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1. Renal function test Dr.A.B.DHADAS

2. Gross anatomy Located in post abd wall (T11-L3) Size - 13 x 6 x 4 cms Wt - 150 gms Blood supply- Renal artery Venous drainage- Renal vein

3. Functional anatomy Functional unit Nephrons ( 1.3 million / kidney ) Parts of nephron - Glomerulus PCT Loop of Henle DCT Collecting duct

4. GLOMERULUS Formed from a specialized capillary network Each capillary develops into 40 glomerular loops Glomerular loop consists of different cell types supported on a specialized basement membrane Cells are epithelial, endothelilial & mesangial Mesangial cells are rich in myofilaments & contract in response to a variety of stimuli e.g. angiotensin vasopressin etc & reduces GFR by reducing the available filtration surface

5. Proximal Tubule 15 cms long lined by cuboidal / columnar cells with luminal brush border Most metabolically active part of nephron Reabsorb 60-80% of glomerular filtrate volume , including 70% of Na + & Cl - , most of K + , glucose, HCO3 - , PO4 -- , SO4 -- Secrete 90% of H + excreted by kidney

6. Loop of Henle Situated deep in renal medulla Consists of – Thin descending limb- lined by flat cells, freely permeable for water Thick ascending limb- lined by cuboidal / columnar, impermeable for water

7. Distal Tubule & Collecting Duct DT 5 cms long CD 20 cms long with epithelium made up of principal cells (P cells) & intercalated cells (I cells) P cells involved in Na + & ADH mediated water reabsoption I cells concerned with acid secretion & HCO3 - transport

8. Kidney functions Excretion of nitrogenous waste ( urea, creatinine, uric acid) Water and electrolyte balance Acid base homeostasis Endocrine functions ( erythropoietin,renin , Vitamin D activation)

9. Important Renal Functions Filtration- Preparation of an ultra filtrate Reabsorption - Glucose, amino acids, electrolytes & protein Homeostasis - ECF vol, acid- base balance, BP & electrolyte balance Metabolic - anabolic: glutathione, glycogenesis & NH3 catabolic: hormones (renin), cytokines Endocrine - Erythropoietin, Vitamin D

10. Excretory & Reabsorptive Function Formation of urine Ultra filtrate 100 microlitre/ nephron 170- 200 L/ day ultrafiltrate 0.4- 2L /day urine formation Reabsorption Enzymatic transporters Ion transporters

11. Classification OF RFT Serum markers- Urea, creatinine, uric acid, electrolytes Clearance tests- creatinine clearance, eGFR Urine analysis- proteinuria, microalbuminuria, 24 hour urine Newer markers- cystatin C

12. CLASSIFIATION OF RFT Glomerular function evaluation Clearance test – Inulin clearance, EDTA clearance,cystatin c clearance,creatinine clearance, urea clearance Calculation of creatinine clearance from predictive equations Blood biochemistry- sr. creatinine, BUN, BUN/Sr creat ratio

13. Test to evaluate tubular function PCT- glycosuria, phosphaturia,uricosuria Generalized aminoaciduria, tubular proteinuria,fractional sodium excretion DCT- specific gravity and osmolality of urine, Water deprivation test, ammonium chloride loading test

14. Sample requisite Sample type – Serum Container- plain vacutainer ( red cap) or serum separator tube Volume- 2 to 3ml Preparation- fasting sample (8-12hrs)

15. Blood biochemistry Blood urea nitrogen Proteins >> Amino acids >> ammonia >> urea>> excretion in urine MW of urea 60 and 28 g of nitrogen are present in gram mole of urea Urea= BUN X (60/28) Level of blood urea affected by number of non renal factors- so utility is limited

16. causes Azotemia – increase in blood level of urea Increased BUN pre renal causes- shock, CCF, salt and water deprivation Renal – impairment of renal function Postrenal - high protein diet ,increased protein catabolism – (trauma, burns,fever ) GI hemorrhage or tissue hematoma

17. Method of estimation Urease –Glutamate dehydrogenase UV kinetic method sample tube Is placed in analyzer’s sample rack Analyzer uses a sample probe with a disposable tip to aspirate (5-10 uL ) Analyzer dispenses sample into cuvette and adds reagent 1 and reagent 2 depending on kit

18. Reaction sequence R1- Buffer ( phosphate ), urease enzyme R2- alpha –Ketoglutarate, NADH ,Glutamate dehydrogenase, stabilizers 1 ) urease reaction- U rea + H2 O>>( urease)>> 2NH4 + CO2 2 )Coupled GLDH reaction N ADH >>> NAD accompanied by absorbance at 340nm

19. Optical measurement The cuvette is carried to optical measuring unit A UV lamp/LED source passes light at 340nm through the reaction mixture Rate of decrease in absorbance is proportional to the concentration of urea F inal BUN concentration is calculated and expressed in mg/dl N ormal 15-40 mg/dl

20. error cause Effect on test troubleshooting Wrong sample type Using fluoride oxalate tube ( inhibits urease) Falsely low BUN Use serum or lithium heparin plasma only hemolysis Poor phlebotomy, delayed separation Release of intracellular enzymes and ammonia Reject grossly hemolyzed samples Delayed separation Plasma/ serum not separated from cells within 2hours Cellular metabolism increases ammonia lead to falsely high BUN Centrifuge and separate quickly Improper storage Sample kept at room temp for >4hrs Bacterial urease can increase BUN artificially store at 2-8 c if delay, freeze for longer storage contamination Ammonia contamination (environment ,anticoagulants) Falsely high Use clean,dry tubes and proper anticoagulant Patient related High protein meal before collection,dehydration Physiological high BUN misinterpretated as pathological Advice fasting sample whenever possible

21. Error Cause Effect troubleshooting Reagent deterioration Expired, improperly stored, NADH instability Inaccurate /low absorbance Check reagent integrity, use fresh kits Temp variations Reaction not at 37c Slower or unstable enzyme activity Ensure analyzers incubator is calibrated Interfering substances Hb, bilirubin ,lipemia Spectral interference at 340nm ,high value Run serum blank, use sample pretreatment Carryover contamination Probe not washed properly between samples Cross contamination lead to falsely high Regular probe wash cycles, maintenance Linearity exceeded Very high BUN Reaction saturated, falsely low Analyzer flags, auto dilute and rerun Air bubbles in cuvette Pipetting error Erratic absorbance readings Ensure probe/cuvette system is bubble free Instrument calibration drift Calibration not updated Random high/low values Recalibrate with fresh standards run QC

22. Serum creatinine Principle – jaffe method , enzymatic creatinine method Creatinase hydrolyzes creatinine to creatine Creatinase hydrolyzes creatine >>> sarcosine + urea Sarcosine oxidase = sarcosine >>> glycine + formaldehyde +H2O2

23. Peroxidase reacts with H2O2 + Chromogen>>> colored compound Intensity of color is directly proportional to serum creatinine concentration Analyzer measures absorbance photometrically (usually at 510-550nm ) End point method

24. Serum creatinine Creatinine is nitrogenous product formed in muscle from creatinine phosphate. Endogenous production of creatinine is proportional to muscle mass and body wt. Serum creatinine is more specific indicator of renal function Because of significant kidney reserve , increase in serum creatinine level in blood does not occur until about 50% of kidney function is lost.

25. Causes Increased level Prerenal,renal and post renal azotemia Large amount of dietary cooked meat Muscular body Active acromegaly and gigantism Decreased level – female , vegetarian diet,malnutrition ,muscle wasting,increasing age Reference range- male – 0.7- 1.3 mg/dl female – 0.6- 1.1mg/dl

26. error cause effect troubleshooting Wrong sample tube EDTA,Citrate , fluoride(chelates creatinine or interference with enzymes False low creatinine Use serum/lithium heparin plasma only hemolysis Rough collection, small needle False increase creatinine Avoid hemolysis, proper phlebotomy Icterus Liver disease sample May cause false low absorbance Use bichromatic reading or enzymatic method lipemia Postprandial fatty sample Turbidity lead to high value Ultracentrifuge or serum blank Delayed separation Enzymatic degradation Altered values Separate serum within 1 hr Diet and drugs High meat intake, cephalosporins False increase creatinine Avoid sample post heavy meals

27. Error cause effect troubleshooting Reagent deterioration Expired or improperly stored reagents Low or high bias Use fresh reagent, store 2-8c Calibration error Improper calibration, expired standard Systemic bias Recalibrate analyzer Optical interference Bilirubin , Hb, lipids False increase Dual wavelength Carry over contamination Probe not wash properly Cross contamination Regular probe washing, maintenance

28. BUN/CREATININE RATIO Normal is 12:1 to 20 :1 Causes of increased BUN/Creatinine ratio =>20:1 Pre renal azotemia ( reduced renal perfusion ) High protein diet Increased protein catabolism GI hemorrhage

29. causes Increase of both BUN and serum creatinine with disproportionately greater increase of BUN Postrenal azotemia (obstruction to outflow of urine Causes of decreased BUN/creatinine ratio <10:1 Acute tubular necrosis, low protein diet,starvation,severe liver disease

30. Uric acid Principle – uricase method Uric acid >>> alantoin + H2O2 Clinical uses – gout, tumor lysis syndrome, renal stones Reference value- 3.5- 7mg/dl men 2.5- 6 mg/dl woman

31. Error Cause Effect troubleshooting Wrong sample tube EDTA,Citrate,fluoride inhibit uricase False decrease uric acid Use serum or lithium heparin plasma hemolysis RBC contents interfere with color reaction False increase in uric acid Avoid hemolyzed samples Lipemia Turbid sample scatters light false increase Use serum blank/centrifuge Alcohol intake Increases uric acid transiently False increase Take history,repeat sample fasting Delayed separation RBC metabolism alters levels False decrease Separate serum within 1 hour Diet High purine meal ( meat,seafood ) Transient increase In uric acid Collect fasting sample

32. E rror Cause Effect troubleshooting Reagent deterioration Improper storage of uricase or chromogen Low values Store at 2-8c, use fresh reagent Calibration error Wrong std or curve drift Systemic error (all increase or decrease) Recalibrate with std Optical interference Bilirubin, hb , lipids Falsely low or high Use bichromatic readings/blank Enzyme inhibition Drugs (allopurinol, ascorbic acid) inhibit uricase Falsely low uric acid Take drug history,interpret carefully QC failure Analyzer drift Random error Run internal QC daily, EQAS

33. Electrolytes Na+, K+, Cl-, HCO3 Ion selective electrode – direct or indirect Flame photometry Clinical use – detect imbalances in renal disease

34. Calcium, phosphate, magnesium Calcium – Arsenazo III method/ o- cresolphthalein Phosphate – molybdate UV method Magnesium – calmagite method Imbalances – CKD- MBD(mineral bone disorder)

35. Error cause effect troubleshooting Wrong sample EDTA,Citrate , oxalate tubes chelates ca and Mg Falsely low ca/mg Use serum or lithium heparin plasma hemolysis RBC rupture releases Mg and phosphate False increase Mg and Phosphate Avoid hemolyzed samples Lipemia / icterus Turbidity interferes with absorbance False low/high depending on method Use serum blank/ultracentrifuge Prolonged tourniquet hemoconcentration False increase Ca and Mg Release tourniquet within 1 min Delay in separation Glycolysis decreases phosphate False decrease phosphate Separate plasma within 1 hr Fasting status Postprandial increase phosphate variability Collect fasting sample

36. Error Cause Effect troubleshooting Reagent instability Dye degradation ( calmagnite , arsenazo ) Erratic values Store at 2-8c ,use freshb reagents Protein interference Ca binds proteins >> turbidity False increase Ca Use serum blank, bichromatic reading Drugs interference Diuretics, antacids affect Ca/Mg Falsely high /low Take drug history ISE errors Improper calibration,electrode fouling Drift in values Regular electrode maintainence ,2 –point calibration QC failure Analyzer drift Random/systematic error Run 2 levels of QC daily

37. Cystatin C Low mol wt protein Marker of GFR ,independent of muscle mass Enzymatic immunoassays available Useful in pediatric and elderly patients

38. Measurement of GFR Glomeruli serve as filter of blood Lab evaluation of Glomerular function aimed at determining no. of functioning glomeruli , usually determined by GFR Measurement GFR is usually consider as Glomerular function test Measurement of GFR can be made by determining the rate of clearance of substance filtered by glomeruli

39. Clearance Def : Amount of plasma that has been completely cleared of a substance in a unit time. Or Volume of Blood or Plasma which contains the amount of the substance which is excreted in the urine in one minute

40. Characteristic of substance used to measure GFR Freely filtered Neither reabsorbed nor secreted by tubule Not metabolized Not stored in kidney Not toxic No effect on filtration rate Preferably easy to measure in plasma & urine

41. Substances used to measure GFR Exogenous Inuline –ideal but used for research only Cr EDTA Iohexol Endogenous Urea Creatinine RBP β 2 – Microglobulin Cystatin C -require an immunoassay

42. Clearance Formula : U x V B U = Conc in urine ( mg/dl) V = Volume of urine ( ml/min) B = Conc in blood (mg/min) Unit of Clearance = ml/min)

43. Urea clearance test Introduced by Van Slyke Urea clearance is defined as no. of ml of blood which contain the urea excreted in minute by kidney Normal urea clearance 75 ml/min at the rate of excretion of urine > 2 ml/min With urine vol of 1 ml/min == 54ml /min The of clearance with decrease of urine vol was found to proportional to square root of V

44. Procedure of urea clearance Best carried out in morning Pt allowed to have breakfast as usual Carry out test over two hr between 9am to 1200 noon At 9 am give a cup of water to pt 0905 pt empty bladder completely. Urine discarded 1005bladder again emptied completely. Urine specimen 1 1010 specimen of blood taken 1105 bladder emptied completely. Urine specimen 2

45. Calculation Two samples are taken to check reliability of collection Sufficient water given before test to promote a reasonable flow of urine Interpretation Max clearance 60 to 95 ml /min Std (V=1)clearance 40 to 65 ml/ min Dividing line between normal & abnormal is 70% 0f average normal

46. % of average normal clearance Renal function >70 Normal 70-40 Mild deficit 40-20 Moderate deficit 20-5 Severe deficit <5 Uremic coma

47. Creatinine clearance test Most widely used test for estimating GFR & measured as index of it It is filtered by glomeruli & not reabsorbed by tubule but secreted by PCT At normal plasma creatinine level only about 10% of urine creatinine is derived from secretion Administration of 1200 mg cimetidine to prevent almost all secretion & make cretinine clearance a good measure of GFR

48. Method No spl precaution is necessary Collect 24 hr specimen of urine with preservative 10 ml conc Hcl Take 10 ml blood without anticoagulant Estimate serum & urine creatinine & calculate clearance Normal range 120+20 ml/min

49. Because of accurate measurement of urine is very difficult in clinical practice, actual measurement of Cr clearance is decreasing in clinical practice S/P creatinine can be measured more accurately & can be used to predict Cr clearance (GFR) using algorithms Cr clearance ( ml/min/1.73 m2 )= (140- age in yrs) x 2.12 x Wt (kg) x K S creatinine x BSA K= 0.85 for women & 1.00 for men BSA= body surface area

50. Estimation of GFR by Equations GFR depend on age,sex and body wt and serum creatinine levels National kidney foundation formula eGFR = 175 x ( sr.creatinine in mg/dl) x age in year x 0.742 if female

51. Case 1 52 yr old male Complaints- fatigue, ankle swelling,nocturia , reduced urine output for 1 week Past history- hypertension since 12 yrs ,irregular medication No history of gout or kidney ds

52. investigations Serum creatinine- 3.8 mg/dL( increased) Blood urea – 90 mg/dl (Increased) Uric acid – 6.0 mg/dl (N) Sodium – 132 mmol/L(decreased) Potassium – 5.8 mmol/l (increased) Calcium 8.1mg/dl (decreased) Phosphate 5.6 mg/dl (increased ) eGFR 22 ml/min/1.73m (CKD stage 4)

53. Interpretation Advanced chronic kidney disease with low eGFR Electrolyte imbalance – hyponatremia,hyperkalemia , ,hypocalcemia, hyperphosphatemia (suggests CKD-MBD ) Uric acid normal- rules out acute urate nephropathy

54. Possible errors Pre analytical Was fasting sample collected? (post meal phosphate falsely increase) Was sample transported quickly ? (delay>> decrease phosphate) Hemolysis ? ( false increase potassium , phosphate,Mg )

55. Analytical Lipemic/icteric interference? Was creatinine measured by Jaffe method? (possible false increase in icterus,ketoacidosis ,high glucose) Were electrolytes measured by ISE? (protein/lipid interference lead to pseudohyponatremia

56. troubleshooting Recheck potassium with non-hemolyzed sample (to confirm true hyperkalemia) Confirm creatinine with enzymatic method ( to rule out Jaffe interference) Add interpretative comment – CKD stage4 with electrolyte imbalance , suggest nephrology referral and management for hyperkalemia and bone mineral ds Communicate urgent potassium result (>5.5 mmol/L)

57. Case 2 50 year old male with CLL Lab result – Potassium reported as 7.0 mmol/l (critical high) Clinical picture- patient asymptomatic, normal ECG

58. Error suspected- WBC count > 200,000/ uL . Fragile leukocytes rupture during clotting Serum potassium falsely increase (in vitro release of K+) Troubleshooting – Repeat potassium in heparinized plasma >> result 4.5 mmol/L (normal) Comment- pseudohyperkalemia due to leukocytosis interpret cautiously

59. Case 3 42 yr old female with obstructive jaundice Lab result – serum creatinine reported as 0.22 mg/dl ( falsely low) Error cause – Jaffe method >> bilirubin interferes ( negative bias) True creatinine likely normal.

60. troubleshooting Re-run using enzymatic creatinine method. Add footnote : creatinine by Jaffe method may be underestimated in icteric samples

61. Case 3 35 yr old male, sample collected post heavy meal + alcohol Lab result : uric acid -10.5mg/dL (suggestive of Gout) Error cause : dietary effect (purine rich meal) + alcohol intake increases uric acid transiently. Also lipemia caused turbidity interfering with uricase method

62. Troubleshooting Repeat in fasting state after 12 hrs >> result 6.5mg/dL (normal) Use serum blank if lipemia present

63. Case 4 65 yr old diabetic male Lab result : urea = 20 mg/dL (low normal) Creatinine = 8.0 mg/dL ( very high) Error cause Analytical interference in Jaffe method (drugs; cephalosporins, ketoacids) Biological inconsistent (true renal failure would increase both)

64. Troubleshooting Re run creatinine with enzymatic method >> result 1.0mg/dL (normal) Report corrected , clinician notified.

65. Signs & Symptoms of Renal Diseases Symptoms of uremia Disorders of micturition Disorders of urine volume Alteration in urinary composition Pain Edema

66. Routine Urinalysis Specimen Evaluation- Proper labeling Morning voided urine Gross/physical examination Chemical screening Sediment examination

67. Gross / Physical Examination Colour Urochrome, urobilin , uroerythrin Hydration status & urine concentration Red – hematuria, haemoglobinuria, drugs, dyes, beets Yellow brown/ green brown- bilirubin Orange red/orange brown- urobilin Dark brown/black- rhabdomyolysis, HGA, melanin

68. TUBULAR FUNCTION TEST Tests for proximal tubular function- Measurement of phosphate reabsorption Urinary amino acid chromatography Detection of glycosuria Tests for distal tubular function- Tests of urinary concentration & dilution Tests for urinary acidification

69. Test for conc. Ability 6pm- Meal with 200ml water given Empty bladder before retiring to rest Discard this urine Next morning 8am – empty bladder . Specimen 1

70. 9am - again empty bladder. Specimen 2 10 am- collect a further specimen . Specimen 3 Measure sp gr of all three specimen by urinometer Interpretation Sp gr of at least one specimen should exceed 1.022 A value < 1.022 indicate impaired renal function

71. Clinical Significance To differentiate between central & nephrogenic Diabetes Insipidus

72. Dilution Test No water after mid night Empty bladder at 7am 1200ml of water given over 30 minutes Empty bladder hourly for next 4 hours Volume & sp gr of each sample is measured Specific gr of at least 1 sample should be < 1003 With renal dysfunction smaller volume of urine is passed & sp gr of 1003 is not achieved

73. Osmolality Indicates no. of solute per unit of solution With normal fluid intake –urine osmolality is 500-800 m Osm/ kg of water Freezing point depression method is commonly employed to estimate osmolality by an instrument called osmometer

74. Test for urinary acidification 0.1gm/kg of NH4Cl/ CaCl2 is given over a period of 1hr Collect urine hourly for next 8hrs and estimate pH of urine and the NH 3 present pH of Urine sample should be < 5.5 in normal subject In DTA urinary pH remains >6.5

75. PG viva questions Difference between osmolality and osmolarity? Structure and size of glomerular filtration barrier Cystic diseases of kidney Causes of high level of creatinine Difference between nephrotic and nephritic syndrome

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