1. KIDNEY FUNCTION TESTS

2. Kidney functions 1- Eliminations of toxic substances and metabolites:
2- Regulation of acid-base balance and electrolyte balance:
3- Secretion of hormones:

3. Evaluation of kidney function tests
I- Evaluation of Elimination:
A- Clearance tests:
Def.: volume of blood from which a sub. is completely cleared by the kidney per unit time.
Criteria of the ideal material:
1-freely filtered at glomerular barrier
2-Not reabsorbed by the tubules

4. 3-not secreted by tubules
4-present at stable plasma conc.
Factors affecting clearance:
*Size of the nephron:
*Pregnancy:
*D.M.:
*Kidney disease:
Types of clearance:
1-Inulin clearance:
2-Creatinine clearance:

5. 3-Urea clearance:
4-Estimated:
5-Calculated:
6-Radio labeled substance clearnce:
Clearance of a substance: UxV/P x1.73/A
1-Inulin clearnce:
Adv:
Disadv.:
Determination:
a- Chemical method:
b- Enzymatic method:

6. Inulin Inulinase fructose.
Fructose + NADH sorbitol dehydrogenase Sorbitol + NAD
2- Iohexol clearnace:
3-Creatinine clearance:
Range:
Adv:
Disadv:
Mild renal impairment: ml/min
Moderate impairment: ml/min
Severe impairment: ml/min
Renal failure: <15 ml/min

7. 4- Urea Clearance:
Adv:
Disadv:
If urine flow >2 ml/min: Maximum urea clearance:
UxV/P
If urine flow <2 ml/min: Standard urea clearance:
U x √V /P
Result is given in ratio: Clearnce obtained
Max. or Std. clearnce
5- Estimated glomerular filtrate:
Adv.:

8. 6- Calculated clearance:
(140- age in years) x wt/kg
72 x serum creatinine
7- Radiolabeled substance clearance:
Cr51-EDTA – I125 – Radioactive vit. B12.
Adv:
Disadv:
Values of clearance test:

9. II- Estimation of NPN substances: (1) Urea:
is a metabolic product derived from the catabolism of either exogenous or endogenous proteins.
Protein →A.A. → Ammonia → Urea.
N.V.:
serum: mg/dl
Causes of ↓ Urea: ↓ protein intake-severe liver disease-vomiting and diarrhea.
Causes of ↑ Urea:

10. I-Pre-renal causes: II-Renal: III-Post-renal: BUN:
Methods of determination:
I-Enzymatic methods:
1-Nesslerization method: (old-not specific nor sensitive)
2-Berthelote reaction: (reagent not stable – color stable- obey Bear’s law)
3-Double enzymatic reaction (reference method):
Urea Urease Ammonia +α ketoglutamate GDH Glutamate NADH NAD
II-Chemical methods:

11. (2) Creatinine: 1- Urea + orthophthaldehyde → isoindoline derivative
isoindoline derivative + methoxy quinoline → red color.
2- Diacetyle monoxime heat diacetyle + hydroxylamine
Diacetyle + urea ferric ammonium sulfate diazin
(2) Creatinine:
*end product of creatine
*N.V.:
*causes of increased creatinine level:
A-Non-pathological causes:
B-Pathological causes:
1-decreased renal perfusion:
2-loss of functioning nephron:
3-Prostate enlargement

12. **Urea/Creatinine ratio: 25-30/1 **BUN/Creatinine ration: 10-12/1
*Creatinine results should be supplemented with urea to confirm result and to give idea about etiology of the disease.
**Urea/Creatinine ratio: 25-30/1
**BUN/Creatinine ration: 10-12/1
Benefits:
**1/Creatinine: reflect healthy renal tissues.
Methods of determination of creatinine:
1-Jaffe reaction:
Creatinine + picric acid NaOH Alk. Picrate
Methods to avoid positive interference:
A- Slot method:
B- Lioyd’s reagent:
C- 2 points assay:

13. II-Enzymatic methods:
1- Creatininase-Creatinase method:
Creatinine creatininase creatine Creatinase sarcosin + urea
Sarcosin sarcosin oxidase H2O2 + glycine + formaldehyde
H2O2 peroxidase O2 + H2O
O2 + phenol + para aminoantipyrine red color
2- coupled enzymatic reaction:
Creatinine creatininase creatine
Creatine + ATP CK Creatine phosphate + ADP
ADP + phosphoenolpyruvate (PEP) PK Pyruvate + ATP
Pyruvate + NADH LD lactate + NAD
III-Other methods:
Reference method: HPLC
Method used in test strips: creatinine dinitrobenzoic acid red col.

14. (3) Uric acid: N.V.: Causes of hyperuricemia:
1- increased production:
2-decreased elimination:
Methods of determination:
I-chemical method:
Phosphotungestic acid reduction technique:
PTA + uric acid alkali allantoin + tungistin blue (680 nm)
Caraway method – Brown method
II-enzymatic methods:
1- one step uricase method (Reference method)

15. Uric acid + O2 uricase alantoin + H2O2
2- Coupled enzymatic reaction:
a) Uric acid + O2 uricase alantoin + H2O2 (trender reaction)
b) Uric acid + O2 uricase alantoin + H2O2
H2O2 + ethanol catalse acetaldehyde
acetaldehyde + NAD aldehyde dehydrogenase NADH + compound acetate measured at 340 nm
c) Uric acid + O2 uricase alantoin + H2O2
H2O2 + methanol formaldehyde.
formaldehyde + acetyl acetone diacetyl 1-4dihydro-lutidin (yellow color at 410 nm)

16. New Markers for Evaluating Glomerular functions
I- B2-Microglobulin:
-small nonglycosylated peptide (M.W.: daltons) found on the surface of most nucleated cells.
-levels of B2-microglobulin remain stable in normal persons.
-elevated levels in serum indicate increased cellular turnover as im myeloproliferative and lymphoproliferative disorders, inflam., and renal failure.
-As a small, endogenous peptide, B2-M is easily filtered by the glomerulus and about 99.9% is then reabsorbed by the proximal tubules.
-Measurement of B2-M is used clinically to assess renal tubular function in renal transplant patients.

17. -elevated levels of B2-M in renal transplant patients indicate organ rejection.
-it is more efficient marker of renal transplant rejection than serum creatinine as it does not depend on lean muscle mass.
II- Cystatin C:
It is a low molecular-wt protein produced by nucleated cells.
It is freely filtered by the glomeruli and catabolized by the proximal tub.
It is produced at a constant rate and remain stable if kidney function is normal.
It is useful in indicating early changes in kidney functions but expensive and time consuming as it is measured by immunoassay methods.
Arrangement of markers of GFR:
1-Gold standard marker: Inulin continuous infusion urinary clearance.
2-Silver standard marker: Inulin single bolus pl. clearnce – Radioisotope labeled markers - Iohexol.
3-Bronze standard marker: Cystatin C – Creatinine.
4-Of uncertain clinical use: Cr. Cl., urea, RBPr, A1-MG

18. II- Evaluation of Renal Ability to Regulate Acid-base Balance & Electrolyte Balance:
1- Specific Gravity:
Def.: Ratio of wt of certain volume of urine to the wt equal volume of water at a fixed temp.
- it measures the ability of kidney to concentrate the glomerular filterate.
It varies directly with the grams of solutes excreted / lit.
The physiological range is but for 24 hs collected urine =
Factors affecting: Solutes (pr & glucose) – urine vol.
In renal tubular diseases, the concentrating ability of the kid. is one of the first functions lost.
Increases in: - decreases in:

19. 2- Osmolality: Methods of determination:
1- Hydrometer: urinometer is a hydrometer designed to fit into and float in a narrow cylinder filled with urine with a specific gravity scale arround it from 1000 to 1040
It should be calibrated by testing it with a sol. of known specific gravity (1000 with water and 1030 with 75 ml zylene and 25 ml bromozene)
How to adjust Sp.G. (temp – Pr – glucose)
2- Refractometer:
Principle: refraction of light in air differs than in solutes.
3-Dipsticks: the strip consists of a polyelectrolyte & an indicator (bromothymol blue) that changes color as H+ is displaced by Na+ or K+ in the patient’s urine.
2- Osmolality:
Def.: M.W. of a sub. In grams dissolved in 1 Kg water.

20. Osmolarity: M.W. of a sub. In grams dissolved in 1 lit.
Osmolality = Ǿ nc
Ǿ = osmotic coefficient (% of particles dissolved)
n = number of atoms after dissolving
c = conc. In mol.
Calculated osmolality= 1.86 (Na) + glucose/18 + BUN/2.8
Osmolal gap (delta osmolality): difference between measured & calculated osmolality.
Normally, measured osmolality > calculated osmolality by up to 30 mosmol/Kg (the difference is due to accumulation of osmotically active metabolites other than Na, gluc. & urea).
Causes of increased osmolal gap:
Lactic acidosis – ketoacidosis – ethanol overdose – CRF – methanol, isopropanol & ethylene glycol poisoning.

21. Measured osmolality: Serum: 285-310 mosmol/kg
Urine: mosmol/kg
Calculated osmolality: mosmol/kg (only serum)
One osmolal sol.: sol. Containing 1 mol of solute in 1 kg water
One osmolar sol.: sol. Containing 1 mol of solute in 1 lit. water
When substances are dissolved in a solvent, 4 physicochemical parameters are altered:
1- ↑ osmotic pressure ↑ in boiling pont
3- ↓ in vapour pressure 4- ↓ in freezing point
Methods of determination of osmolality:
1- Measurement by Osmometer: freezing point depression technique.
2- Calculation (serum only)
U/S osmol ratio: 1-3
*if U/S osmol ratio >1.2 + U. Na <20 mmol/l (prerenal causes of renal impairment)

22. 3- Urine concentration test:
If U/S osmol ratio <1.2 + U. Na >40 mmol/l (intrinsic causes of renal impairment).
3- Urine concentration test:
For the kidney to concentrate urine we must have active secretion of ADH and good response of the kidney to ADH.
Causes of abnormal conc. Test:
4- Urine dilution test:
Represent ability of kidney to dilute urine.
5- Fractional Na excretion:
Urine Na X Plasma creatinine
Plasma Na Urine creatinine
6- Renal failure index:
<1 = prerenal impairment >2 = tubular impairment

23. Plasma creatinine Urinary Na X 7- Ability of kidney to acidify urine:
Urine creatinine
<1 = prerenal impairment >2 = tubular impairment
7- Ability of kidney to acidify urine:
The kidney acidify urine by:
a- Reabsorbtion of HCO3-
b- Excretion of H+ produced during tissue metabolism as:
NH4+ - H+ exchange – with buffer anions as inorganic phosphate
T.A.= the amount of base needed to titrate a specimen of urine to PH 7.4.
So, the net excretion of H+ by kidney= T.A. + NH4+ - HCO3-
Ammonium chloride test:
Patient is fasting overnight.
At 8 a.m., empty the bladder.

24. - Urine is collected every hour (8 collections)
- At 10 a.m. give ammonim chloride by mouth (0.1 gm/kg) in gelatin capsule.
- Urine is collected every hour (8 collections)
- PH is determined in each urine collection.
- Blood sample before amm. Chloride and 2 hours after are taken to determine HCO3-
Normal response: (contraindicated in pt.s with liver disease)
In response to amm. Chloride load, the PH of urine falls to <5.3 in at least one sample but be sure that satisfactory acidosis has been induced by amm. Chloride (plasma total HCO3- decrease by about 4 mmol/L) in 2 hours period after amm. Chloride.

25. Diseases of the kidney I- Acute renal failure:
Rapid loss of renal functions which is reversible. (CRF is irreversible with slow loss of renal functions)
Causes:
1- Prerenal: (decreased renal perfusion)
due to shock – hge – hypotension – HF …………..
Lab investigations:
Urea – creatinine – UA – K – Na – Mg – Phosphate – Ca – sec. hyperparathyroidism – metabolioc acidosis – HCO3 – urine/serum osmol ratio >1.2 with U. Na <20 mmol/L
In acute RF, kidney excrete small vol. of concentrated urine with low Na conc. – Urinr urea/serum urea = >20 – RFI=<1 – Fractional Na excretion = <1

26. 2- Renal causes:
A- Nephrotoxic drugs (Cyclosporine – NSAID)
B- Kidney diseases (GN-acute tubular necrosis)
C- Systemic diseases (SLE-rheum. Arthritis)
Lab. Inv.:
as prerenal but with: urine Na > 40 mmol/L – urine osmolality <400 - U/S osmolality <1.2 – fractional Na excretion >2 – RFI >2 – urine urea/serum urea <10
3- Post renal causes:
Obstruction to urine pathway (stone – tumor – prostatic)
As pre-renal.

27. II- Chronic renal failure: (Irreversible – slowly progressing)
↑ urea, creatinine, K, phosphate, Mg
↓ creatinine clearance, Na, Ca, calcitriol, proteins, pH
Fixed Sp. G. – Polyurea.
III- Nephrotic Syndrome:
Diagnosed by: Massive proteinuria – Hypoproteinemia – Hypercholesterolemia.
1- Massive proteinuria >5 g/day
2- Hypoproteinemia.
3-Hypercholesterolemia
4- Hypoalbuminemia
5- ↑ high MW proteins ( A2 macroglobulin – lipoprot…….)
6- ↓ low MW proteins
7- false hyponatremia

28. 8- Hypocalcemia
9- Urea, creatinine, GFR are normal.
10- Electrophoresis
11- Urine albumin / creatinine ratio >3