Biochemical kidney function tests with their clinical applications

Biochemical kidney function Tests with their
clinical applications:1
Dr. Rohini C Sane

Classification of Kidney Function Tests
❖Classification of Kidney Function Tests:
1. Tests to measure glomerular function rate→ glomerular function tests : all
Clearance tests(insulin ,creatinine , urea), glomerular filtration rate(GFR),
glomerular permeability, proteinuria are included in this group.
2. Tests to measure Renal Tubular function → Tubular function tests: include urine
concentration or dilution tests, urine acidification test(acid excretion test),
measurement of Tubular secretory mass , reabsorption studies
3. Analysis of blood /serum : estimation of blood urea , serum creatinine, serum
protein, serum electrolytes , urine protein, urine electrolytes, beta 2-
microglobulin (all these tests are often useful to assess renal functions).
4. Urine output/examination: simple routine examination of Urine for volume , pH,
specific gravity, osmality and presence of certain abnormal constituents (proteins,
blood , ketone bodies , glucose etc.) help to assess kidney functions /diseases to
limited extent.
5. Renal blood flow

Classification of Kidney Function Tests
TestforAssessmentof
glomerularfunctions
TestforAssessmentof
tubularfunctions
Urineanalysis Testtomeasurerenal
plasmaflow(RPF)
1. Renalclearancetest
2.Bloodanalysisof urea,
uricacidandcreatinine
3.Proteinuria
4.Hematuria
1.Urineconcentration
test(fluiddeprivationtest)
2.Urinedilutiontest(excess
fluidintaketest)
3.Tubularproteinuria(Urine
proteinelectrophoresis)
4.Aminoaciduria
5.GlycosuriaPresenceof
reducingsubstances
6.Aminoaciduria
7.Phenolsulfothalein
Test(PSP)
8.Acidloadtest(ammonium
chlorideloadingtest)
1.Physicalexamination
2.Chemical
examination
3.Microscopic
examination
1.Para-aminoHippurate
test
In addition ,renal biopsy is performed to
confirm the diagnosis of renal disease.

Clinical applications of Kidney Function Tests
❖Clinical applications of Kidney Function Tests:
1. Establish presence & absence of renal disease
2. Define the nature of disease
3. Assessment of the extent of renal damage
4. Define the degree of renal dysfunction
5. Monitoring progression of renal disease
6. Monitoring and adjusting the dose of renal toxic drugs which are mainly
excreted by the kidney.
7. Monitoring the response of treatment

Choice of kidney Function Test
• The assessment of kidney function starts with routine urine examination
followed by serum creatinine and /or blood urea estimations and final the
specific tests to measure the glomerular function and Tubular functions
(clearance tests).
• It is to be noted that two- third of tissue must be functionally damaged to
show an abnormality in these tests.

Jaffe’s reaction for Qualitative analysis of urine

Routinely performed Biochemical Kidney Function Tests
❖Biochemical Kidney Function Tests include :
1. Measurement of NPN in blood (serum creatinine , uric acid and blood urea).
The major route of excretion of all these compounds is urine.
2. Clearance tests
3. Renal plasma flow
4. Concentration & dilution tests
5. Urinary & plasma osmolality
6. Tests to assess renal acidification
7. Excretion of extraneous compounds like PSP
8. Routine complete urine analysis
9. Serum protein , albumin ,globulin ,A/G ratio
10. Measurement of serum and urine electrolytes(Tests to assess renal handling
of sodium ions)

Plasma/serum creatinine
❖Plasma /serum Creatinine :
a) Is derived from breakdown of tissue creatine.
b) Is related with muscle mass.
c) Is not significantly influenced by high protein diet and hypercatabolic shock.
d) Increase (Serum Creatinine level > 1.5 mg/dl) indicates renal impairment.
e) Plasma /Serum creatinine : is the most specific and sensitive index of renal
function . It is better indicator than blood urea although it is less sensitive
than Clearance tests to assess kidney functions .
f) urinary concentration of creatinine:70% of Serum concentration of creatinine.
g) Serum Creatinine level usually parallel to severity of renal disease .
h) The kidneys reserve is much that about 50% kidney function must be lost
before Serum creatinine is raised.

Estimation of Serum Creatinine by Jaffe’s Method
• Principle : Serum creatinine reacts with alkaline picrate to form orange color
complex of creatinine picrate . The intensity of color complex is directly
proportional to the concentration of creatinine present in serum. The intensity
of color is measured using green filter (530 nm) & compared with creatinine
standard treated similarly.
• Normal range for serum creatinine (adult male) = 0.7 -1.4 mg/dl(GFR 95- 115
ml/min)
• Normal range for serum creatinine (adult female) = 0.6 -1.3mg/dl (GFR 85-
110 ml/min)
• Normal range for serum creatinine (children) = 0.4 -1.2 mg/dl

Principle of Estimation of Serum Creatinine by Jaffe’s Method
Principle : Serum creatinine reacts
with alkaline picrate to form orange
color complex of creatinine picrate .
The intensity of color complex is
directly proportional to the
concentration of creatinine present in
serum.
The intensity of color is measured
using green filter (530 nm) &
compared with creatinine standard
treated similarly.
.

Principle of Estimation of Serum Creatinine by
enzymatic method

Clinical interpretation of serum creatinine estimation
❖Normal Range for serum creatinine : 0.6-1.2mg/dL
❖Hence serum creatinine estimation is an important kidney function test.
❖High levels of serum creatinine are also seen in early stages of muscular
dystrophy ,but as the disease progresses serum creatinine level falls.
Clinical conditions associated with High Serum creatinine levels include
Urinary tract infection
Chronic nephritis
Renal failure

Factors affecting Serum Creatinine
FactorsdecreasingSerumCreatinine Factors increasing Serum Creatinine
Low muscle mass Old age
Female Male
Malnutrition Dehydration
Medicines : Thiazide ,Vancomycin Medicines :Amphotericin, Cephalosporins, Kanamycin
Glomerulonephritis
Pyelonephritis
Renal diseases/ failure
Congestive cardiac failure

Estimation of Urine Creatinine By Jaffe’s Method
• Principle: Creatinine (from Diluted urine) is treated with alkaline picrate solution to form
orange color complex of creatinine picrate. The intensity of color complex is directly
proportional to the concentration of creatinine present in urine. The intensity of color is
measured using green filter (530 nm) & compared with creatinine standard treated similarly.
The intensity of color is directly proportional to concentration of Creatinine in serum.
• Dilution of urine : Dilute urine 1: 100 with distilled water
• Concentration of creatinine in sample (gm/L) = Y
• Urinary excretion of creatinine in24 HRS (gm/ 24 Hr.)= Y X volume of urine excreted in
liters over period of 24hr (e.g. 1.2 L)
• Normal Range for urine creatinine : 0.8-1.5gm/24 Hours

Clinical interpretation of urine creatinine estimation
Normal Range for urine creatinine : 0.8-1.5gm/24 Hours
Clinical conditions associated with
High urine creatinine levels
Clinical conditions associated with
Low urine creatinine levels
Diabetes Mellitus Advanced renal disease
Acromegaly Paralysis
Gigantism Muscle atrophy
infections

Creatinine coefficient
❖Creatinine coefficient is Urine Creatinine expressed in mg/body Kg weight .
• Normal range of Creatinine coefficient (male)= 20 -28 mg/kg
• Normal range of Creatinine coefficient (female)= 15 -21 mg/kg
• Creatinine coefficient value increases in muscular dystrophy .

Plasma beta-2-microglobulin
❖Beta-2-microglobulin :
a) Is a small peptide found on the surface of mast cells.
b) Forms the part of class I antigen histocompatibility complex.
➢Plasma Beta-2- microglobulin :
1. is the reliable index of GFR because it is completely filtered by the glomeruli
and reabsorbed . It is catabolized by the cells of the PCT .
2. is not influenced by diet or muscle mass .
3. is increased in inflammatory diseases and malignancies .

Beta-2-microglobulin
Beta -2 microglobulin is a small peptide found on the surface of mast cells. It forms the
part of class I antigen histocompatibility complex.

Renal threshold of substances:1
• Renal threshold of substances : is defined as its concentration in blood (or
plasma) beyond which it is excreted into urine.
❖While calculating the renal threshold of a particular compound ,it is assumed
that both the kidneys are optimally functioning ,without any abnormality .
❖This is not always true (conditions in which renal threshold is altered) .
❖Renal Glycosuria : is associated with reduced threshold for glucose due to its
diminished tubular reabsorption.

Renal threshold of substances:2
• Renal threshold of substances : is defined as its concentration in blood ( or
plasma) beyond which it is excreted into urine.
Substance Renal threshold of substances
Glucose 180 mg/dl
Ketone bodies 3 mg/dl
Calcium 10 mg/dl
Bicarbonate 30 mequ /L

Properties of the substances used to measure GFR
❖Properties of the substances used to measure GFR include:
1. Freely filtered by glomerulus.
2. Should not be reabsorbed/ secreted.
3. Should not be metabolized by the kidney.
4. Should not be toxic .
5. Should not be affected by its dietary intake.
❖Endogenous substances used for clearance test : Creatinine and Urea
❖Exogenous substance used for clearance test : Inulin

Advantages clearance Tests for measuring glomerular filtration
❖Renal clearance determination may be most helpful in the early stages of
progressive renal disease while blood analysis may be more sensitive when
renal failure has advanced.
❖Creatinine clearance test and Urea clearance tests:
1. Evaluate the actual excretory capacity of the kidney since they measure the
amount of a substance excreted in the urine as compared to concentration
of the same substance in the plasma .
2. are sensitive and clinically more useful than the tests measuring retention
of substance as urea ,creatinine as these substances may not elevate in
blood until there is a significant decrease in glomerular filtration kidney
function (below 50% normal clearance).

Calculation of Renal Clearance
❖Renal clearance depends on the plasma concentration of the substance ,its
excretory rate ,which in turn depends on the GFR and renal plasma flow.
❖GFR can be measured by determining the excretion rate of a substance which
is filtered through the glomerulus but neither reabsorbed nor secreted by the
renal tubules.
❖ Clearance (C) of a substance(in ml /min) = U X V / P
U= mg/L concentration of a substance in urine
V = volume of urine in ml excreted per min (24hr urine volume/24x60 )
P = mg/L concentration of a substance in plasma/serum
Concentration of a substance in urine and plasma /serum should be expressed
in same units (mg/dl or mmols /L).
Clearance of a given a substance is determined by mode of excretion .
❖The maximum rate at which the plasma can be cleared of any substance is
equal to the GFR.

Renal conditions associated with lower GFR
• Acute tubular necrosis
• Glomerulonephrosis
• Acute and chronic renal failure

Inulin Clearance tests
❖Inulin :
1. a plant carbohydrate(polysaccharide composed of fructose units) .
2. Not metabolized by the human body/renal tubules.
3. Inulin is freely filtered by the glomerulus and is neither reabsorbed neither
secreted in the tubules of kidney. Therefore GFR can be calculated by measuring
Clearance of Inulin(close to GFR).
4. Inulin Clearance =125 ml/min
5. Inulin is administered intravenously(exogenous) to measure GFR. Therefore Inulin
Clearance tests is not preferred but it is the method of choice when accurate
determination of GFR is required.
❖51Cr-EDTA satisfy similar criteria. The GFR is calculated from the rate of fall of
plasma radioactivity as the isotope is cleared .
• Creatinine and Urea are employed and preferred for measurement of Clearance as
they are present in the blood (endogenous).

Procedure of Inulin Clearance tests
❖Procedure of Inulin Clearance tests :
1. About 100ml sterile 10 % solution of inulin is given as slow intravenous drip
within 2 hr.
2. Urine specimen formed during this period is collected totally .
3. Blood sample is taken in the middle of test .
4. Inulin is estimated by resorcinol giving a red color .
5. The test needs continuous infusion of inulin so as to keep plasma levels
adequate.
6. Since it involves administration of exogenous compound , this procedure is
not used routinely .
❖Inulin Clearance – Urea Clearance /glomerular filtration rate= 125-75/ 125=0.4
(40 % of urea present in glomerular filtrate is reabsorbed in the tubules) .

Disadvantages of measurement of inulin clearance Test
❖Disadvantages of measurement of inulin clearance Test :
1. The need for its intravenous administration.
2. The technical difficulty of its analysis.

Creatinine Clearance tests
❖Creatinine Clearance tests:
• Creatinine is excretory /waste product derived from Creatine phosphate present in
muscles . This conversion is spontaneous ,non enzymatic .
• As the production of Creatinine is continuous ,the blood level will not fluctuate
much . Blood may be collected at any time.
• Excretion of Creatinine : constant , is not influenced by dietary/external factors ,age
or body metabolisms/exercise .
• Women and children excrete less Creatinine than ,because of smaller muscle mass.
• Creatinine is freely filtered by the glomeruli but not reabsorbed by the tubules. It is
marginally secreted by the tubules. Because these properties the clearance can be
used to estimate GFR.
• The value of Creatinine Clearance is close to GFR . The measurement is a sensitive
and good approach to assess the renal glomerular function.
• 24 hr. urine sample should actually contain total urine volume especially when
collected from children or mentally retarded person .
Advantages of Creatinine Clearance tests.

Calculation of Creatinine Clearance
• Creatinine Clearance may be defined as the volume (ml)of plasma that would be
cleared of creatinine per minute . Clearance of Creatinine refers to the milliliters of
plasma which contains the amount of that Creatinine excreted by kidney per minute.
• Creatinine Clearance (C) = U X V / P
U= urine concentration of Creatinine (mg/L)
V = urine output in ml/min (24 hr. urine volume /24 x60)
P = plasma concentration of Creatinine(mg/L)
Concentration of Creatinine in urine and plasma should be expressed in same units
(mg/dl or mmols/L).
• Normal range of Creatinine Clearance (for males)= 85-125 ml/min (marginally higher
than GFR as it is partially secreted by tubules)
• Values of Creatinine Clearance are lower in women due to lower muscle mass
compared to males. (75-115ml / min).
❖ Creatinine Clearance can be expressed in terms of body surface area.

Normal reference Creatinine clearance values
Category Serum creatinine GFR
Adult male 0.7-1.4 mg/dL 95-115 mL/min
Adult female 0.6-1.3 mg/dL 85-110 mL/min
children 0.5-1.2 mg/dL
Patient with graver prognosis ≤ 50 mL /min
For 1.73 Square meter body surface area

Correction of creatinine clearance for body surface area
• It is useful to correct the clearance value for surface area especially in
children ,person with short and tall frame .
• creatinine clearance = U X V X 1.73/ P X A
• After Correction for body surface area, creatinine clearance becomes
comparable for male , female and children which is 100 ml/min/1.73 sq.
meter.

Diagnostic importance of creatinine clearance tests
• A decease in creatinine clearance (value < 75% normal)serves as a sensitive
indicator of deceased GFR due to renal damage.
❖This test is useful for an early detection of impairment in kidney functions (often
before the clinical manifestations are seen e.g. acute or chronic damage to
glomerulus or any of its components ) .
❖Reduced blood flow to the glomeruli may also produce a decreased creatinine
clearance.
❖Changes in plasma creatinine which may not apparently indicate abnormal renal
function , may show gross changes in the value of creatinine clearance.
❖Rise in plasma creatinine by another mg (= 2 mg/dl) will decease the clearance value
by 50 %.
❖Other substances will not show such drastic drop in the clearance value.
❖The test may be helpful in long term monitoring of patients with renal insufficiency
under protein restricted diet.

Relationship between glomerular filtration rate (GFR)and
Serum creatinine
• It is observed that the GFR must fall to about 50% of its normal value before
significant increase in Serum creatinine occurs .
• Normal Serum creatinine level dose not necessarily mean that all is well with
the kidney.
• Loss of 50% of the functions of nephrons leads to doubling of Serum creatinine
concentration(approximate).

Therelationshipbetweenglomerularfiltrationrate(GFR)and
SerumCreatinine

Advantages of creatinine clearance
1. External factors will rarely interfere. The effect of fluid intake and excretion
on creatinine clearance is much less than that of urea.
2. Conversion of creatine phosphate to creatinine is spontaneous and non
enzymatic.
3. As the production of creatinine is continuous , blood levels will not
fluctuate. Blood may be collected any time of day for the test.
4. It is not affected by diet or exercise . The blood creatinine values relatively
stable.
5. No need of intravenous administration of creatinine as it is synthesized
endogenously.
6. It fulfils all the requirements of renal substance i.e. freely filtered and not
reabsorbed by the tubules.

Disadvantages of Creatinine Clearance test
• Creatinine is filtered by glomeruli and actively excreted by the tubules .
• When GFR is severely reduced ,external excretion increases .Then major route
becomes the degradation by intestinal bacterial flora . This will violate results.
The Creatinine Clearance is overestimate GFR by about 10-20 ml/min.
• Very early stages of decease in GFR may not be identified by Creatinine
Clearance(Creatinine blind area).
• Other prerenal, renal and postrenal causes influence Creatinine Clearance .

Limitations of Creatinine Clearance test
❖Creatinine Clearance tests is not reliable because
a. Creatinine is partially secreted by tubules.
b. The test is incapable of differentiating prerenal , renal and postrenal causes
of renal failure .

Blood Urea
❖Blood Urea :
1. is the end product of protein metabolism.
2. is synthesized in the liver.
3. After being filtered by the glomeruli ,it is partially passively reabsorbed by
the renal tubules at low rates of urine flow.
4. plasma Urea levels is an useful as an index of glomerular function.

Blood Urea level
• Normal Blood Urea level : 20- 40 mg/dL
• Estimation of Blood Urea level is done by enzymatic method using urease and
glutamate dehydrogenase .
• Blood Urea level is expressed in terms of its nitrogen(nitrogenous substances
analyzed by kjeldahl method).
• Blood Urea level = Blood Urea nitrogen (BUN) x 2.14
• Molecular weight of urea = 60 and each 60 gram of Urea contains 28 gram of
nitrogen→ 60/28 
Since route of urea excretion is through kidney ,blood urea level is estimated to
assess kidney function.
As urea level is affected by dietary protein and other pre-renal factors ,serum
creatinine estimation should also be done simultaneously.

Diagnostic importance of Blood Urea level :1
Blood urea level decreases rarely.
Low Blood Urea level is seen in :
1. Pregnancy
2. Starvation
3. Hepatic failure (terminal stages of liver failure)
4. Diet grossly low in proteins
Blood Urea level increase with:
a. age
b. Lower range in young adults
c. higher range in elderly people .

Diagnostic importance of Blood Urea level :2
❖Causes of increased Blood Urea levels :
Prerenal Renal Postrenal Medication
Dehydration Acuteglomerulonephritis* Stonesinurinarytract ACEinhibitors
Severevomiting Nephrosis Enlargedprostrate Acetaminophen
Intestinalobstruction Malignanthypertension Strictureoftheurethra Aminoglycosides
Diarrhea Chronicpyelonephritis Tumorsofbladder
affectingtheureteric
junction
Amphotericin B
Diabeticcoma Renaltuberculosis Diuretics
Severeburns Mercurialpoisoning NSAIDs
Fever Nephritis
Severeinfections * Valuesmaybeashighas
300mg/dl

Ratio of blood urea nitrogen and serum creatinine
Condition Ratio of blood urea nitrogen and serum creatinine
Normal(physiological) 24-40
Retention of blood urea Nitrogen
due to pre-renal causes
≥ 40
Absence of pre-renal conditions but
early stages of renal disease
24-40(Normal since tubules secrete raised serum
creatinine up to a level )
Post renal obstruction ≤14 ( depending upon degree of obstruction of urea
excretion)
Severe renal tubular damage 10
Serum creatinine values ≥ 2 mg/dL suggestive of severe kidney damage.

Calculation of Urea Clearance
• Urea Clearance may be defined as the volume (ml )of plasma that would be
cleared of Urea per minute . Clearance of Urea refers to the milliliters of
plasma which contains the amount of that Urea excreted by kidney per minute.
• Urea clearance = UV/P X 1.73/A
• Maximum Urea Clearance (Cm) = U X V / P*
U = urine concentration of Urea (mg/ml )
V = urine output in ml/min (24 hr. urine volume /24 x60)
P = plasma/serum concentration of Urea (mg/ml)
A = body surface area of the patient ( 1.73= standard average surface area of the
normal individual)
* The above calculation is applicable if urine output is more than 2 ml/min.
Normal range of Maximum Urea Clearance = 75 ml/min(60-95ml) .It is lower
than GFR as it is partially reabsorbed by tubules.

Standard Urea Clearance Test
❖Urea Clearance drastically changes when volume of urine is less than 2 ml/min
This is known as Standard Urea Clearance(Cs).
• Standard Urea Clearance (Cs) = U X V / P*X 1.73/A
U= urine concentration of Urea (mg/ml)
V = urine output in ml/min (24 urine volume / 24 x60)
P = plasma /serum concentration of Urea (mg/ml)
A = body surface area of the patient ( 1.73= standard average surface area of the
normal individual)
✓The above calculation is applicable if urine output is less than 2 ml/min.
➢Normal range of Standard Urea Clearance = 54 ml/min(40-65ml) .

Diagnostic importance of Urea clearance test
• Urea Clearance < 75% of the normal (normal value = 75%) suggestive of renal
failure .The Clearance values fall progressively with failing renal function .
A decease in Urea clearance serves as a sensitive indicator of deceased GFR
due to renal damage( < 20 % in severe renal failure).
In Uremic coma ,Urea Clearance is < 5% of the normal .
• Blood Urea levels as such are in normal physiological range and is found to
increase only when the Urea clearance falls below 50% normal i.e. the Urea
Clearance values may be abnormal even though serum Urea values are within
normal limits .
• Pre-renal conditions : increase in blood urea, the urea clearance value may not
fall significantly ,due to high excretion of urine in urine.
❖Creatinine clearance is a better indicator of renal function than Urea clearance
The sensitivity of urea clearance test is much less than creatinine clearance .

Limitations of Urea Clearance Test
❖Urea Clearance although useful in assessing renal failure ,it is not true
measure of GFR (Urea Clearance < GFR) because :
a) influenced by protein content of diet, protein catabolism , protein loss by
hemorrhage, dehydration, fluid intake ,infection ,surgery and liver diseases
(can affect plasma Urea levels) .
b) 40% of filtered Urea is partially passively reabsorbed by both PCT and DCT
of the renal tubules (hence tubular function affects Urea Clearance).
❖Urea Clearance is not as sensitive as creatinine clearance for assessing renal
functions.

The relationship between glomerular filtration rate (GFR) ,
Blood Urea Nitrogen and Serum Creatinine

Specimen for serum uric acid estimation

Principle of Estimation of Serum Uric Acid by enzymatic method
(Uricase/ Peroxidase method):1
Serum uric acid undergoes hydrolysis in presence of enzyme uricase to liberate
allantoin , carbon dioxide & hydrogen peroxide . Hydrogen peroxide so formed
undergoes to hydrolysis in presence of enzyme peroxidase to liberate nascent
oxygen. Nascent oxygen being highly reactive, reacts with 4-amino pyrene &
dichlorophenol sulphonate (DCFS) to form a color complex.
The intensity of color complex is measured with green filter at 540 nm. The
intensity of unknown is compared with standard treated similarly against the
blank. The intensity of color is directly proportional to concentration of uric acid
in serum.

Principle of Estimation of Serum Uric Acid by enzymatic
method(Uricase/ Peroxidase method):2

Procedure of Estimation of Serum Uric Acid by enzymatic
method(Uricase/ Peroxidase method)
Label three test tubes as BLANK(B), STANDARD (S), TEST (T)
Pipette reagent as follows:
Mixwell.Incubateatroomtemperaturefor10mins.MeasureOD(absorbance)usinggreenfilterat520nm.
Calculations of Concentration of Serum Uric Acid (mg/dl) =
OD TEST(T) – OD OF BLANK (B)
OD STANDARD (S) - OD BLANK (B)
BLANK (B) STANDARD (S) TEST(T)
Distilled water 100 µl -- --
Standard (6 mg/dl) --- 100 µl ---
Sample --- --- 100 µl
Working reagent 1ml 1ml 1ml
X 6

Principle of estimation of serum uric acid by phosphotungstic acid
(Caraways method)
Uric acid from protein free filtrate of serum reduces phosphotungstic acid
reagent in presence of sodium carbonate to form blue colored
phosphotungstous salt.
The intensity of color complex is measured with green filter at 540 nm. The
intensity of unknown is compared with standard treated similarly against the
blank.
The intensity of color is directly proportional to concentration of uric acid in
serum.

Normal values of serum uric acid by method(enzymatic)
• Normal concentration of serum uric acid = 2-6 mg/dL (357 micromoles / L)
• Men –3.5-7.2 mg/dl (210-420 mg/dL)
• Women ---2.6-6.0 mg/dl (150-350 mg/dL)

Diagnostic importance of Serum uric acid levels
• Normal range of serum uric acid : 2-6 mg/ dL
• Increased serum uric level (hyperuricemia)may occur due to increased
formation of uric acid ,decreased its excretion by kidney (therefore
Kidney/renal function Test) or combination of both.
Hyperuricemia Hypouricemia
Glomerulonephritis Fanconi’s syndrome
Gout(primary and secondary gout) Wilson’s disease
Diabetic ketoacidosis Severe liver disease
Lesch-Nyhan’s syndrome
Von-Gierke’s disease Xanthuria(congenital xanthine
oxidase deficiency)
Lactic acidosis
Leukemia, Multiple myeloma
Polycythemia
Gout

Azotemia
• Impairment of renal function results in elevation/ increase in blood levels of
non-protein nitrogen (NPN) i.e. Blood urea (normal range20-40mg/dL) and
serum creatinine ( 0.5-1.2mg/dL) .An increase of these end products in blood is
called Azotemia .It is hallmark of kidney failure .
• BUN / mg Creatinine Ratio = 12-20 (Normal adult on normal diet)
Low BUN/mgCreatinineRatio HighBUN/mgCreatinineRatio
Acute renal tubular necrosis Prerenal Azotemia
Lower protein intake High BUN/mg Creatinine Ratio with High urea and high
creatinine indicates Prerenal Azotemia superimposed on
chronic kidney disease (CKD) or post renal obstruction
Starvation
Severe liver disease

Uremic syndrome
❖Uremic syndrome: is the terminal manifestation of renal failure .
❖Effects of Uremic syndrome:
1. Increased urea levels causes Carbamoylation of proteins.
2. Increased uric acid causes uremic pericarditis(thickened pericardium).
3. Excess polyols is the basis of peripheral neuropathy.
4. Increased Beta-2-micoglobullins is the reason for amyloidosis.

Uremic syndrome
Uremic peripheral neuropathy Uremic pericarditis
Uremic syndrome : Excess polyols is the basis of peripheral neuropathy . Increased uric acid
causes uremic pericarditis.

Cystatin C
❖Cystatin C is
• produced at constant rate and expressed in all organs of body.
• freely filtered by kidney glomeruli.
• completely reabsorbed but degraded in the tubules.
• Serum Cystatin C not affected by age ,sex ,muscle mass or inflammatory
processes.
• Sensitive to changes in the creatinine blind area of GFR(40-70 ml/min/1.73 m2)
• There is no tubular secretion of Cystatin C ,it is sensitive to minor changes in
GFR in the earliest stages of chronic kidney diseases .

Cystatin C as a glomerular filtration maker
• Cystatin C:
• a protein maker of kidney Function and more sensitive than creatinine .
• is non glycosylated protein and has 120 amino acids (13 KD) .It is most
abundant extracellular cysteine protease inhibitor.
• High concentration of Serum Cystatin C in all fluids e.g. milk, tears , saliva,
semen .
• Serum Cystatin C reference range : 0.8-1.2 g/dl (mg/L)
• Even minor changes in GFR in the early stages of chronic kidney diseases are
associated with increased Serum Cystatin C levels .

Cystatin C
Serum Cystatin C is not affected by age ,sex,
muscle mass or inflammatory processes.

Tests determining renal blood flow

Tests determining renal blood flow
Renal blood flow : the volume of blood passing through kidney per minute.
Characteristics of substances used for tests determining renal flow :
1. foreign to the human body
2. Being filtered through the glomeruli of nephron
3. Removed by the kidneys via tubules by secretion
e.g. p-amino Hippurate (PAH) is removed about 90% from the plasma in a single
passage through kidney.
Total renal blood flow of p-amino Hippurate (PAH): 1100ml/min
Clearance of p-amino Hippurate (PAH): 700ml /min (by considering the fact that
about 10% of blood circulating through kidneys does not come into contact with
functional cells).

Diodrast and Para amino hippuric acid (PAH)
• Diodrast: ( di-idopyridone acetic acid): is used as a contrast medium to take
urinary tract X-rays.
• Diodrast is partly filtered by glomerulus and mostly excreted by the tubules.
• Diodrast and para amino hippuric acid (PAH): excreted by a single passage of
blood through the kidneys.
• Para amino hippuric acid (PAH) has a clearance 700 ml/min or1200 ml/min if
expressed as blood ( 1/4th of total cardiac output) .
• Thus clearance of Para amino hippuric acid (PAH) represents renal plasma flow.
• Since , renal plasma flow is 700 ml/min and GFR is 125 ml/min →1/5th of the
plasma brought to glomeruli becomes glomerular filtrate. This is called the
filtration fraction.

Substance filtered Reabsorption by
renal tubular cells
Secretion by renal
tubular cells
Result
Creatinine/Inulin Nil Nil GFR = clearance
Uric acid 70 % by proximal
convoluted tubules
(PCT)
by distal convoluted
tubules( DCT)
GFR  clearance
Urea 40 % by PCT Nil GFR > clearance
Diodrast /PAH Partial Nil GFR < clearance
Relationship of GFR with renal clearance

Common tests to assess kidney functions
Constituent of urine Normal Blood levels Normal urine levels Factorsaffectingurinary
excretion
Urea 15-40 mg/dL 15-30g/day Dietaryproteins,protein
catabolism,renalbloodflow
Creatinine 0.7-1.4 mg/dL(male)
0.3-1.3 mg/dL(female)
1-2 g/day Age,sex,musclemass,GFR,
Tubularsecretion
Uric acid 3-7 mg/dL(male)
2-5 mg/dL(female)
0.5-0.8 g/day Purinemetabolism,tubular
excretion
Sodium 135-142 mmol/L 40-220 mmol/L Stateofhydration,dietary
sodium,renalfunction
Potassium 3.5-4.5 mmol/L 25-125 mmol/L dietarypotassium,renal
function,acid-basebalance
Calcium(total) 9-11mg/dL 200 mg/day dietarycalcium,Calcitonin,
renalfunction,vitaminD,PTH

Pre-renal ,renal and post-renal conditions
Pre-renal conditions Renal conditions Post-renal conditions
Patients in cardiac failure
frequently have diminished
kidney functions ,which may
result in accumulation of
metabolic products in the
blood.
Reduction of kidney functions
may develop as a consequence
of damage to the epithelium of
the capillaries , which make up
the glomerular tuft.
Sometimes the solutes
present in the urine are
precipitated either within the
pelvis of the kidney or in the
ureter or tubules .These
results in appearance as
calculi.

Early symptoms of Kidney failure
❖Early symptoms of the Kidney failure include :
1. Polyuria (passing more volume and frequency of urine)
2. nocturia (passing more urine during night)
3. High blood pressure
4. Unexpected Anemia
5. Fatigue ,lassitude and tiredness
6. Microalbuminuria
7. Mild elevation of serum creatinine

Tests for assessment of tubular functions

Biochemical Tests for tubular functions
❖Assessment of the concentrating and diluting ability of kidney can provide the
most sensitive means of detecting early impairment in renal function since the
ability to concentrate or dilute urine is dependent upon :
1. Adequate GFR
2. Renal plasma flow
3. Tubular mass
4. Healthy tubular cells
5. Presence of antidiuretic hormone (ADH)
❖ The urinary specific gravity and osmolality are used to measure the
concentrating and diluting ability of the tubules.

Urine concentration test(fluid deprivation test)
❖Urine concentration test helps toassess renal tubular function.
• Urine concentration Test issimple and involves accurate measurement of Specific Gravity
of urine (after overnight water and food deprivation) which depends on concentration of solutes
inurine.
• One day before the test(15 hr.) ,fluid is deprived after breakfast ,but food is allowed .
• Inaddition to early morning specimen , urine samples are collected at 60 and120 minutes.
• Concentrating ability of kidney depends on the ability of the posterior pituitary toelaborate
antidiuretic hormones (ADH) in response towater deprivation and on tubular response toADH.
➢Ifthe Specific Gravity of urine is above 1.020 for at least one of the sample collected or osmolality
exceed 850 mosmol/kg ,the renal tubular function(renal concentrating ability) isconsidered
tobe normal .
➢Inkidney diseases ,the maximum Specific Gravity of urine is less than 1.020 in any of the three
samples .
➢The test is useful in differential diagnosis of psychogenic polydipsia and diabetes insipidus.
➢The ADH test is preferred to test concentrating ability .

Urine concentrating Test(fluid deprivation Test)in dehydration
• Dehydration maximally stimulates ADH secretion . If kidney is normal ,water is
selectively reabsorbed resulting in excretion of urine of high solute
concentration and urine osmolality should be at least three times that of
plasma (286mosmol/kg).

Clinical interpretation of urine concentration Test
In case the urine doesn’t have specific gravity 1.025 or osmolality 850
mosmol/kg ,it is sure that renal concentrating ability is impaired either due to:
• tubular defect or
• Decreased secretion of ADH (diabetes insipidus).
So, ADH test must be carried out.
❖If the renal tubules and collecting ducts are working efficiently and if ADH is
present they will be able to reabsorb water .
❖In polyuria of diabetes insipidus ,where hormone ADH is lacking ,or in
nephrogenic diabetes insipidus(lack of response to ADH),the osmolality will
remain constant even after fluid deprivation.
❖ In practice fluid deprivation test is extremely unpleasant for the patients .
It is potentially dangerous if there is severe inability to retain water.
❖ Clinically ,the loss of concentrating ability is manifested by nocturia ,
(passage of urine at night) and polyuria.

Urine dilution test (water load test)
❖Urine dilution test (water load test/ excess fluid intake test ) helps to
assess renal tubular function.
❖Procedure of urine dilution test: after emptying the bladder , 1000 ml to
1200ml of water is given to patient . Urine specimens are then collected
every hour for next 4 hours.

Clinical interpretation of urine dilution test
❖Clinical interpretation of urine dilution test:
➢If functioning of renal tubule is normal , the urinary specific gravity should fall
to 1.005 or less or an osmolality of less than 100mosm/kg.
❖If the renal tubules are diseased , the concentration of solutes in urine will
remain constant irrespective of excess water intake.
➢When the Specific Gravity of urine doesn't fall to 1.003 or below after a water
load , it indicates impairment of renal function.
➢With impaired renal function such low specific gravity is not reached and
smaller volume of urine are passed (urine volume <100 ml with specific gravity
of 1.10 in more advanced cases).
➢Renal or cardiac failure may be cause of decreased urine output. If
concentrating ability is normal the cause may be extra –renal.
➢However ,this test is not routinely performed.

Applications of measurement of Osmolarity of urine
• Osmolarity : a measure of the concentration of dissolved/free particles( ions/
unionized particles) in the specimen/solution.
• Osmolarity =molarity x number of particles (ions or unionized molecules)
resulting from ionization.
• The concentrating activity of the renal tubules is regulated by osmoreceptors
which are sensitive to changes in the solute concentration rather than to
changes in the specific gravity .
• The simultaneous determination of serum and urine osmolarity is considerably
more accurate way of measuring the concentrating ability of the tubules.
• Application of measurement of Osmolarity of urine : determination gives
better idea of concentrating ability of the renal tubules.

Osmolality in response to ADH
Condition Urinary
osmolarity
Plasma
osmolarity
response to ADH
Diabetes insipidus ( central ) Decreased increased Normal
Diabetes insipidus ( nephrogenic) Decreased increased No response
Compulsive water drinking Decreased Decreased Normal
Osmotic diuresis Normal Normal Normal

Urinary acid load test
❖Urinary acid load test / urinary acid excretion test/ Ammonium chloride
loading Test :
➢This test measures acid excretion by the kidney following administration of 0.1g of
ammonium chloride/Kg weight after an overnight fast.
➢Urine samples are collected before and at 2 ,4 and 6hr. following ammonium
chloride administration.
➢Normal Urinary acid excretion:30-90 mequ ammonia/minute with a urinary pH of
5.3 .
➢In renal failure ,there is impairment in Urinary acid excretion .

Phenolsulfonaphthalein(PSP)Testfortubularsecretoryfunctionofkidney
Phenolsulfonaphthalein (PSP) or phenol red test : a non –toxic and safe dye. It is
exclusively excreted by kidney. Hence the dye of choice for excretory function of
kidney .
Application of Phenolsulfonaphthalein (PSP) Test : gives measure secretory capacity
of the tubules. In the absence of renal disease , test may be used as a measure of
renal blood flow.
➢Test is contradicted in azotaemia and patients with bladder retention.
Removal of (PSP) by a human body Percentage of removal of dye
From plasma during one passage through kidney 60-70
Liver 20
Portion of dye removed by kidney Mechanism of excretion
6 % Glomerular filtration
94% Tubular excretion

Test results of Phenolsulfonaphthalein(PSP)Testfortubularsecretoryfunction
ofkidney
Phenolsulfonaphthalein(PSP) appears in urine based on action of tubular epithelial
cells as well as the renal blood flow.
➢Intravenous test are preferred over intramuscular as they are more valid
because it eliminates the uncertainties of absorption which exist in
intramuscular test.
➢After intravenous injection of 6mg of PSP in 1ml of saline, urine specimens are
collected at 15,30,60 and 120 minutes.
Time of excretion a dye by the kidney for a
normal individual after its intravenous injection
Percentage of excretion of dye
First 15 minutes 25 or more
120 minutes (2 hours) 70 or less
First 15 minutes <23(suggestiveofimpairedexcretoryfunctionof
kidney).2hoursexcretionmaybenormal

Biochemical kidney function tests with their clinical applications

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