever ends earlier. Subsequent episodes of fever within a week or so after the initial fever are not usually accompanied by proteinuria. In one study of 196 children with fever, proteinuria occurred in 6% of the cases /51/.

Physical exertion: proteinuria and hematuria can occur after physical exertion. The degree of proteinuria and hematuria depend on the level and duration of the exertion. In most cases, there is an increase in the ratio UProtein/UCreatinine. The proteinuria generally disappears 48 hours after the end of the physical exertion. In the event of acute, severe short-term exertion, high-molecular weight proteins such as IgG, albumin, and transferrin are excreted in larger quantities than the lower-molecular weight proteins, which is indicative of increased glomerular permeability /52/. On the other hand, proteinuria does not increase so sharply during prolonged physical exertion, and mostly albuminuria is evident. Thus, in 16 healthy individuals who completed a 100 km hill walk, the total protein levels rose by an average of 2.3 g/mol creatinine to 3.7 g/mol creatinine, and albumin rose on average by 0.85 g/mol creatinine to 1.61 g/mol creatinine /53/.

Orthostatic proteinuria /14/

Healthy individuals with normal protein excretion experience an increase in protein excretion when standing. Patients with proteinuria also experience increased protein excretion when standing. Orthostatic proteinuria is defined as an elevated protein excretion that occurs only when an individual is standing. This form of proteinuria usually occurs in individuals under 30, and accounts for 60% of cases of childhood proteinuria. This proportion is even higher among adolescents. In children under 16 years, the incidence of orthostatic proteinuria is higher in girls than in boys. In some individuals, orthostatic proteinuria is stable and reproducible, while in others it is only transient or intermittent. Orthostatic proteinuria is usually associated with total protein excretion of no more than 1 g/24 h. For confirmation, a 12 hour daytime urine specimen and a 12 hour night-time specimen (when the patient is inactive) should be collected. The protein excretion in night-time specimens from children should be less than 4 mg/m2/h, and from adults, less than 100–150 mg of protein. The protein excretion in daytime urine specimens should be 2–4 times higher than the excretion in the night-time specimens to confirm orthostatic proteinuria. If the protein excretion from specimens taken while the individual is at rest exceed 200 mg, or if the excretion from the night-time specimen is the same as the daytime excretion, the proteinuria is not orthostatic. This is also the case if the total protein excretion rate is over 1 g/24 h.

Persistent asymptomatic proteinuria

Isolated proteinuria is defined as persistent proteinuria in an otherwise healthy individual. This mostly affects children with no evidence of renal disease, either from clinical examination or from laboratory testing. A constant, persistent protein pattern cannot always be found. Therefore, proteinuria is considered persistent asymptomatic proteinuria if 80% or more of urine specimens are positive for proteinuria. Studies show that persistent asymptomatic proteinuria in children and adolescents is not associated with progressive renal disease /54/. In other studies, renal biopsies reveal significant glomerulopathies such as focal glomerulosclerosis, IgA nephropathy, membranous glomerulopathy and mesangial proliferative glomerulonephritis /55/. The following criteria point to nephropathy in cases of persistent non-orthostatic proteinuria in childhood and therefore indicate the need for further invasive diagnostic testing on the kidneys: congenital proteinuria, EPH gestosis in the mother, family history of glomerulonephritis or chronic renal failure.

Table 12.9-3 Etiology of proteinuria

Functional/transient

  • Fever
  • Severe physical exertion
  • Cold exposure
  • Congestive heart failure
  • Seizures
  • Emotional stress

Isolated proteinuria

  • Orthostatic proteinuria
  • Persistent asymptomatic proteinuria

Selective glomerular proteinuria

  • Minimal change glomerulopathy
  • Membranous glomerulonephritis, grade I
  • Focal segmental glomerulonephritis, stage I
  • IgA nephritis
  • Early stage diabetic nephropathy

Non-selective glomerular proteinuria

  • Rapidly progressing glomerulonephritis
  • Proliferative glomerulonephritis (vasculitis)
  • Membranoproliferative glomerulonephritis
  • Membranous glomerulonephritis, grades II and III
  • Focal segmental glomerulonephritis, grades II and III
  • Diabetic nephropathy, stages III and IV
  • Arterial hypertension, benign nephrosclerosis
  • Pre-eclampsia

Non-selective glomerular and tubular proteinuria

  • Renal amyloidosis
  • Gold nephropathy, D-penicillamine-induced glomerulonephritis
  • Diabetic nephropathy (stages IV and V)
  • Membranoproliferative glomerulonephritis
  • Systemic vasculitis with renal involvement
  • Acute kidney graft rejection

Tubular proteinuria

  • "Pyelonephritis", interstitial nephritis
  • Analgesic nephropathy
  • Tubulotoxic nephropathy (aminoglycosides, cisplatin, cadmium, mercury, lead, lithium)
  • Fanconi syndrome(s), type II renal tubular acidosis
  • Myeloma kidney
  • Chromoprotein kidney (malaria tropica, rhabdomyolysis)

Pre-renal proteinuria

  • Excretion of free light chains (multiple myeloma, immunocytoma, chronic lymphatic leukemia, lupus erythematosus, Sjörgren’s syndrome)
  • Intravasal hemolysis (hemolytic anemia, paroxysmal nocturnal hemolysis, march hemoglobinuria, erythrocyte enzyme defect)
  • Rhabdomyolysis
  • Lysozymuria in monocytic leukemia

Post-renal proteinuria

  • Urinary tract infection
  • Lithiasis
  • Kidney, bladder and prostate tumors
  • Injury
  • Menstruation
  • Münchausen syndrome (artificial admixture of protein)

Table 12.9-4 Recommendations of the National Kidney Foundation for albuminuria as a clinical marker of kidney damage /32/

1. Populations at increased risk for chronic kidney disease (CKD), (i.e., those with diabetes mellitus, hypertension, or family history of CKD) should be screened for albuminuria, at least annually, as part of their regular health examination. The determination of albumin is preferable to that of total protein. Total protein should, nonetheless, be determined in children to detect, apart from albuminuria, low molecular weight proteinuria as well.

2. If the total protein/creatinine ratio in spontaneous urine is above 0.5–1 g/g of creatinine, the determination of total protein instead of albumin is also acceptable.

3. Timed urine collections should not be used. Rather, the ratio of concentrations of urine albumin (in milligrams per deciliter) to urine creatinine (in grams per deciliter) on a spot untimed urine specimen should be used. First-morning spot collections are best for children and adolescents to avoid confounding the effect of orthostatic proteinuria.

4. Patients should refrain from vigorous exercise for 24 hours before sample collection.

5. Repeat assays at later points in time, at least in patients with diabetes mellitus. Specifically to identify persistent albuminuria, repeat to confirm values greater than the reference range (≤ 30 mg albumin/g creatinine) in 2 or three tested samples.

6. Individuals with documented persistent micro albuminuria (2–3 measurements greater than the reference range) who are undergoing treatment for elevated blood pressure, lipid disorders, or both should be retested within 6 months to determine if treatment goals and reduction of micro albuminuria has been achieved.

  • If treatment has resulted in a significant reduction of micro albumiuria, annual testing for micro albuminuria is recommended.
  • If no reduction of the albuminuria is seen, blood pressure and lipid values should be checked to determine if the target values were achieved, and whether other medications apart from anti-hypertensive therapy interfered with the renin-angiotensin-aldosterone system. The treatment regimen should be modified accordingly.

7. Children should be screened using a standard dipstick on 2 occasions; once before starting school and then in early adolescence (as recommended by the American Academy of Pediatrics). Subsequent testing should be performed as needed, as recommended in the Pediatric PARADE recommendations /57/.

Table 12.9-5 Categories of albumin excretion in chronic kidney disease /1/

Cate-
gory

AER
(mg/24 h)

ACR
(mg/mmol)

ACR
(mg/g)

Elevated

A1

< 30

< 3

< 30

None to mild

A2

30–300

3–30

30–300

Moderate

A3

> 300

> 30

> 300

Strong

AER, excretion in the 24-hour urine; ACR, albumin/creatinine ratio

Table 12.9-6 Albuminuria as a marker of nephropathy and systemic disease

Clinical and laboratory findings

Non-diabetic, non-hypertensive population

According to the PREVEND study /58/ albuminuria is common in the general population with a prevalence of 7.2% and independently associated with cardiovascular risk factors and cardiovascular morbidity. The majority of these micro albuminuric individuals (74.9%) has no reported diabetes or hypertension. After excluding the diabetic and hypertensive individuals micro albuminuria is still prevalent in 6.6% of the individuals.

Urine albumin excretion predicts cardiovascular and non cardiovascular mortality in the general population. According to the PREVEND study /59/ a positive dose-response relationship between increasing urinary albumin concentration (UAC) and mortality was evaluated. A higher UAC increased the risk for cardiovascular (CV) death and non-CV death with the increase being higher for CV mortality than for non-CV-mortality. A twofold increase in UAC was associated with a relative risk of 1.29 for CV mortality and 1.12 for non-CV mortality. Higher albumin excretion, including levels below 30 mg/24 h may reflect the presence of subclinical cardiovascular disease (CVD) among adults without CVD /60/. The results of this study support the hypothesis that increased UAC is an independent marker of increased left ventricular mass.

The quantity of albuminuria is associated with an increased risk: the higher the level of albuminuria, the higher the risk of need for renal replacement therapy and more rapid renal function decline. A urinary albumin concentration of ≥ 20 mg/L identifies individuals who start renal replacement therapy during follow-up, with 58% sensitivity and 92% specificity within the next 9 years /61/. The assumption that an albumin/creatinine ratio (ACR) below 30 mg/g (30 mg/24 h) is normal must, therefore, be doubted.

The Framingham Heart Study /62/ showed in a community-based sample of middle-aged non hypertensive, nondiabetic individuals with an ACR above the sex-specific median (men ≥ 3.9 mg/g; women ≥ 7.5 mg/g) the risk was 3-fold for developing CVD compared with individuals with ACR below the median values.

In the Nurses Health Study, the risk of developing hypertension in women with an ACR of 4.3–24.2 mg/g was 76% higher than in individuals with an excretion below 1.7 mg/g /63/.

Post-menopausal hormone use influences the renin-angiotensin system and renal endothelial function, impacting albumin excretion. Hormone use of > 6 years in women aged 66.8 years, on the average, was associated with lower albumin excretion [18 (10–29) mg/L, ACR 2,5 (1.6–3.9 mg/g)] in comparison to women of the same age without hormone substitution [21 (12–37) mg/L, ACR 3.5 (2.1–5.8 mg/g)] /64/.

Adiposity

Potential links between adiposity, kidney response, low grade albuminuria and CVD are described in Lit. /65/. With increasing fat mass, visceral adipocytes decrease the production of circulating adiponectin and increase the production of adipokines that enhance insulin resistance. The lower adiponectin levels lead to impaired podocyte function, possibly because of increased NADPH oxidase. Podocyte dysfunction will lead to albuminuria and increased levels of H2O2 in the urine. The increased production of H2O2 from renal NADPH oxidase could potentially cause H2O2 to enter the circulation, contributing to systemic inflammation that accompanies low-grade inflammation.

The elderly /66/

In the Cardiovascular Health Study, the relationship between albuminuria, impaired renal function, cardiovascular events and mortality was investigated during a period of 8.3 years in 3291 individuals over the age of 65 years. 34.9% had normal kidney function (12.2% with albuminuria), 46.1% had preclinical kidney disease (17.9% with albuminuria) and 18.9% had chronic kidney disease (47% with albuminuria). An ACR > 23 mg/g in women and > 36 mg/g in men, as well as a reduced GFR (cystatin C > 1.32 mg/L), were each associated with a greater than 2-fold risk for cardiovascular and overall mortality. Individuals with the combination of albuminuria and a reduced GFR manifested a more than 4-fold risk for overall and cardiovascular mortality.

Type 1 diabetes mellitus /67/

Approximately 30–60% of patients with type 1 diabetes develop albuminuria within 10–20 years. Modifiable factors in relation to albuminuria are hyperglycemia, hyperlipidemia, obesity, hypertension, and smoking. Albuminuria is an indicator of glomerular hyper filtration (Fig. 12.9-2 – Relative risk of overall mortality, cardiovascular mortality, progression and end stage in CKD, as a function of the albumin/creatinine ratio and the GFR). Glomerular hyper filtration exists in the initial years after onset of hyperglycemia. The GFR increase is related to both a rise in renal plasma flow and in filtration fraction, caused by afferent but not efferent vasodilatation and increased glomerular capillary pressure. Without treatment, this phase continues for about 10 years before urinary albumin loss commences and rises to a level of 30–300 mg/24 h.

Albuminuria has become firmly entrenched as the primary predictive marker of risk for eventual end-stage renal disease /68/. The findings of extraordinary high risk progression to albuminuria and the association between renal function impairment and albuminuria gave plausibility to a simple model of diabetic nephropathy comprising the sequential stages: micro albuminuria heralds proteinuria, which after long-term exposure initiates the process of renal function loss that leads to end stage renal disease /68/. A new model of diabetic nephropathy has been emerged. The onset of micro albuminuria heralds, in a subset of approximately one-third of patients, a process of progressive early function decline leading to advanced CKD and end stage renal failure that occurs irrespective or in parallel of the progression of micro albumiuria to proteinuria. Micro albuminuria and early function decline may, thus, represent two phenotypes that have separate underlying etiological processes /68/.

Type 2 diabetes mellitus

At diagnosis, 16% of patients with type 2 diabetes have albuminuria. This often precedes the type 2 disease. One possible explanation is the relatively close connection with the metabolic syndrome, which is a pre-diabetic insulin resistant condition. In type 2 diabetes a correlation between albuminuria and severity of the glomerular damage is present. In diabetic patients who develop micro albuminuria, the systolic blood pressure is higher at night than during the day /69/.

Diabetes and cardiovascular risk

In diabetes mellitus, albuminuria is associated with increased cardiovascular risk and is 2–4-fold higher than in non-diabetic individuals. For every of 0.4 mg/mmol (3.5 mg/g) increase in ACR, the risk for a major cardiovascular event increases by 6%. Diabetics without albuminuria have a negligible cardiovascular risk, while in those with macro albuminuria the annual risk is 2.3% /70/. It was shown in the Action in Diabetes and Vascular Disease: Preterax and Diamicron MR Controlled Evaluation (ADVANCE) study that in patients with type 2 diabetes, severe albuminuria and a low GFR are independent risk factors for cardiovascular and renal events. However, the presence of both together carries a high risk. Thus, a GFR below 60 [mL × min–1 × (1.73 m2)–1] and an ACR of > 300 mg/g were associated with 3.2-fold and 22.2-fold greater cardiovascular and renal risks, respectively /32/.

Diabetes and hypertension

The prevalence of hypertension in diabetics is significantly higher than in non-diabetics (40–50% vs. 20%). The co-existence of diabetes mellitus and essential hypertension raises the risk of cardiovascular disease and of mortality by a factor of 3 in elderly diabetics and by a factor of 2 in younger diabetics. Diabetes and hypertension act synergistically with regard to the development of increased albumin excretion and complications of diabetes. In diabetics, high blood pressure accelerates the development of diabetic nephropathy and retinopathy and increases the risk of macro vascular complications such as myocardial infarction, congestive heart failure, and stroke. Hypertension contributes to the complications to an extent of up to 75% /71/. The combination of diabetes mellitus and hypertension is, in the majority of the patients, responsible for the end stage renal failure. In order to decrease the macro vascular complications of diabetes, efficient reduction of blood pressure to values of < 130/80 mmHg is more effective than glycemic control.

Functional/transient proteinuria

Essential hypertension is associated with elevated albumin excretion and increased cardiovascular risk /72/. The prevalence is calculated to be, on the average, 10–20%. In the HARVEST study, which involved patients with stage 1 hypertension, 6.1% of the hypertensive patients had albuminuria of ≥ 30 mg/24 h, and the percentage rose to 14.6% if the upper threshold was decreased to ≤ 29 mg/24 h /73/. In essential hypertension, albumin excretion correlates better with the systolic, rather than the diastolic, blood pressure. An important mechanism leading to micro albuminuria is believed to be elevated intraglomerular pressure.

The reduced GFR and the proteinuria are factors that interfere with the circadian rhythm of the blood pressure, and they are associated with hypertension. In patients with normal GFR and albuminuria, masked hypertension should be taken into consideration /74/.

Table 12.9-7 Proteinuria types of SDS-PAGE /77/

Type I

Non-selective glomerular, MW 50–150 kDa

Nephrotic (> 3 g/24 h), normal GFR

  • Proliferative glomerulonephritis
  • Stage IV diabetic nephropathy
  • Amyloidosis

Mild proteinuria (< 300 mg/L), no hematuria, normal GFR

  • Residual condition following previous glomerulonephritis, monitoring at intervals of 6–12 months

Questionable proteinuria (< 120 mg/L), no hematuria, normal GFR

  • The finding is not indicative of glomerulopathy. Occasional monitoring required.

Type II

Selective glomerular, MW 50–70 kDa, mainly albumin and transferrin

Mostly nephrotic proteinuria (> 3 g/24 h), normal GFR

  • Minimal change glomerulopathy
  • Focal sclerosing glomerulonephritis (early)
  • Early peri membranous glomerulonephritis

Type III

Entirely tubular, MW 10–70 kDa

Mainly moderate proteinuria (0.3–1.5 g/24 h)

  • Interstitial nephritis
  • Pyelonephritis
  • Graft rejection
  • Acute renal failure
  • Hereditary tubulopathy

Moderate to severe proteinuria up to > 3 g/24 h, additionally free light chains

  • Free light chain tubulopathy, myeloma kidney

Type IV

Non-selective glomerular and entirely tubular, MW 10–> 150 kDa

Severe proteinuria (1.5–3 g/24 h), reduced GFR

  • Advanced glomerulonephritis
  • Stage V diabetic nephropathy
  • Advanced amyloidosis

With serum creatinine < 2.5 mg/dL (221 μmol/L) mixed disorder due to glomerular and interstitial kidney damage

  • Hypertensive glomerulosclerosis and pyelonephritis (proteinuria > 1 g/24 h)
  • Diabetic nephropathy and pyelonephritis (proteinuria > 1 g/24 h)

Type V

Non-selective glomerular and partial tubular, MG 30–> 150 kDa

Proteinuria < 1 g/24 h

  • Slight proliferative glomerulonephritis
  • Stage III-IV diabetic nephropathy
  • Hypertensive nephrosclerosis

Proteinuria < 150 mg/24 h

  • Stage III diabetic nephropathy
  • Incipient lupus erythematosus
  • Hypertension?

Proteinuria 150–1,000 mg/24 h, hematuria (monomeric and dimeric Hb)

  • Slight proliferative glomerulonephritis (e.g., IgA nephropathy)

GFR, glomerular filtration rate; MW, molecular weight

Table 12.9-8 Concentration ratios of urinary proteins and their predictive value /42/

Concentration ratio

Clinical and laboratory findings

IgG/albumin

< 0.03

Selective glomerular proteinuria

≥ 0.03

Non-selective glomerular proteinuria

α1-microglobulin/ albumin

≥ 0.1

Mixed proteinuria

< 0.1

Glomerular proteinuria

α2-macroglobulin/albumin

< 0.02

Renal hematuria

≥ 0.02

Postrenal hematuria

CRPSerum/CRPUrine

≥ 1.0

Bacterial infection

< 1.0

Transplant rejection

(Albumin + IgG + α1-microglobulin)/ total protein

≥ 0.6

Renal proteinuria

< 0.6

Suspected free light chain excretion

Free light chains κ/λ

< 0.25

Monoclonal λ chains

0.25–2.17

Polyclonal light chains

> 2.17

Monoclonal κ chains

Table 12.9-9 Glomerular and tubulo-interstitial proteinuria

Clinical and laboratory findings

Acute glomerulonephritis (GN) /75/

Acute GN is a syndrome with clinical symptoms that include macro hematuria, oliguria, acute renal failure with a sudden decline in GFR, fluid retention in the form of edemas and hypertension. Post-streptococcal GN and post-infectious GN are typical forms of acute GN. The now rare post-streptococcal GN begins acutely 7 days to 12 weeks after the streptococcal infection and mainly affects children in the age of 2 to 10 years. Only 10% of the patients are above the age of 40. The clinical symptoms improve spontaneously and diuresis resolves within 1–2 weeks. Although the majority of patients return to full health, some suffer from residual hypertension and recurrent or persistent proteinuria. The incidence of chronic kidney disease is up to 20%.

Laboratory findings: hematuria, mostly macro hematuria, erythrocyte casts, elevated creatinine. Hematuria normalizes within 6 months. Total protein excretion < 3 g/24 h, non-selective glomerular proteinuria. In 15% of patients, mild proteinuria persists after 3 years, while 2% of patients still exhibit proteinuria after years.

Rapid progressive glomerulonephritis (GN) /75/

Rapid progressive GN is a clinical syndrome which is characterized by the signs of glomerulonephritis (hematuria, proteinuria, red blood cell casts) and by a sudden decrease of kidney function to end-stage renal failure within a few days to weeks. Rapid progressive GN comprises 2–4% of the glomerulonephritides, is associated with the formation of glomerular crescents and overlaps many glomerular diseases. The clinical symptoms are unspecific, such as lethargy and malaise.

Laboratory findings: as for acute GN.

Minimal change glomerulopathy

This involves a nephropathy with selective glomerular proteinuria. Increased excretion of medium seized proteins with a MW of approximately 60 kDa such as albumin and transferrin.

Laboratory findings: total protein excretion up to 3 g/24 h. The IgG/albumin ration is < 0.03.

Nephrotic syndrome (NS) /76/

Patients with NS exhibit severe proteinuria, hypoalbuminemia, edema, and various degrees of hyperlipidemia and lipiduria. NS occurs as a complication associated with a variety of systemic diseases such as diabetes mellitus, systemic lupus erythematosus and amyloidosis, but also Hodgkin’s disease, infectious diseases and drug-induced conditions. In adults, the most encountered histological lesions, which are linked to primary NS are focal segmental glomerulosclerosis, membranous glomerulopathy, minimal change glomerulopathy and membranoproliferative glomerulonephritis. The latter is regularly seen with NS. Thromboembolic complications are observed in 20–30% of patients, with arterial thrombosis less commonly seen than venous thrombosis.

Laboratory findings: excretion of total protein > 3.5 g/m2 and 24 h, and in children > 40 mg/m2 per hour; non-selective proteinuria. Lipids: VLDL, IDL, LDL, triglycerides and Lp(a) elevated. The following results are indicative of an increased risk of thrombosis: serum albumin < 25 g/L, proteinuria > 10 g/24 h, elevated plasma fibrinogen level, antithrombin < 75%, hypovolemia.

Chronic glomerulonephritis (GN)

Chronic GN is a syndrome with progressive renal insufficiency in patients with glomerular inflammation, hematuria, and often hypertension. As a rule, chronic GN proceeds for 10 years or more before any replacement therapy becomes necessary. While in adults diabetes mellitus is the primary cause of chronic GN, the primary cause in children from among the acquired glomerulonephritides is focal-segmental glomerulosclerosis.

Laboratory findings: proteinuria can be nephritic (≤ 3 g total protein/24 h) or nephrotic (> 3 g/24 h). The assessment of GFR via creatinine and cystatin C determination as well as the determination of the total protein and marker proteins are important prognostic criteria. Thus, the renal function prognosis significantly worsens with total protein excretion exceeding 1 g/24 h and severely worsens above 5 g/24 h. An increase in the non-selectivity of proteinuria and the consequent increase in the IgG/albumin ratio also point to an unfavorable prognosis.

Tubulo-interstitial nephropathy (TIN)

In the acute form of this disease, both the tubules and the interstitium are initially affected. In the chronic progressive forms, the blood vessels and glomeruli are also affected. The result is tubulo-interstitial fibrosis, which, regardless of the cause of TIN, represents the common final phase of the disorders that cause chronic kidney disease. Since almost all renal diseases involve these types of changes in their advanced stages, more recent classifications of TIN encompass almost all renal disorders. The majority of cases of TIN involve the secondary forms, in which tubulo-interstitial changes are the result of an underlying glomerular disease or primary forms, which are triggered by toxic processes (medications), malignancies, metabolic and immunologic factors. Most of the primary TINs (85%) are induced by medication (analgesics, antibiotics).

Laboratory findings: the urine findings are less pronounced than is the case with nephritic and nephrotic glomerulopathies and are frequently missed by routine laboratory testing with test strips and serum creatinine determinations. An important finding is the leukocyturia without bacteriuria. Total protein excretion is almost always < 1 g/24 h and does not exceed 3 g/24 h. One typical finding is the tubular proteinuria resulting from decreased reabsorption of low-molecular weight proteins. Increased excretion of the tubular proteins can be detected semi quantitatively by SDS-PAGE, a quantitative determination is made by determining the marker proteins (α1-M > 14 mg/g creatinine; β2-M > 0.2 mg/g creatinine). In TIN, there is a significant relationship between tubular damage and proteinuria selectivity. Furthermore, the selectivity index and the α1-M excretion are prognosis factors used to predict future loss of renal function /28/. If the proteinuria is highly selective, the tubulo-interstitial damage is less pronounced and there will be a near total remission. A proteinuria that is only moderately selective or nonselective is indicative of a major tubular damage and the prognosis is accordingly worse. The concurrence of a tubular proteinuria is indicative of a poor prognosis in cases of primary glomerular disease.

Table 12.9-10 Clinical assessment of urine protein marker analysis, modified from Ref. /77/

Proteinuria

Clinical and laboratory findings

Minimal, selective glomerular

  • TP: 150–500 mg/24 h
  • ACR 30–300 mg/g;
    (3–30 mg/mmol)
  • SDS-PAGE: Type III or V

Stage III diabetic nephropathy, hypertensive nephropathy (early stage)

SLE nephropathy (early stage)

Pronounced, selective glomerular

  • TP: 500–3,500 mg/24 h
  • ACR 300–3,000 mg/g
  • IgG/albumin < 0.03
  • α1-microglobulin/albumin < 0.1
  • SDS-PAGE: Type II

Minimal change glomerulopathy, SLE nephropathy (remission)

Early stages and bland courses of focal-sclerosing, peri-

membranous and immune complex-associated glomerulonephritis

IgA nephritis, uncomplicated course

Incipient EPH gestosis (early symptoms)

Gold/penicillamine nephropathy, beginning

Non-selective glomerular

  • TP: 500–3,500 mg/24 h
  • ACR 300–3,000 mg/g
  • IgG/albumin > 0.03
  • α1-microglobulin/albumin < 0.1
  • SDS-PAGE: Type I
  • Possibly hematuria

Acute glomerulonephritis

Rapidly progressing glomerulonephritis

Goodpasture syndrome

SLE nephropathy (acute stage)

Pregnancy-related nephropathy (advanced)

Stress proteinuria, orthostatic proteinuria

Hyperthermic/hypothermic proteinuria

Nephrotic (non-selective glomerular)

  • TP: > 3,000 mg/24 h
    (children > 40 mg/m2/h or 1 g/m2/24 h)
  • serum albumin < 25 g/L)
  • ACR > 3,000 mg/g
  • IgG/albumin > 0.03
  • α1-microglobulin/albumin < 0.1
  • SDS-Page: Type I

Hemolytic-uremic syndrome

Congenital nephrotic syndrome

Goodpasture syndrome

Advanced nephrosclerosis

Mixed proteinuria

  • TP: 500–3,500 mg/24 h
  • ACR 300–3,000 mg/g
  • IgG/albumin > 0.03
  • α1-microglobulin/albumin > 0.1
  • SDS-PAGE: Type IV

Nephropathy: diabetic (stage 4 + 5), hypertensive (beginning nephrosclerosis), malaria quartana, amyloidosis, myeloma kidney (glomerulosclerotic progress), panarteritis nodosa, scleroderma, SLE (acute), gold/penicillin amine (late stage), extensive burn trauma, kidney transplant rejection, chronic pyelonephritis, stress proteinuria, hyper-/hypothermic and orthostatic proteinuria

Tubular proteinuria

  • TP: 0.15–1.5 g/24 h
  • α1-microglobulin
  • ACR < 30 mg/g; < 3 mg/mmol
  • SDS-PAGE: Type III
  • Chronic cases: β-NAG

Interstitial nephritis (Hanta virus), bacterial, allergic (Balkan nephritis), pyelonephritis (acute), tubular toxins (aminoglycosides, cephalosporins, methicillin, cyclosporin A, cisplatinum, methotrexate, X-ray contrast media, Cd, Pb) analgesics (phenacetin, paracetamol, phenylbutazone), hereditary tubulopathies, renal-tubular acidosis, Fanconi syndrome, reflux nephropathy, hypokalemia, hypercalcemia, Wilson’s disease, gout, Sjögren’s syndrome, recovery from acute kidney failure, HIV infection, acute malaria tropica, kidney transplant (normal function)

TP, total protein; Tb, tuberculosis; PNH, paroxysmal nocturnal hematuria; SLE, systemic lupus erythematosus; HIV, human immuno deficiency virus; β-NAG, N-acetyl-β-D-glucosaminidase.

Table 12.9-11 Post-renal proteinuria /77/

Clinical and laboratory findings

Hypersecretion of IgA, Tamm-Horsfall protein and other uromucoids

Since these proteins are not detected by the determination of marker proteins and of total protein (exception: Biuret), this type of post-renal proteinuria remains, for the most part, undetected. This is fully reasonable, since it has no pathological significance. Reduced excretion of IgA and Tamm-Horsfall protein are observed in intercurrent urinary tract infections /79/.

Urinary tract infection

These infections lead to increased secretion of lysozyme, CRP, immunoglobulins and various proteins that protect against infections in the urine. Additionally, the permeability of the uroepithelium is increased, and this leads to an exsudation of albumin and other plasma proteins in the urine. Finally, bacterial proteins are released into the urine. In sum, a variable urinary protein pattern, which can easily be mistaken for a non-selective glomerular proteinuria, is found. Nonetheless, most urinary tract infections are signaled by leukocyturia, by a positive granulocyte esterase, by the presence of nitrite and the typical clinical symptoms (alguria, dysuria, pollacisuria).

Bleeding

Bleeding in the renal pelvis, urinary tract, prostate, and genitalia results in increased loss of erythrocytes and blood plasma in the urine. In contrast to renal proteinuria, the urinary protein pattern with SDS-PAGE is, in this case, identical with that of the plasma. Marker proteins can be used to differentiate between glomerular erythrocyte loss associated with acute GN (glomerular hematuria), and post renal bleeding. These marker proteins occur as components of very large macromolecular complexes such as α2-macroglobulin or apolipoprotein A-I. They can only result from bleeding, because the complexes cannot enter the urine via the glomerular basement membrane. Post-renal hematuria can be assumed, if a sample is clearly positive for hemoglobin, the apolipoprotein A-I level is > 0.4 mg/L /80/ and/or the α2-macroglobulin/albumin ratio > 0.02 /81/.

Münchhausen syndrome

Proteins are occasionally artificially added to urine in order to mimic renal disease for material or psychological advantage. If foreign proteins are used (e.g., egg white or gelatin) this will be evident from the following pattern of results:

  • Test strips: negative or only very weakly positive
  • Biuret quantitative total protein assay: very high result
  • Albumin, IgG, α1-microglobulin: slight or not detectable
  • Free light chains: slight or not detectable
  • SDS-PAGE: Ovalbumin (43 kDa) or gelatin (20–30 kDa) discernable.

If, however, human serum or human albumin preparations are used for deceptive purposes, it is not possible to differentiate these cases from genuine glomerular proteinurias without further investigations.

Table 12.9-12 Toxic nephropathies: urine protein findings and clinical evaluation /77/

Toxins, pharmacons

Clinical and laboratory findings

Aminoglycosides, cephalosporins, methicillin, amphotericin B, methotrexate, cisplatin, cyclosporin

lithium

Proteinuria type: acute onset of tubular proteinuria, α1-M ↑↑, β-NAG ↑↑

Clinical findings: acute toxicity. The obligatory dose-dependent rise in tubular proteins in the urine is an expression of a reversible impairment of tubular function. Therapy can include (e.g., nephroprotective measures, dose reduction) but is adjusted to GFR (e.g., creatinine clearance, or the plasma drug concentration).

Phenacetin Paracetamol N-acetyl salicylic acid

Non-steroidal anti-inflammatory drugs

Proteinuria type: tubular proteins that increase over the course of years, enzymuria and sterile leukocyturia, α1-microglobulin, β-NAG ↑↑, leukocyte esterase

Clinical findings: chronic abacterial, interstitial nephritis due to the cumulative effect of prostaglandin synthetase inhibitors. The tubular kidney damage is irreversible, and reflects the cumulative effect of approximately 1 kg of phenacetin or equivalent doses of other cyclooxygenase inhibitors.

Lead. Cadmium

Proteinuria type: tubular proteinuria and enzymuria, α1-microglobulin , β-NAG

Clinical findings: damage due to tubular deposits of renally eliminated heavy metals and toxins. The excretion of the marker proteins of proximal tubular function correlates with the degree of exposure and the excretion of heavy metals in the urine. The tubular proteinuria precedes the renal function impairment by years /82/.

Gold D-penicillamine Mercury compounds Probenecid

Proteinuria type: non-selective glomerular or mixed proteinuria, microhematuria

Clinical findings: membranous glomerulonephritis. Deposition of immune complexes in tubular epithelial cells and the basal membrane. In the range of 0.2–0.4 g TP/24 h, the gold-penicillamine therapy can be continued. With higher values treatment must be interrupted and the urine findings have to be checked 2–4 months later /83/. Gold-penicillamine nephropathy is reversible within 21 months /84/.

X-ray contrast media

Proteinuria type: acute tubular or mixed glomerular and tubular proteinuria, α1-microglobulin , β-NAG , (albumin )

Clinical findings: X-ray contrast media are both tubulotoxic and glomerulotoxic. The tubulo-toxicity is the decisive factor in the prognosis of contrast media induced nephropathy. The most sensitive indicator of tubulo-toxicity is tubular proteinuria. Low-osmolality contrast media (600–800 mmol/kg) are less tubulo-toxic than high-osmolality media (> 1,500 mmol/kg).

TP, total protein; β-NAG, N-acetyl-β-D-glucosaminidase

Table 12.10-1 Factors that increase the risk of renal stones

Risk of renal stones

Purine-rich food

Animal products such as liver, entrails, fowl, fish and meat products, consumed in excess, cause the increased formation of uric acid and increase the risk of forming uric acid stones.

Excess protein in the daily diet

Due to excess consumption of meat and foods that contain meat extracts, the sulfuric amino acids cysteine and methionine are increasingly absorbed. The sulfur is excreted as sulfuric acid and acidifies the urine. If the formation of HCO3 and NH4+ is not sufficient for compensation, this can result in the formation of uric acid stones and calcium oxalate stones.

Metabolic syndrome

Metabolic syndrome is associated with higher prevalence of nephrolithiasis. The odds of renal stone disease significantly increase with the number of metabolic syndrome traits (e.g., abdominal obesity, arterial hypertension, diabetes, dyslipidemia) /10/. In persons with calcium nephrolithiasis the prevalence of hypertension, diabetes, overweight (BMI ≥ 25 kg/m2 ) and dyslipidemia was 17, 2, 42 and 38%, respectively. In a large group of Caucasian stone formers, hypertension was the only associated urinary calcium excretion, while no metabolic syndrome trait was associated with increased oxalate excretion /10/.

Excess consumption of chocolate, tea and vegetables

The daily intestinal intake of oxalic acid can be 50–1,000 mg. Cocoa, tea and some vegetables such as spinach, red beets, mangold and rhubarb are rich in oxalic acid. The formation of oxalate stones is promoted.

Alcohol

The metabolism of alcohol and purines in beer increases the formation of acids which are excreted with the urine. The urine becomes acidic, which favors the calcium oxalate and uric acid urolithiasis.

Stress

Stress leads to a rise in the hormones prolactin, TSH and arginine vasopressin. The latter causes the increased reabsorption of water in the distal tubulus and in the collecting ducts, thus to a concentration of soluta and, consecutively, the urinary stone diathesis.

Obesity

Epidemiologic studies have demonstrated that the stone risk incidence increases with body mass index through multiple pathways. The underlying pathophysiology is thought to be related to insulin resistance, dietary factors and a lithogenic urinary profile. Uric acid stones and calcium oxalate stones are observed frequently in these patients. Insulin resistance is thought to alter the renal acid-base metabolism, resulting in a lower urine pH, and increasing the risk of uric acid stone disease. Obesity is also associated with excess nutritional intake of lithogenic substances (e.g., calcium, phosphate, uric acid, oxalate, cystine) and with an increase in urinary tract infection incidence /40/.

Heritable factors

Close relatives of nephrolithiasis patients have a high risk of stone formation. A study /27/ showed that nephrolithiasis offspring carried several urinary metabolic risks predisposing to stone formation which are similar to their parents and about one in every five neprolithiasis children had nephrolithiasis level urinary supersaturation. In Thailand family members of stone patients had a relative risk 3.18 times higher than the general population /41/.

Table 12.10-2 Composition and frequency of renal stones /8/

Chemical name

Mineralogical
name

Incidence
(%)

Calcium oxalate

60–75

  • Monohydrate

Whewellite

35

  • Dihydrate

Weddellite

40

Carbonate apatite

Dahllite

2

Calcium phosphate

Brushite

1

Magnesium
ammonium
phosphate

Struvite

10–20

Cystine

2–3

Uric acid

Uricite

5–10

Xanthine

Very rare

Dihydroxyadenine

Oxypurinol

Calcium carbonate

Calcite

Silicon dioxide

Calcite quartz

Table 12.10-3 X-ray appearance of renal stones /9/

Shadow-producing

Weakly shadow-
producing

Not shadow-producing

Calcium oxalate

  • Whewellite
  • Weddelite

Magnesium ammonium phosphate (struvite)

Cystine

Uric acid (uricite)

Urate

Xanthine

2.8-dihydroxyadenine

Drug-induced calculi

Calcium phosphate

  • Carbonate apatite
  • Brushite

Table 12.10-4 Further investigation in addition to the basic program in renal stone formers /9/

Ca oxalate stone

Ca phosphate, carbonate apatite, brushite

Uric acid- and urate stone

Infection stone, brushite

Cystine stone

2.8-DHA, Xanthine stone

Nephro­calcinosis

Parathyroid hormone Sodium Potassium Chloride

Parathyroid hormone Sodium Potassium Chloride

Uric acid

C-reactive protein

 

 

Sodium, potassium,
chloride, magnesium

Parathyroid hormone

25(OH)D, 1,25 (OH)2D

Vitamin A

Volume Osmolality Calcium Oxalic acid Uric acid Citrate Magnesium Daily pH profile

Volume Osmolality Calcium Phosphate Citrate

Daily pH profile

Volume Osmolality Uric acid Daily pH profile

Daily pH profile

Volume Osmolality Cystine

Daily pH profile

Volume Osmolality Uric acid Daily pH profile

Volume,
osmolality

Calcium
phosphate

Oxalate
Uric acid

Citrate

Magnesium

Daily pH profile

Serum tests, upper table; urine examinations, lower table, two 24-hour urine samples should be investigated. Daily urinary pH profile: at every miction, but at least 4 individual circadian measurements.

Table 12.10-5 Reference intervals /9/ and abnormal excretions associated with nephrolithiasis /27/

Parameter

Reference
intervals

Nephrolithiasis
(Adult ≥ 18 years)

Nephrolithiasis
(< 18 years)

Volume

≥ 1 liter

pH value

≥ 5.0

6–7

TmPO4/GFR

0.80–1.35
mmol/L

Phosphate

1,100 mg/day

1.47 mg/
mg creatinine

Calcium

M: 2.00–7.50
mmol/24 h

F: 2.00–6.25
mmol/24 h

≥ 4 mg/kg/day

M: ≥ 300 mg/day

F: ≥ 250 mg/day

≥ 4 mg/kg/day

Oxalate

< 0.50
mmol/24 h

≥ 45 mg/
1.73 m2/day

≥ 20 mg/day

≥ 45 mg/
1.73 m2/day

≥ 20 mg/day

Uric acid

M: < 4.80
mmol/24 h

F: < 4.50
mmol/24 h

≥ 700 mg/day

0.76 mg/
mg creatinine

Citrate

1.52–7.00
mmol/24 h

M: ≤ 365 mg/
1.73 m2/day

F: ≤ 310 mg/
1.73 m2/day

M: ≤ 365 mg/
1.73 m2/day

F: ≤ 310 mg/
1.73 m2/day

Magnesium

1.70–6.80
mmol/24 h

≤ 0.8 mg/kg/day

≤ 0.8 mg/kg/day

Sodium

> 250
mmol/24 h

1.6–3.2 g/
1.73 m2/day

Cystine

< 1,000
μmol/24 h

Creatinine

M: ≥ 10.6
mmol/24 h

F: ≥ 7.0
mmol/24 h

Creatinine
clearance

M: 135–200
mL/1.73 m2

F: 120–180
mL/1.73 m2

M, male; F, female

Table 12.10-6 Renal stones /939/

Clinical and laboratory findings

Calcium oxalate stone – Whewellite,Weddellite

Four urinary risk factors lead to the formation of calcium oxalate stones: the molar concentrations of calcium, citric acid and oxalic acid, as well as the pH. In the Western countries, hyper oxaluria and the low solubility product of calcium oxalate are the cause of renal stones in 70–80% of the cases. Calcium oxalate stones occur in two forms, namely, as calcium oxalate monohydrate (CaC2O4 × 1 H2O), also termed whewellite, and as calcium oxalate dihydrate (CaC2O4 × 2 H2O), with the name of weddellite. Whewellite grows slowly and is compact. In slices, the surface resembles that of a tree trunk with annual rings. Patients with primary hyper oxaluria have only whewellite stones. Weddellite grows quickly and forms loose crystal-like structures. Patients with weddellite stones often have hyper calciuria.

Uric acid stones, urate stones

In Germany, approximately 10% of patients with renal stones have a uric acid urolithiasis; in gout patients, the proportion is 20–40%. Urate stone diathesis is of secondary genesis and is due to protein-rich nutrition and to alcohol consumption. Patients with obesity, metabolic syndrome and insulin resistance are particularly affected. Not uncommonly, uric acid deposits form in association with the mixed whewellite stones.

While uric acid stones form at pH < 5.8, urate stones are produced at pH > 6.8. They form at these pH values if uric acid H-ion dissociates and is substituted by cations such as Na+, K+ and NH4+ . Urate stones do contain uric acid, but they are not uric acid stones.

Phosphate stones – Brushite, Carbonate apatite

Calcium phosphate occurs in the mineral form as brushite and carbonate apatite. The recurrence rate is 80%. During the course of the day, the urinary pH in the patients is slightly acidic (pH 6.5–6.8) to alkaline. Stone formation is favored by distal renal tubular acidosis and primary hyperparathyroidism.

Brushite stones (CaHPO4 × 2 H2O) are rare; their frequency in stone formers is 2%. The pH of the urine and hypercalcemia are responsible for their formation. Brushite stones are hard and lithotripsy is not always successful, since residual stones can remain in the renal pelvis.

Carbonate apatite (dahllite) stones occur at alkaline pH, mostly in combination with magnesium ammonium phosphate (struvite).

Infection stones (Struvite)

Struvite, carbonate apatite and ammonium urate are infection-associated urinary stones. An infection with urease-forming bacteria is usually present at the same time. The bacteria release ammonium and bicarbonate into the urine, whereby the urine becomes alkaline. In this way, the crystallization of carbonate apatite and magnesium ammonium phosphate is favored.

Table 12.10-7 Calcium/creatinine ratio in healthy individuals and patients with hyper calcuria (HCU)in the Ca absorption test /18/

 

Healthy
individuals

Absorptive
HCU

Resorptive
HCU

A

0.08–0.23

0.16–0.27

0.36–0.64

B

0.27–0.46

0.56–0.98

0.73–0.96

The urinary calcium/creatinine molar relationship is given. A, 2-hour sample following fasting; B, 2-hour sample following calcium loading (1 g) with milk, bread, butter and calcium syrup.

Figure 12.1-1 Structure of the renal filtration mechanism. A, slit diaphragm; B, podocyte foot processes; C, adhesion proteins of the slit diaphragm; D, glomerular basal membrane.