KIDNEY FUNCTION TESTS

-kidneys are retroperitoneally on either side of the spinal column *2 regions: Outer (Cortex) and Inner (Medulla) *5 basic parts of nephron: a. Glomerulus b. Proximal convoluted tubule c. loop of Henle d. Distal convoluted tubule e. collecting duct I. TESTS FOR GLOMEULAR FILTRATION RATE Glomerular Filtration Rate - clearance of normal molecules that are not bound *150L of glomerular filtrate is produced daily *GFR decreases by 1.0ml/min/yr after 20-30years 1. Clearances -removal of substance from plasma into urine *mL/minute = volume of plasma that would be totally cleared of the solute in 1min. -Plasma concentration & clearance is inversely proportional! Formula:

*assess GFR among paediatrics and elderly - diabetic nepropathy, renal failure Reference values: adults 0.5-1.0 >65yrs old 0.9-3.4 II. TESTS FOR RENAL BLOOD FLOW A. BLOOD UREA NITROGEN (BUN) *major end product of protein & amino acid catabolism *45% of total NPN *80% of the nitrogen excreted -synthesized in liver from CO2 & ammonia (from Krebs cycle) st *1 metabolite to increase in kidney diseases *about 25g urea is excreted daily BUN:Creatinie ratio: 10:1-20:1 -compute urea from BUN: BUN x 2.14 = urea(mg%) Reference value: 8.23mg/dL (2.9-8.2mmol/L) Methods: Fluoride or citrate will inhibit urease Thiosemicarbazide & ferric ions enhance color rxn 1. Direct/Chemical Method Diacetyl Monoxime Yellow 2. Indirect/Enymatic Method a. Hydrolysis of urea by Urease -urease from jack beans *Berthelot rgts to measure ammonia b. Coupled Urease/Glutamate Dehydrogenase -UV enzymatic method 3. Isotope Dilution Mass Spectrophotometry - REFERENCE METHOD Notes: -Serum urea drops () in severe hepatic disease -Plasma contains 20-35mg/dL NPN 45% Urea 20% Amino Acid & 20% Uric Acid 5% Creatinine 1-2% Creatine 0.2% Ammonia - BUN in celiac disease, pregnancy B. CREATININE -end product of muscle metabolism -from creatine (a-methyl guanidoacetic acid) -also produced by methionine, arginine and lysine -not affected by protein diet -not easily removed by dialysis -not reused, solely as waste product *Monitor renal fxn, index of overall renal fxn *measure of completeness of 24hr urine collection *Evaluate fetal kidney maturity 2mg/dL in amniotic fluid Reference Value: 0.5-1.5mg/dL (44-133umol/L)

A. INULIN CLEARANCE *reference method -not routinely done due to continous IV infusion *values higher in male Reference values: male-127ml/min Female 118ml/min B. CREATININE CLEARANCE -related directly to muscle mass -excretion not affected by diet *1.2-1.5g creatinine excreted per day *estimate of the amount of plasma that flowed thru the kidney glomeruli/minute *Excellent measurement of renal function! *Measure of the completeness of a 24-hr urine collection Reference value: male-85-125ml/min Female 75-112ml/min C. UREA CLEARANCE -demonstrate progression of renal dse or therapy response -does not give reliable estimates of GFR variably reabsorbed -the faster the urine flow, the less urea is reabsorbed -about 50% of creatinine clearance (50% GFR) 2. Cystatin C -low molecular weight protease inhibitor -produced by all nucleated cells -freely filtered, not secreted, completely reabsorbed *presence in urine denotes damage of tubules *Plasma level when glomerular filtration * more rapidly than crea in the early stage of GFR impairment -not affected by muscle, mass, age, gender

Methods of Creatinine -Avoid hemolyzed and icteric samples *Serum & urine creatinine = indices of renal fxn *24hr urine sample <0.8g/day creatinine indicates that some of the urine was probably discarded - impaired renal fxn, myasthenia - pregnancy I. ENZYMATIC METHOD -eliminate nonspecificity of Jaffe -more specific than Jaffe A. Creatinine Aminohydrolase-CK method B. Creatinase H202 method -has potential to replace Jaffe method! -w/o interference from acetoacetate/cephalosporins *Creatinase also known as Creatinine aminhydrolase II. DIRECT JAFFE METHOD -formation of red tautomer of creatinine picrate - due to ascorbate, glucose, uric acids, a-ketoacids Jaffes Reagent: Saturated picric acid + 10% NaoH A. Folin Wu B. Lloyd or Fullers Earth Method -sensitive and specific *Lloyds (Na aluminium silicate) *Fullers (Na Mg silicate) -elution techniques are utilized

DISEASE CORRELATION: UREMIA -marked in plasma urea and other nitrogenous products -accompanied by academia & electrolyte imbalance -kidneys fail to eliminate waste products in urine *Features: Anemia, Uremic Frost, Edema, Foul breath, urine like sweat BUN-Crea Ratio with normal creatinine: Prerenal Azotemia BUN-Crea Ratio with Creatinine: Postrenal, Pre renal, Renal failure C. BLOOD URIC ACID *Major product of purine (adenine+guanine) catabolism -final breakdown of nucleic acids catabolism *Formed from xanthine by the action of xanthine oxidase -filtered, partially reabsorbed, secreted *weak acid: pH 7.4; 95% above exists as monosodium urates *1g uric acid is excreted daily Derived from 3 sources: Ingested nucleoproteins catabolism Catabolism of endogenous nucleoproteins Direct transformation of endogenous purine nucleotides Reference Values: (Uricase) Male: 3.5-7.2mg/dL Female: 2.6 6mg/dL DISEASE CORRELATION: Hyperuricemia 1. Gout rd th -primarily in males; dx between 3 & 5 decade -acute inflammatory arthritis *Definitive Diagnosis: birefringent crystals in synovial fluid -highly susceptible to nephrolithiasis 2. Nuclear Metabolism -important monitoring to avoid nephrotoxicity *Allopurinol used for treatment 3. Chronic Renal Disease - GFR and tubular secretion - >10mg/dL levels cause urinary tract calculi 4. Lesch-Nyan Syndrome (inborn error of purine metabolism) -deficiency of Hypoxanthine-guanine phosphoribosyl transferase (HGPRT) Uric Acid: ethanol consumption, 2 to glycogen storage dse Uric Acid: Wilsons disease, Hodgkins disease, Fanconis syndrome (renal type aminoaciduria) Methods: -uric acid stable in serum & urine for 3days at room temp -Potassium oxalate as anticoagulant cannot be used *Ascorbic acid & Bilirubin = major interferences

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III. KINETIC JAFFE METHOD -popular, inexpensive, rapid & easy -automated equipment for precision *serum + alkaline picrate = rate of change in absorbance is measured between 2 points -Interferences: a-ketoacids and cephalosporins IV. ISOTOPE DILUTION MASS SPECTROMETRY (ID-MS) -definitive method for creatinine *ID-MS & Isocratic Ion Exchange HPLC w/ UV detection (candidate reference method)

DISEASE CORRELATION: AZOTEMIA -elevated level of nitrogenous substances GFR with normal renal fxn renal blood flow = GFR = Pre-Renal Azotemia reabsorption = slower filtrate flow *Dehydration = urea + normal plasma creatinine - (GFR) damaged w/in kidneys *BUN: >100mg/dL Renal Azotemia *Crea: 20mg/dL *Uric Acid: 12mg/dL GFR due to urinary tract obstruction Post-Renal Azotemia Urea > creatinine due to back-diffusion urea and creatinine in blood

I. CHEMICAL METHODS (Principle: REDOX Reaction) *Lagphase: incubation period after alkali addition to inactivate non-uric acid reactants II. ENZYMATIC METHODS (Uricase Method) -simplest and most specific -uric acid peak absorbance: 293nm -allantoin does not have a UV peak Principle: -uricase oxidizes uric acid to form allantoin -resultant product allantoin has no absorption - in absorbance is proportional to uric acid conc III. TESTS MEASURING TUBULAR FUNCTION A. Excretion Tests 1. Para-Amino Hippurate Tests (Diodrast Test) -measures renal plasma flow -requires clearance of dye Reference Range: 600-700mL/min 2. Phenosulfonpthalein Dye Test -measures dye excretion propotional to renal tubular mass -6mg PSP is administered to IV Reference Range: 1200ml blood flow/min B. Concentraion Test -reflects fxn of collecting tubules & loops of Henle -assess the quantity of solutes present in urine; reflects the ability of kidneys to produce conc urine *3 most prevalent solutes excreted: urea, Cl, Na *Specimen for tests: FIRST MORNING URINE 1. Specific gravity simplest test of renal concentrating ability *Fixation of SG at 1.010 + 290mOsm/kg osmolality = severe loss of concentrating ability of kidneys! Reference Values: 1.005-1.030 2. Osmolality - total no. of solute paricles present/kg solvent (moles/kg) -more accurate than SG in assessing renal tubular fxn *Urine osmolality is primarily due to UREA *Serum osmolality is primarily due to CHLORIDE -not affected by high molecular weight substances -proteins & lipids do not contribute to osmolality Methods: -Osmolality is determined by msq of colligative properties - osmolality = boiling point & osmotic pressure - osmolality = freezing point & vapour pressure Reference Values: serum: 275-295 mOsm/kg Urine: 300-900mOsm/kg A. DIRECT METHOD Freezing Point Osmometry Popular Mtd Vapor Pressure Osmometry Seebeck effect

B. INDIRECT METHOD -use glucose or urea in osmolality Formula:

Interpretation of results: *Conc. Urine specimen = 1.025 SG > 800mOsm/kg *Loss of conc. Renal ability = 1.2:1; D. Insipidus = <1.1 * Urine: Serum Osmolality > 1:1 -glomerular disease - solute in the urinary filtrate NOTES: - plasma osmolality = urine flow -serum osmolality is useful for assessing water deficit/excess *Serum osmolality > 2.1-2.3x Serum Na = hyperglycemia, anion gap acidosis, uremia *Tubular Failure = BUN, crea, PO4 + Ca -In DM, 5000mOsm/day requires large amount of H20 intake *Osmolal Gap: difference between measured & calculated plasma osmolality; sensitive indicator of alcohol or drug overdose *Osmolal Gap > 12 mOsm/kg is significant!!!! - DKA, drug overdose, renal failure