Physiology of urine formation and kidney function test swati mam

1. PHYSIOLOGY OF URINE
FORMATION AND
RELEVANT FUNCTION
TEST
Guide- Dr. Nilofer Mujawar
Student- Dr. Swati P.

2. • The kidneys perform their most important functions
by filtering the plasma and removing substance from
the fitrate at variable rates, depending on the needs
of the body.
• The kidneys serve multiple functions, including-
1. Excretion of metabolic waste products and foreign
chemicals,
2. Regulation of water and electrolyte balances,
3. Regulation of body fluid osmolality and electrolyte
concentration,
4. Regulation of arterial pressure,
5. Regulation of acid base balance,
6. Secretion, metabolism and excretion of hormones,
7. Gluconeogenesis.

3. Physiologic Anatomy Of The Kidneys
• Two kidneys- on the posterior wall of the
abdomen, outside peritoneal cavity.
• Each kidney- weighs 150 gms, size- clenched
fist.
• Medial side- hilum (renal artery, vein,
lymphatics, nerve supply and ureter)
• Capsule- tough fibrous, protects inner delicate
structures.

4. Renal blood supply
• Blood flow to both kidneys- 22% of the cardiac
output or 1100ml/ min.
• Renal artery, interlobar, arcuate, interlobular,
afferent, glomerular capillaries, efferent
arterioles.
• Peritubular capillaries, interlobular, arcuate,
interlobar, renal.

5. Nephron- functional unit of the kidney
• Each kidney- 1 million nephrons.
• Kidney can not regenerate new nephrons.
• After 40 yrs- functioning nephrons decreases
about 10% every 10 yrs.
• Each nephron contains-
1. Glomerulus- tuft of glomerular capillaries,
through which large amount of fluid filtered from
the blood,
2. Long tubule- filtered fluid is converted into urine
on its way to pelvis of the kidney.

6. Urine formation
• The rates at which different substances are
excreted in the urine represent the sum of three
renal processes:
1. Glomerular filtration
2. Reabsorption of substances from the renal
tubules into the blood
3. Secretion of the substances from the blood in
the renal tubules.
Expressed mathematically:
Urinary excretion rate= filtration rate- reabsorption
rate + secretion rate.

8. GLOMERULAR FILTRATION
• Glomerular filtrate: the fluid that enters the
capsular space. (female-150lit, male-180lit).
• Filtration fraction: the fraction of blood
plasma in the afferent arteriole in the kidneys
that become glomerular filtrate (0.16-0.2).

9. Filtration membrane
• The glomerular capillaries and the podocytes,
which completely encircles the capillaries,
form a leaky barrier known as filtration
membrane.
• Substance filtered from blood crosses three
filtration barriers:
1. Glomerular endothelial cells,
2. Basal lamina,
3. Filtration slit formed by podocytes.

11. Principle of filtration
• The use of pressure to force fluids and solutes
through a membrane is same in glomerular
capillaries and elsewhere in the body.
• The volume of the fluid filtered in the renal
corpuscle is much larger in other capillaries of the
body for three reasons:
1. Larger surface area, mesangial cells relax
increased GFR and contracts decreased GFR.
2. Filtration membrane- thin and porous,
thickness- 0.1mm, 50 times leakier.
3. Glomerular capillary blood pressure is high.

12. Net filtration pressure
• Pressures which promotes filtration: GBHP,
CHP.
• Pressure which oppose filtration: BCOP.
• NFP=GBHP-CHP-BCOP.

14. GFR
• The amount of filtrate formed in all renal
corpuscles of both the kidneys each minute is the
GFR.
• Male- 125ml/min, female- 105ml/min.
• GFR- too high decreased reabsorption, too low
increased reabsorption.
• Mechanism that regulates GFR operate in two
main ways:
1. By adjusting blood flow into and out of
glomerulus,
2. Altering the glomerular capillary surface area
available for filtration.

15. Regulation of GFR
• Renal regulation of GFR
1. Myogenic mechanism
2. Tubuloglomerular feedback
• Neural regulation of GFR
• Hormonal regulation of GFR

17. TYPE OF
REGULATION
MAJOR STIMULUS MECH AND SITE OF
ACTION
EFFECT
ON GFR
Renal Myogenic mech Increased stretching of
smooth muscle fibres in
afferent arterioles walls
due to increased BP
Stretched smooth muscle
fibres contracts,
narrowing lumen of the
arterioles
Decrease
Renal Tubuloglomerul
ar feedback
Rapid delivery of Na and
Cl to the macula densa
due to high systemic BP
Decrease in release of
NO by JGA causes
constriction of the afferent
arterioles
Decrease
Neural Neural Increase in the activity
level of the renal
sympathetic nerves
releases norepinephrine
Constriction of afferent
arterioles through
activation of alpha 1
receptor and increased
release of renin
Decrease
Hormon
e
Angiotensin II Decrease blood volume
or BP stimulates
production of Angiotensin
II
Constriction of afferent
and efferent arterioles
Decrease
Hormon
e
ANP Stretching of atria of
heart stimulates
secretion of ANP
Relaxation of mesangial
cells in glomerulus
increases capillary
surface area available for
increase

18. Tubular reabsorption and tubular secretion
RENAL CORPUSCLES
GFR: 105-125ml/min of fluid
that is isotonic to blood.
Filtered substances: water
and all solutes present in the
blood(except proteins)
including ions, glucose, aa,
creatinine, uric acid.

19. PCT
 Reabsorption (into blood) of
filtered:
• Water- 65% (osmosis)
• Na- 65% (sod pot pumps)
• K-65% (diffusion)
• Glucose-100% (symporters and
facilitated diffusion)
• Cl- 50% (diffusion)
• HCO3- 80-90% (facilitated
diffusion)
• Urea- 50% (diffusion)
• Ca, Mg- variable (diffusion)
 Secretion:
• H- variable (antiport)
• NH4- variable, increase in
acidosis
• Urea- variable (diffusion)
• Creatinine- small amount

20. LOOP OF HENLE
 Reabsorption (into blood) of:
• water- 15% (osmosis in
descending limb)
• Na- 20-30% (symporters in
ascending limb)
• K- 20-30% (symporters in
ascending limb)
• Cl- 35% (symporters in
ascending limb)
• HCO3- 10-20% (facilitated
diffusion)
• Ca, Mg- variable (diffusion)
 Secretion:
• Urea- variable (recycling from
collecting duct)

21. Early DCT
 Reabsorption (into blood)
of:
• Water- 10-15% (osmosis)
• Na- 5% (symporters)
• Cl- 5% (symporters)
• Ca- variable (stimulated by
parathyroid hormone)

22. Late DCT and CD
 Reabsorption (into blood) of:
• Water- 5-9% (insertion of water
channel stimulated by ADH)
• Na- 1-4% (sod pot pumps and sod
channel stimulated by
aldosteron)
• HCO3- variable amount depends
on H secretion
• Urea- variable (recycling to loop
of henle)
 Secretion (into urine) of:
• K- variable amount to adjust for
diatery intake (leaky channels)
• H- variable amounts to maintain
acid base balance

25. Renal function tests
INDICATIONS FOR RENAL FUNCTION TESTS
1. Early identification of impairment of renal function
in patients with increased risk of chronic renal
disease
2. Diagnosis of renal disease
3. Follow the course of renal disease and assess
response to treatment.
4. Plan renal replacement therapy (dialysis or renal
transplantation) in advanced renal disease.
5. Adjust dosage of certain drugs (e.g.
chemotherapy) according to renal function

26. Classification of renal function tests
Tests to evaluate glomerular
function
1. Clearance tests to measure
glomerular filtration rate:
Inulin clearance, 125I-
iothalamate clearance, 51Cr-
EDTA clearance, CystatinC
clearance, Creatinine
clearance, and Urea
clearance
2. Calculation of cr clearance
from prediction equation
3. Blood biochemistry: sr. cr,
BUN, BUN/sr. cr
4. Microalbuminuria and
albuminuria
Tests to evaluate tubular
function
1. Tests to assess proximal
tubular function:
• Glycosuria, phosphaturia,
uricosuria
• Generalized aminoaciduria
• Tubular proteinuria
• Fractional sod excretion
2. Test to asses distal tubular
function:
• Specific gravity and
osmolality of urine
• Water deprivation test and
water loading test
• Ammonium chloride loading
test

27. Glomerular filtration rate (GFR)
• Best test for assessment of excretory renal function
• Varies according to age, sex, and body weight of an
individual; a normal GFR also depends on normal renal
blood flow and pressure.
• Normal GFR in young adults is 120-130 ml/min per 1.73
m2.
• Creatinine clearance is commonly used as a measure of
GFR. Equations can be used to estimate GFR from
serum creatinine value.
• GFR declines with age (due to glomerular
arteriolosclerosis)

28. GFR is measured to
(i) detect suspected incipient kidney disease (i.e. early detection),
(ii) monitor course of established kidney disease,
(iii) plan renal replacement therapy in advanced renal disease, and
(iv) adjust dosage of certain drugs which are nephrotoxic.
Based on GFR, chronic kidney disease is divided into following stages
(US National Kidney Foundation Kidney Disease Quality Outcomes
Initiative Classification of Chronic Kidney Disease, 2002):
• Stage 1: Kidney damage with normal or increased GFR (GFR ≥ 90
ml/min/1.73 m2 )
• Stage 2: Kidney damage with mildly reduced GFR (GFR 60-89
ml/min/1.73 m2)
• Stage 3: Moderately reduced GFR (GFR 30-59 ml/ min/1.73 m2)
• Stage 4: Severely reduced GFR (GFR 15-29 ml/min/ 1.73 m2)
• Stage 5: Kidney failure (GFR < 15 ml/min/1.73 m2)

29. Following methods are used to measure GFR:
(1) Clearance tests and
(2) Prediction equations.
• Glomerular filtration rate refers to the rate in ml/min at which a
substance is cleared from the circulation by the glomeruli.
• If a substance is not bound to protein in plasma, is
completely filtered by the glomeruli, and is neither secreted
nor reabsorbed by the tubules, then its clearance rate is equal
to the glomerular filtration rate (GFR).
• Clearance of a substance refers to the volume of plasma, which is
completely cleared of that substance per minute; it is calculated
from the following formula:
• Clearance = UV/P where,
U = concentration of a substance in urine in mg/dl;
V = volume of urine excreted in ml/min; and
P = concentration of the substance in plasma in mg/dl.

30. • The agent used for measurement of GFR should
have following properties:
(1) It should be physiologically inert and preferably
endogenous,
(2) It should be freely filtered by glomeruli and
should be neither reabsorbed nor secreted by
renal tubules,
(3) It should not bind to plasma proteins and should
not be metabolized by kidneys, and
(4) It should be excreted only by the kidneys.
However, there is no such ideal endogenous agent

31. The agents used for measurement of GFR are
• Exogenous: Inulin, Radiolabelled
ethylenediamine tetraacetic acid (51Cr- EDTA),
125I-iothalamate
• Endogenous: Creatinine, Urea, Cystatin C

32. • One major problem with clearance studies is
incomplete urine collection.
• Abnormal clearance occurs in:
(i) pre-renal factors: reduced blood flow due to
shock, dehydration, and congestive cardiac
failure;
(ii) renal diseases; and
(iii) obstruction to urinary outflow.

33. Inulin Clearance
• Inulin- inert plant polysaccharide (a fructose polymer)
• filtered by the glomeruli
• neither reabsorbed nor secreted by the tubules
• an ideal agent for measuring GFR.
• Method- A bolus dose of inulin (25 ml of 10% solution IV) is
administered followed by constant intravenous infusion (500
ml of 1.5% solution at the rate of 4 ml/min). Timed urine
samples are collected and blood samples are obtained at the
midpoint of timed urine collection.
• the ‘gold standard’ (or reference method) for estimation of
GFR.
• Disadvantages- time consuming, expensive, constant
intravenous infusion of inulin is needed to maintain steady
plasma level, and difficulties in laboratory analysis.
• values- males- 125 ml/min/1.73 m2 , females-110
ml/min/1.73 m2, children < 2 years and in older adults,
clearance is low.
• This test is largely limited to clinical research.

34. Clearance of Radiolabeled Agents
• Urinary clearance of radiolabeled iothalamate
(125Iiothalamate) correlates closely with inulin
clearance.
• Drawbacks- expensive with risk of exposure to
radioactive substances.
• Other radiolabelled substances used are 51Cr-EDTA and
99Tc-DTPA.

35. Cystatin C Clearance
• cysteine protease inhibitor of MW 13,000, which
is produced at a constant rate by all the nucleated
cells.
• It is not bound to protein, is freely filtered by
glomeruli and is not returned to circulation after
filtration.
• It is a more sensitive and specific marker of
impaired renal function than plasma creatinine.
• Its level is not affected by sex, diet, or muscle
mass.
• It is measured by immunoassay.

36. Creatinine Clearance
• most commonly used test for measuring GFR.
• produced constantly from creatine in muscle.
• completely filtered by glomeruli and not reabsorbed by tubules, a small
amount is secreted by tubules.
• A 24-hour urine sample is preferred to overcome the problem of diurnal
variation of creatinine excretion and to reduce the inaccuracy in urine
collection.
• After getting up in the morning, the first voided urine is discarded.
Subsequently all the urine passed is collected in the container provided.
After getting up in the next morning, the first voided urine is also collected
and the container is sent to the laboratory.
• A blood sample for estimation of plasma creatinine is obtained at midpoint
of urine collection.
• Creatinine clearance is calculated from
(1) concentration of creatinine in urine in mg/ml (U),
(2) volume of urine excreted in ml/min (V) and
(3) (3) concentration of creatinine in plasma in mg/dl (P).

37. • Creatinine clearance in ml/min per 1.73 m2 is then derived
from the formula UV/P.
• Because of secretion of creatinine by renal tubules, the
above formula overestimates GFR by about 10%.
• In advanced renal failure, secretion of creatinine by tubules is
increased and thus overestimation of GFR is even more.
• (Jaffe’s reaction and cimetidine-enhanced creatinine clearance).
• not an ideal test for estimation of GFR because of following
reasons:
1. A small amount of creatinine is secreted by renal tubules that
increase even further in advanced renal failure.
2. Collection of urine is often incomplete.
3. Creatinine level is affected by intake of meat and muscle mass.
4. Creatinine level is affected by certain drugs like cimetidine,
probenecid, and trimethoprim (which block tubular secretion of
creatinine).

38. Urea Clearance
• filtered by the glomeruli
• 40% of the filtered amount is reabsorbed by the tubules.
• The reabsorption depends on the rate of urine flow.
• not a sensitive indicator of GFR.
• clearance tests are more helpful in early cases.
• If biochemical tests are normal and renal function
impairment is suspected, then creatinine clearance test
should be carried out.
• If biochemical tests are abnormal, then clearance tests
need not be done.

39. Blood Biochemistry
• Two biochemical parameters are commonly
used to assess renal function:
1. blood urea nitrogen (BUN)
2. serum creatinine
• insensitive markers of glomerular function.

40. • Methods for estimation of BUN:
• Two methods are commonly used.
1. Diacetyl monoxime urea method: This is a direct
method. Urea reacts with diacetyl monoxime at high
temperature in the presence of a strong acid and an
oxidizing agent. Reaction of urea and diacetyl monoxime
produces a yellow diazine derivative. The intensity of
color is measured in a colorimeter or spectrophotometer.
2. Urease- Berthelot reaction: This is an indirect method.
Enzyme urease splits off ammonia from the urea
molecule at 37°C. Ammonia generated is then reacted
with alkaline hypochlorite and phenol with a catalyst to
produce a stable color (indophenol). Intensity of color
produced is then measured in a spectrophotometer at
570 nm. Reference range for BUN in adults is 7-18
mg/dl. In adults > 60 years, level is 8-21 mg/dl.

41. • Causes of increased BUN:
1. Pre-renal azotemia: shock, congestive heart
failure, salt and water depletion
2. Renal azotemia: impairment of renal function
3. Post-renal azotemia: obstruction of urinary tract
4. Increased rate of production of urea:
• High protein diet
• Increased protein catabolism (trauma, burns,
fever)
• Absorption of amino acids and peptides from a
large gastrointestinal hemorrhage or tissue
hematoma

42. Serum creatinine
• Creatinine is a nitrogenous waste product formed
in muscle from creatine phosphate. Endogenous
production of creatinine is proportional to muscle
mass and body weight.
• Serum creatinine is a more specific and more
sensitive indicator of renal function as compared
to BUN because:
1. It is produced from muscles at a constant rate
and its level in blood is not affected by diet,
protein catabolism, or other exogenous factors;
2. It is not reabsorbed, and very little is secreted
by tubules.

43. • Methods for Estimation of Serum Creatinine
• The test for serum creatinine is cheap, readily available, and
simple to perform.
• There are two methods that are commonly used:
1. Jaffe’s reaction (Alkaline picrate reaction): This is the most
widely used method. Creatinine reacts with picrate in an
alkaline solution to produce a yellow-red color. The color is
measured in a spectrophotometer at 485 nm. Certain
substances in plasma (such as glucose, protein, fructose,
ascorbic acid, acetoacetate, acetone, and cephalosporins)
react with picrate in a similar manner; these are called as
non-creatinine chromogens (and can cause false elevation
of serum creatinine level). Thus ‘true’ creatinine is less by
0.2 to 0.4 mg/dl when estimated by Jaffe’s reaction.
2. Enzymatic methods: These methods use enzymes that
cleave creatinine; hydrogen peroxide produced then reacts
with phenol and a dye to produce a colored product, which is
measured in a spectrophotometer

44. • Causes of Increased Serum Creatinine Level
1. Pre-renal, renal, and post-renal azotemia
2. Large amount of dietary meat
3. Active acromegaly and gigantism
• Causes of Decreased Serum Creatinine Level
1. Pregnancy
2. Increasing age (reduction in muscle mass)

45. BUN/Serum Creatinine Ratio
• discriminate pre-renal and post-renal azotemia from renal
azotemia.
• Normal ratio is 12:1 to 20:1.
• Causes of Increased BUN/Creatinine Ratio (>20:1):
1. Increased BUN with normal serum creatinine:
• Pre-renal azotemia (reduced renal perfusion)
• High protein diet
• Increased protein catabolism
• Gastrointestinal hemorrhage
2. Increase of both BUN and serum creatinine with
disproportionately greater increase of BUN:
• Post-renal azotemia (Obstruction to the outflow of urine)
Obstruction to the urine outflow causes diffusion of urinary
urea back into the blood from tubules because of
backpressure.

46. • Causes of Decreased BUN/Creatinine Ratio
(<10:1)
– ATN
– Low protein diet, starvation
– Severe liver disease

47. Test to assess tubular function
A. Test to asses proximal tubular function
1. Glycosuria
2. Aminoaciduria
3. Tubular proteinuria
4. Urinary concentration of sodium
5. Fractional excretion of sodium

48. B. To asses distal tubular function
1. Urine specific gravity: Normal specific gravity is
1.003 to 1.030.
i. Causes of increased specific gravity:
a. Reduced renal perfusion (with preservation of
concentrating ability of tubules),
b. Proteinuria,
c. Glycosuria,
d. Glomerulonephritis.
e. Urinary tract obstruction.
ii. Causes of reduced specific gravity:
a. Diabetes insipidus
b. Chronic renal failure
c. Impaired concentrating ability due to diseases of
tubules

49. 2. Urine osmolality
3. Water deprivation test
4. Water loading ADH suppression test
5. Ammonium chloride loading test (acid loading
test)

50. REFERENCES:
1. TORTORA & DERRICKSON
2. GUYTON
3. ESSENTIALS OF CLINICAL PATHOLOGY-
KAWATHALKAR.