1. Kidney Function Testing
DR. Said Al ghora

2. An Introduction to the Urinary System
Produces urine
Transports urine towards bladder
Temporarily store urine
Conducts urine to exterior

3. The Function of Urinary System
Excretion & Elimination:
removal of organic wastes products from body fluids (urea, creatinine, uric acid)
Homeostatic regulation:
Water -Salt Balance
Acid - base Balance
Enocrine function:
Hormones B) C)

4. Kidney – basic data Urine excreted daily in adults: cca 1.5L
Kidney only ca 1% of total body weight, despite it
The renal blood flow= 20% of cardiac output
Plasma renal flow= PRF ca 600 mL/Min./1.73 M2
Reflects two processes
Ultrafiltration (GFR): 180 L/day
Reabsorption: >99% of the amount filtered

5. Renal threshold
Renal threshold of a substance is the concentration in blood beyond which it is excreted in urine
Renal threshold for glucose is 180mg/dL
Tubular maximum (Tm): maximum capacity of the kidneys to absorb a particular substance
Tm for glucose is 350 mg/min

6. Kidney Function
A plumbers view

7. How do you know it’s broken?
Decreased urine production
Clinical symptoms
Tests

8. Where can it break? Pre-renal Renal (intrarenal)
Post-renal (obstruction)

9. Causes of kidney functional disorders
Pre-renal e.g. decreased intravascular volum
Renal e.g. acute tubular necrosis
Postrenal e.g. ureteral obstruction
Oliguria is a significant finding in a patient. An example is provided by Case 3 in the Chem Path tutorials. The traditional classification of causes is into prerenal, renal and postrenal. Usually the cause of the oliguria is obvious and can be appropriately managed.

10. Signs and Symptoms of Renal Failure
Symptoms of Uraemia (nausea, vomiting, lethargy)
Disorders of Micturation (frequency, nocturia, dysuria)
Disorders of Urine volume (polyuria, oliguria, anuria)
Alterations in urine composition (haematuria, proteinuria, bacteriuria, leukocytouria, calculi)
Pain
Oedema (hypoalbuminaemia, salt and water retention)

11. Lab findings Rising creatinine and urea Rising potassium Decreasing Hb
Acidosis
Hyponatraemia
Hypocalcaemia
Other laboratory findings are progressive acidosis, hyperkalemia, hyponatremia, and anemia. Acidosis is ordinarily moderate, with a plasma HCO3 content of 15 to 20 mmol/L. Serum K concentration increases slowly, but when catabolism is markedly accelerated, it may rise by 1 to 2 mmol/L/day. Hyponatremia usually is moderate (serum Na, 125 to 135 mmol/L) and correlates with a surplus of water. Normochromic-normocytic anemia with an Hct of 25 to 30 % is typical.
Hypocalcemia is common and may be profound in patients with myoglobinuric ARF, apparently due to the combined effects of Ca deposition in necrotic muscle, reduced calcitriol production, and resistance of bone to parathyroid hormone (PTH). During recovery from ARF, hypercalcemia may supervene as renal calcitriol production increases, the bone becomes responsive to PTH, and Ca deposits are mobilized from damaged tissue.

12. Why Test Renal Function?
To identify renal dysfunction.
To diagnose renal disease.
To monitor disease progress.
To monitor response to treatment.
To assess changes in function that may impact on therapy (e.g. Digoxin, chemotherapy).

13. When should you assess renal function?
Older age
Family history of Chronic Kidney disease (CKD)
Decreased renal mass
Low birth weight
Diabetes Mellitus (DM)
Hypertension (HTN)
Autoimmune disease
Systemic infections
Urinary tract infections (UTI)
Nephrolithiasis
Obstruction to the lower urinary tract
Drug toxicity
Nephrolithiasis: The process of forming a kidney stone, a stone in the kidney

14. Biochemical Tests of Renal Function
Measurement of GFR
Clearance tests
Plasma creatinine
Urea, uric acid and β2-microglobulin
Renal tubular function tests
Osmolality measurements
Specific proteinurea
Glycouria
Aminoaciduria
Urinalysis
Appearance
Specific gravity and osmolality pH
osmolality
Glucose
Protein
Urinary sediments

15. Biochemical Tests of Renal Function
Measurement of GFR
Clearance tests
Plasma creatinine
Urea, uric acid and β2-microglobulin

16. Biochemical Tests of renal function
In acute and chronic renal failure, there is effectively a loss of function of whole nephrons
Filtration is essential to the formation of urine  tests of glomerular function are almost always required in the investigation and management of any patient with renal disease.
The most frequently used tests are those that assess either the GFR or the integrity of the glomerular filtration barrier.

17. Measurement of glomerular filtration rate
GFR can be estimated by measuring the urinary excretion of a substance that is completely filtered from the blood by the glomeruli and it is not secreted, reabsorbed or metabolized by the renal tubules.
Clearance is defined as the (hypothetical) quantity of blood or plasma completely cleared of a substance per unit of time.
Clearance of substances that are filtered exclusively or predominantly by the glomeruli but neither reabsorbed nor secreted by other regions of the nephron can be used to measure GFR.
Inulin
The Volume of blood from which inulin is cleared or completely removed in one minute is known as the inulin clearance and is equal to the GFR.
Measurement of inulin clearance requires the infusion of inulin into the blood and is not suitable for routine clinical use
V is not urine volume, it is urine flow rate

18. Biochemical Tests of Renal Function
Measurement of GFR
Clearance tests
Plasma creatinine
Urea, uric acid and β2-microglobulin

19. Creatinine
1 to 2% of muscle creatine spontaneously converts to creatinine daily and released into body fluids at a constant rate.
Endogenous creatinine produced is proportional to muscle mass, it is a function of total muscle mass the production varies with age and sex
Dietary fluctuations of creatinine intake cause only minor variation in daily creatinine excretion of the same person.
Creatinine released into body fluids at a constant rate and its plasma levels maintained within narrow limits  Creatinine clearance may be measured as an indicator of GFR.

20. Clinical Significance
Elevated Creatinine is found in
Impaired renal function
Very high protein diet
Vary large muscle mass: body builders, giants, acromegaly patients
Rhabdomyolysis/crush injury
Drugs:
Trimethoprim
Amiloride

21. Clinical Significance
For renal transplant patients, an increase in serum creatinine of 2 mg/L has been used as a criterion of establishing rejection.
In other persons a change in creatinine of 2 mg/L would represent a 20% loss in renal function.

22. Specimen One can analyze serum, plasma, or diluted urine.
The common anticoagulants (fluoride and heparin) do not cause interference, though heparin, which can be formulated as the ammonium salt, must be avoided in enzymatic methods that measure ammonia production.
Storage
7 days at 4-25oC
At least 3 months at -20oC

23. Specimen Urine should be diluted 1:100
Bacterial contamination has been found to falsely lower creatinine values measured using the Jaffé reaction.
The mechanism of this interference appears to be bacterial production of a substance that retards the rate of the Jaffé reaction.

24. Creatinine aminohydrolase Lactate dehydrogenase
Enzymatic Method
Creatinine + H2O Creatine
Creatine +ATP Creatine-P + ADP
ADP + Phosphoenolpyruvate ATP + Pyruvate
Pyruvate + NADH Lactate + NAD+
The difference in absorbance at fixed times during conversion is proportional to the concentration of creatinine in the sample
Creatinine aminohydrolase
Creatine Kinase
Pyruvate Kinase
Lactate dehydrogenase

25. Creatinine Clearance
Creatinine clearance is used to estimate the glomerular filtration rate (GFR).
One method of determining GFR from creatinine is to collect urine (usually for 24-hours) to determine the amount of creatinine that was removed from the blood over a given time interval.
Clearance is defined as the (hypothetical) quantity of blood or plasma completely cleared of a substance per unit of time.
The most frequently used clearance test is based on the measurement of creatinine.
Creatinine is chosen because it is freely filtered at the glomerulus and is not reabsorbed by the tubules.

26. However, a small amount of the creatinine (about 5%) in the final urine of healthy persons is derived from tubular secretion.
To do the test, one needs a precisely timed urine collection and a blood sample taken during the collection period.
Best results are obtained from a 24-h urine collection.
The test is initiated by having patients empty their bladder at the beginning of the timed period.
Urine is collected throughout the period, the bladder is again emptied at the end of the time period.

27. The 'clearance' of creatinine from plasma is directly related to the GFR if:
The urine volume is collected accurately
There are no ketones or heavy proteinuria present to interfere with the creatinine determination.
It should be noted that the GFR decline with age (to a greater extent in males than in females) and this must be taken into account when interpreting results.

28. Creatinine Clearance
Creatinine determinations are performed on both samples. The creatinine clearance is calculated from the following formula:
A person has a plasma creatinine concentration of 0.01 mg/ml and in 1 hour produces 60ml of urine with a creatinine concentration of 1.25 mg/mL.

29. Creatinine clearance (mL/min)= (UV)/P X 1.73/S
where U is urinary creatinine (mg/L), V is volume of urine (mL/min), P is plasma creatinine (mg/L), S is the calculated surface area of the patient, and is the surface area (m2) of a standard 70 kg person.
The range of creatinine clearance in healthy persons corrected to a surface area of 1.73 m2 is 90 to 120 mL/min.
At low filtration rates, the creatinine clearance does not parallel true glomerular filtration rate because a relatively large portion of the urine creatinine is secreted rather than filtered.

30. Effect of Muscle Mass on Serum Creatinine
Kidney
Output
Plasma
Pool
Content
Creatinine
Input
Normal
Muscle
Mass
Kidneys
Diseased
Increased
Reduced

31. Biochemical Tests of Renal Function
Measurement of GFR
Clearance tests
Plasma creatinine
Urea, uric acid and β2-microglobulin

32. Measurement of nonprotein nitrogen-containing compounds
Catabolism of proteins and nucleic acids results in formation of so called nonprotein nitrogenous compounds.
Protein
 Proteolysis, principally enzymatic
Amino acids
 Transamination and oxidative deamination
Ammonia
 Enzymatic synthesis in the “urea cycle”
Urea

33. Plasma Urea
Urea is the major nitrogen-containing metabolic product of protein catabolism in humans,
Its elimination in the urine represents the major route for nitrogen excretion.
More than 90% of urea is excreted through the kidneys, with losses through the GIT and skin
Urea is filtered freely by the glomeruli
Plasma urea concentration is often used as an index of renal glomerular function
Urea production is increased by a high protein intake and it is decreased in patients with a low protein intake or in patients with liver disease.

34. Plasma Urea
Many renal diseases with various glomerular, tubular, interstitial or vascular damage can cause an increase in plasma urea concentration.
The reference interval for serum urea of healthy adults is 5-39 mg/dl. Plasma concentrations also tend to be slightly higher in males than females. High protein diet causes significant increases in plasma urea concentrations and urinary excretion.
Measurement of plasma creatinine provides a more accurate assessment than urea because there are many factors that affect urea level.
Nonrenal factors can affect the urea level (normal adults is level 5-39 mg/dl) like:
Mild dehydration,
high protein diet,
increased protein catabolism, muscle wasting as in starvation,
reabsorption of blood proteins after a GIT haemorrhage,
treatment with cortisol or its synthetic analogous

35. Clinical Significance
States associated with elevated levels of urea in blood are referred to as uremia or azotemia.
Causes of urea plasma elevations:
Prerenal: renal hypoperfusion
Renal: acute tubular necrosis
Postrenal: obstruction of urinary flow

36. Increased dietary protein Severe tress: fever, etc Rhabdomyolysis
Increased protein catabolism:
Increased dietary protein
Severe tress: fever, etc
Rhabdomyolysis
Upper GI bleeding
Causes of urea plasma decrease
Decreased dietary protein
Increased protein synthesis ( Pregnant women , children )
severe liver disease
Overhydration (IV fluids)
Rhabdomyolysis is the rapid breakdown (lysis) of skeletal muscle (rhabdomyo) due to injury to muscle tissue. The muscle damage may be caused by physical (e.g., crush injury), chemical, or biological factors

37. Specimen
Serum and heparinized plasma can be used for the urease/GLDH methods.
Fluoride will inhibit the urease reaction; therefore methods employing urease cannot use serum preserved with fluoride.
Ammonium heparin also cannot be used as an anticoagulant for urease methods.
Stability in serum or plasma:
7 days at 4–8°C
1 year at -20°C
Because of urea’s susceptibility to bacterial degradation, serum and urine samples should be kept at 4° to 8° C until analysis.

38. Urease/GLDH Method
The method is optimized for 2-point kinetic measurement.
Decrease in absorbance at 340 nm is proportional to concentration of urea

39. BUN / Creatinine Ratio Normal BUN / Creatinine ratio is 10 – 20 to 1
Creatinine is another NPN
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
Renal decreased BUN / Creat ratio
Low dietary protein or severe liver disease

40. Uric acid
In human, uric acid is the major product of the catabolism of the purine nucleosides, adenosine and guanosine.
Purines are derived from catabolism of dietary nucleic acid (nucleated cells, like meat) and from degradation of endogenous nucleic acids.
Overproduction of uric acid may result from increased synthesis of purine precursors.
In humans, approximately 75% of uric acid excreted is lost in the urine; most of the reminder is secreted into the GIT

41. Uric acid
Renal handling of uric acid is complex and involves four sequential steps:
Glomerular filtration of virtually all the uric acid in capillary plasma entering the glomerulus.
Reabsorption in the proximal convoluted tubule of about 98 to 100% of filtered uric acid.
Subsequent secretion of uric acid into the lumen of the distal portion of the proximal tubule.
Further reabsorption in the distal tubule.
Hyperuricemia is defined by serum or plasma uric acid concentrations higher than 7.0 mg/dl (0.42mmol/L) in men or greater than 6.0 mg/dl (0.36mmol/L) in women

42. Greater-than-normal levels of uric acid (hyperuricemia) may be due to:
Alcoholism
Diabetes
Gout
Hypoparathyroidism
Lead poisoning
Leukemia
Nephrolithiasis
Renal failure
Toxemia of pregnancy
Purine-rich diet
Excessive exercise
Chemotherapy-related side effects

43. Lower-than-normal levels of uric acid may be due to:
Fanconi syndrome
Wilson's disease
Syndrome of inappropriate antidiuretic hormone (SIADH) secretion
Multiple Sclerosis
Low purine diet
Fanconi syndrome: is a disorder of the kidney tubes in which certain substances normally absorbed into the bloodstream by the kidneys are released into the urine instead
Wilson's disease or hepatolenticular degeneration is an autosomal recessive genetic disorder in which copper accumulates in tissues;
syndrome of inappropriate antidiuretic hormone hypersecretion (SIADH) is a condition mostly found in patients diagnosed with small cell carcinoma of the lung, pneumonia, brain tumors, head trauma, strokes, meningitis, and encephalitis. This is a syndrome characterized by excessive release of antidiuretic hormone
Multiple sclerosis (abbreviated MS, also known as disseminated sclerosis or encephalomyelitis disseminata) is an inflammatory disease in which the fatty myelin sheaths around the axons of the brain and spinal cord are damaged,

44. Gout
Gout is a kind of arthritis that occurs when uric acid builds up in the joints.
In Gout increased serum levels of uric acid lead to formation of monosodium urate crystals around the joints.
Acute gout is a painful condition that typically affects one joint.
Chronic gout is repeated episodes of pain and inflammation, which may involve more than one joint.

45. Specimen
Serum or plasma may be used; slight but insignificant positive bias (0.2 mg/dL) has been noted in plasma specimens as compared with serum.
Stability in serum / plasma:
6 months at -20°C
7 days at 4-8°C
3 days at 20-25°C

46. Enzymatic Colorimetric
Uricase
Uric acid + H2O + O Allantion + CO2 + H2O2
TBHBA + 4- Aminoantipyrine + 2H2O Quinoneimine + 3 H2O
Uric acid is oxidized to allantoin by uricase.
The generated hydrogen peroxide reacts with 4-aminophenazone/ESPT to quinoneimine.
POD

47. Acute Renal Failure Metabolic features: Retention of:
Urea & creatinine
Na & water
potassium with hyper-kalaemia
Acid with metabolic acidosis
Classification of Causes:
Pre-renal
reduced perfusion
Renal
inflammation
infiltration
toxicity
Post-renal
obstruction

48. Pre-renal versus intrinsic ARF

49. Reference Ranges BUN 10 - 20 mg / dl Creatinine 0.5 - 1.5 mg /dl
Uric Acid mg / dl
Creatinine Clearance ml / min
Ammonia ug / dl
BUN / Creat Ratio to 1

50. Case Study—Renal Failure

51. Case Study #1
HSL is a 63 year-old male with HTN, CAD, and hyperlipidemia; routine physical examination reveals asymptomatic hematuria.
What do you do?

52. Case Study 2
Ms. Garcia, a 54 yr old Hispanic female, dx with IDDM for 10 years. Admitted to the hospital with CHF, ESRD, altered lab values (K+=6.2; BUN 45, Creatinine 3.5; Hgb 6.2; Hct 18.6).
States that her “breathing keeps getting worse and worse, …can’t get around, bones break…decreased appetite but keep gaining weight…funny taste in mouth…blood sugar real high…legs jump at night”. States that a doctor told her she had “bad kidneys”.
1. What lab work is typically done?
2 If ESRD, what lab results would be anticipated?
3. What S&S of ESRD does Ms. Garcia display and WHY?
4. What conservative measures might have delayed ESRD? Discuss dietary, fluid, medications, etc…

53. Case Study
1. What lab work is typically done? Chem 12; H&H ; 24 hr creatinine clearance most helpful; know normals!
2 If ESRD, what lab results would be anticipated? ^ BUN, serum creatinine; low H&H; ^ K+, metabolic acidosis from kidneys inability to excrete acid load (especially NH3) and from defective reabsorption of bicarbonate.

54. Case Study
3. What S&S of ESRD does Ms. Garcia display and WHY? Metabolic Acidosis due to decreased ability to excrete acid metabolites therefore Kussmauls breathing in effort to blow off excess CO2. musculoskeletal system affected with renal osteodystrophy as GFR dec., kidney cannot eliminate phosphate; high phosphate binds with Ca which is drawn from bone; in CRF, kidneys do not metabolize vitamin D to its active form which is required for reabsorption of Ca from intestinal tract; weight gain from Na and water retention; uremic damage causing peripheral neuropathy..plus other symptoms including anemia from decreased production of erythropoietin; and HTN..

55. 4. What conservative measures might have delayed ESRD
4. What conservative measures might have delayed ESRD? Discuss dietary, fluid, medications, etc…Control HTN usually by Na and fluid restriction and antihypertensives, esp by ace inhibitors; restrict phosphate intake and use phosphate binders and give with meals; inc. Ca levels by adm. of active Vit D; monitor K levels; adm erythropoietin; avoid use of nephrotoxic drugs…such as aminoglycosides; protein restriction in diet.

56. Labs Urinalysis Biochemical test 2+ protein 3+ occult blood
>60 RBC per HPF
Biochemical test
K+ 4.0
BUN 19
sCr 1.5
Glucose 116

57. Repeat Labs One month ago… 6 months ago… One year ago…
sCr 1.7 (MDRD 45)
Glucose 88; HBA1c 5.9
Hct 40.4
LDL 92
CrCl 46 ml/min
24 hr Uprot mg
Renal U/S: normal
Modification of Diet in Renal Disease
One month ago…
sCr 1.6 (49)
6 months ago…
sCr 1.3 (59)
One year ago…
sCr 1.5 (54)

58. To summarize:
1. Use the Creatinine Clearance as the best estimate of GFR
2. Use the Serum Creatinine to follow renal function over time
3. Use the Creatinine Index to check the adequacy of a urine collection
4. Use the BUN to help assess GFR, volume status, and protein intake