1. Clinical Chemistry Renal Assessment

2. Creatinine Metabolic product cleared entirely by glomerular filtration Not reabsorped In order to see increased creatinine in serum, 50% kidney function is lost Correlates with muscle mass – Male values higher than females

3. Creatinine: serum Increased Urinary tract obstruction Decreased glomerular filtration – Chronic nephritis Decreased Muscular dustrophy

4. Creatinine: Urine Increased Muscle Disease Decreased Kidney Disease

5. Creatinine: Methodology Jaffe reaction – basic reaction for creatinine – Kinetic Principle: Protein-free filtrate(serum/urine) mixed with alkaline picrate solution forms a red “tautomer” of creatinine picrate which absorbs light at 520 nm, proportional to the amount of creatinine present Issues – Subject to interferences from cephalosporins and alpha-keto acids – Enzymatic New technology involving coupled reactions

6. Reference Range: Creatinine Serum 0.5-1.5mg/dL Urine 0.8-2.0gm/ 24 hour

7. Clearance Measurements Evaluation of renal function relies on waste product measurement, specifically the urea and creatinine Renal failure must be severe, where only 20- 20% of the nephron is functioning before concentrations of the waste products increase in the blood The rate that creatinine and urea are cleared from the body is termed clearance

8. Clearance Definition – Volume of plasma from which a measured amount of substance can be completely eliminated into urine per unit of time – Expressed in milliliters per minute Function – Estimate the rate of glomerular filtration

9. Creatinine Clearance Used to determine GFR ( glomerular filtration rate) Most sensitive measure of kidney function Mathematical derivation taking into effect the serum creatinine concentration to the urine creatinine concentration over a 24- hour period

10. Creatinine Clearance Specimen requirements 24-hour urine – Keep refrigerated Serum/Plasma – Collected during 24-hour urine collection Instructions for urine collection Empty bladder, discard urine, note exact time Collect, save and pool all urine produced in the next 24-hours. Exactly 24 hours from start time, empty bladder and add this sample to the collection

11. Creatinine clearance - Procedure – Determine creatinine level on serum/plasma - in mg/dL – Determine creatinine level on 24 hour urine measure 24 hr. urine vol. in mL, take a aliquot make a dilution (usually X 200) run procedure as for serum multiply results X dilution factor – Plug results into formula

12. Formula U cr (mg/dL) X V Ur (mL/24 hour) X 1.73 P Cr (mg/dL) X 1440 minutes/ 24 hours A U cr= urine creatinine P cr= serum creatinine 1.73= normalization factor for body surface area in square meters A= actual body surface area

13. Nomogram 1.Left side, find patient’s height( in feet or centimeters) 2.On right side, find patient’s weight (lbs or kg) 3.Using a straight edge draw a line through the points located 4.Read the surface area in square meters, on the middle line

14. Reference ranges Males – 97 mL/min- 137 mL/min Females – 88mL/miin-128 ml/min

15. Creatinine Clearance Exercise Female Patient: 5'6“ & 130 lbs. – Urine Creatinine – 98 mg/dL – Serum Creatinine – 0.9 mg/dL – 24 Hour Urine Volume – 1,200 mL – Set up calculation

16. Microalbumin Important in management of diabetes mellitus Perform an albumin/creatinine ratio

17. Urinalysis In-depth renal assessment Refer to UA notes for review of individual tests

18. Other Tests To Monitor Kidneys Measurement of the non-protein nitrogen substances – BUN – Uric Acid

19. BUN Blood urea nitrogen – Urea is the nitrogenous end-produce of protein / AA metabolism. – Urea is formed in the liver when ammonia (NH 3 ) is removed and combined with CO 2. – Most widely used screening test of kidney function

20. Blood urea nitrogen (BUN) Serum normal values – 5.0-20.0 mg/dL Decreased concentration seen late in pregnancy and in protein starvation. If concentration exceeds 20.0 mg/dL, term azotemia applies. – Azotemia – nitrogen in the blood not always kidney’s fault, excessive hemorrhage, shock, and other reasons does not imply clinical illness, but can progress to symptomatic illness.

21. BUN: Methodology Kjeldahl – a classical method for determining urea concentration by measuring the amount of nitrogen present Berthelot reaction - Good manual method - that measures ammonia – Uses an enzyme (urease – from Jack Bean meal) to split off the ammonia Diacetyl monoxide ( or monoxime) – Popular method but not well suited for manual methods because ➵ Uses strong acids and oxidizing chemicals

22. Disease correlations: BUN Prerenal  BUN Prerenal  BUN ( Not related to renal function ) – Low Blood Pressure ( CHF, Shock, hemorrhage, dehydration ) – Decreased blood flow to kidney = No filtration – Increased dietary protein or protein catabolism Prerenal  BUN Prerenal  BUN ( Not related to renal function ) – Decreased dietary protein – Increased protein synthesis ( Pregnant women, children )

23. Disease Correlations: BUN Renal causes of  BUN Renal causes of  BUN Renal disease with decreased glomerular filtration – Glomerular nephritis – Renal failure from Diabetes Mellitus Post renal causes of  BUN ( not related to renal function ) Post renal causes of  BUN ( not related to renal function ) Obstruction of urine flow – Kidney stones – Bladder or prostate tumors – UTIs

24. BUN / Creatinine Ratio – Normal BUN / Creatinine ratio is 10 – 20 to 1 – Pre-renal increased BUN / Creat ratio – BUN is more susceptible to non-renal factors – Post-renal – Post-renal increased ratio BUN / Creat ratio – Both BUN and Creat are elevated – Renal – Renal decreased BUN / Creat ratio – Low dietary protein or severe liver disease

25. Uric acid Source – Final breakdown product of nucleic acid catabolism - from both the food we eat, and breakdown of body cells. – Uric acid is filtered by the glomerulus ( but 98 – 100 % reabsorbed ) Increased levels – Not a primary test for kidney function - useful as a confirmatory or back - up test. – *Most useful for diagnosis and monitoring gout – Also seen during toxemia of pregnancy

26. Uric acid diseases Gout – Increased plasma uric acid – Painful uric acid crystals in joints – Usually in older males ( > 30 years-old ) – Associated with alcohol consumption – Uric acid may also form kidney stones Other causes of increased uric acid – Leukemias and lymphomas (  DNA catabolism ) – Megaloblastic anemias (  DNA catabolism ) – Renal disease ( but not very specific )

27. Uric Acid: Methodology 1.Phosphotungstic Acid Reduction — This is the classical chemical method for uric acid determination. In this reaction, urate reduces phosphotungstic acid to a blue phosphotungstate complex, which is measured spectrophotometrically. 2.Uricase Method — An added enzyme, uricase, catalyzes the oxidation of urate to allantoin, H2O2, and CO2. The serum urate / uric acid may be determined by measuring the absorbance before and after treatment with uricase. (Uricase breaks down uric acid.) 3.ACA — Uric acid, which absorbs light at 293 nm, is converted by uricase to allantoin, which is nonabsorbing at 293 nm. – Uric acid + 2H2O + O2 Uricase > Allantoin + H2O2 + CO2 (Absorbs at 293 nm) (Nonabsorbing at 293 nm)

28. Uric Acid Normal values – Men3.5 - 7.5 mg/dL – Women 2.5 - 6.5 mg/dL

29. Laboratory Evaluation of Renal Function

30. Proteinuria Case 1 A 20 year old patient is referred to you for,he has been diabetic for 6 years,he was told to have some kidney problem by his MD.He wants to know the cause of renal dysfunction. GPE:BP 145/90,otherwise exam is normal How would you proceed ? BUN 15mg/dl, creatinine 1.0mg/dl,U/A shows SG 1.024,trace protein,a few hyaline casts What test would you order next ? 24h protein collection, U protein/U creatinine ratio or both?

31. Case 1 continued Urine protein /Urine creatinine returns 15mg/150mg ratio(<0.1) Does this patient have abnormal proteinuria ? Patient wants to know if he has microalbuminuria,you order urine micro albumin result is :60mg micro albumin /gm creatinine. Is this abnormal, does this patient have diabetic nephropathy?

32. Urine Protein:Categories of persistent proteinuria Overflow: Capacity to reabsorb normally filtered protein in proximal tubules over whelmed due to overproduction:e.g.light chains,hemoglobinuria and myoglobinuria Tubular proteinuria: Decreased reabsorption of filtered proteins by tubules due to tubulointerstitial damage ; usually <2 gm Glomerular proteinuria: Microalbuminuria to overt proteinuria usually>3.5 gm

33. Screening for Urine protein Dipstick: Gives green color, does not check for light chains Negative – 10 mg/dl Trace – 15-25 mg/dl 1-2+ – 30-100 mg/dl 3+ – 300 mg/dl nSulfosalicylic acid: white precipitate

34. Urine protein :Quantitative measurement n 24 hour collection of urine for protein normal excretion is <150 mg/24 hour n Spot urine protein/urine creatinine ratio : (as 24 h urine creatinine excretion is a function of muscle mass i.e. 15 mg/kg for females and 20mg/kg for males ) a normal ratio is 150/1500 or 3 indicates nephrotic range proteinuria n Case 1 has normal urine protein excretion, trace protein on u/a is due to highly concentrated urine,pt may still have microalbuminuria

35. Microalbuminuria Urine albumin excretion below detection by regular dipstick First clinical sign of diabetic nephropathy Incidence increases with the duration of diabetes and may be present at the diagnosis of NIDDM Transient albuminuria may occur with fever,infection,exercise,decompensated CHF Associated with poor glycemic control and elevated BP

36. Detection of Micro albuminuria: 24 hour urine collection Normal urine protein excretion : <150mg (20% of this is albumin) Therefore, normal urinary albumin excretion is < 30 mg/day Microalbuminuria :urinary albumin excretion 30-300 mg/day

37. Microalbuminuria :Detection by Spot Urine Albumin to Urine Creatinine ratio Easier than cumbersome 24 hr.collection If we assume daily creatinine excretion to be 1000 mg and normal urine albumin excretion <30 mg; albumin / creatinine ratio should be less than 0.03 or 30mg/g creatinine Thus case 1 has micro albuminuria which is likely due to diabetic nephropathy.How would you manage him now?

38. Why and When to Screen Patients for Microalbuminuria ? BP control with Ace_I and ARB’s have been known to reduce microalbuminuria and delay the progression of kidney disease in diabetics IDDM patients should be screened yearly,beginning 5 years after the onset of disease Patients with NIDDM should be screened at presentation

39. Proteinuria Case 2 A70 year- old male is referred for chronic azotemia PMH: unremarkable GPE: BP120/60, LE edema Labs: U/A SG 1.010 pH 6.0, protein neg, glucose 2+, Uprotein /U creatinine ratio 4 BUN 30mg/dl creat.3.0, Blood Sugar 78mg/dl albumin 2.8, Hb 10 gm What other tests would you order to diagnose cause of his renal dysfunction ? UPEP,why?

40. Clinical Assessment of Renal Function: Glomerular Filtration Rate(GFR) Parameters used Blood urea nitrogen Serum creatinine Endogenous creatinine clearance

41. Case 3 Azotemia A 55 year old diabetic female is admitted with intractable vomiting and low urine output Exam: BP 120/60 with postural hypotension Labs: BUN 60, Creat. 2.0 mg/dl ( baseline 1.0mg/dl), Hb 16gm,U/A: SG 1.020, sediment: hyaline casts,U Na : 10 mmol/L,UOsm: 600 mosm/kg,Ucreat.150mg/dl,Fe Na < 0.5 Q.What is the cause of her high BUN to creatinine ratio and her renal failure? What are the other causes of high BUN to creatinine ratio

42. Blood Urea Nitrogen (BUN) Catabolism of aminoacids generates NH 3 NH 2 2 NH 3 + CO 2 = C = 0 + H 2 O NH 2 Urea Mol wt : 60 BUN Mol wt. : 28 Normal BUN 10-20 mg/dl After filtration › 50% is reabsorbed by the tubule BUN level is related to: Renal function, protein intake, and liver function

43. Creatinine Formed at a constant rate by dehydration of muscle creatine Normally 1–2% of muscle creatine is broken into creatinine Mol. Wt. 113 Creatinine is freely filtered by the glomerulii and is not reabsorbed 10–15% is secreted into proximal tubule

45. Creatinine Normal serum level 1–2 mg/dl 24 hour creatinine excretion 20 mg/kg/day for males 15 mg/kg/day for females Children, females, elderly, spinal cord injured have low serum and urine creatinine

46. BUN/Creatinine ratio 10:1 Normal Chronic renal failure

47. D/D in Case 3 with BUN Creatinine ratio >10:1 Decreased perfusion » Hypovolemia » Congestive heart failure Increased urea load – GI bleed – Glucocorticoids -Tetracycline – Hyper catabolic states – High Protein diet Obstructive uropathy Decreased muscle mass

48. Pathophysiology of Pre-renal Azotemia in Case 3 Decreased “Effective” Intravascular ADH Volume + Renal Hypoperfusionactivation of RAS Diminished GFRaldosterone Low urine volume and U sodium and high Uosmolality

49. Case 3 :Diabetic patient continued.. Vomiting stopped,BP improved and BUN/creat lowered to 35/1.8mg/dl. 24 hours later she developed UTI, trimethaprim/sulfamethoxazole was started Next day 24 hr urine output 800 mL Exam: Unremarkable BUN: 20 mg/dl Creat: 3.0 mg/dl U osm : 600 mosm/kg,U Na : 10 mom/l, FeNa: <1% Urine Sediment: Hyaline casts What is the cause of < 10: 1,BUN to creat ratio now?

50. BUN/Creatinine ratio ‹ 10:1 Decreased urea load Low protein diet Liver failure Inhibition of creatinine secretion Cimetidine Trimethoprim Probenecid – Increased removal: Dialysis

51. BUN/Creatinine ratio ‹ 10:1 Increased creatinine load Ingestion of cooked meat Rhabdomyolysis Interference with creatinine measurement Ketosis Cefoxitin Increased muscle mass Anabolic steroids Muscular development

52. Case 3 continued… 6 months later Pt was discharged with normal BUN and creatinine,6 months later she is admitted with vague abdominal pain, an US done shows 6 cm abdominal aortic aneurysm, she undergoes resection with cross-clamping of aorta for 2 hours. Post surgery she is oliguric (u/o less than 70ml in 8 hours).On exam well hydrated. U/A: SG 1.015,”Dirty brown sediment “U Na 40 mEq /L U osmolality 350 mOsm/l,Fe Na 2% What is your diagnosis after reviewing the lab data ? How would you manage?

53. “Dirty Brown” Sediment in ATN

54. Urinary Indices in Diagnosis of Acute Renal Failure Pre renal ATN Uosm(mosm/kgH 2 0) >500 <350 Urine sodium (mmol/l) 40 Urine/plasma urea nitrogen >8 <3 Urine/Plasma Creatinine >40 <20 Fractional Excretion of Sodium 1% Sediment normal “dirty brown”

55. Fractional Excretion of filtered Sodium(FeNa) FeNa= Amount of Na excreted Amount of Na filtered FeNa=UNa x Urine volume PNa x GFR FeNa = UNa x V PNa x[(UCr x V) /PCr] FeNa % =UNa x PCr X 100 PNa x UCr

56. Case 4 20 y/o male is seen at West point,on admission physical : wt 70Kg, BUN 10mg/dl, serum creatinine 1.0mg/dl, GFR was 100ml/min and he excreted 1500mg creatinine /day in the urine. 2 months later he develops acute glomerulonephritis with RBC and fatty casts.His serum creatinine increases to 2mg/dl and remains at 2mg/dl at 1 year follow up.Wt is 72kg What is his estimated GFR by Cockcroft and Gault formula and by serum creatinine? What would be the creatinine excretion now at 1 year ?

57. Concept of Clearance ? Measurement of GFR by Creatinine Clearance(Ccr) Urine is collected for 24 hours and plasma creatinine is measured the next day 1. Filtered creatinine = Excreted creatinine 2. GFR x Pcr = Ucr x Volume 3. GFR = Ucr. mg/dl x V ml Pcr.mg/dl Normal GFR = 100 ml/min GFR declines by 1 ml/min/year after age 40

58. GFR Estimation by Plasma Creatinine nCockcroft and Gault Formula* Calculated creatinine clearance = (140–age) x wt (kg) 72 X serum creatinine(mg/dl) For females, subtract 15% (or multiply by 0.85); for paraplegics multiply by 0.8, for quadriplegics, multiply by 0.6 Est GFR for this pt is.. (140-20)x70 72x2 *Applicable only when patient is in a steady state, not edematous and not obese

59. GFR Estimation by Plasma Creatinine(Pcr) In steady state Creatinine excretion = creatinine production=constant Creatinine excretion =Urine creatinine x Urine volume Filtered creatinine =GFR x Plasma creatinine As creatinine production is a function of muscle mass and remains constant Thus plasma creatinine values vary inversely with GFR GFR1/2 X 2 Pcr = GFR x Pcr = constant A rise in Pcr almost always represents a fall in GFR

60. In case 4,serum creatinine increased from from 1 to 2 mg/dl and remained at that level, his 24urine creatinine will remain the same Another example :70 kg man with serum creat. of 1 mg/dl and GFR of 100 ml/min was excreting 1500 mg creatinine/day,if you remove his one kidney, next day his GFR will be 50ml/min,urine creatinine excretion will be 750 mg /day.Over the next few days creatinine will accumulate in the blood and level will increase to 2 mg /dl and thus filtered and excreted amount will be the same

61. Summary How to evaluate a patient with renal disease How to interpret u/a,urine protein to creatinine ratios Interpretation of urea nitrogen and creatinine ratios Estimation and measurement of GFR& to see when a patient would need renal replacement therapy Interpret urine indices in evaluation of various causes of ARF

62. Reading of renal function

63. Glomerular filtration rate Clearance of inulin Clearance of creatinine:normal range Clearance of creatinine – Male:120±25 mL/min – Female:95±20mL/min – Infant:17 mL/min/1.73M 2

64. P[Inulin] × GFR = U[Inulin] × urine volume

65. Difference between inulin and creatinine Age effect: age >40y/o -> Ccr decrease 1mL/min/yr Urine Cr collection: – Age ≦ 60y/o:male: 20-25mg/kg; female:15- 20mg/kg – Age>60y/o:10mg/kg

66. Plasma Cr

67. Condition associated with PCr increased and not changed GFR Increased Cr production – Rhabdomyolysis – Meat Decreased Cr excretion – Cimetidine, triamterene, probenecid, amiloride, trimethoprim, spironolactone Measured bias – Endogeneous: ketone, ketoacids, glucose, bilirubin, urate, urea, fatty acid – Exogeneous: cephalosporines, 5-FU, phenylacetyl urea, acetoheximide

68. Estimate Ccr Cockcroft and Gault equation: CCr=[(140-age(yr)) ×BW(kg)] ÷[72×Pcr(mg/dl)] Female: above data×0.85 1/Pcr EsGFR(ml/min/1.73M 2 )=KL(body length, cm) ÷ Pcr – K LBW:0.33 NB-1yr:0.45 2yr-adolescent girls: 0.55 2yr-adolescent boys:0.77

70. BUN Reverse relationship with GFR, but many confounding factors Urea nitrogen can reabsorb paralleling with Na and H2O resorption BUN ： Pcr = 15-20:1

71. Urinalysis Urine sample: fresh (30-60min) 3000rpm, 3-5min -> suspension with pellet Color

73. Urine protein Daily urinary protein:150mg/day Microalbuminuria Detection: dipstick – Tetrabromophenol blue dye –albumin – Sulfosalicylic acid Sulfosalicylic acid

74. Protein(mg/dL)dipsticksulfosalicylic acid 00no turbid 1-10traceslight turbid 15-30+1turbid 40-100+2white without ppt 150-350+3white with ppt >500+4coarse ppt

75. Urine protein 24 hr daily protein loss Spot UTP/UCr

76. Urine pH and osmolality Normal range:4.5-8.0 How about alkalization urine? Urine sp. Gr. To estimate urine osmolality Plasma osmolality & urine osmolality

78. Urine Na excretion Urine excretion = intake Na amount Urine [Na]<20meq/L Urine [Na]>40meq/L Significance of %FENa

79. ARF with %FENa <1% Prerenal factor ATN – Non-oliguric ATN (10%) – Chronic prerenal disease- – Contrast media – Sepsis – Myoglobulinuria or hemoglobulinuria AGN or vasculitis Obstructive nephropathy

80. Urinary cast Hyaline castconc. Urine or diuretics Red cell castGN or vasculitis WBC castTIN, APN, GN Epithelial castATN, GN Fatty castGN with proteinuria, NS Granular castproteinuria, degenerative cells Waxy castCRF

81. Renal acidification evaluation Urinary pH: Net acid excretion: Net acid excretion Urinary anion gap: Urinary anion gap Acidification loading test: Acidification loading test

82. Urine pH Fresh urine Collect in the morning Must rule out UTI Many confounding factors- proton pump, electro-gradient of membrane, buffer conc., diet, et. al.

83. Net acid excretion Total acid excretion=titratable acid + NH 4 + Net acid excretion=total acid excretion – HCO 3 - excretion Titratable acid= buffer solution of H 3 PO 4 with urea nitrogen Def. of titratable acid excretion:the amount of NaOH(meq) to elevate UpH to 7.4

84. Urinary anion gap Total conc. Of anions = total conc. Of cations Na + +K + +NH 4 + +Ca +2 +Mg +2 =Cl - +H 2 PO 4 - +SO 4 - +organic anions Na + +K + +NH 4 + =Cl - +80 Urinary anion gap:Na + +K + -Cl -

85. Urinary acid loading tests Acid loading test Sodium sulfate infusion test or furosemide test Sodium sulfate infusion test or furosemide test Buffer loading test

86. Acid loading test NH4Cl 0.1g(1.9meq)/kg, po -> collection urine pH and net acid excretion for 2-8hr.(normal: UpH<5.5) CaCl 2 Arginine HCL Diamox test Normal urine CO 2 >80mmHg U-B[PCO 2 ]>30mmHg

87. Increase distal tubule Na conc. Test – for proton pump or voltage- dependent defect Furosemide test: 1mg/kg, collect urine pH, net acid excretion and U[k], po 5hr or iv 3hr – Reading:UpH increase in 1hr and then UpH down to 5.5 in future 2-4hrs; U[k] and acid increase 2 fold Sodium sulfate

88. Buffer loading test IV drip or 2-3ml/min NaHCO3 100-150mEq(total) till plasma NaHCO3 ≧ 30meq/L – Then check blood and urine pH, [HCO 3 - ], CO 2 – Calculate %FEHCO3 - 3-5% >15% – U-B[PCO 2 ] >20-30mmHg, when U[HCO3 - ] >100- 150meq/L