- 28 Aug 2017
Tests to Assess Proximal Tubular Function
Renal tubules efficiently reabsorb 99% of the glomerular filtrate to conserve the essential substances like glucose, amino acids, and water.
1. Glycosuria: In renal glycosuria, glucose is excreted in urine, while blood glucose level is normal. This is because of a specific tubular lesion which leads to impairment of glucose reabsorption. Renal glycosuria is a benign condition. Glycosuria can also occur in Fanconi syndrome.
2. Generalized aminoaciduria: In proximal renal tubular dysfunction, many amino acids are excreted in urine due to defective tubular reabsorption.
3. Tubular proteinuria (Low molecular weight proteinuria): Normally, low molecular weight proteins (β2 –microglobulin, retinol-binding protein, lysozyme, and α1 –microglobulin) are freely filtered by glomeruli and are completely reabsorbed by proximal renal tubules. With tubular damage, these low molecular weight proteins are excreted in urine and can be detected by urine protein electrophoresis. Increased amounts of these proteins in urine are indicative of renal tubular damage.
4. Urinary concentration of sodium: If both BUN and serum creatinine are acutely increased, it is necessary to distinguish between prerenal azotemia (renal underperfusion) and acute tubular necrosis. In prerenal azotemia, renal tubules are functioning normally and reabsorb sodium, while in acute tubular necrosis, tubular function is impaired and sodium absorption is decreased. Therefore, in prerenal azotemia, urinay sodium concentration is < 20 mEq/L while in acute tubular necrosis, it is > 20 mEq/L.
5. Fractional excretion of sodium (FENa): Measurement of urinary sodium concentration is affected by urine volume and can produce misleading results. Therefore, to avoid this, fractional excretion of sodium is calculated. This refers to the percentage of filtered sodium that has been absorbed and percentage that has been excreted. Measurement of fractional sodium excretion is a better indicator of tubular absorption of sodium than quantitation of urine sodium alone.
This test is indicated in acute renal failure. In oliguric patients, this is the most reliable means of early distinction between pre-renal failure and renal failure due to acute tubular necrosis. It is calculated from the following formula:
(Urine sodium × Plasma creatinine) × 100%
(Plasma sodium × Urine creatinine)
(Plasma sodium × Urine creatinine)
In pre-renal failure this ratio is less than 1%, and in acute tubular necrosis it is more than 1%. In pre-renal failure (due to reduced renal perfusion), aldosterone secretion is stimulated which causes maximal sodium conservation by the tubules and the ratio is less than 1%. In acute tubular necrosis, maximum sodium reabsorption is not possible due to tubular cell injury and consequently the ratio will be more than 1%. Values above 3% are strongly suggestive of acute tubular necrosis.
Tests to Assess Distal Tubular Function
1. Urine specific gravity: Normal specific gravity is 1.003 to 1.030. It depends on state of hydration and fluid intake.
- Causes of increased specific gravity:
a. Reduced renal perfusion (with preservation of concentrating ability of tubules),
e. Urinary tract obstruction.
- Causes of reduced specific gravity:
a. Diabetes insipidus
b. Chronic renal failure
c. Impaired concentrating ability due to diseases of tubules.
As a test of renal function, it gives information about the ability of renal tubules to concentrate the glomerular filtrate. This concentrating ability is lost in diseases of renal tubules.
Fixed specific gravity of 1.010, which cannot be lowered or increased by increasing or decreasing the fluid intake respectively, is an indication of chronic renal failure.
2. Urine osmolality: The most commonly employed test to evaluate tubular function is measurement of urine/plasma osmolality. This is the most sensitive method for determination of ability of concentration. Osmolality measures number of dissolved particles in a solution. Specific gravity, on the other hand, is the ratio of mass of a solution to the mass of water i.e. it measures total mass of solute. Specific gravity depends on both the number and the nature of dissolved particles while osmolality is exact number of solute particles in a solution. Specific gravity measurement can be affected by the presence of solutes of large molecular weight like proteins and glucose, while osmolality is not. Therefore measurement of osmolality is preferred.
When solutes are dissolved in a solvent, certain changes take place like lowering of freezing point, increase in boiling point, decrease in vapor pressure, or increase of osmotic pressure of the solvent. These properties are made use of in measuring osmolality by an instrument called as osmometer.
Osmolality is expressed as milliOsmol/kg of water.
Urine/plasma osmolality ratio is helpful in distinguishing pre-renal azotemia (in which ratio is higher) from acute renal failure due to acute tubular necrosis (in which ratio is lower). If urine and plasma osmolality are almost similar, then there is defective tubular reabsorption of water.
3. Water deprivation test: If the value of baseline osmolality of urine is inconclusive, then water deprivation test is performed. In this test, water intake is restricted for a specified period of time followed by measurement of specific gravity or osmolality. Normally, urine osmolality should rise in response to water deprivation. If it fails to rise, then desmopressin is administered to differentiate between central diabetes insipidus and nephrogenic diabetes insipidus. Urinary concentration ability is corrected after administration of desmopressin in central diabetes insipidus, but not in nephrogenic diabetes insipidus.
If urine osmolality is > 800 mOsm/kg of water or specific gravity is ≥1.025 following dehydration, concentrating ability of renal tubules is normal. However, normal result does not rule out presence of renal disease.
False result will be obtained if the patient is on low-salt, low-protein diet or is suffering from major electrolyte and water disturbance.
4. Water loading antidiuretic hormone suppression test: This test assesses the capacity of the kidney to make urine dilute after water loading.
After overnight fast, patient empties the bladder and drinks 20 ml/kg of water in 15-30 minutes. The urine is collected at hourly intervals for the next 4 hours for measurements of urine volume, specific gravity, and osmolality. Plasma levels of antidiuretic hormone and serum osmolality should be measured at hourly intervals.
Normally, more than 90% of water should be excreted in 4 hours. The specific gravity should fall to 1.003 and osmolality should fall to < 100 mOsm/kg. Plasma level of antidiuretic hormone should be appropriate for serum osmolality. In renal function impairment, urine volume is reduced (<80% of fluid intake is excreted) and specific gravity and osmolality fail to decrease. The test is also impaired in adrenocortical insufficiency, malabsorption, obesity, ascites, congestive heart failure, cirrhosis, and dehydration.
This test is not advisable in patients with cardiac failure or kidney disease. If there is failure to excrete water load, fatal hyponatremia can occur.
5. Ammonium chloride loading test (Acid load test): Diagnosis of renal tubular acidosis is usually considered after excluding other causes of metabolic acidosis. This test is considered as a ‘gold standard’ for the diagnosis of distal or type 1 renal tubular acidosis. Urine pH and plasma bicarbonate are measured after overnight fasting. If pH is less than 5.4, acidifying ability of renal tubules is normal. If pH is greater than 5.4 and plasma bicarbonate is low, diagnosis of renal tubular acidosis is confirmed. In both the above cases, further testing need not be performed. In all other cases in which neither of above results is obtained, further testing is carried out. Patient is given ammonium chloride orally (0.1 gm/kg) over 1 hour after overnight fast and urine samples are collected hourly for next 6-8 hours. Ammonium ion dissociates into H+ and NH3. Ammonium chloride makes blood acidic. If pH is less than 5.4 in any one of the samples, acidifying ability of the distal tubules is normal.
- 27 Aug 2017
Normally, a very small amount of albumin is excreted in urine. The earliest evidence of glomerular damage in diabetes mellitus is occurrence of microalbuminuria (albuminuria in the range of 30 to 300 mg/24 hours). An albuminuria > 300-mg/24 hour is termed clinical or overt and indicates significant glomerular damage. (See “Proteinuria” under Article “Chemical Examination of Urine”).
- 27 Aug 2017
Two biochemical parameters are commonly used to assess renal function: blood urea nitrogen (BUN) and serum creatinine. Although convenient, they are insensitive markers of glomerular function.
Blood Urea Nitrogen (BUN)
Urea is produced in the liver from amino acids (ingested or tissue-derived). Amino acids are utilized to produce energy, synthesize proteins, and are catabolized to ammonia. Urea is produced in the liver from ammonia in the Krebs urea cycle. Ammonia is toxic and hence is converted to urea, which is then excreted in urine (Figure 842.1).
Figure 842.1 Formation of urea from protein breakdown
The concentration of blood urea is usually expressed as blood urea nitrogen. This is because older methods estimated only the nitrogen in urea. Molecular weight of urea is 60, and 28 grams of nitrogen are present in a gm mole of urea. As the relationship between urea and BUN is 60/28, BUN can be converted to urea by multiplying BUN by 2.14, i.e. the real concentration of urea is BUN × (60/28).
Urea is completely filtered by the glomeruli, and about 30-40% of the filtered amount is reabsorbed in the renal tubules depending on the person’s state of hydration.
Blood level of urea is affected by a number of non-renal factors (e.g. high protein diet, upper gastrointestinal hemorrhage, liver function, etc.) and therefore utility of BUN as an indicator of renal function is limited. Also considerable destruction of renal parenchyma is required before elevation of blood urea can occur.
The term azotemia refers to the increase in the blood level of urea; uremia is the clinical syndrome resulting from this increase. If renal function is absent, BUN rises by 10-20 mg/dl/day.
Causes of increased BUN:
- Pre-renal azotemia: shock, congestive heart failure, salt and water depletion
- Renal azotemia: impairment of renal function
- Post-renal azotemia: obstruction of urinary tract
- Increased rate of production of urea:
• High protein diet
• Increased protein catabolism (trauma, burns, fever)
• Absorption of amino acids and peptides from a large gastrointestinal hemorrhage or tissue hematoma
Methods for estimation of BUN:
Two methods are commonly used.
- Diacetyl monoxime urea method: This is a direct method. Urea reacts with diacetyl monoxime at high temperature in the presence of a strong acid and an oxidizing agent. Reaction of urea and diacetyl monoxime produces a yellow diazine derivative. The intensity of color is measured in a colorimeter or spectrophotometer.
- Urease- Berthelot reaction: This is an indirect method. Enzyme urease splits off ammonia from the urea molecule at 37°C. Ammonia generated is then reacted with alkaline hypochlorite and phenol with a catalyst to produce a stable color (indophenol). Intensity of color produced is then measured in a spectrophotometer at 570 nm.
Reference range for BUN in adults is 7-18 mg/dl. In adults > 60 years, level is 8-21 mg/dl.
Creatinine is a nitrogenous waste product formed in muscle from creatine phosphate. Endogenous production of creatinine is proportional to muscle mass and body weight. Exogenous creatinine (from ingestion of meat) has little effect on daily creatinine excretion.
Serum creatinine is a more specific and more sensitive indicator of renal function as compared to BUN because:
- It is produced from muscles at a constant rate and its level in blood is not affected by diet, protein catabolism, or other exogenous factors;
- It is not reabsorbed, and very little is secreted by tubules.
With muscle mass remaining constant, increased creatinine level reflects reduction of glomerular filtration rate. However, because of significant kidney reserve, increase of serum creatinine level (from 1.0 mg/dl to 2.0 mg/dl) in blood does not occur until about 50% of kidney function is lost. Therefore, serum creatinine is not a sensitive indicator of early renal impairment. Also, laboratory report showing serum creatinine “within normal range” does not necessarily mean that the level is normal; the level should be correlated with body weight, age, and sex of the individual. If renal function is absent, serum creatinine rises by 1.0 to 1.5 mg/dl/day (Figure 842.2).
Figure 842.2 Relationship between glomerular filtration rate and serum creatinine. Significant increase of serum creatinine does not occur till a considerable fall in GFR
Causes of Increased Serum Creatinine Level
- Pre-renal, renal, and post-renal azotemia
- Large amount of dietary meat
- Active acromegaly and gigantism
Causes of Decreased Serum Creatinine Level
- Increasing age (reduction in muscle mass)
Methods for Estimation of Serum Creatinine
The test for serum creatinine is cheap, readily available, and simple to perform. There are two methods that are commonly used:
- Jaffe’s reaction (Alkaline picrate reaction): This is the most widely used method. Creatinine reacts with picrate in an alkaline solution to produce spectrophotometer at 485 nm. Certain substances in plasma (such as glucose, protein, fructose, ascorbic acid, acetoacetate, acetone, and cephalosporins) react with picrate in a similar manner; these are called as non-creatinine chromogens (and can cause false elevation of serum creatinine level). Thus ‘true’ creatinine is less by 0.2 to 0.4 mg/dl when estimated by Jaffe’s reaction.
- Enzymatic methods: These methods use enzymes that cleave creatinine; hydrogen peroxide produced then reacts with phenol and a dye to produce a colored product, which is measured in a spectrophotometer.
Adult males: 0.7-1.3 mg/dl.
Adult females: 0.6-1.1 mg/dl.
Serum creatinine alone should not be used to assess renal function. This is because serum creatinine concentration depends on age, sex, muscle mass, glomerular filtration and amount of tubular secretion. Thus, normal serum creatinine range is wide. Serum creatinine begins to rise when GFR falls below 50% of normal. Minor rise of serum creatinine is associated with significant reduction of GFR (Figure 842.2). Therefore early stage of chronic renal impairment cannot be detected by measurement of serum creatinine alone.
BUN/Serum Creatinine Ratio
Clinicians commonly calculate BUN/creatinine ratio to discriminate pre-renal and post-renal azotemia from renal azotemia. Normal ratio is 12:1 to 20:1.
Causes of Increased BUN/Creatinine Ratio (>20:1):
- Increased BUN with normal serum creatinine:
• Pre-renal azotemia (reduced renal perfusion)
• High protein diet
• Increased protein catabolism
• Gastrointestinal hemorrhage
- Increase of both BUN and serum creatinine with disproportionately greater increase of BUN:
• Post-renal azotemia (Obstruction to the outflow of urine)
Obstruction to the urine outflow causes diffusion of urinary urea back into the blood from tubules because of backpressure.
Causes of Decreased BUN/Creatinine Ratio (<10:1)
- Acute tubular necrosis
- Low protein diet, starvation
- Severe liver disease
One can estimate GFR from age, sex, body weight, and serum creatinine value of a person from the following formula (Cockcroft and Gault):
Creatinine clearance in ml/min = (140 - Age in years) × (Body weight in kg)
(72 × Serum creatinine in mg/dl)
(72 × Serum creatinine in mg/dl)
In females, the value obtained from above equation is multiplied by 0.85 to get the result.
It is recommended by National Kidney Foundation (USA) to calculate creatinine clearance by Cockcroft and Gault or other equation from serum creatinine value rather than estimating creatinine clearance from a 24-hour urine sample. This is because the latter test is inconvenient, time-consuming, and often inaccurate.
Glomerular filtration rate refers to the rate in ml/min at which a substance is cleared from the circulation by the glomeruli. The ability of the glomeruli to filter a substance from the blood is assessed by clearance studies. If a substance is not bound to protein in plasma, is completely filtered by the glomeruli, and is neither secreted nor reabsorbed by the tubules, then its clearance rate is equal to the glomerular filtration rate (GFR). Clearance of a substance refers to the volume of plasma, which is completely cleared of that substance per minute; it is calculated from the following formula:
Clearance = UV
where, U = concentration of a substance in urine in mg/dl; V = volume of urine excreted in ml/min; and P = concentration of the substance in plasma in mg/dl. Since U and P are in the same units, they cancel each other and the clearance value is expressed in the same unit as V i.e. ml/min. All clearance values are adjusted to a standard body surface area i.e. 1.73 m2.
The agents used for measurement of GFR are:
- Exogenous: Inulin, Radiolabelled ethylenediamine tetraacetic acid (51Cr- EDTA), 125I-iothalamate
- Endogenous: Creatinine, Urea, Cystatin C
The agent used for measurement of GFR should have following properties: (1) It should be physiologically inert and preferably endogenous, (2) It should be freely filtered by glomeruli and should be neither reabsorbed nor secreted by renal tubules, (3) It should not bind to plasma proteins and should not be metabolized by kidneys, and (4) It should be excreted only by the kidneys. However, there is no such ideal endogenous agent.
Clearance tests are cumbersome to perform, expensive, and not readily available. One major problem with clearance studies is incomplete urine collection.
Abnormal clearance occurs in: (i) pre-renal factors: reduced blood flow due to shock, dehydration, and congestive cardiac failure; (ii) renal diseases; and (iii) obstruction to urinary outflow.
Inulin, an inert plant polysaccharide (a fructose polymer), is filtered by the glomeruli and is neither reabsorbed nor secreted by the tubules; therefore it is an ideal agent for measuring GFR. A bolus dose of inulin (25 ml of 10% solution IV) is administered followed by constant intravenous infusion (500 ml of 1.5% solution at the rate of 4 ml/min). Timed urine samples are collected and blood samples are obtained at the midpoint of timed urine collection. This test is considered as the ‘gold standard’ (or reference method) for estimation of GFR. However, this test is rarely used because it is time consuming, expensive, constant intravenous infusion of inulin is needed to maintain steady plasma level, and difficulties in laboratory analysis. Average inulin clearance for males is 125 ml/min/1.73 m2 and for females is 110 ml/min/1.73 m2. In children less than 2 years and in older adults, clearance is low. This test is largely limited to clinical research.
Clearance of Radiolabeled Agents
Urinary clearance of radiolabeled iothalamate (125Iiothalamate) correlates closely with inulin clearance. However, this method is expensive with risk of exposure to radioactive substances. Other radiolabelled substances used are 51Cr-EDTA and 99Tc-DTPA.
Cystatin C Clearance
This is a cysteine protease inhibitor of MW 13,000, which is produced at a constant rate by all the nucleated cells. It is not bound to protein, is freely filtered by glomeruli and is not returned to circulation after filtration. It is a more sensitive and specific marker of impaired renal function than plasma creatinine. Its level is not affected by sex, diet, or muscle mass. It is thought that cystatin C is a superior marker for estimation of GFR than creatinine clearance. It is measured by immunoassay.
This is the most commonly used test for measuring GFR.
Creatinine is being produced constantly from creatine in muscle. It is completely filtered by glomeruli and is not reabsorbed by tubules; however, a small amount is secreted by tubules.
A 24-hour urine sample is preferred to overcome the problem of diurnal variation of creatinine excretion and to reduce the inaccuracy in urine collection.
After getting up in the morning, the first voided urine is discarded. Subsequently all the urine passed is collected in the container provided. After getting up in the next morning, the first voided urine is also collected and the container is sent to the laboratory. A blood sample for estimation of plasma creatinine is obtained at midpoint of urine collection. Creatinine clearance is calculated from (1) concentration of creatinine in urine in mg/ml (U), (2) volume of urine excreted in ml/min (V) (this is calculated by the formula: volume of urine collected/collection time in minutes e.g. volume of urine collected in 24 hours ÷ 1440), and (3) concentration of creatinine in plasma in mg/dl (P). Creatinine clearance in ml/min per 1.73 m2 is then derived from the formula UV/P.
Because of secretion of creatinine by renal tubules, the above formula overestimates GFR by about 10%. In advanced renal failure, secretion of creatinine by tubules is increased and thus overestimation of GFR is even more.
Jaffe’s reaction (see serum creatinine) used for estimation of creatinine measures creatinine as well as some other substances (non-creatinine chromogens) in blood and thus gives slightly higher result. Thus effect of tubular secretion of creatinine is somewhat balanced by slight overestimation of serum creatinine by Jaffe’s reaction.
To provide values closer to the actual GFR, cimetidine (which blocks secretion by renal tubules) can be administered before commencing urine collection (cimetidine-enhanced creatinine clearance).
Creatinine clearance is not an ideal test for estimation of GFR because of following reasons:
- A small amount of creatinine is secreted by renal tubules that increase even further in advanced renal failure.
- Collection of urine is often incomplete.
- Creatinine level is affected by intake of meat and muscle mass.
- Creatinine level is affected by certain drugs like cimetidine, probenecid, and trimethoprim (which block tubular secretion of creatinine).
Urea is filtered by the glomeruli, but about 40% of the filtered amount is reabsorbed by the tubules. The reabsorption depends on the rate of urine flow. Thus it underestimates GFR, depends on the urine flow rate, and is not a sensitive indicator of GFR.
BUN and serum creatinine, by themselves, are not sensitive indicators of early renal impairment since values may be normal e.g. if baseline values of serum creatinine is 0.5 mg/dl, then 50% reduction in kidney function would increase it to 1.0 mg/dl. Thus clearance tests are more helpful in early cases. If biochemical tests are normal and renal function impairment is suspected, then creatinine clearance test should be carried out. If biochemical tests are abnormal, then clearance tests need not be done.
Renal biopsy refers to obtaining a small piece of kidney tissue for microscopic examination. Percutaneous renal biopsy was first performed by Alwall in 1944. In renal disease, renal biopsy is helpful to:
- Establish the diagnosis
- Assess severity and activity of disease
- Assess prognosis by noting the amount of scarring
- To plan treatment and monitor response to therapy
Renal biopsy is associated with the risk of procedure-related morbidity and rarely mortality. Therefore, before performing renal biopsy, risks of the procedure and benefits of histologic examination should be evaluated in each patient.
Indications for Renal Biopsy
- Nephrotic syndrome in adults (most common indication)
- Nephrotic syndrome not responding to corticosteroids in children.
- Acute nephritic syndrome for differential diagnosis
- Unexplained renal insufficiency with near-normal kidney dimensions on ultrasonography
- Asymptomatic hematuria, when other diagnostic tests fail to identify the source of bleeding
- Isolated non-nephrotic range proteinuria (1-3 gm/24 hours) with renal impairment
- Impaired function of renal graft
- Involvement of kidney in systemic disease like systemic lupus erythematosus or amyloidosis
- Uncontrolled severe hypertension
- Hemorrhagic diathesis
- Solitary kidney
- Renal neoplasm (to avoid spread of malignant cells along the needle track)
- Large and multiple renal cysts
- Small, shrunken kidneys
- Acute urinary tract infection like pyelonephritis
- Urinary tract obstruction
- Hemorrhage: As renal cortex is highly vascular, major risk is bleeding in the form of hematuria or perinephric hematoma. Severe bleeding may occasionally necessitate blood transfusion and rarely removal of kidney.
- Arteriovenous fistula
- Accidental biopsy of another organ or perforation of viscus (liver, spleen, pancreas, adrenals, intestine, or gallbladder)
- Death (rare).
- Patient’s informed consent is obtained.
- Ultrasound/CT scan is done to document the location and size of kidneys.
- Blood pressure should be less than 160/90 mm of Hg. Bleeding time, platelet count, prothrombin time, and activated partial thromboplastin time should be normal. Blood sample should be drawn for blood grouping and cross matching, as blood transfusion may be needed.
- Patient is sedated before the procedure.
- Patient lies in prone position and kidney is identified with ultrasound.
- The skin over the selected site is disinfected and a local anesthetic is infiltrated.
- A small skin incision is given with a scalpel (to insert the biopsy needle). Localization of kidney is done with a fine bore 21 G lumbar puncture needle. A local anesthetic is infiltrated down to the renal capsule.
- A tru-cut biopsy needle or spring loaded biopsy gun is inserted under ultrasound guidance and advanced down to the lower pole. Biopsy is usually obtained from lateral border of lower pole. Patient should hold his/her breath in full inspiration during biopsy. After obtaining the biopsy and removal of needle, patient is allowed to breath normally.
- The biopsy should be placed in a drop of saline and examined under a dissecting microscope for adequacy.
- Patient is turned to supine position. Vital signs and appearance of urine should be monitored at regular intervals. Patient is usually kept in the hospital for 24 hours.
Kidney biopsy can be divided into three parts for light microscopy, immunofluorescence, and electron microscopy. For light microscopy, renal biopsy is routinely fixed in neutral buffered formaldehyde. Sections are stained by:
- Hematoxylin and eosin (for general architecture of kidney and cellularity)
- Periodic acid Schiff: To highlight basement membrane and connective tissue matrix.
- Congo red: For amyloid.
For electron microscopy, tissue is fixed in glutaraldeyde. In immunohistochemistry, tissue deposits of IgG, IgA, IgM, C3, fibrin, and κ and λ light chains can be detected by using appropriate antibodies. Many kidney diseases are immune-complex mediated.