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 Decreased Muscular dustrophy • Urinary tract obstruction • Decreased glomerular filtration • Chronic nephritis

4. Creatinine: Urine Increased Decreased Kidney Disease • Muscle 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 Urine 0.8-2.0gm/ 24 hour • 0.5-1.5mg/dL

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 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 • 24-hour urine • Keep refrigerated • Serum/Plasma • Collected during 24-hour urine 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 Ucr(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 Left side, find patient’s height( in feet or centimeters) On right side, find patient’s weight (lbs or kg) Using a straight edge draw a line through the points located 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 (NH3) is removed and combined with CO2. • 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( 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( Not related to renal function ) • Decreased dietary protein • Increased protein synthesis ( Pregnant women , children )

23. Disease Correlations: 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 ) • 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 increased ratio BUN / Creat ratio • Both BUN and Creat are elevated • Renaldecreased 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 • Men 3.5 - 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 • Sulfosalicylic acid: white precipitate

34. Urine protein :Quantitative measurement • 24 hour collection of urine for protein normal excretion is <150 mg/24 hour • 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 <0.1 . A ratio >3 indicates nephrotic range proteinuria • 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,UNa: 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 NH3 NH2 2 NH3 + CO2 = C = 0 + H2O NH2 • 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 Hypoperfusion activation of RAS Diminished GFR aldosterone 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 • Uosm: 600 mosm/kg ,UNa: 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