Chapter six

Renal Function Testing and Non-protein

Nitrogen Substances

1
 Objectives
Upon completion of this chapter the student will be able to:
• Discuss the anatomy, physiology and pathophysiology of
the renal system
• Define the non protein nitrogenous (NPN) compounds
• Discuss about, NPN compounds mainly of Creatinine,
urea, and uric acid and their source, metabolism, and
clinical significance
• Explain about Creatinine, urea, and uric acid tests
– principle of the test, source of error, Iterpretation, limitation of the test
• Discuss renal clearance tests such as creatinine, and others
• Calculate renal clearance tests result, and urea creatinine ratio
Outline
• Anatomy, physiology and pathophysiology of the
renal system
• Non- protein nitrogenous (NPN) compounds
• Urea and Blood urea Nitrogen (BUN)
• Creatinine
• Uric acid
• BUN/creatinine ratio
• Clearance tests
Major Structures of Urinary
System
• Kidney
• Ureters
• Bladder
• Urethra
Urinary System
Kidney: Structure
– Bean shaped paired organs
– Outer layer = cortex; composed primarily of glomeruli, PCT, DCT
– Inner layer = medulla; composed primarily of the loop of Henle
and collecting ducts
– Renal pelvis: collects urine into the ureters
• Ureters: urine flows from renal pelvis into the ureters, then into
the bladder
• Bladder: urine stored here until voided
• Urethra: urine voided through urethra to outside of body
 Physiology of kidney
1. Removal of unwanted substances(waste and surplus)
– Elimination of Non protein Nitrogenous (NPN) substances (urea,
creatinine, uric acid)
2. Homeostasis function
– Water balance
– Ionic (electrolytes) Equilibrium
– Acid – base balance
3. Endocrine regulation
– Secretion of erythropetin ,rennin and prostagladin.
– Kidney is the target organ for catabolism of insulin,
glucagons, aldosterone and vitamin D. 6
Major Components of Kidney
• Nephron: This is the functional unit of the kidney
• Each kidney contains approx 1 million nephrons
• Composed of three main units

• Arterioles: afferent and efferent

• Glomeruli
• Tubules: PCT, Loop of Henle, DCT,
Collecting Ducts (tubules)
Major Components of Kidney cont’d
• Arterioles
• Afferent arteriole: supplies blood to the glomerulus
• Efferent arteriole: outgoing blood supply from the
glomerulus to peritubular capillaries or vasa recta which
surround the tubules
• Glomerulus
• Tuft of porous capillaries that is formed from the afferent
arteriole and drained by the efferent arteriole
• Function
• Filtration based on molecule size and charge
• Water and small diameter/molecular weight molecules rapidly pass
through the filtration barrier with little or no resistance
Major Components of Kidney cont’d
• Bowman’s Capsule
• Surrounds the glomerulus, opening into the proximal
convoluted tubule
• Tubules
• Proximal Convoluted Tubule (PCT): reabsorb essential
substances (water)
• Loop of Henle: concentrate urine
• Distal Convoluted Tubule (DCT): homeostatic regulation
• Collecting Tubules (Ducts): directs urine flow into renal
pelvis; responsive to the hormones ADH and aldosterone
10
 The Glomerulus

Blood pressure inside of the glomerulus is very high.

Because of differences in the resistance between the afferent and
efferent arterioles. 11
Forces the fluids and some solids out of the blood into the glomerular
capsule.
 Urine Formation
Urine formation requiers :

a) Glomerular Filtration
• Due to differences in pressure water,
small molecules move from the
glomerulus capillaries into the glomerular
capsule
b) Tubular reabsorption
•Many molecules are reabsorbed
from the nephron into the capillary
(diffusion, facilitated diffusion,
osmosis, and active transport)
Tubular secretion
c) Substances are actively removed from blood
and added to tubular fluid (active transport)
ie. H+, creatinine, and some drugs are moved 12
by active transport from the blood into the
distal convoluted tubule
 Glomerular Filtration
• Filtration: the forcing of fluids and
dissolved substances through a
membrane by pressure occurs in the
renal corpuscle of the kidneys across
the endothelial capsular membrane
(Bowman's) capsule.

• The resulting fluid is called the

filtrate.

• Filtrate lacks large molecular weight

substances such as protein,protein-
bound substances, cells
13
• Filtration is a passive process.
Proximal convoluted tubule
• Primary site of reabsorbtion
• Returns valuable substance back to the blood
circulation.
• About 70% of water, Na+, Cl-, all of the glucose (up to the
renal threshold), almost all of the amino acid, vitamins and
protein, and various amounts of mg2+, Ca2+, K+, HCO3 and urea
are reabsorbed
• The process is active except for water and Cl-(diffuse
in the wake of Na+)
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Loop of Henle
• In descending Loop of Henle
• hypo osmolar compared to the surrounding interstium
• Permeable to water.
• In ascending loop of Henle/diluting segment
• the filtrate is hyperosmolar compared to the intestitium
• impermeable to water
• Permeable to Na+ and Cl-

15
Distal convoluted Tubule
• Make a small adjustment to achieve electrolyte and acid-base
homeostasis
• Controlled by aldosterone
– It is produced by adrenal cortex
– Its secretion is triggered by decreased blood flow (decreased renal blood
volume or decreased blood pressure).
– It is regulated by the Reinin – Angiotensin mechanism and to lesser extent
by ACTH.
– Stimulates Na+ reabsorption by distal tubule and K+ and H+
secretion.
• Hydrogen ion secretion is linked to bicarbonate regeneration and
ammonium secretion which occur here.
16
• Small amount of Cl- are reabsorbed
Collecting Duct
• The upper portion of the collecting duct is still under the control
of aldosterone
• Cl- and urea are reabsorbed here
• Controled by antidiuretic hormone (ADH)
• Secreted by the posterior pituitary
• Its secretion is triggered by increased blood osmolality.
• Stimulates water reabsorption

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18
Renal Threshold
• Defined as the plasma concentration of a substance that
when exceeded, the kidney tubules will not reabsorb any
more into the bloodstream, resulting in the substance
being excreted into the urine
• Substances are reabsorbed into the bloodstream
dependent upon their blood concentration and the
body’s needs
• When the plasma concentration of a substance is higher
than a certain ‘threshold value’, reabsorption of the
substance is no longer possible
• The substance is then spilled into the urine
• Example: glucose renal threshold is ~160-180 mg/dl
Renal Pathophysiology
• Glomerular disease
• Acute glomerulonephritis
• Chronic glomerulonephritis
• Nephrotic syndrome
• Tubular diseases
• Decreased excretion/reabsorption of certain substances or
reduced urinary concentrating capability.
• Renal tubular acidosis(RTA)
• Urinary tract infection
• Renal obstruction
• Renal calculi
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• Renal failure: acute and chronic
Laboratory Tests for the Evaluation of Kidney
Function
• Practical evaluation of kidney status include examining:
• The Nephron functions of glomerular filitration
• The secretory capacity for particular endogenous and exogenous compounds
• the reabsorptive capacity for water and electrolytes

• The diagnosis of renal disease using clinical signs and

symptoms are minimal or entirely absent.
• Panel of kidney function tests give valuable information about the status of
the kidney function.

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Renal function tests
• Glomerular function tests:
• All the clearance tests (innulin, creatinine, urea)
• Tubular function test:
• Urine concentration or dilution test or urine acidification test
• Osmolality measurements, Specific proteinurea, Glycouria,
Aminoaciduria
• Analysis of blood/serum
• blood urea, serum creatinine, protein and electrolytes
−Urinalysis (Macroscopic Examination, Chemical Analysis/Urine
Dipstick, Microscopic Examination)
• Appearance, Specific gravity and osmolality, pH, Glucose, 22
Protein, ketone bodies, Urinary sediments
Factors affecting Renal function tests
• Pre-renal factors
• Affect blood volume, blood flow or blood pressure
• Include:
• Dehydration due to pyloric & intestinal obstruction and diarrhea, shock
• Excessive loss of blood due to severe intestinal bleeding
• Cardiac failure: the defect lies in the blood supply before if reaches the
kidney due to cardiovascular system failure and consequent
hypovolemia

23
Factors affecting RFT cont’d

• Renal Factors
• The defect on the kidney
• Include:

• Disease affecting the GFR and tubular function

mainly due to damage of the nephrones from
glomerulus nephritis, nephrotic syndrome
• Changes in the renal vascular system that
decrease the blood flow

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Factors affecting RFT cont’d
• Post Renal factors
• The defect lies in the urinary tract after it exits kidney
• Due to any obstruction of the urine flow such as:
• Enlargement of the prostate gland
• Stones in the urinary tract
• Carcinomas or tumors that may compress the ureters, urethra or bladder
opening.

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Non-protein Nitrogenous (NPN)
substances
• These are compounds that contain nitrogen, but are
not proteins
• Formed in the body as a result of the derivative
metabolism of nucleic acids, amino acids and
proteins
• The kidneys play an essential role in the excretion of
these metabolic waste products.
• Measurement of these compounds can be used to assess kidney
function

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Non-Protein Nitrogen Compounds

• Include >15 compounds

• Amino acids: from protein catabolism (breakdown)
• Ammonia: from amino acid catabolism
• Urea: from ammonia catabolism
• Creatinine: from creatine breakdown in the muscle
• Uric acid: from nucleic acid catabolism

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Urea
• Urea constitutes half of the NPN substances in blood
• Synthesized in the liver from CO2 and the ammonia, from
the deamination of amino acids in the reactions of the urea
cycle.
• Major excretory product of protein metabolism.
• It is neither actively reabsorbed nor secreted by the
tubules
• Readily filtered from the plasma by the glomerulus.
• 40% is reabsorbed by passive diffusion.
– Reabsorption depends on urine flow rate and degree of
hydration.
Urea
• More than 90% of urea excreted through the kidney and < 10% urea
excreted through GIT and skin.
• Plasma concentration depends on:

– Renal function and perfusion

– Protein content of the diet
– The amount of protein catabolism
Urea Vs blood urea
nitrogen(BUN)
• Urea is different from BUN
• The structure of urea is NH2-CO-NH2 with a molecular weight of 60g,
the two Nitrogen atoms represent 28g,
Mol. Wt of urea = 60/28 = 2.14
At wt of Nx2
• (BUN in mg /dl) (2.14)= Urea in mg/dl

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Clinical significance of urea
• Azotemia: an elevated level of urea in the blood
• Uremia or uremic syndrome: Very high levels of plasma urea
accompanies by renal failure
• Cause of decreased urea levels
• Decreased protein intake
• Severe liver disease
• Conditions that leads to increased protein synthesis such as normal
pregnancy and infancy?
• Severe vomiting & diarrhea

31
Clinical significance of urea cont’d
Increased urea level
• Pre renal
• Decreased renal blood flow due to congestive heart failure
shock, hemorrhage dehydration.
• Increased protein catabolism and intake of high protein diet
• Renal
• Acute & chronic renal failure
• Glomerular nephritis
• Tubular necrosis
• Post renal 32

• Obstructions to the flow of urine any where in urinary tract

Clinical significance of urea cont’d
• In conjunction with serum creatinine, BUN/creatinine
ratio used to differentiate between pre renal & post renal
azotemia
• Urea clearance is a poor indicator of GFR.
• urea clearance generally underestimates GFR

33
Specimen collection/handling for urea
• Urine
• Timed collection preferred
• Must be diluted prior to measurement1:10, 1:20
• Stability: up to 1 week stored in refrig when pH<5
• Some bacteria are able to hydrolyze urea to ammonia resulting in
falsely decreased urine urea levels
• As ammonia increases in the urine, the pH becomes more alkaline
Urea Specimens
• Serum or heparinized plasma
• Stability: up to 24 hours at room temperature; 1 week at 2-4 oC
• Specimen Collection and Handling Requirement for
BUN
• Serum or heparinized plasma, non-hemolyzed; fasting not required
Methods of Urea Measurement
• Enzymatic (indirect) method
• Chemical (direct) method
1. Enzymatic methods (indirect method)
• Based on the preliminary hydrolysis of urea with urease
(specific enzyme) to liberate ammonium ions, followed by a
secondary reaction that measures the amount of
ammonium ion spectrophotometrically or by electrode
conductivity
• Called ‘indirect’ methods because these methods measure
the amount of ammonia ‘liberated’ from the urea molecule
present in the sample
 Enzymatic urea methods

• Spectrophotometric measurement of NADH  NAD (ABS at 340

nm).
• The amount of NAD produced is directly proportional to the
amount of ammonium ion which is directly proportional to the
amount of urea present
• Disadvantage: endogenous ammonia will interfere
Methods cont…
2. Chemical methods
• Called ‘direct’ methods because urea in sample
reacts directly with reagent causing a color
change that is spectrophotometrically measured
• Color reagent: diacetyl-monoxime
• Advantage: endogenous ammonia does not interfere

• Based on the condensation of urea with diacetyl

monoxine;
Limitations: Urea Methods
• Sources of error????
Reference range for Blood Urea N (BUN)

• For adults (Serum/plasma)……………….. 6-20 mg/dl

• New born up to one week( Serum/plasma)… 3-
25mg/dl
• Adult over 60 (Serum/plasma) …….8-23mg/dl
• Urine: 12-20 g/24hrs

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Creatinine
• Formation and Excretion
• Spontaneously derived from creatine in muscle
• High energy ATP storage and use in muscle
• Produced at a constant rate day to day
• Excreted into urine through glomerular filtration; not significantly
reabsorbed or secreted by tubules
Creatinine
• Excreted into the circulation at a relatively constant rate,
proportional to the muscle mass of the individual
• Removed from the circulation by glomerular filtration and it is
excreted into the urine without being reabsorbed by the tubules
to any significant extent.
• Normally, creatinine clearance exceeds the glomerular filtration
rate by 10% to 40%.
• A small but significant amount of creatinine is secreted by the
PCT,
• better indicator of renal function than urea
• serum creatinine is not affected by diet, exercise, or hormones,
42
factors that influence the levels of urea
Clinical significance
• Elevated level of creatinine is mainly associated with abnormal
renal function, especially as it relates to GFR.
• However, it is relatively insensitive monitor and may not be
measurably increased until renal function has deteriorated
more than 50%.
• Its serum level increases in disease associated with extensive
muscle destruction
• Excellent analyte for the assessment of renal function
• Strong relationship between plasma creatinine and the GFR
• Plasma creatinine level are relatively constant and unaffected by diet
• The serum creatinine level in combination with BUN is used to 43
differentiate between pre renal & post renal cause of azotemia.
Creatinine Specimen
Collection and Handling
• Urine
• Time collection preferred; random acceptable
• Stability: up to 4 days in refrigeration
• Longer when frozen
Creatinine
• Serum or heparinized plasma
• Avoid hemolysis
• Avoid lipemia
• Stability: one week at refrigeration temps
Methods of Creatinine Measurement

1. Chemical method: Jaffe Reaction

• Creatinine + alkaline picrate  Janovski
complex
(yellow) (red-
orange color)
• This reaction lacks specificity:
– Falsely increased results with high levels of: protein,
ascorbic acid, ketones, glucose, pyruvate and uric acid
– Falsely decreased results: bilirubin???
Methods of Creatinine measurement
• Several modifications have been used to increase the specificity
of this reaction:
• Prepare a protein-free filtrate (PFF):
• React with tungstic acid to precipitate out protein
• Absorb protein onto Fuller’s earth or Lloyd’s reagent
• Kinetic Jaffe reaction:
• detection of the rate of color formation is timed such that interfering
substances do not interfere.
• This is common with most automated methods of analysis.

c. Most frequently used methods are based on the Jaffe reaction,

even though bias present due to interference: inexpensive, rapid
and easy method to perform
Methods of Creatinine measurement
• Coupled enzymatic method with spectrophotometric end
point:

Creatininase
Creatinine + H2O ---------------- Creatine

Creatinase
Creatine +H20 ------------------ Sarcosine + urea

sarcosine oxidase
Sarcosine + O2 + H2O ------------ formaldehyde + Glycine + H2O2

peroxidase
Oxygen accptor + H2O2 --------- Colored products

• This method may use a manual spectrophotometer, semi-

automated spectrophotometer or automated Clinical Chemistry
analyser to measure creatinine
• Has potential to replace Jaffe reaction as most widely
method used
• Less interference as compared to Jaffe reaction
Creatinine Method
Limitations
• Sources of Error:
• Falsely increased results with high levels of:
protein, ascorbic acid, ketones, glucose,
pyruvate and uric acid
• Falsely decreased results: bilirubin??
• Specimen hemolysis or lipemia
• Note the modifications mentioned to limit
sources of error: PFF and kinetic
Creatinine cont’d
• Reference Range
• Serum Adult male: 0.6-1.1 mg/dl
Adult female: 0.5-0.8 mg/dl
Child: 0-0.6 mg/dl

• Urine Male: 800-2000 mg/24hr

Female: 600-1800 mg/24hr

• Amniotic fluid: 1-2 mg/dl

50
Uric Acid
• Formation and Excretion
• End product of purine (adenine and guanine) metabolism by
the liver
• Purines are precursors of the nucleic acids ATP and GTP
(adenosine diphosphate and guanosine triphosphate)
• Readily filtered by the glomerulus, but then undergoes a
complex cycle of reabsorption and secretion by the tubules
Clinical Significance of Uric Acid
• Increased uric acid (hyperuricemia)
– Gout: at plasma pH uric acid is readily insoluble and at
concentrations >6.4 mg/dl the plasma is saturated resulting in crystal
deposition in tissues and joints
– Increased catabolism of nucleic acids
• Patients on chemotherapy for proliferative disease such as
leukemia, lymphoma, multiple myeloma
• Must monitor uric acid levels to avoid nephrotoxicity
• Allopurinol treatment is used to interrupt the uric acid synthesis
pathway in these patients, avoiding nephrotoxicity
– Renal disease: filtration and secretion are impaired
Clinical Significance of Uric Acid

• Decreased uric acid (hypouricemia)

• Severe liver disease
• Defective tubular reabsorption
• Over treatment with allopurinol, uricosuric drugs
Specimen Collection and Handling
Requirement for Uric Acid
• Serum or heparinized plasma; fasting not
required
• Remove from cells quickly
• Avoid hemolysis and gross lipemia
• Salicylates (aspirin) may cause increased results
• Stability: 3-5 days at 2-4oC
• Urine
• Timed urine collection preferred
• Sample should be refrigerated to inhibit bacterial
growth
• Stability: 3-5 days at 2-4C
Methods of Uric Acid
Measurement
• Caraway method
• Phosphotungstic acid (PTA) reduction by uric acid
• Historical
• lacks specificity
• uric acid is a reducing substance, thus other
reducing substances will also react in this
method
Uric Acid methods
• Uricase method: increased specificity
Uric acid uricase Allantoin+H2O2
4-AA+DCBS+H2O2 POD quinoneimine + 4 H20
• Modification of this reaction uses a coupled enzymatic reaction that
measures the quinoneimine dye produced.

4-AA…..4-amino antipyrine
DCBS….3,5 Dicholoro-2 hydroxy benzene sulfonate
Uric acid: Interferences
• Bilirubin and ascorbic acid will interfere by destroying H2O2;
• thus reagents often contain potassium ferricyanide and
ascorbate oxidase to minimize these effects
• Hemolysis
• Lipemia
• Ascorbic acid
Reference Range Uric
acid
• Serum Adult male: 3.5-7.2 mg/dl
Adult female: 2.6-6.0
mg/dl
Child: 2.2-5.5
mg/dl

• Urine 250-750 mg/24 hr

BUN/Creatinine Ratio
• Used to differentiate causes of azotemia:
• Pre-renal
• Post-renal

• Calculated: serum BUN (mg/dl)

serum creatinine (mg/dl)

• Normal ratio: 10-20 with majority around

12-16
60
BUN/Creatinine Ratio
• Differentiation of azotemia

• Increased ratio with

Tend to be caused by pre-renal
increased BUN, conditions:
normal creatinine
Congestive Heart Failure
Shock, hemorrhage
Dehydration
Increased protein metabolism
61
Increased protein catabolism
BUN/Creatinine Ratio
• Differentiation of azotemia

• Increased ratio with

Tend to be caused by - renal
increased BUN, conditions that decrease kidney
increased creatinine function:
Acute renal failure
Chronic renal failure
Glomerulonephritis
Tubular necrosis 62
BUN/Creatinine Ratio
• Decreased ratio
with decreased Tend to be caused by
BUN conditions of decreased urea
production:
Low protein diet
Liver disease

63
Reading assignment
• What will be the BUN/ Cr ratio during post renal azotemia??
• How do we calculate GFR?
Renal clearance tests / Estimate of GFR
• Extremely useful, effective and sensitive way of measuring the
actual excretory capacity of the kidney.
• Detect much earlier stages of the disease
• Clearance tests are more sensitive and clinically more useful than the
other function tests.
• The concentration of a substance excreted in the urine is
measured and compared to the concentration of the same
substance in the plasma
• The renal clearance of a substance is defined as the volume of
plasma from which the substance is completely cleared by the
kidney per unit time
65
Clearance test cont’d
• The amount of substance cleared by the kidney is generally
expressed as a volume of plasma which contains the quantity of the
substance excreted in the urine.
ml plasma cleared = Uc x V , Uc=conc of subs in urine in mg/dl
Pc Pc= conc of subs in plasma in mg/dl
V=urine volume excreted per minute in ml
(urine flow rate)
• The clearance rate is proportional to the size of the kidney and the
patients body surface area.
ml plasma clearance/ min=Uc x V x 1.73

Pc A
1.73= A factor generally accepted average body surface area in m 2 66
A=the body surface area of the patient
Markers used for renal clearance test
• Substance used to monitor GFR must meet the following
criteria
• Freely filterable at the glomerular barriare
• Not reabsorbed by the tubules
• Not secreted by the tubules
• Present at a stable plasma concentration
• Include:
• Exogenous markers: inulin, manitol& iohexol
• Endogenous markers: creatinine, urea & low molecular
proteins
67
• For the benefit of patients and ease of procedure creatinine is
the best choice for renal clearance tests
Creatinine clearance
• Used to approximate GFR.
• Creatinine is ideal substance for the measurement of clearance
• It is an endogenous metabolic product synthesized at a
constant rate for a given individual
• It is cleared essentially only by glomerular filtration (it is not
reabsorbed and only sightly secreted by the PCT)
• It can be analyzed inexpensively by readily available
colorimetric methods.
• Creatinine clearance has become the standard laboratory assay
for determination of Early renal failure 68
Procedure
• Hydrate the patient by administering a minimum of 600ml of
water
• Caffeine containing compounds (coffee, tea), heavy exercise
and drugs should be avoided
• The water intake promote good urine flow
• Have the patient void and discard the urine specimen
• Collect a 4, 12 or 24 hrs specimen and record the exact times of
starting and completing each collection.
• Although a full 24 hr period is ideal, the excretion rates of an endogenous
marker (creatinine) is sufficiently constant that a 4-h period may be used
• Blood sample can be collected at any time during the urine
collection (ideally taken at mid point of 24 hour urine collection) 69
• Determine urine & blood creatinine and Calculate the clearance
Creatinine clearance cont’d
• Normal values
• Males 85-125ml/min
• Female 75-115ml/min

• Decreased creatinine clearance

• It is sensitive indicator of decreased GFR
• It can be caused by acute or chronic damage to the
glomerulus, reduced blood flow to the glomeruli, and acute
tubular damage.

70
 Review Questions
• Describe the process of urine formation
• What are the major components of nephron
• What is the principles of the enzymatic methods of analysis of urea, uric
acid and creatinine?
• What is the principle of the classic creatinine method?
• What are sources of error in the urea method?
• Why is BUN/ creatinine ratio useful?
• What is the formula for calculating creatinine clearance?