Renal Function Tests

Specimen Collection

Specimen collection requirements depend on the procedure or test requested. Generally, no additional patient preparation is required for serum creatinine and blood urea nitrogen (BUN) levels, and a random blood sample is sufficient. However, recent high protein ingestion may significantly increase serum creatinine and urea levels. In addition, hydration status can have a considerable impact on BUN measurement.

For timed urine collections such as the 24-hour urine creatinine clearance, accurate collection over the entire period is crucial, as under- or over-collection can significantly affect the results. Hence, a 5- to 8-hour timed collection may be preferable to a 24-hour collection, especially outside a hospital setting.[5][6][7]

Collecting midstream urine for urine analysis is preferred as this sample is less likely to be contaminated by epithelial cells and commensal bacteria.

Procedures

Assessment of Renal Function

Several clinical laboratory tests help investigate and evaluate kidney function. Clinically, the most practical tests for assessing renal function are those that estimate the glomerular filtration rate (eGFR) and quantify proteinuria (albuminuria).

Glomerular Filtration Rate

The best overall indicator of the glomerular function is the glomerular filtration rate (GFR). GFR is the rate in milliliters per minute at which substances in plasma are filtered through the glomerulus; in other words, the clearance of a substance from the blood. The normal GFR for an adult male is 90 to 120 mL/min. However, this number varies significantly by age. Some studies suggest a decrease of 7.5 mL/min/1.73m² after 30 years due to aging processes. Therefore, an otherwise healthy 70-year-old individual may have a GFR of 60 mL/min/1.73m².[8][9][8]

The characteristics of an ideal marker of GFR are as follows: 

• It should appear endogenously in the plasma at a constant rate
• It should be freely filtered at the glomerulus
• It should be neither reabsorbed nor secreted by the renal tubule
• It should not undergo extrarenal elimination.

As no such endogenous marker currently exists, exogenous markers of GFR are used. Assessment of GFR using inulin, a polysaccharide, is considered the reference method for estimating GFR. This method involves the infusion of inulin and then the measurement of blood levels after a specified period to determine the inulin clearance rate. Other exogenous markers used are radioisotopes such as chromium-51 ethylenediaminetetraacetic acid (51 Cr-EDTA) and technetium-99-labeled diethylenetriaminepentaacetic acid (99 Tc-DTPA). The most promising exogenous marker is the non-radioactive contrast agent, iohexol, especially in children.

The inconvenience associated with the use of exogenous markers, specifically that the testing has to be performed in specialized centers with the ability to assay these substances, has encouraged the use of endogenous markers.

Creatinine

The most commonly used endogenous marker for assessing glomerular function is creatinine. The calculated creatinine clearance is used to provide an indicator of GFR. This process involves collecting urine over 24 hours or over another accurately timed period of 5 to 8 hours, as 24-hour collections are unreliable. Creatinine clearance is calculated using the equation:

C = (U_Cr × V) / P_Cr

C=clearance, U=urinary concentration (mg/dL), V=urinary flow rate (volume/time in mL/min), and P=plasma concentration (mg/dL)

Creatinine clearance should be corrected for body surface area. Improper or incomplete urine collection is a major issue affecting the accuracy of this test; hence, accurately timed collection is essential. Furthermore, due to tubular secretion, creatinine overestimates GFR by around 10% to 20%.

Creatinine is the by-product of creatine phosphate in muscle, and it is produced at a constant rate by the body. For the most part, creatinine is cleared from the blood entirely by the kidney. Decreased clearance by the kidney results in increased blood creatinine. The amount of creatinine produced per day depends on muscle mass. Thus, there are different creatinine ranges in males and females and lower creatinine values in children and those with decreased muscle mass. Dietary factors also influence creatinine levels. Creatinine can change as much as 30% after ingesting red meat. During pregnancy, GFR increases, leading to lower creatinine levels in pregnant women. In addition, serum creatinine indicates renal impairment at a relatively late stage—renal function is decreased by up to 50% before a rise in serum creatinine is observed.

Serum creatinine is also used in GFR estimating equations such as the Modified Diet in Renal Disease (MDRD) and the Chronic Kidney Disease-Epidemiology Collaborative Group (CKD-EPI) equations. The CKD-EPI group has also developed complex equations incorporating serum creatinine and cystatin C, using a population comprising healthy individuals and CKD patients; these equations are preferred when estimating GFRs in multi-ethnic populations due to their reduced bias.[10][11][12] A recent review by Inker et al presented new equations using cystatin C and creatinine without race that show an improved correlation between measured and calculated GFR. Further details of these complex equations can be found in this study and supplementary materials.[13] 

Some support the use of race as a qualitative factor to estimate muscle mass, but the trend is toward removing race from GFR calculations. These equations were generally formulated for White Americans with a modification factor for Black Americans, supposing increased GFR for the same creatinine level. Race can be categorized as Black or non-Black, or additional race categories can be considered, such as Asian, Hispanic, or Native American. Some studies have found that Blacks in Europe do not have a significantly elevated GFR for a given creatinine level, and other studies have found that adding a race variable to creatinine-based equations in Africans or Asians does not improve the correlation between estimated and calculated GFR.[14][15][16][17] No clear consensus is present, and many guidelines suggest using cystatin C as a marker instead of creatinine, as cystatin C is not muscle-mass dependent. In addition, further research is required to identify additional compounds consistent across age, sex, and race to estimate GFR.[16][18][19]

The CKD-EPI formulas have undergone several iterations.[20] Although some experts may prefer eGFR equations using cystatin or both cystatin and creatinine, in practice, cystatin is not widely measured. Therefore, the National Kidney Foundation (NKF) and the American Society of Nephrology (ASN) Task Force recommend using the CKD-EPI creatinine-based equation.[15] This formula has been adopted by most major laboratories for estimated GFR, including Quest and Labcorp.

The CKD-EPI equation: eGFR = 142 × min(SCr/κ,1)^α × max(SCr/κ,1)^−1.200 × 0.9938^Age × 1.012 (if female)

(Abbreviations/units: SCr is serum creatinine in mg/dL, κ=0.7 for females and 0.9 for males, α=−0.329 for females and −0.411 for males, min=the minimum of S/κ or 1, and max=the maximum of S/κ or 1)

For estimating GFR in children, the Chronic Kidney Disease in Children Study (CKiD or Schwartz bedside) equation is commonly used, which uses serum creatinine (mg/dL) and the child's height (cm).[21] Another formula, the Schwartz-Lyon equation, has also been used for children (younger than 18) and is believed to be more accurate compared to CKD-EPI when measured GFR is lower than 75 mL/min/1.72 m².[22][23] The CKD-EPI equation cannot be used in children, and it overestimated GFR in young adults aged 18 to 39. Modifications to the CKD-EPI formula using sex-specific creatinine growth curves for children and adults aged 18 to 40 allow a well-validated improvement of eGFR; this formula is also referred to as CKD-EPI40.[22]

GFR is classified into the following stages based on kidney disease. Stage 1 CKD refers to patients with a normal GFR but other signs of possible renal structural damage, such as proteinuria.

Kidney Disease Improving Global Outcomes (KDIGO) stages of CKD are as follows:

• Stage 1: GFR greater than 90 mL/min/1.73 m²  
• Stage 2: GFR 60 to 89 mL/min/1.73 m²
• Stage 3a:  GFR 45 to 59 mL/min/1.73 m²
• Stage 3b: GFR 30 to 44 mL/min/1.73 m²
• Stage 4: GFR 15 to 29 mL/min/1.73 m²
• Stage 5: GFR less than 15 mL/min/1.73 m² (end-stage renal disease)

These stages provide an easier estimation of GFR without requiring urine collection or the use of exogenous materials. However, as they use serum creatinine, they are also affected by the issues around serum creatinine measurement; hence, the correction for different variables is required. In addition, these equations using a single serum creatinine presuppose a stable creatinine; therefore, they are often inaccurate in the frequent situation of rapidly changing creatinine levels.

Blood Urea Nitrogen

Urea, or BUN, is a nitrogen-containing compound formed in the liver as the end product of protein metabolism and the urea cycle. About 85% of urea is eliminated through the kidneys, whereas the rest is excreted through the gastrointestinal (GI) tract. Serum urea levels increase in conditions where renal clearance decreases, such as acute and chronic renal failure or impairment. Urea may also increase in other conditions not related to renal diseases, such as upper gastrointestinal bleeding, dehydration, catabolic states, and high protein diets. Urea may be decreased in starvation, low-protein diet, and severe liver disease. Serum creatinine is a more accurate assessment of renal function compared to urea; however, urea is increased earlier in renal disease.

When BUN levels are increased, the BUN-to-creatinine ratio can be useful to differentiate pre-renal from renal causes. In pre-renal disease, the ratio is close to 20:1, whereas in intrinsic renal disease, it is closer to 10:1. Upper gastrointestinal bleeding can be associated with a very high BUN to creatinine ratio (sometimes >30:1).

Cystatin C

Cystatin C is a low-molecular-weight protein that functions as a protease inhibitor produced by all nucleated cells in the body. Cystatin C is formed at a constant rate and freely filtered by the kidneys. Serum levels of cystatin C are inversely correlated with the GFR. In other words, high values indicate low GFRs, whereas lower values indicate higher GFRs, similar to creatinine. The renal handling of cystatin C differs from creatinine. Although glomeruli freely filter both, once cystatin C is filtered, it is reabsorbed and metabolized by proximal renal tubules, unlike creatinine. Thus, under normal conditions, cystatin C does not enter the final excreted urine to any significant degree. Cystatin C is measured in serum and urine. The advantages of cystatin C over creatinine are that it is not affected by age, muscle bulk, or diet, and many reports have indicated that it is a more reliable marker of GFR compared to creatinine, particularly in early renal impairment when creatinine levels are within normal range. Cystatin C has also been incorporated into eGFR equations, such as the combined creatinine-cystatin KDIGO CKD-EPI equation.

Cystatin C concentration may be affected by the presence of cancer, thyroid disease, and smoking. Evidence suggests that thyroid hormones increase cystatin C production.[24]

Albuminuria and Proteinuria

Albuminuria refers to the abnormal presence of albumin in the urine. The term microalbumin is now considered obsolete as there is no such biochemical molecule; the condition is now referred to only as increased urine albumin. Albuminuria is used as a marker to detect incipient nephropathy in diabetics. Albuminuria is an independent marker for cardiovascular disease as it connotes increased endothelial permeability, and it is also a marker for chronic renal impairment. Urine albumin may be measured in 24-hour urine collections or early morning or random specimens as an albumin/creatinine ratio. The presence of albuminuria on 2 occasions, excluding a urinary tract infection, indicates glomerular dysfunction. The presence of albuminuria for 3 or more months is indicative of CKD. Frank proteinuria is defined as greater than 300 mg/d of protein. Normal urine protein is up to 150 mg/d (approximately 30% to 40% albumin, 10% to 20% globulins, and 50% uromodulin [Tamm-Horsfall protein]).[25]

Increased amounts of protein in the urine may be due to the following:

• Glomerular proteinuria: Caused by defects in the selectivity of the glomerular filtration barrier to plasma proteins, such as glomerulonephritis and nephrotic syndrome
• Tubular proteinuria: Caused by incomplete tubular reabsorption of proteins, such as interstitial nephritis
• Overflow proteinuria: Caused by increased plasma concentration of proteins, such as Bence-Jones protein and myoglobinuria
• Urinary tract inflammation or tumor

Urine protein may be measured using either a 24-hour urine collection or a random urine protein-to-creatinine ratio (an early morning sample is preferred as it is a close representative of the 24-hour sample).

The KDIGO classification defines 3 stages of albuminuria: 

• A1: Less than 30 mg/g creatinine
• A2: 30 to 300 mg/g creatinine
• A3: Greater than 300 mg/g creatinine

In nephrotic syndrome, urine protein excretion exceeds 3.5 g/d and is associated with edema, hypoalbuminemia, and hypercholesterolemia.

Tubular Function Tests 

The renal tubules play a crucial role in reabsorbing electrolytes and water and maintaining acid-base balance. Electrolytes such as sodium, potassium, chloride, magnesium, phosphate, and glucose can be measured in urine. Measurement of urine osmolality allows for assessment of the concentrating ability of urine tubules. A urinary osmolality higher than 750 mOsm/kg H₂O implies a normal concentrating ability of tubules. A water deprivation test can be used to exclude nephrogenic diabetes insipidus. In distal renal tubular acidosis, an ammonium chloride test can be used to confirm the diagnosis of distal renal tubular acidosis with failure to acidify the urine to a pH of less than 5.3. In Fanconi syndrome, aminoaciduria, glycosuria, phosphaturia, and bicarbonate wasting (proximal renal tubular acidosis) are present.

Urine Analysis

Urine analysis involves evaluating urine characteristics to aid in disease diagnosis, through physical observation, chemical, and microscopic examination. The physical inspection involves assessing color and clarity. Normal urine is straw-colored, whereas darker urine may indicate dehydration. Red urine may indicate hematuria or porphyria or could represent the dietary intake of food such as beets. Cloudy urine may be observed in pyuria due to urinary tract infection. Specific gravity indicates renal concentrating ability, which can be measured using refractometry or, chemically, a urine dipstick. The physiological range for specific gravity is 1.003 to 1.030. Specific gravity is increased in concentrated urine and decreased in dilute urine. 

The urine dipstick provides qualitative analysis of different analytes in urine using chemical analysis.

The urine dipstick uses dry chemistry methods to detect protein, glucose, blood, ketones, bilirubin, urobilinogen, nitrite, and leukocyte esterase and can be performed as a point-of-care test. The color changes following the urine's interaction with the chemical reagents impregnated on the dipstick paper are compared to a color chart guide to interpret the results.

In healthy urine specimens, urine protein is negative. Bilirubin is not detected in normal urine. Glucose is not detected in healthy patients but may be observed in diabetes mellitus, pregnancy, and renal glycosuria. Ascorbic acid (vitamin C) supplements may cause false-negative results for hemoglobin, leukocyte esterase, nitrite, and glucose.[26]

Blood may be present after renal tract injury or infection, with ascorbic acid causing a falsely negative result. The urine dipstick detects the globin portion of hemoglobin and thus cannot detect the difference between myoglobin and hemoglobin in the urine.

In addition, both intact red blood cells (RBCs) and hemoglobinuria are detected. The presence of blood on a urine dipstick test with normal RBC indicates rhabdomyolysis and can help differentiate it from hematuria, where RBCs are also detected on the urine dipstick. In normal urine, RBC per high-power field is between 0 and 3, and white blood cells (WBCs) are between 0 and 5. Ketones are present in fasting, severe vomiting, and diabetic ketoacidosis. Urine dipstick only detects acetoacetate and acetone, not the ketone beta-hydroxybutyrate. Bilirubin is detected in the presence of conjugated hyperbilirubinemia. Urobilinogen may typically be present, but it is absent in conjugated hyperbilirubinemia and increased in the presence of prehepatic jaundice and hemolysis. Nitrite and leucocyte esterase are indicators of urinary tract infection. Some bacteria, for example, Enterobacteriaceae, convert nitrates to nitrites.

The microscopic urinalysis involves a wet-prep analysis to identify cells, casts, crystals, and microorganisms. RBC casts typically denote glomerulonephritis, whereas WBC casts are consistent with pyelonephritis. The presence of WBCs and WBC casts indicates infection; RBCs indicate renal injury; and RBC casts indicate tubular damage or glomerulonephritis. Hyaline casts consist of protein and may occur in glomerular disease. Fatty casts are observed in nephrotic syndrome. Crystals may also be identified in urine and are indicative of the following conditions:

• Triple phosphate crystals resemble a coffin lid found in alkaline urine and urinary tract infections.
• Uric acid crystals are needle-shaped and are associated with gout.
• Oxalate crystals are envelope-shaped and are present in ethylene glycol poisoning or primary and secondary hyperoxaluria.
• Cystine crystals are hexagonal and are observed in cystinuria.

The best specimen for urine analysis is a freshly voided midstream urine sample. Midstream urine is less likely to be contaminated by commensal bacteria and epithelial cells.

Acute Versus Chronic Renal Impairment

Acute renal impairment or acute kidney injury refers to the sudden onset of kidney injury within a period of a few hours or days. CKD is caused by long-term diseases such as hypertension and diabetes. Causes of acute kidney injury can be divided into the following:

• Causes that result in decreased blood flow to the kidneys (pre-renal causes), such as hypotensive and cardiogenic shock, dehydration, and blood loss from major trauma
• Causes that result in direct damage to the kidneys (renal/intrinsic causes), such as damage to kidneys by nephrotoxic medications and other toxins, sepsis, cancers such as myeloma, autoimmune diseases or conditions that cause inflammation, or damage to the kidney tubules
• Causes that result in blockage of the urinary tract, such as bladder, prostate, or cervical cancer, large kidney stones, and blood clots in the urinary tract

Importantly, pre-renal kidney injury may progress to acute tubular necrosis and cause intrinsic renal injury.

Urine output is a useful tool for evaluating kidney function and is used in guidelines to define acute kidney injury. Patients with acute kidney injury present with oliguria, defined as less than 400 mL/d or less than 0.5 mL/kg/h for more than 6 hours.[27] The RIFLE classification (risk, injury, failure, loss of kidney function, and end-stage kidney disease) is based on serum creatinine, GFR changes, and urine output determinants. The Acute Kidney Injury Network (AKIN) classification criteria for acute kidney injury also uses serum creatinine changes and urine output; however, it does not rely on GFR changes and does not require a baseline serum creatinine.

Investigations that assist in determining if the renal injury is pre-renal, renal, or post-renal include measuring urine specific gravity, which is increased (greater than 1.020) in dehydration and pre-renal causes. The presence of WBCs and RBCs, tubular epithelial cells, casts, or crystals in the urinary sediment under light microscopy can assist in the differential diagnosis.

Fractional excretion of sodium (FeNa) is useful in distinguishing acute tubular necrosis from pre-renal uremia and requires the measurement of creatinine and sodium in spot urine specimens. Fractional excretion is calculated using the following formula:

FeNa = 100 × (urinary sodium × serum creatinine)/(serum sodium × urinary creatinine).

A value of less than 1% indicates a pre-renal cause, and values greater than 2% indicate intrinsic causes. Spot urine sodium concentrations of less than 20 mmol/L indicate pre-renal acute kidney injury. Fractional excretion of urea calculated similarly to FeNa using serum urea and urine urea instead of sodium can also be used to determine the presence of pre-renal versus intrinsic acute kidney injury, with values less than 35% suggesting pre-renal injury. In general, urine osmolality of more than 500 mOsm/kg is associated with pre-renal causes, whereas an osmolality similar to serum (approximately 300 mOsm/kg) reflects an intrinsic cause. However, in patients receiving diuretic therapy, the FeNa is not reliable.[28]

Novel Biomarkers

Several new biomarkers have been reported to be useful for determining acute kidney injury and have utility in differentiation between acute kidney injury and stable CKD and pre-renal and intrinsic acute kidney injury. These biomarkers include low-molecular-weight proteins and fall into 2 main categories as follows:

• Proteins and chemicals present in the systemic circulation, which undergo glomerular filtration
  • Cystatin C
  • Beta-2-microglobulin
  • Retinol-binding protein
  • Recently recognized filtration markers pseudouridine, acetylthreonine, myoinositol, phenylacetylglutamine, and tryptophan [14]

• Proteins that are produced in response to cellular or tissue injury
  • Neutrophil gelatinase–associated lipocalin (NGAL)
  • Kidney injury molecule 1 (KIM-1)
  • L-type fatty acid–binding protein (L-FABP)
  • Fibroblast growth factor 23 (FGF23)
  • Beta-trace protein

The optimum clinical utility of these substances is further clarified through ongoing research.

Indications

Indications for assessing renal function vary, ranging from acute emergency to chronic settings. Primarily, renal function tests are performed to identify renal disease, determine appropriate patient management, and prevent further deterioration of renal function. Further indications in patients in whom renal disease has been identified are to stage the level or type of renal disease, monitor the progression of renal disease to ensure optimal management occurs timeously, and monitor response to interventions. In other scenarios, renal function tests may be required to establish and monitor renal function where a known or possibly nephrotoxic therapeutic agent is initiated for patient management. Renal function tests are also indicated in those who are transplant donors to assess the initial donor suitability and, after that, to detect any significant deterioration of renal function post-donation. Renal function tests can also be used to identify which area of the functional unit of the kidney (nephron) is affected, for example, glomerular versus tubular disease.[29][30]

Potential Diagnosis

Renal function tests can assess overall renal function by directly measuring or estimating the glomerular filtration rate. In addition, they can determine whether the renal disease is acute or chronic. Urine albumin levels can be used to detect incipient nephropathy in at-risk patients, such as those with diabetes.

Disorders of tubular function, such as Fanconi syndrome, can be detected using more detailed renal function tests, particularly urine amino acids, glucose, phosphate, and pH.

Normal and Critical Findings

The normal GFR for an adult is 90 to 125 mL/min in individuals older than 2.[31] A GFR of less than 15 mL/min is considered to be end-stage renal failure requiring renal replacement therapy. A normal GFR does not exclude renal disease, which may be evidenced by albuminuria/proteinuria or imaging studies.

Reference intervals for serum creatinine and urea are dependent on age and gender.

The presence of electrolytes in urine depends on the hydration status and duration of the urine collection, apart from pathological factors. Reference intervals are often wide and dependent on the clinical context.

Interfering Factors

Creatinine

Preanalytical issues such as high-protein intake and increased muscle mass may lead to elevated creatinine levels, but they are not representative of an individual's renal function. Likewise, serum creatinine as a marker of renal function is often unreliable in those with decreased muscle mass, such as older people, amputees, and individuals affected by muscular dystrophy. Creatinine is commonly measured on automated analyzers using either a colorimetric reaction known as the Jaffe reaction or an enzymatic assay. The Jaffe reaction involves the formation of an alkaline picrate. It is subject to negative (for example, bilirubin) and positive interferences (for example, ketones and proteins). Various modifications to the Jaffe reaction have been made to overcome some of these issues.

BUN

Serum urea/BUN concentrations may also be raised in the presence of a high-protein diet or with patients using oral corticosteroids.

Urine Albumin and Protein

Urine albumin or protein may be increased in the presence of conditions not related to renal disease, such as posture, fever, and exercise. Furthermore, in the presence of a urinary tract infection, urine protein levels may be raised without any intrinsic renal pathology present.

Complications

Complications of the majority of tests of renal function are rare, apart from those related to venipuncture. Measurement of GFR using isotopes may cause minimal radiation exposure. However, it is not advised to have repeated exposures over short periods. Some patients may experience allergic reactions to radiocontrast agents containing iodine.

Patient Safety and Education

For GFR measurement using radioactive isotopes, patients should be advised regarding the small amounts of ionizing radiation involved and the need to exclude pregnancy in any female of childbearing age before the test.

In general, patients undergoing blood drawns should be advised regarding potential issues of bruising and pain.

Twenty-four-hour urine collection bottles may contain small amounts of preservatives such as thymol, and direct contact with skin and mucous membranes must be avoided. Collection bottles must be kept out of reach of small children who may accidentally ingest the preservatives contained inside.

Patients should also be advised to maintain regular fluid intake before these tests.

Clinical Significance

Creatinine

Serum creatinine levels are elevated when the glomerular filtration rate significantly reduces or when urine elimination is obstructed. About 50% of kidney function must be lost before a rise in serum creatinine can be detected. Thus, serum creatinine is a late marker of acute kidney injury. This limitation is why other substances that may be elevated earlier in the disease process are being studied as markers for kidney disease.

BUN

Serum urea or BUN levels increase in acute and chronic renal disease. Equations for eGFR are used to determine the presence of renal disease and the stage of CKD and to monitor response to treatment.