1. RENAL FUNCTION TESTS
MARIA VICTORIA E. GASATAYA, M.D.

2. KIDNEY FUNCTION
 A plumbers view Input
Arterial
Filter
Processor
Output Output
Venous Urine

4. RELATIONSHIP OF NEPHRON AREAS TO RENAL
FUNCTION TESTS
PAH Osmolarity
Clearance
tests Ammonia
Titratable
acidity
Free water
clearance
Osmolarity

5. EVAULUATION OF RENAL FUNCTION
 Various aspects of renal function are
-GFR(Glomerular Filtration Rate)
-RPF(Renal Plasma Flow)
-Reabsorption & Excretion of various substances like Na+,
K+, Ca+2, inorganic phosphate, glucose, urea, a.a, H2O &
osmoles.
 In clinical practice
-determination of Creatinine Clearance is a measure of
GFR
-water deprivation & vasopressin administration tests to
determine urinary concentrating ability, &
-bicarbonate & ammonium chloride loading test to examine
urinary acidification are usually sufficient for diagnostic
evaluation & measurement of kidney function. 5

6. CONT…
 The results of these tests are important in assessing the
excretory function of the kidneys. For example, grading of
chronic renal insufficiency and dosage of drugs that are
excreted primarily via urine are based on GFR (or creatinine
clearance).
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7. GLOMERULAR FILTRATION TESTS
 Standard Test – Clearance Test
 Substance analyzed must not be reabsorbed or
secreted by the tubules
 Other factors to consider:
- stability of subst. in 24 hr. urine collection
period, consistency of the plasma
level, substance availability to the
body, availability of the tests for analysis of the
substance

8. PRINCIPLE OF CLEARANCE
 Some substances when filtered enter the tubules
are not reabsorbed and so 100% excreted=
GFR (inulin= gold standard for
GFR, creatinine (but this one partially
reabsorbed, particularly in uremia, then
clearance <GFR
 Some substances are filtered, enter tubules, and
more of the substance is secreted enters the
tubules by excretion. Clearance>GFR
 Some substances are filtered, enter tubules, but
are completely reabsorbed, so they did not
reach the final urine (e.g. cystatin C)

9. TEST SUBSTANCES FOR GLOMERULAR
FILTRATION TESTS
1. Urea
2. Creatinine
3. Inulin
4. Beta 2 microglobulin
5. Microglobulin
6. Cystatin C
7. Radioisotopes

10. UREA
 Used historically as marker of GFR
 Freely filtered but both re-absorbed and
excreted into the urine
 Re-absorption into blood increased with
volume depletion; therefore GFR
underestimated
 Diet, drugs, disease all significantly
effect Urea production

11. CYSTATIN C
 CYSTATIN C : It is a LMW nonglycosylated protein produced
at a constant rate by all nucleated cells in the body, freely
filtered by the glomeruli, not secreted, but totally
reabsorbed by the renal tubules.
 Little or no cystatin is excreted in urine.
 Normal adults have circulating level of approx. 1mg/l.
 This is better indicator of renal function as compared to
creatinine in early stages of GFR impairment as it is
independent of age, gender, body composition & muscle
mass.
 Cystatin C can be estimated in blood by enzyme
immunoassays or immuno-turbidometry. Both techniques
are currently kit based & expensive.
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12. SERUM CREATININE & CREATININE CLEARANCE
 Creatinine is derived from the metabolism of creatine &
phosphocreatine, the bulk of which is in muscle.
 Since creatinine is chiefly excreted by glomerular filtration
serum creatinine levels reflects changes in GFR.
 Currently, routine lab. measurements of GFR use creatinine
as a test substance
 Serum creatinine values are low when the muscle mass is
decreased, as in malnutrition.
 Billirubin interferes with creatinine measurements.
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13. DISADVANTAGES – USE OF CREATININE
1. Some creatinine is secreted by the tubules,
secretion increases as bld. levels increase
2. Chromogens react in the chemical analysis
3. Medications inhibit secretion – gentamycin,
cephalosporins, cimetidine – false low serum
levels.
4. Bacteria breaks down urinary creatinine (room
temp. for extended periods)

14. DISADVANTAGES – USE OF CREATININE
5. Diet heavy in meat eaten during 24 hr. urine
collection affect results, if plasma drawn before
collection.
6. In muscle wasting diseases, not reliable
indicator

15. FORMULA FOR URINE VOLUME
amount of urine
V = time of collection
Ex. Urine vol. for a 2-hour specimen (120 min)
measuring 240 ml.
240 ml./120 min. = 2 ml/min.

16. GLOMERULAR FUNCTION TEST
 The concept of clearance is based upon the fact that the
rate of removal of a substance from the plasma must equal
its simultaneous rate of excretion in urine.
 Thus if the urinary excretion rate & plasma concentration of
a substance are known, we can calculate the volume of
plasma from which that substance would have been
completely removed.
 The standard formula for clearance is : C = U x V
P
C = clearence/min(ml/min)
U = urinary concenteration(mg/dl)
P = plasma concenteration(mg/dl)
V = urine volume/min(ml/min)
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17. SAMPLE PROBLEM FOR GFR
C=UxV
P
Urine creatinine = 120 mg/dl
Plasma creatinine = 1.0 mg/dl
1440 ml urine from 24 hr collection (1440 min)
Urine volume: ___1440 ml____ = 1 ml/min.
1440 min.

18. C=UxV
P
C = 120 mg/dl (U) X 1 ml/min (V)
1 ml/min.
= 120 ml/min.

19. GFR ADJUSTMENTS
Average person = 1.73 m2 body surface
Approx. amt. of plasma filtrate produced per min
= 120 ml.
Variations in size and muscle mass – adjust
clearance for body size: formula is:
C = UV x 1.73
P A
- With A being the actual body size in sq. m. of
surface

20. CALCULATION OF ACTUAL BODY SIZE
Log A=(0.425 X log wt.)+(0.725 X log Ht.) – 2.144
where height is in meters and weight is in
kilograms
Or BSA (m2) = (W0.425 x H0.725) x 0.007184
where BSA is body surface area

21. CLINICAL SIGNIFICANCE OF GFR
 GFR is determined not just by the number of
functioning nephrons but the functional
capacities of these nephrons.
 Does not detect early renal disease
1. determines extent of nephron damage in
known cases of renal disease.
2. monitor effectiveness of treatment
3. determine feasibility of giving medications

22. CALCULATED GLOMERULAR FILTRATION ESTIMATES
 What is the currently recommended method to estimate GFR?
 The National Kidney Disease Education Program (NKDEP) of the
National Institute of Diabetes and Digestive and Kidney Diseases
(NIDDK), National Kidney Foundation (NKF), and American Society of
Nephrology (ASN) recommend estimating GFR from serum creatinine.
 Two commonly used equations are the Modification of Diet in Renal
Disease (MDRD) Study equation and Cockcroft Gault equation. Both
equations use serum creatinine in combination with age, sex, weight,
or race to estimate GFR and therefore improve upon several of the
limitations with the use of serum creatinine alone.
 Epidemiology Collaboration (CKD-EPI) equation is a new equation
based on serum creatinine.

23.  What is the Cockcroft-Gault formula?
 The Cockcroft-Gault formula was developed
in1973 using data from 249 men with creatinine
clearance (CCr) from approximately 30 to130
mL/m2. It is not adjusted for body surface area.
CCr={((140-age) x weight)/(72 SCr)} x 0.85 if female
 where CCr is expressed in milliliters per minute,
age in years, weight in kilograms, and serum
creatinine (SCr) in milligrams per deciliter

24.  What is the MDRD Study equation? (modification of diet in renal disease)
 The 4-variable MDRD Study equation was developed in 1999 using data
from 1628 patients with CKD with GFR from approximately 5 to 90 milliliters
per minute per 1.73 m2. It estimates GFR adjusted for body surface area4
and is more accurate than measured creatinine clearance from 24-hour
urine collections or estimated by the Cockcroft-Gault formula. The equation
is:
 GFR = 186 x (SCr)-1.154 x (age)-0.203 x (0.742 if female) x
(1.210 if African American)

 The equation was re-expressed in 2005 for use with a standardized serum
creatinine assay, which yields 5% lower values for serum creatinine
concentration:
 GFR = 175 x (Standardized SCr)-1.154 x (age)-0.203 x (0.742 if
female) x (1.210 if African American)

 GFR is expressed in mL/min/1.73 m2, SCr is serum creatinine expressed in
mg/dL, and age is expressed in years.

25. RADIOISOTOPES
 SINGLE INJECTION TECHNIQUE : in clinical
practice, radionuclides are often used to estimate total GFR
or to measure difference in clearance bet. one kidney
compared to other in the same patient.
 The technique is based on use of a single injection, plasma
disappearance curves to estimate the true GFR.
 Briefly, the radionuclide dye is injected & the signal from
radiolabelled form is used to obtain measurement.
 The most commonly used Radionuclides for GFR are
-DTPA (Diethyl triamine Penta-acetic acid)
-EDTA (ethylene diamine tetra acetic acid) &
-Iothalamate
 Iohexol, a non ionic non radioactive LMW radiocontrast
agent, as an alternative to inulin, measured easily by
HPLC(high performance liquid chromatography)
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26. CONT…
 Renal Clearance of INULIN is the Gold Standard for
determination of GFR.
 51Cr-EDTA clearance closely resembles Inulin clearance & it
is the radionuclide of choice for GFR estimation in Europe.
 However, 99m Tc-DPTA is often the preferred agent, because
99m Tc-DPTA is inexpensive, easily available & renal imaging
can be simultaneously performed.
 GFR can be estimated based upon either plasma clearance
or upon the tracer uptake by the kidneys.
 When a substance is freely filtered & not protein bound,& is
not reabsorbed, secreted or metabolised by the
kidney, then its clearance is similar to GFR.
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27. TUBULAR REABSORPTION TESTS
 Loss of tubular reabsorption is the first function
affected in renal disease.
 Concentration Tests = determine the ability of
tubules to reabsorb essential salt & water
 Ultrafiltrate – specific gravity 1.010
 Fishberg test – 24 hr. water deprivation
 Mosenthat Test – compare vol. & specific gravity of
day and night urine samples to evaluate
concentrating ability
 Specific gravity of 1.025 after 16 hr H2O
deprivation, urine osmolality of 800 mOsm

28. OSMOLARITY
 Specific gravity = number and density of the
particles present in a soln. (urea, Na, Cl)
 Osmolarity = number of particles present
 Osmole = 1 gm. molec. wt. of subst. / number
of particles into which it dissociates
ex. Glucose – 180 MW = 180 g per osmole
NaCl – 58.5 MW = 29.25 g per osmole
Osmolarity of a soln. measured by colliagative
property of the soln.

29.  Solute dissolved in solvent causes change in
the colligative properties
1. lower freezing point
2. higher boiling point
3. increased osmotic pressure
4. lower vapor pressure
Water is the solvent in both urine & plasma =
comparison to pure water.
Clinical lab. instruments measure freezing point
depression and vapor pressure depression.

30. FREEZING POINT OSMOMETERS
1. Measured amt. of sample cooled to 27 C
2. Sample vibrated to produce crystallization.
3. Heat of fusion due to crystallization raises
temp of sample to freezing point.
4. Temp. sensitive probe measures temp
increase which corresponds to freezing point
5. Info. converted to milliosmoles (comparison
to a known subst.)

31. VAPOR PRESSURE OSMOMETERS
1. Measurement performed is the dew point.
2. Samples absorbed on filter paper discs & placed in a
sealed chamber w/ temp-sensitive thermocoupler.
3. Sample vaporizes & temp. is lowered
4. H20 condenses in chamber & on the thermocoupler.
5. Heat of condensation produced raises temp. of
thermocoupler to dew point temp.
6. Info. converted to milliosmoles (comparison to a
known subst.)

32. FALSE ELEVATED OSMARALITY READINGS DUE:
1. Lipemic serum = displacement of serum water
by insoluble lipids – erroneous results
2. Lactic acid formation occurs if serum samples
are not separated or refrigerated within 20
min. = falsely elevated values with both
methods
3. Volatile substances (become part of solvent
phase) = freezing point osmometers are
elevated.

33. CLINICAL USES OF OSMOLARITY
1. Evaluation of renal concentrating ability
2. Monitoring course of renal disease
3. Monitoring fluid and electrolyte theraphy
4. Diff. Dx of hypernatremia and hyponatremia
5. Evaluating secretion and renal response of
ADH.
Ratio urine to serum osmolarity 1:1

34.  Response to Vasopressin : subsequently,to
confirm the lack of renal concentrating ability &
distinguish Nephrogenic Diabetes Insipidus
(NDI) from Central Diabetes Insipidus (CDI), a
vasopressin test is performed.
 Failure to achieve a ratio of 3:1 ff. injection of
ADH = inability of renal tubules to respond
 Concentration after ADH injection, inability to
produce adequate ADH

35. FREE WATER CLEARANCE (CH2O)
 Use of water deprivation and timed urine
specimen (negative value=less amt. excreted
=dehydration)
 Osmolar clearance Formula
Cosm = Uosm X V
Posm
Ex. Uosm of 600 mOsm, V of 2 ml/min, Posm –
300 mOsm.
CH2O = V- Cosm

36. TUBULAR SECRETION AND RBF TESTS
 PAH (para-amino-hippuric acid = secreted by
prox. tubule
 Before PSP (phenolsulfonphthalein) was used
but standardization difficult due to interference
from medications, elevated waste products,
odtain very accurately timed urine specimens
 PAH test done in specialized renal labs.
 PAH is nontoxic and loosely bound to plasma
proteins thus PAH completely removed as bld.
passes thru peritubular capillaries.

37. RENAL BLOOD FLOW
 Renal blood flow measurements are performed
using the clearance of PAH(para aminohippuric
acid)
 >90% PAH is extracted from the plasma during the
1st pass through the kidneys. Therefore, renal
clearance of PAH is commonly used as an estimate
of renal plasma flow (RPF).
 Plasma clearance following single injection of 131I-
hippuran or 99mTc-mercaptoacetyltriglycine(MAG-
3)is an alternative method. (radioisotopes)
 Other methods-Color Doppler US,Contrast
Enhanced US & MRI.
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38. STANDARD CLEARANCE FORMULA
CPAH (ml/min) = U(mg/dl PAH) X V(ml/min urine
P(mg/dl PAH)

39. TITRATABLE ACIDITY & URINARY AMMONIA
 Distal convoluted tubule secrete H ions and
ammonium ions.
 Excretion of 70mEq/day of acid in the form of
1. titratable acid (H+)
2. hydrogen phosphate ions (H2PO4)
3. ammonium ions (NH4+)
Diurnal variation of alkaline tides (after arising &
postprandially 2pm & 8 pm – lowest at night

40. TESTS OF URINARY ACIDIFICATION
 Renal acidification mechanism are usually examined in
patients with suspected renal tubular acidosis.
 RTA inability to produce an acid urine in metabolic acidosis
 Urine pH : The pH of a fresh specimen of urine is tested
with pH meter. A concentrated, fasting, morning sample of
urine is acidic. A pH of <5.5 virtually excludes distal renal
tubular acidosis.
 Urine Anion Gap : Urine anion gap is the difference between
concentration of principal urinary cations (Na + k) & anion
(Cl) This difference is expected to estimate the unmeasured
anions & cation, which normally include ammonium &
bicarbonate. Since the latter is mostly reabsorbed ,urine
anion gap is chiefly measure of ammonium excretion.
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41. CONT…
 In presence of systemic metabolic acidosis, ammonium
excretion is expected to be stimulated while the bicarbonate
excretion is minimal, hence, the urine anion gap should be
–ve due to obligatory chloride excretion with ammonium.
 In patients with renal tubular acidosis the gap remains +ve
due to impaired ammonium production.
 Sodium Bicarbonate Loading Test : The Bicarbonate
threshold is the plasma bicarbonate level at which
bicarbonate appears in urine.
-The bicarbonate threshold is determined by infusion of
bicarbonate & increasing the plasma bicarbonate in a
stepwise manner to elevate the serum bicarbonate level to
23-26mEq/l.
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42. CONT…
 Other tests for urine acidification are
-Urine PCO2
-Ammonium Chloride Loading test
Tests can be ran simultaneously on either fresh or toluene
preserved urine specimens collected at 2 hr. intervals from
patients who have been primed with oral ammonium
chloride.
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43. CONT…
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