Renal Function Tests

Anatomic and physiologic

overview
The kidneys are a pair of bean shaped
capsulated organ.
The kidney consists of two distinct regions,
the outer cortex and the inner medulla.
Each kidney is composed of about 1 million
or more small filtration units called nephrons
(functional unit of the kidney).

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 Each nephorn consists of a glomerulus
containing afferent & efferent arterioles,
and tubule containing Bowmans capsule,
proximal convoluted tubules, loop of
henle, distal convoluted tubules, and
collecting duct.

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Anatomy of the kidney
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The Nephron 5
 Functions of Kidney
Kidney maintains water, electrolyte and acid
base balance of the body through filtration
and reabsorption process.
Glomerulus is responsible for filtration and
renal tubules are involved in reabsorption.
In addition renal tubules secretes some
solute molecules.
Kidney clears several non-protein metabolic
waste products like urea, uric acid, creatinine
etc., from circulation.
Kidney produces erythropoietin, renin and
prostaglandins.
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 Renal Disorders
Renal or kidney failure occurs when the
kidneys lose their ability to function.

To treat kidney failure effectively, it is

important to know whether kidney disease
has developed suddenly (acute) or over the
long term (chronic).

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Acute renal failure (ARF)

Failure of renal function that occur rapidly.

This is potentially reversible since, if the
patient survives the acute illness, normal
renal function can be regained.
Causes include renal hypoperfusion, specific
renal diseases and nephrotoxic drugs.

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Chronic renal failure (CRF)
Develops insidiously, often over many years,
and is irreversible, leading eventually to
end-stage renal failure
Causes include diabetes, vascular diseases,
glomerulonephritis and pyelonephritis.
Patients with end-stage renal failure require
either long-term renal replacement
treatment (i.e. dialysis) or a successful renal
transplant in order to survive.

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 Assessment of Renal Function
Mostly done by the measurement of:
a) Glomerular Filtration Rate(GFR)
b) Blood & urine non-protein nitrogenous
substance (NPN)
c) Blood & urine total protein or albumin
RFT can be affected by:
1. Prerenal Factors
Diarrhea
Vomiting
Excessive loss of blood due to intestinal
bleeding
Cardiac failure 11
2. Renal Factors
Damage to the nephron
Change in renal vascular system that
decreases the blood flow

3. Post renal factor

Stones in the kidney( renal calculi)
Enlargement of the prostate gland
Tumors of the kidney

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 Measurement of Glomerular Filtration
Rate(GFR)
The glomerulus is involved in filtration of
blood.
It is a sieve and act as selective permeability
barrier.
Approximately 1,200 to 1,500 ml of blood
flow through the kidney in each minute
(1/4 total blood) and the glomerulus filters.
out 125 to 130 ml of essentially protein
free, cell free fluid per minute (180L/day).
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 The rate at which filtrate is formed in
glomerulus is known as glomerular filtration
rate (GFR) (approximately 120 ml/min) .

Clearence tests are used to assess

glomerular filtration rate.

Clearance is defined as volume of plasma

which is completely cleared of a substance
by kidneys per minute. It is expressed as ml
of plasma cleared per minute (ml/min).
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 Measurement of clearance test detects
much earlier stages of renal disease
Different substance may be used in renal
clearance test:
Creatinine & Urea which are endogenous
substances (normally present in the body)
Inulin & mannitol which are exogenous
substances
Some substances are used in preference to
others to be used in clearance tests based
on the following criterion:

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 The compound should be easily quantified
(Inulin is difficult to quantitate).
Toxicity to the patient- The substance should
not be toxic to patient (All are non toxic).
Ease sampling (from administrator to
collection).
Urea & ceatinine are good
Excretion system should be renal
Inulin & mannitol 100%
Creatinine 99%
Urea 50%
Based on the above criterions creatinine is
the preferred substance. 16
 Creatinine clearance
Creatinine is a waste product that is
produced continuously during normal
muscle breakdown.
The kidneys filter creatinine from the blood
into the urine, and reabsorb almost none of
it.
The amount of blood the kidneys can make
creatinine-free each minute is called the
creatinine clearance.
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 Creatinine clearance in a healthy young
person is about 125 milliliters per minute -
meaning each minute, that person's kidneys
clear 125 ml.
Therefore, it is directly proportional to the
Glomerular filtration rate(GFR)
The GFR can vary depending on age, sex,
and size.
Generally, the creatinine clearance is a good
estimation of the glomerular filtration rate.
So clinically it can be seen as a measure of
GFR.
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Creatinine clearance

Where:-
- C= creatinine clearance
- U= urine creatinine
- V= urine volume
- P= plasma creatinine

Normal value
100- 120
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- Urine creatinine = 120mg/dl
- Plasma creatinine = 1 mg/dl
- Urine volume = 1ml/min

= 120 ml/min

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 GFR & Stages of chronic kidney disease
Stage 1 Normal GFR; GFR >90ml/min with
other evidence of chronic kidney
damage
Stage 2 Mild impairment; GFR 60-89ml/min
with other evidence of chronic damage
Stage 3 Moderate impairment; GFR 30-59ml/min
Stage 4 Severe impairment: GFR 15-29ml/min
Stage 5 Established renal failure (ERF): GFR
<15ml/min or on dialysis
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 Measurement of Non-Protein Nitrogenous
Substances (N.P.N)
NPN are waste products produced in the
body as a result of the metabolism of
nucleic acids, amino acids and creatine.
It includes urea, creatinine and uric acid
There will be an elevation in blood and a
decrease in urine of total NPN if kidney is
damaged
The accumulation of NPN in blood is
called Uremia (azotemia)
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 Determination of total NPN for the
assessment of kidney function is no
more in use since the determination of
the individual analytes provides a more
reliable information about the renal
function.

Measurement of urea & creatinine is

widely used for assessment of renal
function but uric acid is mainly used for
assessment of Gout.

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Urea /Blood Urea Nitrogen (BUN)
Urea is synthesized in the liver from
ammonia, CO2 and some amino acids.
The main purpose of urea synthesis is
chiefly to eliminate any surplus nitrogen
from the body.

Difference between BUN and Urea

BUN is the relative amount of nitrogen
in urea
Molecular weight of urea is 60
Atomic weight of nitrogen=14 24
 BUN is represented by two nitrogen
present in urea
BUN has a molecular weight of 28 gm.
Factor=60/28=2.14
Therefore,
Urea = 2.14 X BUN or
BUN= Urea / 2.14

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Method of determination of urea
There are two groups of methods
1. Colorimetric Method
Bertholet Method
Diacetyl Monoxime Method (DAM)
Nesslerization Method
2. UV-Enzymatic Method

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Bertholet Method-1
Principle:
Urea urease + - NaOH
NH4 HCO3 NH3 + CO2 + H2O

NH3 + phenol catalyst Indophenol (Blue Colored)

Note:
Catalysts =hypochlorite& nitroprusside
The intensity of the blue colored derivative is
directly proportional to the concentration of
urea in the sample
= 560 nm
Assay technique= End point
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Bertholet Method-2
Principle:urease

Urea 2NH4+ + CO2
catalyst

NH4 + Salicylate Dicarboxyindophenol
(Green Colored)
Note:
Catalysts =hypochlorite & nitroprusside

The intensity of the green colored derivative

is directly proportional to the concentration
of urea in the sample
= 578nm

Assay technique= End point 28

Nesslerization Method
Principle:
urease
Urea 2NH4+ + CO2
NH4 + Nesslers reagent yellow colored derivative
Note:
The intensity of the yellow colored derivative
is directly proportional to the concentration
of urea in the sample
= 470nm

Assay technique= End point

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Diacetyl Monoxime Method (DAM)
Principle:

catalyst
Urea + DAM Diazine Derivative (yellow
colored)
Note:
Catalysts =Cadmium (cd+)& thiosemicarbazide
The intensity of the yellow colored derivative
is directly proportional to the concentration of
urea in the sample
= 530nm

Assay technique= End point

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UV-Enzymatic Method
Principle:
Urea urease 2NH + + CO
4 2
NH4 + -ketoglutarate + NADHGLDH
Gltamate +
NAD+ + H2O

Note:
GLDH= Glutamate dehydrogenase
The rate of decrease in absorbance is directly
proportional to the concentration of urea in the
sample
= 340nm
Assay technique= Fixed Time Kinetic

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 Reference Values:
Reference values vary from method to
method but the following values can be
taken for orientation purpose
Urea= 15-45 mg/dl
BUN= 7-21 mg/dl

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Creatinine
Is a nitrogenous waste product formed from
the metabolism of creatine in skeletal muscle.
Creatine serve as energy (ATP) store in
muscles where it is phosphorylated (Creatine-
PO4).
About 10% of muscle mass is creatine.
Males have more muscle mass, therefore
more energetic than females and also
produce more creatinine than females.
When creatine-PO4 is metabolized in need of
energy, it produces creatinine, H2O and
energy (ATP).
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 Creatinine is filtered from the extra cellular
fluid by the kidney and excreted in the urine
without being reabsorbed by the tubules.
A small but significant amount of creatinine
is secreted by the proximal tubule.
Plasma level of creatinine increases with
kidney disease.
The measurement of creatinine is
recommended in preference to the
measurement of urea because creatinine is
less affected by age, dehydration, catabolic
states (fever, internal bleeding, and diet).
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Methods for the Determination of creatinine
There are two groups of methods
1. Chemical Method
Jaffee Method
2. Enzymatic Method
Note:
Though, most chemical methods are non
specific and are being replaced by
enzymatic methods, Jaffee is still the
method of choice in most clinical
laboratories.
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Jaffee Method
Principle
NaOH
Cretinine + Picric Acid Alkaline Picrate
(Golden Brown colored).
Note:
The intensity of the golden brown colored
derivative is directly proportional to the
concentration of creatinine in the sample
= 492nm

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 Reference Values:
Reference values vary from method to
method but the following values can be
taken for orientation purpose
Children: 0.3-0.7 mg/dl
Adult:
Male: 0.6-1.4 mg/dl
Female: 0.5-1.1 mg/dl

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Uric acid
It is a major catabolic product of purine
(Adenine & guanine) which are components
of DNA and RNA.
Can be obtained from ingested food (meat)
and destruction of tissue cells.
Purine is converted to uric acid in liver and
transported to kidney to be excreted (75%
excreted by the kidney & the other excreted
by the GI tract)
Uric acid >6.4 mg/dl in plasma become
saturated & precipitate in tissues as urate
crystals and causes Gout. 38
 Measurement of uric acid is important for
the diagnosis of renal disease and Gout.

Gout
Is a painful condition due to accumulation
of uric acid crystals in joints
Types of Gout
1. Primary-disorder in purine metabolism
2. Secondary- Renal damage, leukemia
etc.

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Methods for the Determination of Uric Acid
There are two groups of methods
1. Chemical Method
Folin-Denis Method

2. Enzymatic Method
Uricase-Peroxidase Method

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Folin-Denis Method
Principle:
First prepare protein free filtrate (PFF) using
20% TCA
PFF + Phosphotungstic acid NaOH
Allantoin
+ CO2 + H2O + Tungsten Blue
Note:
The intensity of the blue colored derivative is
directly proportional to the concentration of
uric acid in the sample
= 710nm

Assay technique= End point

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 Uricase-Peroxidase Method (PAP) 1,2
Determination of uric acid by reaction with
uricase, the formed hydrogen peroxide
reacts under catalysis of peroxidase with
DCPS or DCHBS and PAP to give red violet
quinoneimine dye as indicator.

- PAP= 4-amino phenazone

- DCPS= dichlorophenolsulphonate
- DCHBS = dichlorohydroxybenzenesulfonate
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Uric acid Uricase
Allantoin + CO2 + H2O2
H2O2+PAP + (DCPS or DCHBS ) Peroxidase

quinoneimine (pink colored) + H2O

Note:
The intensity of the pink colored
derivative is directly proportional to the
concentration of uric acid in the sample
= 520-546nm
Assay technique= End point
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Reference Values:
Reference values vary from method to
method but the following values can be
taken for orientation purpose
Male: 3.0-7.0 mg/dl
Female: 2.5-6.0 mg/dl

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Determination of Total serum protein
There are many classic and reference
methods for quantification of serum
protein, the biuret reaction has become the
most commonly used method in the clinical
laboratory.
Total protein level decreases in renal
disease
Principle of Biuret Method:
Cupric Ions react with protein in alkaline
solution to form a purple complex.
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 The intensity of the purple colored complex
is proportional to the protein concentration
in the sample, and is measured at 520-546
nm
Sodium potassium tartrate is a component
of the reagent and functions to maintain
copper in the correct valence state and in
an alkaline solution, while potassium iodide
is present as an antioxidant.
Reference Range: Adult 6.48.3 g/dL

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Determination of serum Albumin
Routine Serum albumin analysis employs
anionic dye binding method, however there
are more sensitive and specific
immunological assays used in clinical
laboratories. The dye binding method is
based upon albumins ionic charge at an acid
pH and binding to anionic dyes such
bromcresol green (BCG) or bromcresol
purple (BCP).
Serum albumin level decreases in renal
disease
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Principle dye binding method
In this methods, bromocresol green (BCG) or
or bromcresol purple (BCP) react with
albumin in citrate buffer forming a colored
complex.
The absorption of this complex is directly
proportional to the albumin concentration in
the sample, and is measured at 578 nm

Reference Range: Adult 3.55.2 g/dL.

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