Assessment of Renal Function1

Urinalysis is the basic test for evaluation of all patients with renal disease. The timing of the collection, preparation of the patient, and the storage of specimens are important factors influencing the accuracy of urine examination.

Certain precautions should be observed when collecting urine for examination. The patient should avoid strenuous physical exercise in the hours preceding the collection, since these activities may cause proteinuria and/or hematuria. In women, the collection of urine during menstruation should be avoided. Urine should be collected after washing the external genitalia with water. In females, it is best done after separating the labia and in males after withdrawing the foreskin. The second voided sample of urine in the morning is the most suitable one for microscopy. The midstream specimen should be obtained after discarding first portion of the urinary stream. Mid-stream collection prevents contamination from the genitalia and the urethral meatus. Urine should be collected in an appropriate sterile container with a capacity of at least 50–100 mL and a diameter opening of at least 5 cm to allow easy collection. The collected urine should be transported to the laboratory as soon as possible. If the urinary pH is alkaline or the specific gravity is low, elements such as RBCs and WBCs may undergo lysis. To prevent cell lysis, the samples are kept in the refrigerator at +2 to +8°C, but at these temperatures phosphates and urates may precipitate, which makes the samples difficult to examine. Standardized methods like the use of measured samples, constant duration and speed of centrifugation, removal of a fixed volume of supernatant (avoiding pouring off), gentle and thorough resuspension of the sediment, transfer of fixed volumes of sediment to the slide, and the application of cover slips with a defined surface help in the semi quantification of sediments. Addition of thymol, borate, formalin and toluene to urine or refrigeration at 4°C has been advocated to preserve the urine for longer periods. Some of these can interfere with chemical reactions. The best way to avoid such errors is to analyse urine within one hour of collection.

The color of the urine must be looked for under natural lighting in a transparent container and viewed against a white background. The color ranges from pale to dark yellow to amber, depending on the concentration of urochrome. In gross hematuria, the urine is pink or cola colored. The number of erythrocytes, the amount of hemoglobin, pH, and the duration of contact between the hemoglobin and urine determine the color. The lower the pH and the longer the contact, the darker the color. Urine is also of a variable red color in hemoglobinuria, myoglobinuria, after eating beetroot by some genetically susceptible people or after ingestion of rifampicin and few other drugs. Jaundice and other conditions associated with hyperbilirubinemia are associated with dark yellow to brownish urine. In conditions like porphyria, melanoma or alkaptonuria, the urine color is normal when freshly passed but darkens 2on standing. Drugs influence the color of urine. Nitrofurantoin intake is associated with dark yellow or brown color, pyridium with pink or red color, levodopa with brown to black color. Amitryptiline and methylene blue give green or blue color to urine. Imipenam–cilastatin can cause brownish urine upon standing.

Normal, freshly voided urine is usually clear and transparent. High concentrations of leucocytes, erythrocytes, epithelial cells, bacteria, candida, crystals, or contaminants are associated with a cloudy specimen. Clear, fresh urine may become cloudy after standing, especially when kept in a refrigerator, because of the precipitation of phosphates or urates. Chyluria is a rare condition characterized by ‘milky’ appearance of freshly passed urine and is due to a mixture of lipids, erythrocytes, leucocytes and fibrin. When allowed to stand, chylous urine forms three layers: a white top layer that contains fatty material, a middle pinkish layer often containing clots and a bottom layer containing cells and cell debris. Turbidity may be intermittent and may be precipitated by fatty meals. The microscopic examination of chylous urine reveals large amounts of fat globules, lymphocytes and erythrocytes. Chyluria is due to a fistulous connection between the lymphatic and urinary system. In most instances this is caused by the obstruction of the lymphatic system by the filarial parasite Wuchereria bancrofti, which is endemic in several tropical regions. Very rarely, chyluria may have non-parasitic causes, such as trauma, tuberculosis, neoplasms, or renal surgery. Pneumaturia is the passage of air in the urine. The gas is odorless and is observed by patients with a fistulous connection between the bowel and the urinary tract. However, it may also be associated with urinary tract infection including emphysematous pyelonephritis or following instrumentation of the urinary tract.

Freshly passed normal urine is odorless. It gets a characteristic pungent odor due to the release of ammonia on exposure to air. Ketones cause a sweet or fruity odor in urine. Urinary tract infection is often associated with a pungent smell in a freshly passed sample. Some rare diseases confer a characteristic smell to the urine. These are maple syrup urine disease (maple syrup odor due to a substance called sotolone), phenylketonuria (mousy odor) and isovaleric acidemia (sweaty feet odor).

Relative density can be measured by any of the four methods: specific gravity, osmolality, refractometry and dry chemistry (dipsticks).

In the normal individual, urinary pH may range from 4.5–8.0. It usually averages from 5.0–6.0, with variations mainly caused by food intake. The pH is generally checked by dipsticks utilizing two indicators, methyl red and bromothymol blue, which cover the values between pH 5 and 9. In most cases, the dipsticks for pH are sufficiently reliable. When a more accurate measurement of the pH is needed, a pH meter with a glass electrode is used.

Hemoglobin is detected by a dipstick and is based on peroxidase activity that catalyzes the reaction of a peroxide and a chromogen to produce a colored product. The dipsticks for hemoglobin are sensitive, showing positive reactions at concentrations as low as 0.1–0.6 mg/L. False-negative results may occur with a high concentration of urinary ascorbic acid, nitrite, urine density, and presence of formaldehyde. False-positive results may be seen in urine containing oxidizing agents, large numbers of bacteria or myoglobin. Myoglobin and hemoglobin can be distinguished by dissolving 2.8 g of ammonium sulfate in 5 mL of urine, followed by filtration or centrifugation. In the presence of ammonium sulfate, hemoglobin precipitates, while myoglobin does not. Clearing of the supernatant suggests hemoglobin. The two pigments can also be distinguished by spectrophotometry, electrophoresis, ultracentrifugation, or immunochemical methods.

Under normal conditions, glucose is not detectable in urine. Urinary glucose is commonly estimated with dipsticks by a coupled, two-stage, enzyme-catalyzed reaction. Glucose is oxidized, with glucose oxidase as catalyst, to gluconic acid and hydrogen peroxide. Then, a peroxidase catalyzes the oxidizing reaction between hydrogen peroxide and colorless chromogen to form a colored product. This test is highly specific and can detect and semiquantitate concentrations between 1–20 g/L. Some commercial sticks are more sensitive (0.5 g/L or less) and have a greater value for detecting glucosuria. False-negative results are seen with ascorbic acid at concentrations greater than 4000 µmol/L and in urinary tract infection. False-positive results are seen due to the presence of strong oxidizing substances, such as hypochlorite and chlorine bleach.

Under physiological conditions, urinary protein excretion does not exceed 150 mg/day for adults. The daily physiological proteinuria contains mucoprotein (e.g. Tamm–Horsfall glycoprotein (70 mg), blood group-related substances (35 mg), albumin (16 mg), immunoglobulins (6 mg), mucopolysaccharides (16 mg) and very small amounts of other proteins such as hormones and enzymes.

Proteinuria is usually detected by dipsticks containing bromocresol blue as indicator. With increasing concentrations of protein in urine the dye indicators undergo sequential color changes from pale green to green and blue. The binding of a protein to the indicators is highly pH dependent. Albumin binds to indicators at pH between 5 and 7. Other proteins bind at lower pH, but with a lower affinity than albumin, while Bence-Jones protein does not bind at any pH. The results are expressed on a scale from 0 to ++++ which correspond to nil, 30, 100, 300 and > 2000 mg per litre protein concentration respectively. Dipsticks exclusively detect albumin at concentrations as low as 250 mg/L. Therefore microalbuminuria, globulins, tubular proteins and Bence-Jones protein are not detected by conventional dipsticks. Therefore, dipsticks must not be used whenever a tubular proteinuria or a Bence-Jones proteinuria is suspected. Underestimation of albuminuria can also occur in diluted urine. False-positive results occur in alkaline urine, since the buffer contained in the dipstick is unable to achieve the optimal pH for the indicator to function adequately.4

These methods evaluate the turbidity occurring after proteins are precipitated by sulfosalicylic acid, trichloracetic acid or by heat and acetic acid–sodium acetate buffer. Turbidimetric methods detect all urinary proteins. Tamm–Horsfall mucoprotein and α₁-acid glycoprotein are not precipitated by sulfosalicylic acid and hence they are not detected by this test. Turbidimetric methods are very sensitive and may detect protein concentrations as low as 2.5 mg/L. With these methods also, the protein concentration is expressed on a semiquantitative scale, from 0 to ++++. Many drugs like penicillin or cephalosporin analogues, miconazole, tolbutamide, sulfonamides and radiographic contrast media can cause false positive results.

As urine can contain a large variety of proteins and also many interfering substances, all the available methods to quantitate proteinuria are imperfect.

Several methods are available for the qualitative analysis of proteinuria. Electrophoresis on cellulose acetate or agarose after protein concentration or using very sensitive staining (silver or gold stains) is one of the most widely used method. Better resolution is obtained by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), which detects urinary proteins on the basis of their molecular weight. For proteinuria due to the excretion of monoclonal light-chains, electrophoresis reveals the presence of a homogeneous band. Immunofixation electrophoresis is used to characterize the monoclonal component. Specific proteins like β₂-microglobulin, retinol-binding protein, or α₁-microglobulin can be measured directly to diagnose and follow-up tubulointerstitial diseases. Very sensitive gold and silver stains are sometimes used.

The selectivity of proteinuria is evaluated by the ratio of the clearance of IgG (molecular weight 160,000) to the clearance of transferrin (molecular weight 88,000). When the ratio is less than 0.1 the proteinuria is defined as selective. Non-selective proteinuria is characterized by the excretion of large molecular weight proteins. Identification of selective proteinuria may indicate less severe degree of glomerular damage or greater responsiveness to steroid therapy in nephrotic patients.

Microalbuminuria is defined as a urine albumin concentration between 30–300 mg/24 h. It is not detected by the conventional urine tests. The most common methods to measure microalbuminuria are by special dipstick, radioimmunoassay, enzyme immunoassay, nephelometric and turbidimetric methods. The dipsticks detect albumin at concentration of 10–20 mg/L, and have 570–90 percent specificity. The 24 hour urine collection is considered the ‘gold standard’ method for the measurement of microalbuminuria. It can also be evaluated by spot urine samples by doing urine albumin creatinine ratio. The conditions causing microalbuminuria include diabetic nephropathy, high blood pressure, obesity and hypertriglyceridemia, smoking, oral contraceptive use, hormone replacement therapy and old age.

Normal individuals excrete small amounts of enzymes located in the cells of the renal tubules in their urine. Increased excretion of these enzymes is a sensitive index of renal damage, but is nonspecific. Some of these enzymes include alkaline phosphatase, leucine aminopeptidase, γ-glutamyltransferase, α-glucosidase, N-acetyl glucosamine and neutral endopeptidase.

For microscopic examination, 10 mL of freshly passed urine is taken in a graduated centrifuge tube and centrifuged at 1500–3000 rpm for 5 min. The supernatant 9 mL is removed for further biochemical testing. The sediments are gently resuspended in the remaining 1 mL of urine and the resuspended urine sediment is studied under the microscope using the WBC counting chamber for quantification. The results are expressed as number per unit volume. Both pH and osmolality are taken into consideration before interpreting the findings in urine microscopy. At higher pH the number of casts tends to decrease, and neutrophils tend to undergo lysis whereas at low osmolalities, erythrocytes tend to lose their hemoglobin content thus becoming less identifiable. At osmolalities of less than 360 mOsm/kg of water (specific gravity of less than 1.010), the sample is no longer reliable since erythrocytes and leucocytes tend to undergo lysis. The phase-contrast microscopy with or without polarizing filters is used to identify dysmorphic RBCs, lipids and crystals.

The cellular elements on microscopic examination include RBCs, WBCs, epithelial cells and tubular cells. Normal individuals excrete up to 2 million RBCs per day. Hematuria is defined as the presence of more than 2–5 RBCs/hpf in a centrifuged sample of urine. The evaluation of erythrocyte morphology is an important tool in the management of patients with isolated microscopic hematuria of unknown origin. Hematuria is defined as ‘non-glomerular’ when at least 80 percent of the erythrocytes show a regular (or ‘isomorphic’) appearance on microscopy and ‘glomerular’ when 80 percent of erythrocytes are ‘dysmorphic’. Pyuria is defined as the presence of more than 3 WBCs/hpf in males and more than 5 WBCs/hpf in non-menstruating females in clean catch midstream centrifuged urine specimen. WBCs or pus cells are a typical finding in urinary tract infections. However, they are also found in patients with proliferative glomerulonephritis, acute or chronic interstitial nephritis, tuberculosis and urological disorders. Neutrophils may be found as a consequence of urine contamination from genital secretions in women. In such cases, they are associated with large amounts of squamous epithelial cells and bacteria. The gradual or abrupt appearance of lymphocytes in renal transplant recipients is an early and sensitive marker of acute cellular rejection. Eosinophiluria can occur due to interstitial nephritis atheroembolic renal disease and urinary schistosomiasis. Tubular cells are found in acute tubular necrosis, interstitial nephritis, cellular allograft rejection and nephritic or nephrotic syndrome.

Casts are elongated and have a basic cylindrical shape. Variations in shape occur during their passage through the tubules. Casts are formed as a result of aggregation of Tamm Horsfall glycoprotein with formed elements in the distal tubules and collecting ducts. Tamm Horsfall glycoprotein forms the matrix of the casts. Cells, granules, lipids, crystals or microorganisms are transported along the nephron by the tubular fluid and as they come across the forming cast, they are entrapped and excreted in urine. Cast formation is favored by increased urinary concentration of electrolytes, hydrogen ions, and proteins. The formation of casts is also favored by the interaction between the Tamm Horsfall protein with hemoglobin or myoglobin, Bence-Jones protein or radiocontrast media. Hyaline casts are present in variable number in normal individuals, but are increased in renal diseases. Larger numbers of hyaline casts may also be observed in patients with fever, in 6those receiving furosemide or ethacrynic acid, after strenuous physical exercise or in cardiac failure. Other types of casts include waxy casts and granular casts. Waxy casts are found in patients with chronic or rapidly progressive renal disease.

Urine can contain several types of crystals. Some of which are found only or predominantly in acid urine while others prevail in alkaline urine. In addition, some crystals are birefringent under polarized light while others are not. By knowing crystal morphology, urine pH, and polarizing features, most urine crystals can be identified using a conventional equipment. However, in some instances this is not sufficient and more sophisticated techniques such as infrared spectroscopy are necessary.

Bacteria: These are frequently seen in urine sediments, either as rods or cocci. In the presence of improper collection and handling of specimen, bacteria may be present due to contamination rather than infection. The presence of leucocytes increases the probability of a real infection, especially in women, but leucocytes and bacteria may contaminate urine from genitalia.

Fungi: Candida is the most frequent yeast found in urine. Candidae appear as refractile, pale-green cells, often nucleated and with smooth and well-defined walls. The most frequent cause of candida in the urine is contamination from the genitalia, but candida can also grow in the urinary tract, mostly in patients with diabetes mellitus, structural abnormalities, indwelling catheters, prolonged antibiotic treatment or immunosuppression. In addition to bacteria and fungi, protozoa like trichomonas and parasites like schistosoma may also be present.

Individual functions of the kidneys are assessed by various methods. The ability of the kidney to eliminate solutes from the body depends on the renal blood flow, glomerular filtration, selective tubular reabsorption and tubular secretion. Clearance of a solute is defined as the amount of plasma that is completely cleared off of the solute in unit time. The formula C = V × U/P is used to calculate the clearance of a substance where, C is the clearance in mL/min, V is the volume of urine mL/min, 7U is the urinary concentration of the substance in mg/mL and P represents the plasma concentration in mg/mL.

If a substance is completely removed from the plasma by glomerular filtration and tubular secretion during one passage through the kidney, the clearance of that substance will be equal to the renal plasma flow. p-aminohippuric acid (PAH) is completely extracted from the plasma by a single pass through the kidney. A loading dose of PAH is injected intravenously followed by a constant infusion. PAH plasma concentration reaches a steady state within an hour when the rate of infusion and rate of removal are equal. Urine and venous blood samples are collected over successive time-controlled periods and the clearance calculated. PAH clearance is considered as the ‘gold standard’ to measure RPF, but many other markers like ortho-iodo-hippuran, a radioiodine labelled PAH analogue, 5-hydroxyindoleacetic acid, the serotonin metabolite and ^99mTc-mercapto-acetyl-triglycerine (^99mTc-MAG3) are also used to measure RPF. To avoid continuous infusions of radiomarkers and urine collections, alternative methods based upon the administration of a single intravenous dose of tracer followed by multiple blood samplings are employed. Renal blood flow can be calculated from RPF using the formula:

If a substance is freely filtered at the glomerulus, not reabsorbed or secreted by the renal tubules and is excreted unchanged in the urine, the clearance of that substance equals the glomerular filtration rate. The quantity of substance that appears in urine per unit time equals the quantity of substance that is filtered at the glomeruli. Two non-toxic substances fulfilling these criteria are inulin and polyfructosan. These substances are not endogenously present in humans and must be infused intravenously throughout the GFR measurement. In practice, after a bolus injection, the marker is infused at a constant rate to obtain a stable plasma concentration and samples are taken to calculate the inulin clearance. Radionuclide markers such as ⁵¹Cr-labelled ethylenediaminetetra-acetic acid (⁵¹Cr-EDTA), technetium-radiolabelled DTPA (^99m Tc-DTPA) or ¹²⁵ I-iothalamate can also be used in renal clearance studies.

Since creatinine is an endogenous substance, it is often used as a marker for clinical assessment although it does not meet all the criteria for accurate measurement of GFR. Creatinine is a metabolic product of creatine and phosphocreatine. It is a small molecule with molecular weight 113 Daltons. It does not bind plasma proteins and is mainly eliminated by the kidney in patients with normal renal function. It is freely filtered by the glomerulus and is not significantly reabsorbed throughout the tubules. The secretion of creatinine is inhibited by some commonly used drugs such as cimetidine, trimethoprim, pyrimethamine, or dapsone. Despite these limitations, creatinine clearance is still widely used clinically and gives a good but not accurate estimate of GFR. It is a relatively simple test and can be performed in most laboratories. If the 24 hour urinary creatinine in mg per day and serum creatinine during the 24 hour period are known, the clearance can be calculated as follows.

Two types of markers can be used to calculate plasma clearance: radionuclide labeled markers or non-radionuclide markers. The most frequently used radionuclide-labeled agents are: ^99mTc-DTPA, ⁵¹Cr-EDTA and ¹²⁵I-iothalamate. The main advantages of the radiolabelled agents are the ease and the accuracy of measurements. Their drawback is the exposure of the bladder and the gonads to radioactivity particularly in children and women in the reproductive age group. To avoid these 8adverse effects, radiocontrast agents iothalamate sodium and iohexol are used to calculate GFR with the plasma clearance technique. These agents carry a risk of allergic reactions due to iodine.

Urea, creatinine, and cystatin C have been used to study GFR. Urea is a small molecule produced from aminoacids. The daily production varies largely with protein intake and protein degradation in the intestine. It is freely filtered by the glomeruli, however, it is also reabsorbed by the tubules and the tubular reabsorption depends on water reabsorption. Thus, plasma urea and urea excretion are affected by many parameters. Urea clearance is not a reliable indicator of GFR. When water reabsorption in renal tubules is increased, or when intravascular volume is depleted, high urea serum concentration ensues even without decrease in GFR.

The tubular functions are more complex than glomerular functions and are different in the various segments of the nephron. Since the tubular functions cannot be studied by any single test, individual tests to assess the integrity of the various segments of the renal tubule are undertaken. Some functions take place in a specific tubular segment but others involve different segments in the same nephron. Most of the investigations of the tubule functions are interpreted in the background of an accurate measurement of the GFR.

The proximal tubule is the first segment of the nephron. This segment reabsorbs about 60 percent of the water, nearly all the filtered bicarbonate, glucose and many of the solutes. The reabsorption in the proximal tubule is iso-osmotic. The functions of the proximal tubule can be studied by assessing reabsorption of glucose, phosphates, uric acid and amino acids.

Under normal circumstances, glucose is completely reabsorbed from the filtrate in the proximal tubule. There is an upper limit to the capacity of the proximal tubule to reabsorb glucose. This limit is the tubular maximum for glucose (TmG). In diabetes mellitus, because of high blood glucose levels the filtered load increases beyond the TmG and glysosuria occurs. Rarely, the ability to 9reabsorb glucose by the proximal tubule is impaired and glycosuria may occur even though the blood sugar and filtered load are normal. This is called renal glycosuria. In this condition, the TmG is lower and the threshold blood level above which glycosuria occurs is lower. The maximal capacity of proximal tubule to reabsorb glucose i.e. TmG can be measured by increasing plasma glucose concentration progressively with glucose infusion. Time-controlled urine collections are taken every 15 or 30 min and venous plasma glucose concentrations are measured at the beginning and the end of each period. The quantity of glucose reabsorbed by the proximal tubule TmG is detected by the difference between the filtered load of glucose and the rate of urinary glucose excretion.

Phosphate is mainly reabsorbed in the proximal tubule. As in the case of glucose, there is a limit beyond which, the tubule is unable to reabsorb phosphate. This threshold is called tubular maximum for phosphorus (Tm_Pi). Renal reabsorption of phosphate is regulated by parathyroid hormone which increases urinary excretion of phosphate. Under physiological conditions, in adults, about 20 percent of phosphate is excreted in urine. This is called fractional excretion of phosphate. To calculate renal phosphate excretion, a nomogram has been devised that permits calculation of Tm_Pi/GFR from plasma and urine concentrations of phosphate and creatinine in the fasting state. Tm_Pi/GFR value determines the plasma phosphate levels and is a useful parameter when investigating patients with hypophosphatemia. Measurement of plasma parathyroid hormone (PTH) concentration is also necessary in such cases.

Ninety-five percent of filtered amino acids are reabsorbed by the proximal tubule through various carriers and a tubular maximum has been defined for each amino acid. In clinical practice only the fractional excretion of amino acids and the amino acid to creatinine ratio in urine are used for the evaluation of aminoaciduria.

Urate transport takes place in the proximal tubule. Urate is both reabsorbed and secreted in the proximal tubule resulting in a fractional excretion (clearance of urate/GFR) ranging between 9 and 15 percent. A reduction in the tubular reabsorption of urate occurs in Fanconi's syndrome. It may also occur as an isolated defect of tubular function causing hypouricemia. Hypouricemia may also occur due to volume expansion (decreased tubular reabsorption), to a decrease in urate production or an increase in urate secretion in proximal tubule.

Sodium reabsorption occurs along the entire nephron by various mechanisms. About 60 percent of the filtered sodium is reabsorbed in the proximal tubule. Sodium reabsorption in the proximal tubule is assessed by the lithium clearance test. In the proximal tubule, lithium is absorbed at the same rate as sodium. Thus, study of the clearance of lithium gives an estimate of proximal reabsorption of sodium. The calculation of lithium clearance requires the measurement of lithium concentration in plasma and urine twelve hours after a 250–500 mg oral load of lithium carbonate. An alternative reliable but expensive method uses atomic absorption spectrophotometry. This method is very sensitive and avoids the necessity of lithium load. If the sodium reabsorption in the proximal tubule is defective, it can be reabsorbed in the thick ascending limb of the loop of Henle or the distal convoluted tubule. Hence, tests for sodium reabsorption in proximal tubule are not done for clinical evaluation.

The distal tubule regulates the final pH and concentration of the urine. Its ability to concentrate and dilute urine as well as tests of acidification are used to diagnose specific disorders affecting the function of the distal tubule. The kidney plays a central role in water homeostasis because of its ability to concentrate or dilute urine according to the water balance. Water is absorbed in the proximal tubule, the thin descending limb, and the collecting tubule. In the thick ascending limb and the distal convoluted tubule no net water reabsorption occurs. Since only solutes are reabsorbed in these segments, the tubular fluid leaving the segment is hypo-osmotic. The final concentration of urine depends on the action of vasopressin and the osmolarity of the medullary interstitium. In the presence of the antidiuretic hormone, the collecting tubule becomes 10permeable to water. Water reabsorption occurs due to the concentration gradient between the tubular lumen and the hypertonic medullary interstitium. This gradient is altered in the early stages of renal insufficiency, and is reduced whenever there is medullary damage such as with polycystic kidneys, sickle cell disease, reflux nephropathy, tubular necrosis, or transplanted kidneys. In the absence of antidiuretic hormone, the collecting duct is not permeable to water and the hypotonic tubular fluid is excreted as dilute urine.

The concentrating capacity of the kidney is assessed by measuring the urine and plasma osmolality simultaneously. The maximal concentrating capacity of the kidney is determined by fluid restriction test or water deprivation test. The patient is denied access to all fluid and food intake from 6:00 pm on the day prior to the investigation. The next morning, plasma and urine osmolalities are measured. If the plasma osmolality ranges between 280 and 295 mOsm/kg H₂O and urine osmolality is above 600 mOsm/kg H₂O, normal concentrating ability can be assumed. In order to test the ability of the kidneys to maximally concentrate the urine or if the above targets are not achieved, it is necessary to prolong water deprivation under close monitoring for 18–24 hours. Blood pressure, pulse rate, urinary flow rate, body weight and urine osmolality are checked every hour and plasma osmolality every 2 hours. Major defects in the urinary concentrating ability can be excluded if the osmolality of any urine sample exceeds 800 mOsm/kg H₂O or when plasma osmolality is above the upper normal values (296 mOsm/kg H₂O). Development of tachycardia, hypotension, weight loss of > 5 percent of initial weight, urine flow rate of more than 60 mL/hour in spite of water deprivation indicate inability to concentrate the urine. Administration of vasopressin at this stage increases the urinary osmolality within 2 hours if the patient has central diabetes incipidus. Patients with nephrogenic diabetes insipidus fail to respond to vasopressin because the basic defect is not deficiency of vasopressin.

The diluting capacity of the kidney is tested by the water load test. This investigation is useful in patients who present with unexplained hyponatremia. At the beginning of the investigation, plasma and urinary osmolality are measured. The patient is made to drink 20 ml/kg body weight of water in 20 min. In normal subjects, the plasma osmolality remains within the normal range after the water load and urine osmolality decreases below 100 mOsm/kg H₂O within 2 hours. Nearly 80 percent of the ingested water is excreted within 4 hours. The diluting capacity may be affected by inappropriate vasopressin secretion, adrenal insufficiency, hypothyroidism, potassium depletion or liver disease.

Patients with various renal disorders often exhibit few signs and symptoms, hence, laboratory assessment of renal function is very crucial for early diagnosis of patients with renal disease. Biochemical derangements in the blood often occur late when the renal disease has already shown significant progression, however, simple tests such as urinalysis may show abnormalities during the early stages. Estimation of glomerular filtration rate by various simple formulae is very helpful in staging the chronic kidney disease and serial estimations are helpful in assessment of response to therapy and progression of disease.