Renal Function Testing

Assessment of Glomerular Filtration Rate 

Measurement of GFR 

•GFR cannot be measured directly

•Remarkably constant in single individual under constant conditions

•Large variation between individuals with large spread of normal values

•Causes of interpatient variability include:

■Body size: GFR conventionally factored by 1.73 m2

■Sex: GFR approximately 8% higher in males

■Race

■Age: age-related decline in GFR, 0.75 to 1.0 mL/min/1.73 m2 (0.01 to 0.02 mL/s/1.73 m2) per year

■Pregnancy: GFR elevated as much as 50% in first trimester and onward; returns toward normal by 4 to 8 weeks postpartum

■Protein intake: GFR higher in patients on high-protein diet

■Diurnal variation: values tend to be about 10% higher in afternoon than at night

■Antihypertensive therapy: secondary to lowering of blood pressure; variable effect not directly predictable

■States associated with hyperfiltration: diabetes, obesity, acromegaly

•Clearance (C): the rate at which an indicator substance is removed from plasma per unit concentration; specifies a volume from which all of a substance is removed per unit time

•For a substance Z cleared by renal elimination:

Where Uz is urinary concentration of z, Pz is plasma concentration of Z, and V is urine flow rate

•If substance z is freely filtered and only excreted by GFR, then: GFR = UzxV/Pz

•Thus, plasma concentration of indicator is inversely related to GFR and GFR can be assessed from plasma concentration

•Requires an ideal filtration marker (Table 1) to ensure that elimination of substance is completely dependent on GFR

Table 1. Characteristics of an Ideal Marker for GFR Measurement

	Constant rate of production (or for exogenous marker can be delivered intravenously at a constant rate)
	Freely filterable at the glomerulus (minimal protein binding)
	No tubular reabsorption
	No tubular secretion
	No extrarenal elimination or metabolism
	Availability of an accurate and reliable assay
	For exogenous marker: safe, convenient, readily available, inexpensive, and does not influence GFR (physiologically inert)

Use of Endogenous Markers to Determine GFR 

•Creatinine (MW, 113 d)

•Metabolism:

■Generated in muscle by nonenzymatic conversion of creatine and phosphocreatinine

■Generation is proportional to muscle mass and is relatively constant

■Important role of liver in formation of creatinine through methylation of guanidine aminoacetic acid

•Levels vary according to diurnal and menstrual variations, race, and diet (and method of meat preparation)

•Excretion rate (urinary creatinine excretion × V) mg/kg/d:

■Males: 28.2 – 0.172 × age

■Females: 21.9 – 0.115 × age

•Serum levels can be affected by numerous factors (Table 2)

Table 2. Factors Affecting SCr

	Increase SCr	Decrease SCr
	Ketotic states, hyperglycemia (Jaffé)⁎ Cephalosporins (Jaffé)⁎ Flucytosine (enzymatic method)⁎ Cimetidine, trimethoprim(block secretion) Vigorous exercise Ingesting cooked meats	Dietary protein restriction Muscle wasting, malnutrition Bilirubin (Jaffé)⁎ Renal disease Advanced age Female sex Advanced liver disease

⁎ These factors affect the measurement process directly.

•Not an ideal marker since it also is excreted by tubular secretion:

■Proportion of total creatinine clearance (Ccr) due to tubular secretion increases as GFR decreases and Ccr leads to GFR overestimation by approximately 10 mL/min/1.73 m2 (0.17 mL/s/1.73 m2)

■In some subjects (eg, those with sickle cell anemia), this GFR overestimation can be much greater

•Because of reciprocal relationship between GFR and serum creatinine (SCr), a large change in GFR is initially required to raise SCr levels from normal; however, once SCr is elevated, small changes in GFR will raise it exponentially more

•1/Scr better reflects magnitude of GFR decline

•Laboratory determination of creatinine:

■Jaffé reaction: creatinine reacts with picrate under alkaline conditions to form a chromogen:

∘Positive interference from glucose, ascorbic acid, uric acid, acetoacetate, pyruvate, ketoacids; results in creatinine value 20% higher than true value

∘Negative interference; high serum bilirubin levels may cause spuriously low SCr values

■Enzymatic kinetic alkaline picrate method; less interference from noncreatinine chromogens and much more accurate

■Enzymatic assays (amidohydrolase method) have similar precision to the kinetic method

■All methods lose precision in lower range of assay; thus, SCr is insensitive in determining small changes of GFR from normal

■Autoanalyzers often are calibrated to different creatinine standards with substantial variation (up to 0.4 mg/dL [35 μmol/L]) between creatinine values from different laboratories

•Creatinine clearance:

■Calculated from 24-hour urine sample and single SCr (assumes steady state)

■Inaccuracies result from incomplete urine collections

■Not valid for patients not in steady state

■Overestimates GFR due to tubular secretion of creatinine (especially important at lower GFRs)

■Cimetidine blocks tubular secretion of creatinine and thus increases accuracy of Ccr; optimal dosing is not clear and blockade of tubular secretion often incomplete, thus limiting this technique

•SCr as a marker of GFR:

■Elevated level usually indicates reduced GFR

■Normal level does not exclude possibility of reduced GFR:

∘Factors may keep creatinine generation rate low (Table 2)

∘SCr may thus remain within normal ranges in these patients despite a significant GFR reduction

∘Especially important in patients with chronic kidney disease where patients restrict protein intake and may blunt rise in SCr despite falls in GFR

∘Liver disease where SCr may be low despite poor GFR

•Formulae for estimating GFR using SCr:

■Equations include factors related to creatinine generation and excretion (age, sex, race, body size)

■Rely on patients being in steady state

■Major limitation is due to variation in measurement of SCr with kinetic Jaffé reaction and autoanalyzers and from intrinsic variability of SCr

■Cockcroft-Gault equation predicts Ccr as Ccr = (140 − age) × (weight)/(72 × SCr) (multiply by 0.85 if female):

∘Overestimates Ccr in vegetarians and patients who are malnourished, on low-protein diet, obese, or edematous

∘In patients with renal disease, predicted Ccr overestimates GFR at low values

■Modification of Diet in Renal Disease (MDRD) equation predicts GFR more accurately than measured Ccr and is preferred method: GFR = 170 × [SCr (mg/dL)]−0.999 × [age]−0.176 × [0.762 if patient is female] × [1.18 if patient is black]:

∘Not validated across diverse ethnic populations, in patients age >60 years or <18 years, with diabetes

∘May underestimate GFR in stage 1 chronic kidney disease and overestimate GFR in stages 4 and 5

∘Failure to calibrate creatinine assay to laboratory that developed the estimating equation can introduce systematic error in estimated GFR, particularly at a high GFR

■Among children, the Schwartz and Counahan-Barratt formulae provide clinically useful estimates of GFR

■Note: Use of SCr to estimate GFR relies on steady state; in certain circumstances, clearance methods may be more reliable:

∘Extremes of age and body size

∘Severe malnutrition or obesity

∘Diseases of skeletal muscle

∘Paraplegia or quadriplegia

∘Vegetarian diet

∘Rapidly changing SCr

•Urea (MW, 60 d):

■One of first indicators used to measure GFR

■Shares few features of ideal marker and is poor measure of GFR

■Freely filtered but reabsorbed in proximal and distal nephron (urea clearance is less than GFR); urea reabsorption is substantial in states of decreased renal perfusion

■Urea production is variable and largely dependent on protein intake

■Many variables affect urea level (Table 3)

Table 3. Factors Affecting Serum Urea Nitrogen

	Increase Serum Urea	Decrease Serum Urea
	Dehydration Reduced renal perfusion (heart failure) Increased dietary protein Catabolic states: Fever Trauma GI bleeding Tetracyclines Corticosteroids	Volume expansion Pregnancy SIADH Restriction of dietary protein Liver disease Advanced renal disease

Abbreviations: GI, gastrointestinal; SIADH, syndrome of inappropriate secretion of antidiuretic hormone.

■State of diuresis affects urea clearance more than Ccr and is useful in differential diagnosis of acute renal failure (ARF) where blood urea nitrogen–creatinine ratio is increased when causes are prerenal

•Cystatin C (MW, 13,000 d):

■Cysteine proteinase inhibitor produced by all nucleated cells at constant rate

■Freely filtered and then absorbed and catabolized by renal tubules

■No significant urinary excretion

■Measurement by particle-enhanced nephelometric immunoassay (PENIA) with high degree of precision and accuracy

■Normal adult values range from 0.54 to 1.55 mg/dL

■Most but not all studies show that serum cystatin C is better index of GFR than SCr alone

■Factors that affect serum level of cystatin and that are independent of GFR are still controversial and possibly include older age, male sex, smoking, higher weight, thyroid disease, and higher levels of C-reactive protein

■Additional studies, across diverse populations to determine value as index of renal function, still required

Additional Reading 

1. Bauer JH, Brooks CS, Burch RN: Clinical appraisal of creatinine clearance as a measurement of glomerular filtration rate. Am J Kidney Dis 2:337-346, 1982

2. Levey AS: Nephrology forum: Measurement of renal function in chronic renal disease. Kidney Int 38:167-184, 1990

3. Nilsson-Ehle P, Grubb A: New markers for the determination of GFR: Iohexol clearance and cystatin C serum concentration. Kidney Int Suppl 46:S17-S19, 1994

4. Levey AS, Bosch JP, Lewis JB, Greene T, Rogers N, Roth D: A more accurate method to estimate glomerular filtration rate from serum creatinine: A new prediction equation. Ann Intern Med 130:461-470, 1999

5. Grubb AO: Cystatin C—Properties and use as diagnostic marker. Adv Clin Chem 35:63-99, 2000

6. Knight EL, Verhave JC, Spiegelman D, et al: Factors influencing serum cystatin C levels other than renal function and the impact on renal function measurement. Kidney Int 65:1416-1421, 2004

7. Coresh J, Astor BC, McQuillan G, et al: Calibration and random variation of the serum creatinine assay as critical elements of using equations to estimate glomerular filtration rate. Am J Kidney Dis 39:920-929, 2002

8. Cockcroft DW, Gault MH: Prediction of creatinine clearance from serum creatinine. Nephron 16:31-34, 1976

9. Luke RG: Urea and the BUN. N Engl J Med 305:1213-1215, 1981

10. Herrington D, Drusano G, Smalls U, et al: False elevation in serum creatinine levels. JAMA 252:2962, 1984 (letter)

11. Ibrahim H, Mondress M, Tello A, et al: An alternative formula to the Cockcroft-Gault and the Modification of Diet in Renal Diseases formulas in predicting GFR in individuals with type 1 diabetes. J Am Soc Nephrol 16:1051-1060, 2005

12. Froissart M, Rossert J, Jacquot C, et al: Predictive performance of the Modification of Diet in Renal Disease and Cockcroft-Gault equations for estimating renal function. J Am Soc Nephrol 16:763-773, 2005

Assessment of Renal Plasma Flow (RPF) 

Additional Reading 

1. Cole BR, Giangiacomo J, Ingelfinger JR, Robson AM: Measurement of renal function without urine collection: A critical evaluation of the constant-infusion technic for determination of inulin and para-aminohippurate. N Engl J Med 287:1109-1114, 1972

2. Smith HW, Goldring W, Chasis H: The measurement of the tubular excretory mass, effective blood flow and filtration rate in the normal human kidney. J Clin Invest 17:263-278, 1938

Assessment of Tubular Function 

Additional Reading 

1. Sweeney TE, Beuchat CA: Limitations of methods of osmometry: Measuring the osmolality of biological fluids. Am J Physiol 264:R469-R480, 1993

2. Zerbe RL, Robertson GL: A comparison of plasma vasopressin measurements with a standard indirect test in the differential diagnosis of polyuria. N Engl J Med 305:1539-1546, 1981

Additional Reading 

1. Halperin ML, Richardson RM, Bear R, et al: Urine ammonium: The key to the diagnosis of distal renal tubular acidosis. Nephron 50:1-4, 1980

2. Batlle DC: Segmental characterization of defects in collecting tubule acidification. Kidney Int 30:546-554, 1986

3. Sabatini S, Kurtzman NA: Pathophysiology of the renal tubular acidoses. Semin Nephrol 11:202-211, 1991

Assessment of Tubular Function in ARF 

•Useful in differential diagnosis of ARF, especially in distinguishing prerenal from renal causes

•Tubular function impaired with acute tubular necrosis

•Fractional excretion (FE) of sodium, urea, and uric acid allow assessment of tubular function (Table 4):

■Fractional excretion of substance x = [(U/plasma concentration of substance x)/(U/Scr)] × 100

■Caveats include:

∘Low FE sodium may be seen early in course of tubular injury accompanying rhabdomyolysis, sepsis, administration of radiocontrast materials, nonoliguric forms of ARF, nonsteroidal anti-inflammatory drug use, acute interstitial nephritis, and with acute glomerulonephritis; also may be seen with acute tubular necrosis in setting of vasoconstrictive states such as congestive heart failure and cirrhosis

∘High FE sodium may be seen in prerenal states in which there is impaired renal tubular reabsorption of sodium such as with diuretic use, bicarbonaturia, glucosuria, recent intravenous contrast administration, salt-wasting nephropathy, and mineralocorticoid deficiency

Table 4. Urinary Indices in ARF

	Urinary Study	Prerenal	Renal
	Specific gravity	>1.020	∼1.010
	Urinary osmolality (mOsm/kg)	>500	<300
	Urine sodium (mEq/L)	<20	>20
	Fractional sodium excretion (%)	<1	>2
	Fractional uric acid excretion (%)	<7	>15
	Fractional urea excretion	<35	>35
	Low-molecular-weight proteins (β2-microglobulin, lysozyme)	Low	High
	Brush-border enzymes (N-acetyl-β-glucosaminidase)	Low	High

NOTE. To convert osmolality in mOsm/kg to mmol/kg, multiply by 1; sodium in mEq/L to mmol/L, multiply by 1.

•Biomarkers of renal tubular injury are available:

■Their current role is more experimental than diagnostic

■No current “gold standard”

■Sample biomarkers include brush border enzymes such as N-acetyl-β-glucosaminidase, adenosine deaminase–binding protein, as well as proteins such as kidney injury molecule 1 (KIM-1), neutrophil gelatinase–associated lipocalin (NGAL), and sodium-hydrogen exchanger 3 (NHE3)

•Measurement of low-molecular-weight proteins that are readily filtered and usually reabsorbed by proximal tubule:

■May appear in urine when there is tubular injury

■These proteins include β2-microglobulin, amylase, lysozyme, and retinol-binding protein

■These measurements are not routinely useful

Additional Reading 

1. Carvounis CP, Nisar S, Guro-Razuman S: Significance of the fractional excretion of urea in the differential diagnosis of acute renal failure. Kidney Int 62:2223-2229, 2002

2. Rabb H: Evaluation of urinary markers in acute renal failure. Curr Opin Nephrol Hypertens 7:681-686, 1998

3. Miller TR, Anderson RI, Linas SL, et al: Urinary diagnostic indices in acute renal failure: A prospective study. Ann Intern Med 88:47-57, 1978

Assessment of Proteinuria 

Measurement of Total Urine Protein: Methodology 

•Semiquantitative tests for total urine protein:

■Precipitation tests use 5% sulfosalicylic acid, concentrated nitric acid, or 10% trichloroacetic acid to precipitate proteins in urine; quantity of precipitate is graded from 0 to +4

■Dipstick test uses a pH indicator dye (tetrabromophenol blue) buffered to pH of 3.0; proteins act to change color of indicator dye with color varying depending on concentration of protein present:

∘Detect total protein >10 to 20 mg/dL

■Tests are insensitive but have high specificity

■Dipstick false-negative results with immunoglobulin light chains, tubular proteins that have positive charges (Table 5)

Table 5. Causes of False-Positive and False-Negative Results in Urinary Measurement of Albumin or Total Protein

	False-Positive Results	False-Negative Results
	Dehydration: increased concentration of protein in urine	Excessive hydration: decreased concentration of protein in urine
	Hematuria Exercise (especially albumin) Urinary tract infections Extremely alkaline urine (pH > 8)	Other proteins that do not react with dipstick (eg, monoclonal protein)

■False-positive results (Table 5)

■They detect an abnormal concentration, not an abnormal excretion rate

■Useful for screening purposes

•Quantitative tests for total urine protein:

■24-hour collection:

∘Urinary protein is precipitated with trichloroacetic or sulfosalicylic acid

∘Turbidity is measured with photometer or nephelometer and compared to a standard

∘Adequacy of collection ensured by concomitant measurement of urinary creatinine excretion:

□Normal value <150 mg/d

□Iodinated contrast material can falsely elevate the protein concentration

■Single voided specimen:

∘Ratio of protein to creatinine concentration in random urine can provide simple estimate of daily protein excretion

∘Best to obtain serial specimens at same time of day given circadian variation of urine protein excretion:

□Ratio corrects for variations in urine concentration

□Kidney Disease Outcomes Quality Initiative recommends first morning or random spot urine to monitor proteinuria

■Good correlation with values obtained by 24-hour specimens once creatinine excretion is known

■24-hour specimens or even overnight collections with calculation of the protein-creatinine ratio may lead to better true estimates of daily urine protein excretion and exclude diurnal variations in protein excretion

Spot Versus Timed Urine Collections in Assessment of Proteinuria/Albuminuria 

•See Table 6

Table 6. Comparisons of Methods for Assessment of Proteinuria

	Random Urine for Albumin-Creatinine Ratio	Morning Urine for Albumin-Creatinine Ratio	Timed Collections for Albumin Excretion
	Convenient	Convenient	Inconvenient
	Lower creatinine excretion in women: higher values of albumin-creatinine ratio		Not an issue
	Lower creatinine excretion in elderly: higher values of albumin-creatinine ratio		Not an issue
	Greater creatinine excretion in African Americans: lower values of albumin-creatinine ratio		Not an issue

•Collections of 24-hour or timed urine specimens are associated with high error rate and are inconvenient

•Studies comparing spot urine albumin-creatinine or protein-creatinine ratio with timed specimens have shown correlation coefficients ranging from 0.6 to 0.96

•First morning urine specimen is preferred and shows best correlation with 24-hour value

•Detailed assessments of precision and bias in the accuracy of spot urine specimens versus timed collections is not adequately known

Interpretation of Proteinuria 

•Electrophoretic pattern of urinary proteins (Fig 1):

■Glomerular proteinuria: albuminuria is hallmark (making up 60% to 90% of total proteinuria)

■Tubular proteinuria: low-molecular-weight proteins predominate with total urine protein rarely >2 g/d:

∘Impaired tubular reabsorption of low-molecular-weight proteins, or

∘Overproduction of low-molecular-weight proteins, such as light chains in myeloma

■Selective proteinuria expressed as clearance of IgG over clearance of transferrin is reliable indicator of severity and reversibility of abnormalities of glomerular proteinuria; patients with highly selective proteinuria have milder tubulointerstitial damage, improved prognosis, and better response to therapy:

∘Selectivity index (SI) = urine IgG/serum IgG × serum transferrin/urine transferrin

∘SI ≤0.10 highly selective; SI ≥0.11 and ≤0.20 moderately selective; SI ≥0.21 nonselective

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Fig 1. Electrophoretic patterns of serum and urine in patients with abnormal protein excretion.

•Patterns of proteinuria:

■Intermittent:

∘Due to hemodynamic alterations in permselectivity

∘Associated with fever, exercise, stress

∘Benign prognosis

■Orthostatic:

∘Proteinuria only in erect position with total proteinuria usually <1 g/d

∘Diagnosis with split 24-hour urine specimen: with 16-hour collection while patient upright and 8-hour collection while recumbent

∘Benign condition

■Persistent:

∘Almost invariably sign of structural renal disease and often requires renal biopsy for definitive diagnosis

Additional Reading 

1. Abuelo JG: Proteinuria: Diagnostic principles and procedures. Ann Intern Med 98:186-196, 1983

2. Weber MH: Urinary protein analysis. J Chromatogr 429:315-344, 1988

3. Ginsberg JM, Chang BS, Matarese RA, Garella S: Use of single voided urine samples to estimate quantitative proteinuria. N Engl J Med 309:1543-1546, 1983

4. Schwab SJ, Dunn FL, Feinglos MN: Screening for microalbuminuria: A comparison of single sample methods of collection and techniques of albumin analysis. Diabetes Care 15:1581-1584, 1992

5. Rodby RA, Rohde RD, Sharon Z, et al: The urine protein to creatinine ratio as a predictor of 24-hour urine protein excretion in type 1 diabetic patients with nephropathy. The Collaborative Study Group. Am J Kidney Dis 26:904-909, 1995

6. Zelmanovitz T, Gross JL, Oliveira JR, et al: The receiver operating characteristics curve in the evaluation of a random urine specimen as a screening test for diabetic nephropathy. Diabetes Care 20:516-519, 1997