Kidney Function Tests

The diagnosis of albuminuria requires the demonstration of increased albumin loss (either increased ACR or increased albumin in a timed collection) in at least two out of three urine samples collected in the absence of infection or an acute medical illness (Fig. 21.1). Establishing the diagnosis of albuminuria or proteinuria has both prognostic and management implications. In the setting of diabetes, the best possible metabolic control should be achieved before patients are examined for albuminuria, and patients should not be screened during other transitory illnesses. Screening should commence 5 years after diagnosis in patients with type 1 diabetes mellitus and at diagnosis in patients with type 2 diabetes, and should continue on an annual basis up to the age of 75 years. Patients demonstrating an ACR of 3.0 mg/mmol or greater should have urine samples sent to the laboratory on two other occasions (ideally within 2 months) for albumin estimation. Patients demonstrating increased ACRs in one or both of these additional samples are said to have persistent albuminuria. Diabetic nephropathy is uncommon in patients who have had type 1 diabetes for less than 5 years, and other causes of kidney disease should be considered.

Samples for urinary albumin (or total protein) measurement may be analyzed fresh, stored at 4°C for up to 1 week, or stored at −70°C for longer periods. Freezing at −20°C appears to result in loss of measurable albumin and is not recommended. For analysis, stored samples should be allowed to reach room temperature and be thoroughly mixed prior to testing.

Analytical Methods and Traceability: Urinary Total Protein

Numerous methods can be used for the measurement of protein in urine, including the original Lowry method, turbidimetry after mixing with trichloroacetic or sulfosalicylic acid, turbidimetry with benzethonium chloride (benzyl dimethyl {2-[2-(p-1,1,3,3-tetramethyl butylphenoxy)ethoxy]ethyl} ammonium chloride), and dye binding with Coomassie Brilliant Blue, pyrogallol red molybdate, and pyrocatecholviolet-molybdate, which is used in dry-slide applications.

Total protein measurement is more difficult in urine than in serum. The concentration of urinary protein is normally low (100 to 200 mg/L) and large sample-to-sample variation in the amount and composition of proteins is common. The concentration of nonprotein potentially interfering substances is high relative to the protein concentration and is highly variable, and the inorganic ion content is high. All these factors affect the precision and accuracy of the various methods.

Because of the need for automation, the benzethonium chloride and dye-binding methods have become the most popular in current clinical use but they do not provide equal analytical specificity and sensitivity for all proteins. Concerns about different responses to different proteins have led to many variants of the published methods. Most methods tend to react more strongly with albumin than with globulin and other nonalbumin proteins. As different methods are responding to different properties of proteins and the abundance of these properties varies between the proteins found in urine, the “quantity intended to be measured” is not the same for different assays. For those reasons, there is no reference measurement procedure or standardized reference material for urinary total protein. The variety of methods in use means that significant between-laboratory variation is inevitable. This variation tends to diminish at higher concentrations of urinary total protein, presumably in part as albumin becomes the predominant protein and thus reduces a component of the between-sample variation. These methodological problems are a major reason why urine albumin is a preferred analyte for the assessment of proteinuria.

FIG. 21.1 Suggested protocol for the further investigation of an individual demonstrating a positive reagent strip test for albuminuria/proteinuria or quantitative albuminuria/proteinuria test. Reagent strip device results should be confirmed using laboratory testing of the albumin-to-creatinine ratio (ACR) on at least two further occasions. Patients with two or more positive (≥3 mg albumin/mmol creatinine) tests on early morning samples 1 to 2 weeks apart should be diagnosed as having persistent albuminuria. (The possibility of postural proteinuria should be excluded by the examination of an early morning urine.) Protein-to-creatinine ratio (PCR) measurement can be substituted for the ACR but is insensitive in the detection of mild to moderately increased albuminuria/proteinuria: Approximate PCR equivalent to an ACR of 30 mg/mmol is 50 mg/mmol. ^aConsider other causes of increased ACR (e.g., menstrual contamination, uncontrolled hypertension, symptomatic urinary tract infection, heart failure, other transitory illnesses, and strenuous exercise), especially in the case of type 1 diabetes present for less than 5 years. The presence of hematuria may indicate nondiabetic renal disease. Note: US guidelines express albuminuria or proteinuria as mg/g creatinine, whereas other guidelines use mg/mmol creatinine. An approximate conversion factor of 0.1136 can be used to convert results in mg/g to mg/mmol. However, for clarity and pragmatism, recent guidelines have accepted decision points that are approximately equivalent: hence, when using this protocol in the United States, 300 mg/g should be substituted for 30 mg/mmol and 30 mg/g for 3 mg/mmol. C&S, Culture and sensitivity; CKD, chronic kidney disease; EMU, early morning urine; MSU, midstream urine.

TABLE 21.1

Characteristics of Some Clinically Important Urinary Proteins
Protein	Mr (kDa)	Free Plasma Concentration (g/L)	Diameter (nm)	pI	Glomerular Sieving Coefficient	Filtered Load >(mg/L)^a	Urinary Concentration (mg/L)^b	% Reabsorbed
IgG	150	10	5.5	7.3	0.0001	1	0.1	99
Albumin	66	40	3.5	4.7	0.0002	8	5	99
α₁-Microglobulin	31	0.025	2.9	4.5	∼0.3	7.5	5	99
Retinol-binding protein	22	0.025	2.1	4.5	∼0.7	17.5	0.1	99
Cystatin C	12.8	0.001	3.0	9.2	∼0.7	0.7	0.1	99
β₂-Microglobulin	11.8	0.002	1.6	5.6	0.7	1.1	0.1	99

Analytical Methods and Traceability: Urinary Albumin

Urinary albumin has been measured using immunoassay since the 1960s when the first such assays became available. Urinary albumin is predominantly measured using quantitative immunoturbidimetric or immunonephelometric approaches capable of detecting albumin at low concentrations. No Joint Committee for Traceability in Laboratory Medicine (JCTLM) listed reference measurement procedure or higher order reference material is currently available for urinary albumin. To date, most urinary albumin assays have been standardized against a serum-based calibrant (ERM-DA-470k/IFCC) distributed by the Institute for Reference Materials and Measurements of the European Commission.

Reference Intervals, Definitions of Proteinuria and Albuminuria

There is no consistent definition of proteinuria. The upper limit of the reference interval ^† for urinary total protein loss varies between 150 and 300 mg/day, depending on the laboratory. Given average daily creatinine excretion of about 10 mmol (0.11 g), an upper limit of normal protein loss of 150 mg/day is equivalent to a urinary PCR of approximately 15 mg/mmol (130 mg/g). The protein in the urine of healthy individuals is made up of albumin (<30 mg/day) and some smaller proteins, together with proteins secreted by the tubules, of which Tamm-Horsfall glycoprotein (THG) predominates. Typical concentrations of proteins found in urine are listed in Table 21.1. Readers should note that the units of expression used for ACR and PCR differ depending on geographical location, typically whether it is inside or outside the United States.^‡

Proteinuria is often detected at the point of care (POC) using urine reagent strip devices, and clinical proteinuria has sometimes been defined as equivalent to a color change of “+” or greater on the relevant pad on the strip. This equates to approximately 300 mg/L of total protein or a PCR of 50 mg/mmol, or protein loss of approximately 500 mg/day (assuming an average urine volume of 1.5 L/day). Indeed, the limits for proteinuria and albuminuria are best described as clinical decision points because they are generally described or defined by expert groups rather than as the product of formal reference interval studies.

In health, relatively small amounts of albumin (<30 mg/day) are lost in the urine. In the international classification of kidney disease (see Chapter 35), proteinuria is categorized based on levels of albumin loss: A1 normal to mildly increased, less than 3.0 mg/mmol (approximately equivalent to <30 mg/g); A2 moderately increased, 3 to 30 mg/mmol (approximately equivalent to 30 to 300 mg/g); and A3 severely increased, greater than 30 mg/mmol (approximately equivalent to ≥300 mg/g). These categories are approximately equivalent to what would have formerly been considered normoalbuminuria, microalbuminuria, and macroalbuminuria (sometimes referred to as “clinical” or “significant” proteinuria), respectively. Microalbuminuria is a term that has been widely used to describe an increase in urinary albumin loss above the reference interval for healthy nondiabetic subjects, but at a level that is not generally detectable by less sensitive clinical tests such as reagent strips designed to measure total protein. The term microalbuminuria is somewhat misleading in that the albumin being measured is identical in form to that circulating in plasma, and the so-called microalbuminuric range refers to increased, not “micro-,” albumin losses. Current guidelines do not support the continuing use of this term, and the term albuminuria is used in this chapter.

Clinical Significance

The physiology and pathophysiology of renal protein handling and the clinical significance of proteinuria in specific clinical situations are discussed in more detail in Chapter 35; more general considerations follow. While reagent strip tests are commonly used in clinical practice, false negative and positive results are common. Most authors agree that positive tests require confirmation by laboratory measurement of the PCR or ACR on an early morning or random urine sample (see Fig. 21.1).

Most commonly, proteinuria reflects albuminuria and it is suggested that urinary total protein measurement can be replaced by urine albumin measurement. Strong evidence has linked increased urinary albumin loss to mortality and morbidity, including kidney disease progression, in people both with and without diabetes: albuminuria is generally held to be a more sensitive indicator of kidney disease than increased total protein loss. However, increases in albumin loss may also reflect overall changes in vascular permeability, and therefore may not indicate explicit deterioration in renal function. Increased urinary albumin loss is common among the general population and is not solely attributable to the presence of diabetes.

Points to Remember

Urinary Albumin

• Albumin (Mr 66 kDa) is predominantly retained in the circulation by the glomerular basement membrane due to its size and negative charge.
• In health, only small amounts of albumin are filtered. This is largely reabsorbed in the proximal tubule.
• Albuminuria may arise due to both glomerular (increased filtration) and tubular (decreased resorption) disease.
• Albumin is the predominant urinary protein in the setting of proteinuria.
• Due to high biological variability and nonrenal influences, albuminuria should be confirmed on at least two occasions, preferably with first morning samples.
• Measurement of the ACR allows the use of a spot sample with correction for variations in urinary concentration in an individual.

Creatinine

Creatininase

Creatininase (EC 3.5.2.10; creatinine amidohydrolase) catalyzes the conversion of creatinine to creatine. The creatine is then detected with a series of enzyme-mediated reactions involving creatine kinase, pyruvate kinase, and lactate dehydrogenase, with monitoring of the decrease in absorbance at 340 nm (see Fig. 21.2A). Initiating the reaction with creatininase allows for the removal of endogenous creatine and pyruvate in a preincubation reaction. The kinetics of the reaction are analytically problematic and a 30-minute incubation is required to allow the reaction to reach equilibrium. This shortcoming has been overcome by a kinetic approach but with a further reduction in the method’s ability to detect creatinine. Consequently, this approach is not widely used.

Creatininase and Creatinase

An alternative approach has been the use of creatinase (EC 3.5.3.3; creatine amidinohydrolase) that yields sarcosine and urea, the former being measured with further enzyme-mediated steps using sarcosine oxidase (EC 1.5.3.1). This produces (1) glycine, (2) formaldehyde, and (3) hydrogen peroxide (see Fig. 21.2B) with the latter being detected and measured with a variety of methods. Care must be taken, however, because of interference (e.g., by bilirubin) in the final reaction sequence. This problem has been minimized by adding potassium ferricyanide (with limited success) or bilirubin oxidase. The potential interference caused by ascorbic acid has been overcome by the inclusion of ascorbate oxidase (L-ascorbate:oxygen oxidoreductase; EC 1.10.3.3). The influence of endogenous intermediate creatine and urea has been minimized by adding a preincubation step and then initiating the reaction with creatininase. This system has been incorporated in a POC testing device using polarographic detection. An alternative detection system involves measurement of the reduction of nicotinamide adenine dinucleotide (NAD) by formaldehyde in the presence of formaldehyde dehydrogenase (see Fig. 21.2C).

FIG. 21.2 (A–D) Determination of creatinine using a variety of enzymatic methods. For additional details, see text. ADP, Adenosine diphosphate; ATP, adenosine triphosphate; NAD, nicotinamide adenine dinucleotide.

Creatinine Deaminase

Creatinine deaminase (EC 3.5.4.21; creatinine imino-hydrolase) catalyzes the conversion of creatinine to N-methylhydantoin and ammonia. Early methods concentrated on the detection of ammonia using either glutamate dehydrogenase or the Berthelot reaction. An alternative approach involves the enzyme N-methylhydantoin amidohydrolase (see Fig. 21.2D).

Dry Chemistry Systems

A number of multilayer dry reagent methods have been described for the measurement of creatinine using enzyme-mediated reactions. An early “two-slide” approach employed creatinine deaminase, with the ammonia diffusing through a semipermeable and optically opaque layer to react with bromophenol blue to give an increase in absorbance at 600 nm. A second multilayer film lacking the enzyme was used to quantitate endogenous ammonia, enabling blank correction. A later single-slide method used the creatininase-creatinase reaction sequence. Lidocaine metabolites have been reported to interfere with this method. The creatinine deaminase system described above has also been used and adapted for use as a POC testing device (see Fig. 21.2D). In all cases, the color produced in the film is quantified by reflectance spectrophotometry.

Other Methods

A definitive method employing IDMS was described by Welch in 1986. Gas or liquid chromatography-IDMS (GC-IDMS, LC-IDMS) are now accepted as the methods of choice for establishing the true concentration of creatinine in serum because of its excellent specificity and low imprecision. GC- and LC-IDMS methods have been approved by the JCTLM as reference measurement procedures for serum creatinine.

Quality Issues and Preanalytical Considerations With Creatinine Methods

Assessment of the methods used for the measurement of creatinine is complex by virtue of the number of variants of the Jaffe reaction and the innovations attempted using enzymatic procedures to overcome the limitations of the former. Although enzymatic methods are more expensive, they are used in dry chemistry systems (with their lower reagent requirement), including some POC testing devices. Kinetic Jaffe approaches generally predominate in wet chemistry analytical systems. Any laboratorian assessing a new creatinine method (e.g., as part of an analyzer purchase) should review the data for that method on common interferences. Despite criticism of the Jaffe methods, good correlation has been noted invariably between them and enzymatic procedures, with differences likely to be due as much to calibration as to interference.

Different methods for assaying serum creatinine have varying degrees of accuracy and imprecision. Mean within-individual biological variation for serum creatinine has been reported as approximately 6.0%, indicating a desirable analytical performance goal of less than 3.0%. Intralaboratory imprecision at a concentration of 88 μmol/L varies between approximately 2.0 and 8.4%. Clearly, many laboratories are therefore outside desirable and even minimum performance standards. Proficiency studies demonstrate that while between-laboratory CVs of approximately 3% are achievable within method groups, overall between-laboratory agreement across methods is much poorer. Further, within- and between-laboratory agreement deteriorates as serum creatinine concentration falls to within and below the reference interval; the exponential relationship between serum creatinine and GFR means that imprecision at lower creatinine concentrations contributes to greater error in GFR estimation than at higher creatinine concentrations.

Over the past 20 years, appreciation of chronic kidney disease (CKD) as a major public health issue and of its identification, staging and monitoring using GFR-estimating equations has led to increased focus on the measurement of creatinine. Creatinine-based estimates of GFR (see later) will clearly vary, depending on how accurate the creatinine measurement used in the calculation is. The more a method overestimates “true” creatinine, the greater will be the underestimation of GFR, and vice versa (see later).

Traceability of Serum Creatinine Measurement

Standardized serum matrix reference materials (SRM 967) with known creatinine concentrations (0.80 mg/dL [71 μmol/L] and 4.00 mg/dL [354 μmol/L]) have been prepared by the National Institute of Standards of Technology (NIST) and included in a list of higher order reference materials by the JCTLM. The material was value-assigned using mass spectrometry and issued in 2007. This material, in combination with GC-IDMS reference methodology, was used by reagent manufacturers to restandardize their methods and since 2009 most clinical laboratory methods have had calibration traceable to the reference measurement procedure and standard.

Although undoubtedly desirable, it must be recognized that standardization is only one arm of the problem. Standardization will not solve the problems of noncommutable calibrator materials and differential reactivity with noncreatinine chromogens across different patient samples, which can be resolved only by the use of highly specific creatinine methods. Wider adoption of enzymatic methods could further improve between-laboratory agreement in creatinine measurement, but these methods are used by a minority of laboratories at present.

Reference Intervals

Reference intervals for serum creatinine are method dependent. A systematic review of creatinine reference intervals in which studies were included only when, in particular, their calibration was traceable to the reference IDMS procedure; proposed adult reference intervals of 0.72 to 1.18 mg/dL (64 to 104 μmol/L) in men and 0.55 to 1.02 mg/dL (49 to 90 μmol/L) in women. These data were derived using an enzymatic (Roche Diagnostics Ltd) assay. Serum creatinine concentration in patients with untreated end-stage renal disease (ESRD) may exceed 11 mg/dL (1000 μmol/L).

Urinary creatinine excretion is higher in men (14 to 26 mg/kg per day, 124 to 230 μmol/kg per day) than in women (11 to 20 mg/kg per day, 97 to 177 μmol/kg per day). Creatinine excretion decreases with age. Typically, for a 70 kg man, creatinine excretion will decline from approximately 1640 to 1030 mg/day (14.5 to 9.1 mmol/day) with advancing age from 30 to 80 years. The measurement of urinary creatinine excretion has been found to be a useful indication of the completeness of a timed urine collection. Creatinine excretion is often used as a method of normalizing the urinary excretion of analytes, that is, the excretion of the test analyte (in millimoles or grams) is divided by the total amount of creatinine (in millimoles or grams) excreted in the same urine specimen. This method is a rough correction for volume differences between patient specimens. Similarly, expressing the concentration of a substance as a ratio to the creatinine concentration is a useful method of adjusting for urinary concentration differences in random (“spot”) urine samples.

Clinical Significance

Serum creatinine concentration is maintained within narrow limits predominantly by glomerular filtration. Consequently, both serum creatinine concentration and its renal clearance (“creatinine clearance”) have been used as markers of the GFR. The application and limitations of these tests are discussed later in this chapter.

Points to Remember

Creatinine

• Creatinine is produced at a fairly constant rate within an individual as a result of a breakdown of creatine within muscle tissue.
• Creatinine is freely filtered at the glomerulus and also secreted in the tubules to a lesser extent.
• Because it is predominantly excreted by the kidneys, plasma creatinine concentration is inversely proportional to GFR (halving the GFR approximately doubles the serum creatinine).
• In addition to GFR, other factors related to creatinine production affect plasma creatinine concentration, including age, gender, race, muscularity, certain drugs, diet, and nutritional status.
• Estimated GFR (eGFR) using the CKD-EPI_creat equation is currently recommended for clinical use (see Table 21.2).

Cystatin C

Biochemistry and Physiology

Cystatin C is a low-molecular-weight (12.8 kDa) protein synthesized by all nucleated cells whose physiologic role is that of a cysteine protease inhibitor. The gene has been sequenced, and the promoter region has been identified as that of the housekeeping type, with no known regulatory elements, and, consequently, the rate of cystatin C release into the circulation was initially considered to be constant. Over time a number of factors have been identified that can affect cystatin C production and thus the circulating concentration (e.g., thyroid hormone concentration). Unlike creatinine, muscle mass does not have a major effect on cystatin C concentration.

Cystatin C is removed from the circulation by the kidneys with no known extrarenal routes of elimination. Due to its small size and high isoelectric point (pI 9.2), it is more freely filtered than some other putative protein markers of GFR. Following filtration, cystatin C is essentially fully resorbed and broken down in the tubules usually leading to very low concentrations in the urine.

In addition to renal function and pathophysiological factors, serum cystatin C concentrations are also affected by age, gender, pregnancy, weight, and height. The influences of age and gender are taken into account with some cystatin C-based GFR estimating equations (see later).

Analytical Methods

Cystatin C is measured by immunoassay, the most practical approaches being particle-enhanced turbidimetric or nephelometric immunoassay, which can run on automated chemistry analyzers or specific nephelometers. Using these assays, a between-day imprecision of CV 3% to 6% can be expected at the upper limit of the reference interval (≅1.00 mg/L) and less than 3% at higher values. The use of immunoassays makes cystatin C relatively free of the interferences that affect Jaffe creatinine assays although the reagents are markedly more expensive than those used for creatinine measurement.

Traceability of Cystatin C Measurement

An international standard, ERM-DA471/IFCC Cystatin C in human serum, has been developed by an International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) working party. The material has verified commutability and is listed on the JCTLM database. Most manufacturers have restandardized their assays against this material, and the use of these traceable assays in research and in the routine laboratory is recommended to allow direct comparability of results.

Reference Intervals

While single reference intervals for adult males and females of all ages are commonly used, typically of the order 0.6 to 1.1 mg/L, it is known that cystatin C concentrations are higher in males than females and climb throughout adult life, reflecting the fall in GFR with advancing age seen in most populations. In a similar manner to serum creatinine, clinical decisions based on cystatin C are more likely to be made using eGFR values derived from cystatin C than on the serum cystatin C concentration itself, making the reference interval less relevant as a decision support tool.

Clinical Significance

Because cystatin C is produced at a relatively constant rate, freely filtered at the glomerulus and neither secreted nor reabsorbed intact in the proximal tubule, its concentration in serum can serve as a marker of GFR, with GFR being related to the reciprocal of serum cystatin C. In a similar manner to serum creatinine, equations have been developed to estimate GFR based on serum cystatin C with or without added demographic variables and with or without serum creatinine; some of these equations are given in Table 21.2. The Chronic Kidney Disease-Epidemiology Collaboration (CKD-EPI) cystatin C equations are well validated in adults.

Generally the use of cystatin C equations provides some, albeit modest, improvement over equations based on serum creatinine alone. An increasing body of evidence suggests that the use of cystatin C GFR-estimating equations gives improved risk prediction of death and kidney failure unrelated to the accuracy of the GFR estimation; this improved predictive ability may relate to non-GFR determinants of serum cystatin C concentration. To delineate risk, KDIGO suggest that, if confirmation of CKD is required, cystatin C should be measured in adults who have creatinine-eGFR between 45 and 59 mL/min per 1.73 m² and who do not have markers of kidney damage.

Points to Remember

Cystatin C

• Cystatin C is a low-molecular-weight protein (12.8 kDa) produced at a fairly constant rate from most body cells.
• A modest increase in production is seen in obesity, hyperthyroidism, and inflammation.
• Cystatin C is removed from the circulation by the kidneys. It is freely filtered at the glomerulus and largely destroyed in the tubules.
• The clinical utility of cystatin C can be improved with a GFR estimating equation—for example, the CKD-EPI_{cystatin C} equation.

TABLE 21.2

Representative Glomerular Filtration Rate Estimating Equations for Use in Adults
Abbreviation	Glomerular Filtration Rate Equation
Cockcroft and Gault	([(140 − age) × weight)] × 1.23)/(Scr) × 0.85 (if female)
MDRD (ID-MS traceable)	GFR (mL/min per 1.73 m²) = 175 × (Scr × 0.01131)^−1.154 × (age)^−0.203 × (1.210 if patient is black) × (0.742 if patient is female)
CKD-EPI_creat	141 × min(Scr × 0.01131/κ, 1)^α × max(Scr × 0.01131/κ, 1)^−1.209 × 0.993^age × 1.018 (if female) × 1.159 (if black), where Scr is serum creatinine, κ 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 of Scr/κ or 1, and max indicates the maximum of Scr/κ or 1.
CKD-EPI_cys	133 × min(Scys/0.8, 1)^−0.499 × max(Scys/0.8, 1)^−1.328 × 0.996^Age × 0.932 (if female), where min indicates the minimum of Scys/κ or 1, and max indicates the maximum of Scys/κ or 1.
CKD-EPI_creat-cys	135 × min(Scr × 0.01131/κ, 1)^α × max(Scr × 0.01131/κ, 1)^−0.601 × min(Scys/0.8, 1)^−0.375 × max(Scys/0.8, 1)^−0.711 × 0.995^Age × 0.969 (if female) × 1.08 (if black), where Scr is serum creatinine, Scys is serum cystatin C, κ 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 of Scr/κ or 1, and max indicates the maximum of Scr/κ or 1.
BIS1	3736 × (Scr × 88.4)^−0.87 × age^−0.95 × 0.82 (if female)
BIS2	767 × Scys^−0.61 × Scr^−0.40 × age^−0.57 × 0.87 (if female)
CAPA cystatin C equation	130 × cystatin C^−1.069 × age^−0.117 − 7

Urea

Biochemistry and Physiology

Urea (CO[NH₂]₂, Mr 60 Da) is the major nitrogen-containing metabolic product of protein catabolism in humans. During the process of protein catabolism, nitrogen derived from amino acids enters the urea cycle via intermediates, which include aspartate and ammonia. Urea is synthesized exclusively in the liver by the enzymes of the urea cycle (Fig. 21.3). The rate of urea production is dependent on the rate of protein catabolism from both dietary and endogenous sources, the latter being largely from muscle. Urea is distributed evenly throughout the total body water due to its ability to diffuse through most cell membranes facilitated by urea transporter proteins, and is therefore described as having a volume of distribution equal to the total body water.

Renal excretion accounts for more than 90% of urea removal from the body, with some losses through the gastrointestinal (GI) tract and skin. Urea is freely filtered at the glomerulus and, while it is neither actively reabsorbed nor secreted by the tubules, over 50% of urea moves passively out of the renal tubule, into the renal interstitium and back into the plasma. The back-diffusion of urea is dependent on urine flow rate, with less entering the interstitium in high-flow states (e.g., pregnancy) and more with low-flow situations (e.g., prerenal reduction in GFR due to fluid losses). Because water is resorbed to a far greater extent than the urea back-diffusion, urea is markedly concentrated during the process of urine formation, being present in urine at about 20 to 100 times the plasma concentration and is therefore the major contributor to urine osmolality. The role of urea in the functioning of the kidney is complex, involving specific urea transporters under the action of vasopressin.

FIG. 21.3 The urea cycle pathway. AAS, Argininosuccinate synthetase; AL, argininosuccinate lyase; AR, arginase; ADP, adenosine diphosphate, ATP, adenosine triphosphate; CPS I, carbamyl phosphate synthetase I (∗N-acetylglutamate as positive allosteric effector); OTC, ornithine transcarbamylase; P_i, inorganic phosphate.

Analytical Methods

The vast majority of urea measurements in clinical laboratories are undertaken using enzymatic methods based on preliminary hydrolysis of urea with urease to generate ammonium ion, which then is quantitated. This approach has been used in end-point, kinetic, conductimetric, and dry chemistry systems.

The analytical specificity of all routine methods is good and typical assay precision is usually small compared with the biological variation of serum urea indicating good analytical performance. Reference materials, both pure and matrix matched, as well as reference methods based on IDMS are available for urea and are listed on the JCTLM database, and assays from different providers generally provide acceptably consistent results.

Reference Intervals

Serum urea concentrations are higher in men than in women, and there is an increase in the upper reference limit throughout adult life by a factor of about 50% from the 20s to the 70s. Pregnancy is associated with lower urea concentrations compared with age-matched nonpregnant women due to the associated glomerular hyperfiltration. In the pediatric age range, from after the first 2 weeks, the first year of life is associated with low serum urea, followed by a rise to slightly above adult concentrations to the age of about 10 years, then falling to young adult values by the late teens.

A reference interval for serum urea in healthy adults is 2.1 to 7.1 mmol/L (6 to 20 mg/dL expressed as urea nitrogen). Specific reference intervals will depend on the age, diet, and gender balance of the reference population (e.g., in adults older than 60 years of age), a higher reference interval (e.g., 2.9 to 8.2 mmol/L [8 to 23 mg/dL]) may be used. On an average protein diet, urinary urea excretion expressed as urea nitrogen is 12 to 20 g/day (430 to 710 mmol/day).

The term blood urea nitrogen (BUN) is used in some countries to refer to serum urea tests, with results reported as mg/dL of BUN. The SI system recommends the use of the term urea, expressed in mmol/L. To convert mg/dL BUN to mmol/L urea multiply the value by 2.8 (or by 0.357 for the reverse).

Clinical Significance

Measurement of blood and serum urea has been used for many years as an indicator of kidney function. However, it is generally accepted that creatinine measurement provides better information in this respect. Serum and urinary urea measurement may still provide useful clinical information in particular circumstances. The measurement of urea in dialysis fluids is widely used in assessing the adequacy of renal replacement therapy (see Chapter 35).

Urea concentration in serum is significantly affected by the rate of production as well as the rate of removal, which not only limits its value as a test of kidney function, but also allows its use for a range of other factors. For example, urea production, and therefore plasma concentration, is increased by a high-protein diet, increased endogenous protein catabolism, such as occurs in many hospitalized patients, reabsorption of blood proteins after GI hemorrhage, and treatment with cortisol or its synthetic analogues.

Urea removal from the circulation can be reduced due to any cause of reduced glomerular filtration, including prerenal, renal, or postrenal factors. The differential response of urea relative to creatinine can be useful diagnostically. In obstructive postrenal conditions (e.g., malignancy, nephrolithiasis, and prostatism), both serum creatinine and urea concentrations will be increased, although in these situations the increase in serum urea is greater than that of creatinine because of increased back-diffusion. These considerations give rise to the main proposed clinical use of serum urea—namely, its measurement in conjunction with that of serum creatinine and subsequent calculation of the urea to creatinine ratio. This can be used as a crude discriminator between prerenal and intrinsic causes of reduced GFR. For a normal individual on a normal diet, the reference interval for the ratio is between about 49 and 81 mmol urea/mmol creatinine (12 and 20 mg urea/mg creatinine). In the setting of known acute kidney injury (AKI), a raised urea to creatinine ratio (>81 mmol/mmol) is thought to be more indicative of a prerenal cause than an intrinsic renal cause, such as acute tubular necrosis.

A low serum urea concentration occurs in the setting of low protein intake—for example, starvation or anorexia nervosa. This is less commonly seen among hospital inpatients who, although often do not consume adequate nutrients, do not exhibit a low serum urea due to the presence of significant catabolism of endogenous protein. Low serum urea concentration can also be seen with end-stage liver disease due to decreased urea synthesis.

Measurement of urinary urea output provides a crude index of overall nitrogen balance and may be used as a guide to replacement in patients receiving parenteral nutrition.

Uric Acid (Urate)

Biochemistry and Physiology

Uric acid (C₅H₄N₄O₃, Mr 168) is the major product of catabolism of the purine nucleosides, adenosine, and guanosine in humans (Fig. 21.4). These precursors are metabolized into uric acid in a series of metabolic steps finishing with the conversion of xanthine to uric acid by the action of xanthine oxidase. The bulk of excreted uric acid arises from the degradation of endogenous nucleic acids (approximately 400 mg/day) with a lesser contribution from dietary sources (300 mg/day). Overproduction of uric acid may result from increased synthesis of purine precursors. In humans, approximately 75% of uric acid excreted is lost in the urine; most of the remainder is secreted into the GI, where it is degraded to allantoin and other compounds by bacterial enzymes.

FIG. 21.4 Metabolism of purines: (A) Synthesis. (B) Catabolism. (C) Salvage pathways. ATP, Adenosine triphosphate.

Renal handling of uric acid is complex and involves four sequential steps: (1) glomerular filtration of virtually all the uric acid in capillary plasma entering the glomerulus; (2) near complete reabsorption in the proximal convoluted tubule; (3) secretion into the distal portion of the proximal tubule; and (4) further reabsorption in the distal tubule. The net urinary excretion of uric acid is 6% to 12% of the amount filtered.

The physicochemical properties of uric acid are important in considering uric acid concentrations in the circulation, in tissue, and in the kidneys. The first pKa of uric acid is 5.75; above this pH, uric acid exists chiefly as urate ion, which is more soluble than uric acid. At a urine pH below 5.75, uric acid is the preponderant form. On the basis of the ionization form in the circulation, it has been proposed that urate should be the preferred term rather than uric acid when describing serum measurements.

Sample Collection

Most blood collection tubes are suitable for sample collection and urate is sufficiently stable that no special collection or sample handling conditions are required. Traditionally, urinary uric acid excretion is determined in a 24-hour urine sample. If analysis is not undertaken promptly, alkalinization of the sample is recommended to maintain uric acid in solution.

Analytical Methods

Methods based on the enzyme uricase ([urate:oxygen] oxidoreductase; EC 1.7.3.3) are the most commonly used routine methods for measuring uric acid. Uricase acts on uric acid to produce allantoin, hydrogen peroxide, and carbon dioxide. The reaction can be observed in either the kinetic or the equilibrium mode. Most current enzymatic assays for serum urate involve a peroxidase system coupled with one of a number of oxygen acceptors to produce a chromogen. The step for quantitation of the hydrogen peroxide produced is sometimes referred to as a Trinder reaction. One popular method measures hydrogen peroxide with the aid of horseradish peroxidase and an oxygen acceptor to yield a chromogen in the visible spectrum. The most common oxygen acceptor used is 4-aminophenazone, together with phenol or a substituted phenol. The JCTLM lists pure substance and matrix matched certified reference materials for uric acid as well as IDMS reference methods and reference measurement services for analysis in serum and urine. Evidence suggests that most routine methods are meeting clinical needs. Interference in Trinder assays can be caused by antioxidants such as ascorbate (e.g., intravenous vitamin C), bilirubin, and unspecified interferants in serum from patients with kidney failure. Rasburicase is a urate-consuming enzyme (urate oxidase) used to treat patients with tumor lysis syndrome. Rasburicase can lower serum urate concentrations in blood collection tubes ex vivo unless both whole blood and serum are cooled before and after separation or treated by acidification.

Reference Intervals

The serum urate concentration increases gradually with age, rising about 10% between the ages of 20 and 60 years. A significant rise is seen in women after menopause, reaching concentrations similar to those found in men. Additionally, higher concentrations of serum urate are found with increases in waist circumference, body mass index, and other components of the metabolic syndrome. A population reference interval may require partitioning on the basis of gender and age, and will be affected by the metabolic status of the population. It can be argued that a clinical decision point for the relevant clinical question may be more useful than a population reference interval given the interaction with common comorbidities. A reference interval for serum urate has been reported to be 3.5 to 7.2 mg/dL (0.21 to 0.43 mmol/L) for males and 2.6 to 6.0 mg/dL (0.16 to 0.36 mmol/L) for females.

During pregnancy, serum urate concentrations fall during the first trimester and until about 24 weeks’ gestation, when values begin to rise and eventually exceed nonpregnant concentrations. Urinary uric acid excretion in individuals on a diet containing purines is 250 to 750 mg/day (1.5 to 4.5 mmol/day). Excretion may decrease by 20% to 25% on a purine-free diet to less than 400 mg/day (2.4 mmol/day).

Clinical Significance

The most common clinical use for urate assessment is risk assessment for gout and determination of therapy adequacy. Other clinical conditions where serum urate measurements have potential utility include cardiovascular risk assessment and a diagnosis of preeclampsia. Urine uric acid measurements may play a role in assessing the cause of hyperuricemia and in assessing the risk of renal stone formation.

Hyperuricemia

The major causes of hyperuricemia are summarized in Box 21.1. Asymptomatic hyperuricemia is frequently detected through biochemical screening, although there is no evidence for clinical benefit resulting from this practice.

Gout

Gout occurs when monosodium urate precipitates in joints and tissues from supersaturated body fluids. These deposits of urate are responsible for the clinical signs and symptoms. Gouty arthritis is caused by urate crystal formation in joint fluid and may be associated with deposits of crystals (tophi) in tissues surrounding the joint. The deposits may occur in other soft tissues as well, and wherever they occur they elicit an intense inflammatory response. The first metatarsophalangeal joint (big toe) is the classic site for gout. Gout is a condition characterized by occasional attacks and long periods of remission. It is important to appreciate that the serum uric acid concentration is often normal during an acute attack and is not a component of the diagnostic criteria. The demonstration of uric acid crystals in joint aspirate fluid is the pathognomonic sign for gout. For details on the diagnostic criteria and associated evidence base, readers are referred to the European League Against Rheumatism (EULAR) guidelines. This report also provides information on the risk factors for the development of gout, which represent those for increasing concentrations of serum uric acid; of these, the major ones are male gender, CKD, hypertension, and obesity.

Gout may be classified as primary or secondary. Primary gout is associated with essential hyperuricemia, which has a polygenic basis. In more than 99% of cases, the cause is uncertain, but the condition is probably due to a combination of metabolic overproduction of purines, decreased renal excretion, and increased dietary intake. Very rarely, primary gout is attributable to inherited defects of enzymes in the pathways of purine metabolism.

Secondary gout is a result of hyperuricemia attributable to several identifiable causes such as CKD; as a consequence of administration of drugs (in particular diuretics); and ketoacidosis or to lactic acidosis (by reducing tubular secretion of urate). Increased nucleic acid turnover and a consequent increase in catabolism of purines may be encountered in rapid proliferation of tumor cells and in massive destruction of tumor cells on therapy with certain chemotherapeutic agents.

Management of an acute attack of gout can be divided into broad strategies: management of the attack and the prevention of subsequent attacks. The main focus for acute management is pain relief. Avoidance of subsequent attacks is based on lifestyle and pharmacotherapy with the aim of reducing plasma urate, which is monitored to assess therapeutic success. An evidence-based treatment target for prevention of gout recurrence is a serum urate concentration less than 0.36 mmol/L.

Kidney Disease

Kidney disease associated with hyperuricemia may take one or more of several forms: (1) gouty nephropathy with urate deposition in renal parenchyma, (2) acute intratubular deposition of urate crystals, and (3) urate nephrolithiasis.

Kidney Stones

Uric acid kidney stones occur in approximately one in five patients with clinical gout. Although serum and urinary uric acid should be measured in stone formers, many uric acid stone formers do not demonstrate hyperuricuria or hyperuricemia. Uric acid stone formation is promoted by the passage of a persistently acid urine with undissociated uric acid (pKa 5.57) being relatively insoluble, whereas urate at pH 7.0 is greater than 10 times more soluble. Pure uric acid stones account for approximately 8% of all urinary tract stones and, unlike many of the calcium-containing stones, are radiolucent. Allopurinol is the mainstay of treatment of uric acid stones.

Preeclampsia

Preeclampsia is pregnancy-induced hypertension associated with proteinuria (>0.3 g/day) and often edema and may become life threatening for the mother or the fetus (see Chapter 44). The role of uric acid measurement in the management of preeclampsia is uncertain: measurement recommendations appear in some guidelines but not others. If assessing urate concentrations in this setting, it is important to use pregnancy-specific reference intervals.

Inherited Diseases

Hyperuricemia can be a feature of several inherited disorders of purine metabolism, most of which are rare, and the diagnosis requires support from a specialist purine laboratory. Readers are referred to specialist textbooks for further information.

Hypouricemia

Hypouricemia, often defined as serum urate concentrations less than 2.0 mg/dL (0.12 mmol/L), is much less common than hyperuricemia. It may be secondary to severe hepatocellular disease with reduced purine synthesis or xanthine oxidase activity, or defective renal tubular reabsorption of uric acid. Defective reabsorption may be congenital, as in generalized Fanconi syndrome, or acquired. Overtreatment of hyperuricemia with allopurinol or uricosuric drugs and cancer chemotherapy with 6-mercaptopurine or azathioprine (inhibitors of de novo purine synthesis) may also cause hypouricemia.

Cardiometabolic Outcomes and Urate

Recent publications have highlighted positive associations between serum urate concentrations and a range of important cardiovascular and health outcomes that are generally associated with the metabolic syndrome (e.g., obesity and insulin resistance, hypertension and renal disease, cardiovascular disease, type 2 diabetes, and all cause and cardiovascular mortality). More important, as well as indicating risk for patients with hyperuricemia, these relationships occur within the usually described “normal range” with the higher urate values associated with increasing risk. While these associations appear to be robust, it is less clear whether or not there is a causal association or whether urate-lowering therapy may be beneficial. The role of measurement of urate in the routine laboratory, validated clinical decision points, and appropriate management of patients based on the results remains to be determined; however, it is likely that there will be more roles for this analyte in the future.

Points to Remember

Urate

• Urate (uric acid) is the major end product of catabolism of the purine nucleosides.
• Excretion of urate is predominantly renal.
• Reference intervals for urate are influenced by many factors, including age, gender, menopausal status, diet, and waist circumference.
• Gout occurs when monosodium urate precipitates in joints and tissues from supersaturated body fluids, eliciting an intense inflammatory response.
• When treating gout, a target urate concentration of <6 mg/dL (<0.36 mmol/L) is used.

Assessment of Kidney Function: Glomerular Filtration Rate

The GFR is widely accepted as the best overall measure of kidney function. Progressive decrease in GFR is associated with the clinical signs and laboratory changes of kidney damage in all forms of kidney disease. Measuring GFR in established disease is useful in targeting treatment, monitoring progression, and predicting the point at which renal replacement therapy will be required. It is also used as a dosage guide to prevent toxicity by drugs excreted by the kidneys. There are a range of methods available to measure and estimate the GFR.

The Concept of Clearance

All tests for GFR are based on the clearance of a substance from the circulation by the kidneys. Renal clearance of a substance is defined as the volume of plasma from which the substance is completely cleared (removed) by the kidneys per unit of time. Provided a substance S is freely filtered in the glomerulus and neither secreted, resorbed, nor metabolized in the tubules, the renal clearance of S is equal to the GFR, and carries the same unit dimension (volume/time). Under these conditions the following equations apply.

where GFR = the flow rate in mL per minute of plasma through the glomerular membranes, equivalent to a clearance in units of mL of plasma cleared of a substance per minute; US = urinary concentration of the substance; V = volumetric flow rate of urine in milliliter per minute; and PS = plasma concentration of the substance (in the same units as the urinary concentration of the substance).

The units of milliliter per minute is the most commonly used unit for GFR and is recommended for universal adoption to avoid confusion from the use of different units.

Kidney size and GFR are roughly proportional to body size, with larger people having a higher GFR and vice versa; to remove this effect it is conventional to adjust GFR to a standard body surface area (BSA) of 1.73 m². There are a number of formulae available to enable BSA estimation (e.g., Du Bois and Du Bois, Haycock). A variety of markers, both exogenous (radioisotopic and nonradioisotopic) and endogenous, have been used to measure or estimate GFR with the choice of marker and procedure based on cost and availability as well as accuracy.

Exogenous Markers of Glomerular Filtration Rate

Protocols for direct measurement of GFR are based on exogenous markers, with urinary clearance of inulin considered to be the “gold standard.” There are many other exogenous markers and also different protocols for GFR determination. Markers may be given intravenously either by continuous infusion or single bolus and measured in serial urine or blood collections. The nonradioisotopic markers include inulin and iohexol, and the radiolabeled markers include labeled EDTA, DTPA, and iothalamate. Determination of GFR with exogenous markers is available in many hospitals and should be considered for clinical use when accurate GFR determination is required (e.g., prior to kidney donation, when dosing particularly toxic medications).

Endogenous Markers of Glomerular Filtration Rate

The most widely used endogenous marker of GFR is creatinine, expressed as its serum concentration, as renal clearance or included in equations for eGFR. Although a range of other low-molecular-weight compounds have been investigated for clinical use, only creatinine and cystatin C are recommended for routine use.

Creatinine

Serum creatinine concentration is a product of the rate of release into the circulation as well as the rate of removal, and awareness of the influence of both of these factors is required for an understanding of the use of creatinine measurements. Creatinine is produced in muscle from creatine at a fairly constant rate proportional to the amount of muscle present. Because muscle mass is affected by age, gender, race, nutrition, exercise, and other factors, the rate of production is variable between different people. The major route of creatinine removal from the circulation is by free filtration at the renal glomerulus although there is some additional loss into the urine by tubular secretion and by loss through the GI tract. Due to the key role of glomerular filtration in creatinine removal, the concentration of creatinine in the circulation is inversely related to GFR—that is, for the same creatinine production, a halving of the GFR will lead to an approximate doubling of the serum creatinine concentration. Importantly, creatinine is convenient and cheap to measure and is widely available.

A range of factors affect creatinine metabolism and measurement, which are important when they are used to assess renal function. Drug effects can be due to blockage or tubular secretion (e.g., cimetidine, trimethoprim) or interference in creatinine assays. In patients with CKD, extrarenal clearance of creatinine becomes important when caused by degradation as a result of bacterial overgrowth in the small intestine, further blunting the anticipated increase in plasma creatinine in response to falling GFR. An important factor for any creatinine-based interpretation is that serum creatinine can remain within the reference interval until notable kidney function has been lost. Additionally, serum creatinine measurement will not detect people with mildly reduced GFR (60 to 89 mL/min per 1.73 m²) and will fail to identify many patients with CKD and category 3A GFR (45 to 59 mL/min per 1.73 m²) (Fig. 21.5). Thus, although an increased serum creatinine concentration generally equates with impaired kidney function, a normal serum creatinine does not necessarily equate with normal kidney function. However, while the interpretation of creatinine concentration against population-based decision points is insensitive for the detection of CKD, changes in serum creatinine within an individual may be used as a sensitive tool for detecting changes in kidney function, whether the results are within or outside population reference intervals. In this setting, changes greater than expected by chance (i.e., that due to biological and analytical variation) are likely to indicate a significant change in GFR in a patient.

Creatinine Clearance

A measured creatinine clearance, performed using a 24-hour urine collection and serum collection, ideally during this period, has been used for many years as a marker of GFR. However, the difficulty in obtaining a complete, timed 24-hour urine collection, together with a high within-subject biological variation in creatinine excretion and the known overestimation of GFR due to creatinine tubular secretion make this test both inaccurate and imprecise as a measure of GFR. In spite of these limitations, creatinine clearance remains in common use in recommendations for drug-dosing decisions (see later).

Estimating Glomerular Filtration Rate

The approximate mathematical relationship between serum creatinine and GFR (GFR α 1/serum creatinine) can be improved by correcting for some of the confounding variables that influence this relationship. Many different equations have been derived that estimate GFR using serum creatinine and inputs of some or all of gender, body size, race, and age. These generally produce a better estimate of GFR than serum creatinine alone or measured creatinine clearance, and professional societies throughout the world have recommended that such estimates should be used in association with serum creatinine. In adults, several such equations have been widely used, including the Cockcroft and Gault equation, the Modification of Diet in Renal Disease (MDRD) Study equations, and the CKD-EPI equations.

The Cockcroft and Gault equation is one of the earliest of these equations (see Table 21.2). It remains widely used in the context of assessing drug dosages for patients with kidney impairment (see later). However, the requirement for the measurement of body weight, the lack of a version validated for standardized creatinine assays, and the availability of demonstrably better equations have limited its ongoing use.

The era of GFR estimation as a widely used simple clinical tool began with publication of the MDRD Study equation in 1999, followed by its abbreviated four variable (creatinine, age, gender, race [black/white]) form in 2000. The original MDRD equation was updated in 2006 to a version suitable for IDMS traceable creatinine assays and this was widely endorsed by national organizations. Generally, the MDRD equation was found to provide a more accurate assessment of GFR than the Cockcroft and Gault equation, with the important advantage of not requiring knowledge of weight, allowing automatic reporting of eGFR with the results for serum creatinine.

In 2009 the Chronic Kidney Disease Epidemiology Collaboration described a further equation (CKD-EPI_creat), again based on serum creatinine, gender, race, and age (see Table 21.2). This equation showed some improvement over MDRD, especially at higher levels of GFR (>60 mL/min per 1.73 m²). KDIGO have recommended that the CKD-EPI_creat equation should be used to estimate GFR.

Because the relationship between creatinine and kidney function varies among individuals, disease conditions, and populations, no GFR equation to date has found universal applicability. This remains an intense area of ongoing research. Other equations that have been developed are the CKD-EPI equations based on cystatin C, either alone or in combination with creatinine, and the Berlin Initiative Study (BIS) Group published equations with claimed superior performance in older people (see Table 21.2).

The relationship between creatinine and GFR has been shown to vary between some ethnicities, presumably due to differences in muscularity and dietary pattern. For example, the MDRD and CKD-EPI_creat equation publications showed a clear distinction between African American and non-African Americans. The requirement to adjust GFR estimating equations for race is a potential limitation to their global application and local implementation.

Limitations to creatinine-based GFR estimating equations include interferences in creatinine measurement and patient-specific factors. Any factor affecting creatinine measurement, such as bias, imprecision, drug effects, hemolysis, icterus, or lipemia, will directly affect the eGFR. Any relevant way in which a patient is different from those included in the trials to establish the equations will also affect the accuracy of the equation at an individual level. These include patients with AKI, in whom serum creatinine concentrations may change rapidly and with extremes of muscularity (e.g., bodybuilders, amputees, and people with muscle wasting disorders). The equations are also not suitable for patients on dialysis and for use during pregnancy; adult equations should not be used with patients under 18 years of age. Notwithstanding this, in general, equations improve the estimation of GFR compared with serum creatinine alone.

Reference Intervals

An average GFR in young healthy individuals is 110 mL/min per 1.73 m² (5th to 95th percentiles approximately 90 to 140 mL/min per 1.73 m²) with a gradual fall with increasing age. The age-related changes are also seen in outputs of eGFR equations. Age-related reference intervals are not widely used in clinical medicine with decision points based on CKD staging or advice for drug dosing providing the usual clinical support.

Glomerular Filtration Rate Measurement and Estimation: Future Considerations

Evaluation of kidney function remains an essential component of medical practice, and much has been achieved in recent years to improve and standardize estimates of GFR. However, there remains room for improvement, and research into new and better markers, equations, and procedures for the measurement of kidney function will continue. The challenge to the clinical lab is to use the best available tools in a coordinated manner so that evidence-based clinical decisions can be made. One particular focus should be collaboration between laboratories so that patients and doctors obtain the same clinical information irrespective the laboratory selected.

Proteinuria: Total Protein and Albumin

Sample Collection for Urinary Total Protein and Albumin Measurement

Analytical Methods and Traceability: Urinary Total Protein

Analytical Methods and Traceability: Urinary Albumin

Reference Intervals, Definitions of Proteinuria and Albuminuria

Clinical Significance

Creatinine

Biochemistry and Physiology

Sample Collection

Analytical Methods

Picrate Concentration

Hydroxide Concentration

Wavelength

Temperature

Compensation

Enzymatic Methods

Creatininase

Creatininase and Creatinase

Creatinine Deaminase

Dry Chemistry Systems

Other Methods

Quality Issues and Preanalytical Considerations With Creatinine Methods

Traceability of Serum Creatinine Measurement

Reference Intervals

Clinical Significance

Cystatin C

Biochemistry and Physiology

Analytical Methods

Traceability of Cystatin C Measurement

Reference Intervals

Clinical Significance

Urea

Biochemistry and Physiology

Analytical Methods

Reference Intervals

Clinical Significance

Uric Acid (Urate)

Biochemistry and Physiology

Sample Collection

Analytical Methods

Reference Intervals

Clinical Significance

Hyperuricemia

Gout

Kidney Disease

Kidney Stones

Preeclampsia

Inherited Diseases

Hypouricemia

Cardiometabolic Outcomes and Urate

Assessment of Kidney Function: Glomerular Filtration Rate

The Concept of Clearance

Exogenous Markers of Glomerular Filtration Rate

Endogenous Markers of Glomerular Filtration Rate

Creatinine

Creatinine Clearance

Estimating Glomerular Filtration Rate

Reference Intervals

Glomerular Filtration Rate Measurement and Estimation: Future Considerations