RENAL FUNCTION TESTS

 Brief Anatomy and Physiology

 Functions of Kidneys
 Renal Disorders
 General Tests for Kidney Function: Blood Urea, Serum Creatinine,
Urinary Proteins
 Tests for Glomerular Function: Clerance Tests, Cystatin C, β-Trace
Protein
 Tests for Tubular Function: Urinary Specific Gravity, Osmolality

The kidneys, the paired fist-sized organs located outside the peritoneal cavity on each side of the
spine, are the body's primary excretory organs, together with the skin and the respiratory system. The kidney
is a highly specialized organ that is crucial for the body homeostasis and maintains the internal environment
of the body by selectively excreting or retaining various substances according to specific body needs. Urine
formed by the kidneys detoxifies the body of the toxic waste substances that include extra nitrogen as urea,
the catabolic end products like uric acid, drug metabolites and xenobiotics, electrolytes like phosphates,
ethereal sulfates and acids. Kidney function is vital to body and without at least one functioning kidney,
death can occur within a few days.

Figure 40.1 Working of a kidney – Nephron is the functional unit of kidneys.

The primary functional unit of the kidney is a ‘nephron’ and each kidney carries about one million nephrons.
The nephron can be divided into three units, i.e., glomerulus, a tubule and the collecting duct system. The
glomerulus is primarily a filtering apparatus, consisting of a capillary network surrounded by a membrane
called Bowman's capsule. The blood supplied by the afferent arteriole (small branches of the renal artery) is

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filtered through the glomerulus to retain the blood cells and larger molecules whereas the smaller ones pass
into the proximal tubule. The capillaries rejoin to form the efferent arteriole at the distal end of the
glomerulus to pour the clear blood into the renal vein (Fig 40.1). Approximately 25% of the cardiac output or
1200 ml of blood per minute is received by the kidneys. One liter of urine, comprised of 95% water and 5%
solids, is the end product of about 1800 liters of blood processed through the kidneys.
The renal tubule could be called as the ‘active’ unit of the nephrons, since it is actively involved with
reabsorption of useful substances and secretion of certain ions and electrolytes (Fig.40.2). Substances like
glucose and sodium are reabsorbed very effectively and urine is mostly devoid of any glucose. But, if the
concentration of glucose in the filtrate is beyond the capacity of the kidneys (renal threshold), glucose may
be excreted into the urine (glycosuria). Secretion allows substances such as hydrogen ions to be eliminated at
a rate that exceeds glomerular filtration. Both reaborption and secretion are controlled by the selective
permeability of different areas of the renal tubule to water, sodium, and urea. The kidney also has a
remarkable ability to dilute or concentrate urine, according to an individual's changing physiological needs,
and to regulate electrolyte excretion. The volume of urinary excretion is proportional to the degree of
hydration of the body and is under endocrine control by hormones such as aldosterone, antidiuretic
hormone, and parathyroid hormone.

Functions of Kidneys
The kidneys are much more than mere filtration organs of the body. The normal function of kidney is vital to
health and life of an individual. The major functions of the kidneys are –
a) Maintenance of the blood volume by regulating the volume of urine.
b) Maintenance of the electrolyte balance – retention/excretion of sodium, potassium and chloride.
c) Calcium homeostasis – reabsorption of calcium and excretion/retention of phosphates. Parathyroid
hormone acts on kidneys to increase the activity of 1α-hydroxylase that converts 25-OH-VitD3 to
calcitriol, which stimulates absorption of calcium from the intestine.
d) Erythropoiesis – Kidney synthesise erythropoietin that is important for maturation of the red blood
cells. Due to decreased synthesis of erythropoietin, anemia is a constant feature of chronic renal
failure.

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 Figure 39.2 Filtration, reabsorption and secretary activities of different parts of a nephron

e) Reabsorption of carbohydrates and amino acids – in some of the renal diseases loss of glucose
(glycosuria) or amino acids (aminoaciduria) in urine, is seen. Proteins with molecular weight equal or
larger than albumin are retained by the glomerulus, but in case of glomerular injury, proteinuria is
observed. Loss of small amount of albumin (microalbuminuria) is considered the earliest indicators
of renal disease.

Tubular Maximum and Renal Threshold

The renal tubules reabsorb metabolites in proportion to the number of carrier (transport)
molecules in the tubular membrane. Therefore, tubules have a limited capacity for
reabsorption of each metabolite – the maximum absorptive capacity of the renal tubules
for a particular substance is known as its ‘tubular maximum’ or Tm.
The Tm determines the renal threshold for the metabolites i.e. if the plasma
concentration of substance ‘X’ exceeds Tm(X), it will be excreted in the urine. The Tm
of glucose 375 mg/min and its renal threshold is 180 mg/dl. It means about 375 mg of
glucose is supplied to the kidneys per minute (at a normal blood flow), when the plasma
concentration of glucose is 180 mg/dl.
Renal threshold for bicarbonate, lactic acid and calcium is 28 mEq/L, 60 mg/dl and 10
mg/dl, respectively.

f) Blood pressure – Kidneys play a major role in maintenance of the blood pressure. They not only
regulate the blood volume but Juxtra-glomerular cells of kidneys also secreted renin in response to
changes in blood pressure (renal plasma flow). The renin enzyme then activates angiotensin I to
angiotensin II in the blood capillaries; the later is responsible for increase in blood pressure.

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 GLOMERULAR FILTRATION RATE
The glomerular filtration rate (GFR) is the pace at which the plasma constituents are
filtered into the bowman’s capsular space. GFR depends upon the balance between
the forces favoring filtration (hydraulic pressure in the glomerular capillaries) and
forces opposing filtration (the sum of hydraulic pressure in Bowman’s space and
colloid osmotic pressure of blood in the glomerular capillaries). The blood supply of
the glomerulus i.e. renal plasma flow and the total surface area (mass) of the
glomerular capillaries also influence the GFR.
Normal Range for GFR is 90 – 120 ml/min for a healthy adult.
Decreased GFR can be observed in - (1) Progressive renal failure - filtration surface
area is reduced over time; (2) Urinary tract obstruction - tubular hydraulic pressure is
elevated; (3) Hemoconcentration due to severe volume depletion, or dysproteinemias
like myelomas - plasma osmotic pressure is elevated; (4) Cardiac failure and renal
artery stenosis - renal plasma flow is reduced; and (5) Diffuse glomerular disease -
permeability is reduced.

Disorders of the Kidneys

As discussed above, normal kidney function is vital for the body; compromise in any of the renal functions
could be detrimental to the survival of an individual. The renal dysfunction manifests in the form of one or
more of the following –
 Reduction in glomerular filtration (resulting in azotemia),
 Changes in urinary volume and frequency of urination (oliguria, anuria and polyuria),
 Abnormal microscopic constituents (Red cells, white blood cells, casts or crystals),
 Abnormal biochemical constituents (proteinuria, glycosuria or aminoaciduria),
 Hypertension and
 Electrolyte disturbances.
The loss of function could be selective to a metabolite, e.g., glucose, amino acids, calcium, phosphate or
water, but generalized dysfunction involving overall reduced function of the renal mass are more common.
Heredity appears to play a major role in a number of renal disorders. A brief account of the disorders is given
here so that students can relate to the renal function tests applied to these conditions.

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Nephrogenic diabetes insipidus
Diabetes inspidus is characterized by increased loss of water in urine due to failure of the kidneys to
reabsorb water. Decreased secretion of antidiureic hormone (ADH or vasopressin) from posterior
pituitary is the primary cause but the disorder could also be due to the renal dysfunction. Patients present
with polyuria (excessive urination), hypernatremia (retention of sodium) and uraemia (increased levels of
urea).
Renal insensitivity to the normal (or even raised) levels of vasopressin can result in nephrogenic diabetes
insipidus. The mutation in the vasopressin hormone receptor has been implicated in the familial (X-
linked) disease.
The nephrogenic diabetes inspidus could also be secondary to a number of causes, viz. -
o Drugs, e.g. lithium, diuretics
o Metabolic: hypokalemia, hypercalcemia.
o Tubulointerstitial disease: partial obstruction, pyelonephritis, cystic diseases, granulomatous
diseases and sickle-cell disease.
Renal glycosuria
Glycosuria, in healthy individuals, occurs when the absorptive capacity of the kidneys is exceeded by the
increased levels of blood glucose. But when glycosuria occurs under conditions of normal blood glucose
levels, it reflects failure of the tubular mechanisms to reabsorb the entire filtered load of glucose and is
termed as ‘Renal glycosuria’.
The isolated form of renal glycosuria is familial with a mixed inheritance pattern. The decreased number,
or defective function, of the glucose transporters (GLUT2) is responsible for the reduced renal threshold
for glucose.
Many patients with chronic renal insufficiency of mild to moderate degree exhibit renal glycosuria,
usually in combination with other disorders of tubular function.
Abnormalities of glucose transport may also be associated with other defects of proximal tubular
transport (Fanconi's syndrome, see below).

Aminoacidurias
There are a variety of disorders that manifest with excretion of aminoacids in urine (aminoaciduria). The
diseases due to inborn errors of metabolism of aminoacids can be associated with isolated or combined
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aminoacidurias. These have been discussed in detail in the chapters on metabolism of proteins. Two
disorders involving the defective absorption of aminoacids from renal tubules (and from intestine, since the
same transporter protein is involved) are Hartnup disease and cystinuria, a brief account of these is as
follows -
 Hartnup disease
o Rare, autosomal recessive disorder resulting in malabsorption of dietary tryptophan, a
pellagra-like syndrome with photosensitive skin lesions, ataxia, and neuropsychiatric
disturbances. Increased renal excretion of neutral aminoacids is characteristic of the disease.
o Tryptophan is one of the precursors of niacin, therefore decreased intestial absorption presents
like (niacin deficiency) pellagra, but is less severe. Oral nicotinamide supplementation helps
alleviating the symptoms.
 Cystinuria
Autosomal recessive disorder due to impaired intestinal and proximal renal tubular reabsorption
of the dibasic amino acids, viz. cystine, ornithine, arginine and lysine.
o Presents with renal calculi causing renal colic, haematuria, urinary obstruction and secondary
pyelonephritis, leading to renal failure.
o Can be diagnosed by amino acid chromatography (COLA test i.e. Cystine, Ornithine, Lysine
and Arginine in urine); Cyanide-nitroprusside urine test is positive.

Phosphate-handling disorders
Kidney is largely responsible for controlling extracellular phosphate levels.
There are several types of phosphate transport defects causing hypophosphataemia and inappropriate
phosphaturia. Hypophosphatemia results in decreased calcification in bones and causes rickets (children)
and osteomalacia (adults). Two type of hypophosphatemic disorders are mentioned here:
 Hereditary hypophosphataemic rickets (Vitamin D-resistant rickets)
X-linked hypophosphatemia is an X-linked dominant disorder characterized by growth
retardation, rachitic and osteomalacic bone disease, hypophosphatemia, and renal defects in
phosphate reabsorption and vitamin D metabolism. The defect lies in proximal tubular phosphate
transport that results in persistent hypophosphataemia and inappropriate phosphaturia. Children
present with rickets associated with hypophosphatemia along with growth retardation and early

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 bone deformity. Patients with the X-linked disorder do not show muscle weakness, tetany, or
hypocalcemia.
Patients do not respond to vitamin D supplimentation but calcitriol therapy is partially effective.
Sometimes there is even resistance to calcitriol therapy due to functional defects of the vitamin D
receptor.
 Hypoparathyroidism and pseudohypoparathyroidism (renal resistance to parathyroid hormone) also
cause reduced renal phosphate excretion.

Calcium handling disorders

Activation of vitamin D3 i.e. conversion of 25-OH-cholecalciferol into 1,25-DiOH-cholecalciferol
(calcitriol) takes place in kidneys. Calcitriol stimulates the gene for calcium binding protein in GIT and renal
tubules and hence is responsible for the absorption/reabsorption of calcium. Parathyroid hormone promotes
the reabsorption of calcium by activating Vit D3.
Parathyroid and Vitamin D dependent reabsorption of calcium is one of the major functions of kidneys. The
disorders that cause hypercalciuria are relatively more common than those associated with hypocalciuria.
Hereditary hypercalciuric nephrolithiasis is a rare disorder associated with proteinuria, nephrocalcinosis,
renal stones and frequently renal failure. The defect lies in the increased absorption of calcium from the
gastrointestinal tract.
Familial hypocalciuric hypercalcaemia is an autosomal dominant disorder following a generally benign
course associated with a defective extracellular sensing receptor. Hypocalciuria and hypercalcaemia are
accompanied by hypermagnesaemia with parathyroid hormone levels in normal range.

Renal Tubular Disease

There are a variety of disorders of tubular function which may be isolated defects restricted to one or more
metabolites or generalised tubular defects, e.g. Fanconi syndrome.

Fanconi syndrome
Fanconi syndrome is a disturbance of renal tubular function resulting in generalized aminoaciduria,
phosphaturia, glycosuria, rickets or osteomalacia and renal tubular acidosis (see below).
Fanconi syndrome could be inherited due to a number of genetic disorders, e.g. cystinosis, tyrosinemia
type I, galactosemia. Von-Gierke’s disease and Wilson’s disease. It could also be acquired due to acute
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 tubular necrosis, hypokalemic nephropathy, myeloma, hyperparathyroidism and kwashiorkor. Heavy
metal poisoning can also lead to Fanconi syndrome.

Renal tubular acidosis

Kidney is the major organ responsible for the acid-base regulation of our body. It excretes H+ ions as well
as the fixed acids like phosphates and sulfates as per the acid-base balance of our body. Renal tubules
control the net excretion of acids or bicarbonate by regulating their reabsorption. Exchange of cations for
H+ is also important for normal acid-base balance, therefore, any impairment in this exchange from distal
tubules can cause renal tubular acidosis. Acidosis associated with renal disease could result from either a
reduced filtration (GFR) or compromised tubular function. If glomerular function is significantly
depressed, there is increased retention of fixed acids that causes high anion gap acidosis. The tubular
form, on the other hand, causes a hyperchloremic metabolic acidosis. Anion gap is used to differentiate
between the two.
RENAL FUNCTION TESTS
A battery of diagnostic tests is offered in the clinical laboratories and one has to select the appropriate test(s)
as per the clinical requirement. The glomerular and tubular function of the kidneys is interlinked and difficult
to separate, except for specific genetic disorders.
A) General Tests for Kidney Function
Blood Urea
Blood urea is a waste product of protein metabolism and is a medium for elimination of nitrogen from the
body. Urea is formed by the liver and carried through blood to the kidneys for excretion. Because urea is
cleared from the bloodstream by the kidneys, a test measuring how much urea nitrogen remains in the blood
can be used as a test of renal function. However, there are many factors besides renal disease that can cause
alterations in blood urea, including protein breakdown, hydration status, and liver failure.
Normal range of serum urea: Adult: 15-40 mg/100 ml; men may have slightly higher values than women.
During pregnancy, urea concentration decreases by about 25% of normal due to increased intravascular
volume. Newborns show values slightly lower than adult ranges whereas elderly people have blood urea
slightly increased due to decreased renal clearance.
Increased blood urea: An increase in the urea level is known as azotemia.
 Diseased or damaged kidneys cause an elevated blood urea because the urea clearance by kidneys is
less than optimum.
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  Conditions in which renal perfusion is decreased, such as hypovolemic shock or congestive heart
failure, urea levels rise.
 A patient who is severely dehydrated may also have high blood urea due to the lack of fluid volume to
excrete waste products.
 Since urea is an end product of protein metabolism, a diet high in protein, such as high-protein tube
feeding, may also cause the urea to increase.
 Extensive bleeding into the gastrointestinal (GI) tract will also cause an elevated blood urea because
digested blood is a source of urea. For example, a hemorrhage of one liter of blood into the GI tract
may elevate the urea up to 80 mg/dl.
Causes of increased blood urea may be divided into three groups -
Pre-renal – Severe vomiting & dehydration, diabetic coma, thrombosis, ulcerative colitis and shock due
to burns, Acute myocardial infarction, congestive heart failure (poor renal perfusion) and hemorrhage
(GIT bleed) etc are other causes of prerenal azotemia.
Renal - Acute glomerular nephritis, chronic renal failure or renal tuberculosis.
Post-renal – Obstruction to urinary flow can lead to accumulation of urea in blood, e.g. enlargement of
prostate, urinary tract stones and bladder carcinoma etc.

Prerenal azotemia can be diagnosed with the help of urinary sodium

In prerenal azotemia, urine sodium is low (the kidney responds to low
blood flow by "trying to retain all the sodium it can.").
In acute tubular necrosis, urine sodium is higher (the renal tubules are
unable to concentrate or dilute the glomerular filtrate effectively.).
Urinary sodium under 20mEq/L suggests prerenal azotemia (or
hepatorenal syndrome, etc.); urinary sodium over 40mEq/L suggests

Decreased Blood Urea: Urea is synthesised in liver and is a product of protein metabolism, therefore,
compromised liver function as seen in severe liver diseases can manifest as decreased blood urea. The other
reason for decreased blood urea could be increased intravascular volume (plasma dilution) as seen during
pregnancy or overhydration. Therefore, a decreased urea may be seen in: Liver failure and cirrhosis,
Malnutrition, Anabolic steroid use, Overhydration (which can result from prolonged intravenous fluids),
Pregnancy (due to increased plasma volume), Impaired nutrient absorption, Syndrome of inappropriate anti-
diuretic secretion (SIADH) – see box.

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 Urea is a gross indicator of renal function since it is also affected by the hydration and blood volume
status of the patient. Creatinine and Creatinine Clearance are more sensitive parameters to assess renal
function.

Syndrome of Inappropriate Secretion of Anti-diuretic hormone (SIADH)

The anti-diuretic hormone is responsible for stimulating the kidney to conserve water.
Increased secretion of ADH in SIADH patients causes excess water to be retained in the
bloodstream rather than being excreted into the urine. SIADH can cause the BUN level,
along with other important substances, to decrease because the fluid volume of the
bloodstream may significantly increase. Hyponatremia and hypervolemia are the
characteristic features.
Causes of SIADH include - cranial injury, cancer (Small cell cancers, esp. lung, produce
ADH), Infections- intracranial, lung infection and some drugs.

Blood Urea Nitrogen (BUN) – Since urea is a vehicle for the excretion of protein nitrogen, blood urea is
more commonly reported as blood urea nitrogen (BUN) in the biochemistry laboratories. Normal range for
BUN is 7-20 mg/100ml.
Urea has MW 60, of which 28 comes from the two nitrogen atoms. Clinical chemists used to measure only
the nitrogen in urea, hence the "urea nitrogen" measurement on lab reports. BUN can be converted to Urea
by the factor of 2.14 –
Urea = BUN x (60/28), or BUN x 2.14.

Serum Creatinine
Serum creatinine is the most commonly used, inexpensive method of evaluating renal dysfunction.
Creatinine is produced from creatine phosphate metabolism in the skeletal muscles. The synthesis is
continuous, non-enzymatic and is proportional to body muscle mass.
Creatinine is freely filtered from the renal glomeruli and therefore the serum creatinine level depends on the
Glomerular Filtration Rate. Renal dysfunction diminishes the ability to filter creatinine; therefore, the serum
creatinine rises in a number of renal disease conditions. The serum creatinine levels are inversely
proportional to GFR (a decrease in GFR by half, roughly increases the serum creatinine two fold), therefore,
creatinine is considered a highly reliable indicator of renal health.
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 BUN:Creatinine Ratio

Serum creatinine and Blood Urea Nitrogen (BUN) are often compared to evaluate
renal function and to distinguish between pre-renal from renal azotemia. While
serum creatinine increases only with nephron damage, the BUN is affected by
hydration, hepatic metabolism of protein and reduced GFR. BUN and creatinine
tests are conducted together, to check for dehydration. The normal BUN:
creatinine ratio should be between 10:1 and 20:1.
High BUN/Creatinine Ratio: In cases of severe dehydration, BUN:creatinine ratio
is increased. Therefore, high BUN/Creatinine Ratio is seen in - dehydration, acute
kidney failure, shock, decreased blood flow to the kidneys or congestive heart
failure.
Low BUN/Creatinine Ratio is seen in - malnutrition, syndrome of inappropriate
antidiuretic hormone secretion (SIADH). This is also sometimes found with lung
diseases, cancer, diseases of the central nervous system, muscle injuries like
rhabdomyolysis, bleeding in the digestive or respiratory tract and liver diseases. It
can also be an indication of pregnancy.
Obstruction to the urine flow increases the BUN and creatinine value to the same
extent and hence the ratio is unaffected.

The serum creatinine level does not rise until at least half of the kidney's nephrons are destroyed or
damaged. Creatinine levels rise and fall more slowly than BUN levels and change only when significant
damage has been caused to the nephrons, creatinine levels are often preferred to monitor renal function on a
long-term basis.

Equations for Calculation of GFR from Serum Creatinine

The creatinine concentration is far from ideal as a GFR marker because it is significantly
influenced not only by GFR but also by factors such as muscle mass, diet, gender, age, and tubular
secretion. To compensate for the inadequacies of the creatinine concentration as a GFR marker,
there have been several successful attempts at constructing GFR prediction equations including
additional parameters. The most widely accepted and used GFR prediction equations for adults are
that proposed by Cockcroft and Gault, which produces absolute GFR values in mL/min; and the
Modification of Diet in Renal Disease (MDRD) equation, which gives relative GFR values in
mL/min/1.73m2.
Cockcroft-Gault GFR = (140-Age) * (Wt in kg) * (0.85 if female) / (72 * Se Creatinine)
MDRD GFR Equation = 186.3 X [Se Creatinine(μmol/L)/88.4]-1.154 X age (years)-0.203
Correction Factors : 0.742 (if female); 1.212 (if African American)

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Normal values for serum creatinine -
 Adult males: 0.8 - 1.4 mg/dl: values are slightly higher in males due to larger muscle mass
 Adult females: 0.6 - 1.1 mg/dl: creatinine clearance is increased in pregnancy, resulting in lower
serum levels
 Children: 0.2 - 1.0 mg/dl: slight increases with age because values are proportional to body mass

Increased serum creatinine – Almost all renal diseases including acute or chronic renal failure and chronic
nephritis are associated with high creatinine values. Increased serum creatinine is also dependent upon the
blood flow to the kidneys, therefore, a decreased renal plasma flow as seen in congestive heart failure results
in increased serum creatinine. Since creatinine levels are also proportional to the muscle metabolism of the
body, increased muscle mass as in conditions like gigentism, acromegaly and enhanced muscle metabolism
(myasthenia gravis). The conditions commonly associated with the increased serum creatinine levels are -
impaired renal function, chronic nephritis, urinary tract obstruction, muscle diseases such as gigantism,
acromegaly, and myasthenia gravis, congestive heart failure and shock.
Increased plasma creatinine levels rough correlate with the proportion of functional loss of nephrons (Table
39.1).
Table 39.1. Relation between serum creatinine levels and the loss of renal function
Creatinine Level Loss of Nephron Function
Normal Up to 25%
> 1.5 > 50%
4.8-5 > 75%
10 90%
A few drugs (probenecid, cimetidine, triamterene, trimethoprim, and amiloride) block the tubular secretion
of creatinine, and will increase serum creatinine levels even though they have not damaged the kidney. This
will be important if the GFR is already very low.
Raised creatinine is also seen in massive rhabdomyolysis / crush injury, when there is so much creatine
released that plasma creatinine rises even though glomeruli are functioning at their full capacity.
Athletes taking oral creatine may show slight increase in serum creatinine levels for the next day or so, but
the effect is slight and unlikely to go above the normal range.
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Decreased serum creatinine levels may be seen in cases of reduced muscle mass as seen in the elderly and
persons with small stature. Although not a product of protein metabolism, inadequate dietary proteins do
influence the creatinine through slower muscle metabolism.
Muscle atrophy can also result in decreased serum creatinine level. If muscle atrophy is suspected,
assessment of serum creatine for normal muscle function may be done.
 Because of the steady rate of creatinine excretion, substances in the urine are conveniently measured
"per gram of creatinine". This is a reasonable alternative to 24 hour urine collection when we're not
concerned about something that's produced sporadically.

Nephrotoxicity
Most of the drugs and their metabolic products are excreted through kidneys. Many
of these drugs and their metabolites are toxic to the kidneys. Filtration, reabsorption
and concentration at nephrons allows nephrotoxins to accumulate and cause injury to
the renal parenchyma. Common nephrotoxic drugs include – ACE inhibitors,
acyclovir, antibiotics particularly aminoglycosides, immunosuppressants like
cyclosporin and lithium. A patient's BUN and creatinine should always be monitored
during the administration of these drugs. It is also important to keep the patient well
hydrated when aminoglycoside antibiotics are given because they are excreted almost
unchanged in the urine.
The commonly prescribed aminoglycoside antibiotics have both potential
nephrotoxic and ototoxic effects. The impaired hearing or dizziness that may result
from ototoxity is more likely if the drug is continued when there is renal dysfunction.

Urinary Proteins
Kidneys are very efficient in retention of the proteins, hardly any protein appears in urine. The proteins
larger than albumin (66kD) are retained by the glomerular membrane and hence only low molecular weight
proteins like Cystatin-C or BTP (β-Trace Protein, see below) could be present in urine. Very low amounts of
albumin (less than 30 mg/L) are excreted into the urine. Although 99% of the filtered proteins are reabsorbed
by the tubules, the presence of detectable amounts of proteins in urine, known as proteinuria, can be seen
under certain conditions.

Albuminuria

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Albumin is the first protein to appear in urine under pathological conditions known as albuminuria. Small
quantity of protein can be seen in urine in acute nephritis, strenuous exercise and during pregnancy. In
nephrotic syndrome, large amounts of albumin (along with other proteins) are lost in the urine. Since
excretion of creatinine is relatively constant in urine, protein/creatinine ratio is used for the diagnosis, a ratio
of >3.5 is diagnostic for nephrotic syndrome.
 Appearance of very small amounts (<300 mg/24 hr) of albumin in urine is termed as
‘microalbuminuria’. Expressed as albumin/creatinine ratio (ACR), microalbuminuria has emerged as
a very useful parameter for the early detection of renal injury in diabetic and hypertensive patients.
Bence-Jones Proteins (BJP)
Multiple myeloma is characterized by monoclonal proliferation of the plasma cells and is, many times,
associated with increased synthesis of light chains of the immunoglobulins. These light chains are very small
molecular weight proteins and hence are excreted in urine known as Bence-Jones proteins. A simple heat test
can be used to diagnose the condition as these proteins get precipitated at a narrow range (60-80 oC) of
temperature. Heating above 80 oC will result in redissolution of B-J proteins.

B)TESTS FOR GLOMERULAR FUNCTION

Clearance Tests
Defined as ‘the volume of plasma that is cleared of the excretory substance found in urine in one minute’,
clearance tests measure the efficiency of the glomerular filtration. Three excretory substances have been used
to determine the clearance, viz. Inulin, Urea and Creatinine.
Effectively, clearance represents the volume of blood or plasma that contains the amount of substance
excreted in urine in one minute. The clearance would be equivalant to glomerular filtration rate if the
filtered substance is neither rebasorbed nor secreted from the renal tubules. The following general formula
can be used to calculate clearance of any substance ‘S’ -

U X V
Clearance of ‘S’ ml/
: B X min
1440
Where – U is concentration (mg/dl) of ‘S’ in urine; V is total urinary volume (ml) in 24 hour; B is the
concentration of ‘S’ in blood, ‘1440’ is the factor for time (number of minutes in 24 Hr)

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Urea Clearance Test
The clearance of urea from plasma into the urine has been labeled as ‘Ambard’s Coefficient’. As per the
above definition, urea clearance could be defined as – ‘The number of ml of plasma that is completely
cleared of urea by the kidneys per minute’.
During every pass through the kidneys, about 10% of the urea in blood is excreted into urine. In normal
health, about 750 ml of blood passes through kidneys every minute and about 20 mg urea is removed from
the plasma in a minute. Assuming a blood urea concentration of 27 mg/dl, this much (20 mg) urea would be
present in ~74 ml of blood, therefore, urea clearance is around 74 ml/min. Normal range for urea clearance is
75±10 ml/min. This value of urea clearance refers to the maximum capacity of the kidneys to clear urea from
plasma and is applicable only when the urinary flow is quite fast (more than 2 ml/min) thus giving less time
for the tubules for reabsorption. Therefore, this clearance is also known as the maximum urea clearance
(Cm).
Rate of removal of urea from blood is proportional to its concentration in blood. Since urea is reabsorbed
from the renal tubules, urea clearance also depends upon the tubular function. If the total urinary output is
less than 2 ml/min then urea clearance is reduced and is known as standard clearance of urea (Cs), which is
given by the formula –

Normal range of standard urea clearance is 54 ±10 ml/min.

Urea clearance and body surface – The urea clearance is proportional to the total body surface and
correction should be applied for large variation in the body surface i.e. in case of very tall/large or short
people or children. The factor is ‘1.73/BS’ – where BS is the body surface derived from the weight and
height of the patient.
Interpretation –
 Mild changes in urea clearance could be seen in low protein diet or other cases of hypoproteinemia.
 Acute Renal failure – there is a decrease in urea clearance proportional to the decreased function of
the nephrons. Generally a 50% decline is observed in acute renal failure followed by recovery
towards normal with the reversal of the renal failure.

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  Chronic Renal Failure – Urea clearance is seen to decrease as per the progression of the disease.
Renal urea clearance keeps the blood urea concentration under control till the clearance falls below
50%. After that blood urea increases and clearance progressively decreases. Values below 20% of
normal signify very bad prognosis. In end stage renal disease, the urea clearance can even fall below
5% of normal.
 Nephrotic Syndrome – Generally urea clearance is normal till the syndrome progresses into overt
renal failure.
Endogenous Creatinine Clearance Test
Creatinine is filtered by the glomeruli very efficiently and is excreted into the urine almost as such. Since
there is no effective secretion or reabsorption of creatinine from the tubules, creatinine clearance into the
urine is equivalent to the glomerular filtration rate (GFR). Creatinine is synthesized endogenously from
muscle creatine, therefore, the test is known as Endogenous Creatinine Clearance Test.
A creatinine clearance test compares the serum creatinine with the amount of creatinine excreted in a volume
of urine for a specified duration of time; a 24-hour time frame is most common. At the beginning of the test,
the patient empties his bladder and the urine is discarded. Then, all urine voided during the specific time
period is collected. Simultaneously, a single blood sample is drawn to determine creatinine in urine and the
serum. Creatinine clearance is then calculated using the clearance equation given above.
Normal creatinine clearance
 Adult males: 97 - 137 ml/min
 Adult females: 88 - 128 ml/min
 Pregnancy: may be as high as 150 - 200 ml/min
 Elderly: GFR declines by about 10% per decade after age 50, therefore, creatinine clearance values
decrease with age in the elderly.
Cockcroft-Gault gave a formula that can be used to calculate the expected creatinine clearance in an
individual taking into account the age and muscle mass –

In case of females the result should be multiplied by 0.85 to adjust for the lesser fraction of muscle mass in
the total body weight.
Decreased creatinine clearance

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A decreased creatinine clearance is an indication of decreased glomerular function and progressive decline in
clearance means diminishing renal glomerular mass. Therefore, creatinine clearance test is used to diagnose
renal dysfunction and is also used to evaluate the progression of renal disease. A minimum creatinine
clearance of 10 ml/min is necessary to maintain life without the use of renal or peritoneal dialysis.
 Creatinine clearance is not a perfect measure of GFR, because creatinine filteration is not perfect and
a small amount of creatinine is secreted into the proximal tubule. These fractions tend to cancel each
other out in health, but when GFR drops below 30 mL/min, tubular secretion approaches or even
exceeds the amount filtered at the glomeruli.

GFR for adults can be estimated by various formulas a common one is –

GFR = 1.12 x (Creatinine Clearance - 20.6)
Inulin Clearance Test
Inulin is a fructosan (homopolymer of fructose) that is freely filtered from the glomeruli and is neither
absorbed nor secreted from the renal tubules. Inulin clearance test, therefore, is a true indicator of
glomerular function and the ideal parameter to measure GFR. The test has been and is still used as the gold
standard to measure GFR.
The procedure involves hospitalisation of the patient. The patient is well hydrated by giving a glass of water
every half an hour. After 30 min of a light breakfast, 10 g inulin in 100 ml saline is administered
intravenously, at a rate of 10 ml/min. Urine is collected at 60 and 120 min post inulin administration along
with the blood samples at 30 and 90 min. Inulin is measured in the urine and blood and the clearance is
calculated (using average values of the two samples) by the usual formula.
Normal range of Inulin clearance is 100 – 125 ml/min.
Unforunately, methods for the estimation of inulin are tedious and expensive and availability of injectable
inulin is difficult. Although it is the best test, inulin clearance is used only in research laboratories as a gold
standard to evaluate/compare the other methods for measurement of GFR.

Iohexol Clearance
Iohexol is a radiographic contrast medium (Omnipaque) which is cleared from the glomeruli without any
significant secretion from the tubules. It has been used for the estimation of GFR in the western countries.
Difficulty with iohexol is its estimation requires HPLC that might not be available in most of the hospitals.

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Cystatin-C
Recently another parameter, Cystatin-C, has emerged as a very good marker for glomerular function.
Cystatin-C is a basic low molecular weight (13 kD) protein with 120 amino acids, is freely filtered by the
glomerulus and almost completely reabsorbed and catabolized by proximal tubular cells. Just like creatinine,
Cystatin also rises in plasma with deterioration of the glomerular function. Advanatage of Cystatin-C is that
it is not affected by age.
The normal levels of Cystatin-C are: 0.51–0.98 mg/L (males) and 0.49–0.94 mg/L (females).
The plasma levels of Cystatin-C change reciprocally to the glomerular function and can be used for the
calculation of GFR. The following equations have been recommended -
For males : GFR [mL/min/1.73 m2]= 83.93 X cystatin C (mg/L)-1.676
For females : GFR [mL/min/1.73 m2] = 86.49 X cystatin C (mg/L)-1.686 X 0.948
Cystatin-C appears to be more affected by gender than age in the calculation of GFR. It has shown excellent
results for the monitoring of patients after renal transplant. Increased sensitivity, compared with creatinine,
for the detection of acute reduction in GFR allows in some cases a more rapid diagnosis of acute rejection or
treatment nephrotoxicity.

β-Trace protein (BTP), also known as prostaglandin D synthase, is a 23- to 29-kD enzyme polypeptide of
168 amino acids, primarily isolated from the cerebrospinal fluid and belongs to lipocalin protein family. The
serum levels of BTP increase in a number of renal diseases. It has been proposed as an alternative marker for
GFR in children and in persons with diabetic nephropathy or various other renal diseases. BTP appears to be
better than creatinine for the estimation of GFR but is not as good as Cystatin-C.
Calculation of GFR based on serum BTP values (mg/L) have, recently, been proposed using the following
equations, if serum urea (eqn 1) or creatinine (eqn 2) concentrations are available.
Eqn 1 : GFR = 112.1 X BTP-0.662 X Urea-0.280 (X 0.880 if female)
Eqn 2 : GFR = 167.8 X BTP-0.758 X Creatinine-0.204 (X 0.871 if female)
(Creatinine concentration in μmoles/L)
C) Tests for Renal Tubular Function
As discussed above, primary function of the tubules is to reabsorb the useful molecules from the filtrate. A
number of inherited disorders have been documented that involve the defective transporters in the renal
tubules. The reabsorption of the specific biomolecule (e.g. amino acid) would be less than optimum due to

By Eido
the mutated tranport carrier, therefore, biomolecule would be lost in the urine (see Hartnup’s disease,
cystinuria and Fanconi’s syndrome above).

Urine Specific Gravity 

Although a crude indicator of fluid status, urine specific gravity is an important parameter to measure the
net excretion from the kidneys. It is a measure of the density of urine compared with the density of water.
High specific gravity would indicate more retention of water and vice versa.
Normal Values: Adult 1.001-1.040 
Infants and children < 2 years 1.001-1.018
Increased Specific Gravity: Seen in cases of dehydration, increased ADH (SIADH), trauma, surgery, stress
and some drugs. High concentration of abnormal urinary constituents like glucose, aminoacids or proteins
would increase the gravity of urine, therefore, high specific gravity is seen in cases of glucosuria,
proteinuria etc.
Decreased Specific Gravity: Excessive dilution of urine due to compromised renal tubular reabsorbtion can
result in decreased specific gravity. Seen in cases of renal failure, glomerulonephritis, pyelonephritis and
decreased ADH secretion (diabetes insipidus).

Serum and Urine Osmolality

‘Osmolality’ refers to the osmotic potential of any fluid and is a measure of the total number of molecules
per unit weight of the solution, without regard to the size or weight of the particles. Sodium, blood urea, and
glucose levels are the major factors in determining serum osmolality. Expressed as mOsm/kg water,
osmolality of urine and serum can yield important information about a patient's ability to maintain a normal
fluid balance status. Normally, body maintains an osmolality of plasma at 290 mOsm/kg. In case of impaired
renal (tubular) function, kidneys are unable to concentrate urine and the urine osmolality will resemble that
of plasma. Serum osmolality values <240 mOsm or >321 mOsm are alarming situations. A serum of
osmolality of 384 mOsm produces stupor, whereas at over 400 mOsm, the patient may have grand mal
seizures. Values greater than 420 mOsm are fatal.
Expected values
Serum osmolality: 282 - 295 mOsm/kg water (a serum osmolality of 285 mOsm correlates with a urine
specific gravity of 1.010).

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 Urine osmolality: It could have very wide range (50 - 1400 mOsm/kg water) depending upon the degree
of hydration, but average is about 500 - 800 mOsm. After an overnight fast, the urine osmolality should
be at least 3 times the serum osmolality. A 24 hour urine osmolality should average between 500 and 800
mOsm/Kg.
Substances such as glucose, proteins, or dyes increase the urinary specific gravity. Therefore, urine
osmolality is a more accurate measurement of urine concentration than specific gravity, and urine
osmolality can be compared with the serum osmolality to obtain an accurate picture of a patient's fluid
balance.
Increased serum and urine osmolality (hyperosmolality) is seen in - Renal disease, Congestive heart failure,
Addison's disease, Dehydration, Hypercalcemia, Diabetes mellitus/hyperglycemia, Hypernatremia, Alcohol
ingestion, Mannitol therapy and Azotemia.
Decreased serum and urine osmolality (hypoosmolality) is seen in - Sodium loss due to diuretic use and a
low salt diet, Hyponatremia, Adrenocortical insufficiency, SIADH and Excessive water
replacement/overhydration/water intoxication.
Mostly, serum and urine osmolalities run parellel with each other, but inverse relationship indicates disease
status. Increased urine osmolality with low serum osmolality is observed in SIADH; the reverse is true in
case of ADH deficiency (Diabetes inspidus) or renal tubular defects.

Concentration and Dilution Tests

Apart from specific reabsorption of glucose, amino acids and other metabolites, renal tubules also absorb
water in response to antidiuretic hormone (ADH). Absorption of water concentrates the urine and its specific
gravity increases. Impaired water absorption from the tubules leads to excessive loss of water through urine
(diuresis). There is a battery of tests known as ‘concentration tests’ and ‘water dilution tests’ that are based
on measuring the specific gravity of urine after deprivation or over-loading of water, respectively.
The tests like ‘Fishberg concentration test’ and ‘Lashmet-Newburg concentration test’ are based on fluid
restriction over a period of time followed by measurement of the specific gravity. Normal tubular function
would retain more water leading to increased specific gravity of urine.
Similarly, water dilution tests involve loading the patient with more than 1200 ml of water over a short
period of time and checking for any decrease in the specific gravity of urine. Although, easy bed side tests,
the concentration and dilution tests are no longer used in the hospitals.

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Excretion Tests
A number of tubular function tests are based on the excretion of foreign substances (dyes) from the
renal tubules. The dyes like Phenol Sulphthalein, Diodone, and p-amino hippurate (PAH) have been tried to
assess the ability of the real tubules to eliminate them into the urine.

Phenol Sulphthalein(PSP) Excretion Test

Most (94%) of the PSP is excreted through the tubular secretion and only 6% is filtered through the
glomeruli, therefore, Rowntree and Geraghty (1912) deviced this test to estimate the tubular secretion.
About 6 mg of the PSP is injected in 1.0 ml water and its appearance and excretion in urine over first 15
minutes is measured. About 30 – 50% of the injected dose of PSP should be excreted in urine within 15
minutes.
The test has been used to estimate individual kidney function by bilateral catheterization. Appearance time of
the dye at the tip of each catheter indicates the function of the respective kidney.

D) Miscellaneous Tests for Renal tubular function

Radioisotope Renography
The concentration of a radiolabeled (gamma emitter) substance by the two kidneys and its subsequent
excretion is followed by a gamma camera positioned over the kidneys. The camera not only gives the visual
image of the radio-pharamaceutical being concentrated by each kidney, but draws a time-activity graph that
shows the transit of the radioactivity through the kidneys.
The Time-Activity curve drawn when the radioactivity is concentrated and then excreted from each kidney is
known as ‘Renogram’ (Fig 40.3).
A number of drugs/dyes have been tried for the studies – most successful have been 51Cr-EDTA and 131
I-
labeled Hippuran. But 131
I, being a beta plus gamma emitter, is harmful to the kidneys, therefore, Tc-
99m

labeled DTPA (Dithylene Tri-amine Penta Acetic acid) and 99m

Tc-MAG3 (mercapto-acetyl-triglycine) are
now used for routine renograms.
The radioisotope renogram can give us the information about kidney function -
a) Renal blood supply – In the few initial seconds after the injection, radiopharmaceutical gets
concentrated in the two kidneys in proportion to the blood supply to each kidney. In case of impaired
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 blood supply (renal artery stenosis) the uptake of radioactivity by the specific kidney would be
slower. The initial rise in the renogram, thus, tells us about the renal plasma flow. A slower uptake
could indicate renal artery stenosis but could also be seen in case of obstructive uropathy due to back
pressure in the tubules.
b) Glomerular mass – The height of the renogram (peak of the radioactivity) over the kidneys, and the
area under the renogran curve (AUC), is proportional to the total renal parenchymal (glomerular)
mass. Any loss of renal tissue (chronic renal failure) would result in lesser uptake of the radioisotope
and decreased AUC.

Figure 39.3 99mTc-DTPA renogram of two patients – A) Normal kidney function; B) Renogram from ‘A’; C)
Compromised Right kidney function; D) Renogram from ‘C’. Red line – Right kidney, Blue line – Left kidney

c) Glomerular Filtration Rate – 99mTc-DTPA renogram is the modern gold standard for the estimation of
GFR. The clearance of the radioisotope from the kidneys results in a downward trend in the
renogram. The slope of this decline (calculated by computer software by using polynomial equations)
gives us the GFR of the two kidneys. Slow decline could be observed in case of renal tubular defects
or urinary obstruction.
d) Diffrential assessment of the kidneys – We get two graphs in the renogram - each line represents the
blood supply, concentraiton and excretion of the respective kidney. Any assymetry in the function of
the two kidneys can be picked up very easily.

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 e) Assessment of Successful Transplant – Careful monitoring of graft function after renal
transplantation is necessary for short- and long-term success. Post renal transplant renogram is done
to assess the perfusion as well as functioning of the transplanted kidney.

Urinary Beta-2 microglobulins (B-2-M) - The short chain of HLA class I proteins is filtered by the
glomeruli and is fully re-absorbed from the proximal tubules. B-2-M appears in urine when levels in the
glomerular filtrate exceed their tubular maximum (more than 4.5 mg/L). Appearance of B-2-M in urine
indicates impaired tubular reabsorption and hence can be used to assess the tubular function.
Urine beta-2-m has found widespread acceptance as a research tool and is considered a very sensitive
marker of renal tubular disease.
N-acetyl-β-D-glucosaminidase ("glucosaminidase", NAG) is a lysosomal enzyme (MW 140,000) found
in serum and urine. Urinary NAG is a proposed marker for tubular disease, especially to diagnose the
cases of subtle industrial poisoning, acute pyelonephritis, early acute tubular necrosis, and early
transplant rejection.

Ammonium Chloride Test - The ammonium chloride test checks for renal tubular acidosis type I (i.e.,
inability of the distal tubule to excrete a load of fixed acid). Ammonium chloride is administered orally
and a person with "RTA I" supposedly will not be able to acidify the urine to a pH of 5.4 or below.

Adenosine Deaminase Binding Protein is an enzyme from the brush borders of the proximal tubule.
Like NAG, its presence in urine indicates renal tubular injury.

Urinary alkaline phosphatase is an enzyme present in proximal tubular brush border cells and in case
of renal tubular injury, the enzyme could appear in the urine.

By Eido