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UROGENITAL SYSTEM Biochemical Investigations of Urogenital Diseases MBBS Year-2 Lecture 26 September 2002 8:30 - 9:30 am Dr. Sidney Tam Hon Clinical Professor Department of Pathology The University of Hong Kong The KIDNEY has 3 major functions: • Regulation of water, electrolyte and acid-base balance • Excretion of waste products of intermediary metabolism, e.g., urea, creatinine, uric acid, phosphate, sulphate and organic acids • Production and elaboration of hormones, e.g., renin, erythropoietin, 1,25 dihydrocholecalciferol Renal Blood Flow (RBF) 1200 mL/min ~ 20% Cardiac Output Glomerular Filtrate Rate 18 L/day 125 mL/min (Filtration Fraction ~ 10%) Urine Formation ~ 1.5 L/day 1 mL/min ~ 99% H2O in glomerular ultrafiltrate reabsorbed by the kidney ~ 65% occurs in the proximal renal tubules accompanied by Na+ and Cl- reabsorption Obligatory Water Loss Urea, SO42-, PO42- & other waste products of metabolism: ~ 550 mOsm/day Maximal Urinary Concentration attainable: ~ 1400 mOsm/L Therefore: 550 mOsm/day Minimal Volume of Urine Water: ----------------------- 400 mL/day 1400 mOsm/L Azotaemia is inevitable with daily U.O. < 400 mL Assessment of Glomerular Function: • The capacity of the kidneys to filter plasma at the glomeruli can be assessed by measuring the Creatinine Clearance, which approximates to the GFR • Plasma Creatinine concentration is an insensitive index of renal function, as it may not appear to be elevated until the GFR has fallen by 50% • Once the plasma Creatinine concentration is elevated, changes in its level reflect changes in GFR Glomerular Filtration Rate (GFR): 1 • GFR is the volume of glomerular filtrate (an ultrafiltrate of plasma) produced by both kidneys (110 - 140 mL/min in adults). • Maintenance of a normal GFR depends on an adequate number of nephrons with intact glomerular function and a normal renal perfusion • Destruction of nephrons or reduced renal blood flow lead to a fall in GFR resulting in the retention of metabolic wastes, reflected by raised Creatinine and Urea levels in plasma Glomerular Filtration Rate (GFR): 2 • Measuring the concentration of a substance which is freely filtered by glomeruli but not reabsorbed nor secreted by the tubules (e.g., inulin) in a timed collection of urine and a concomitant plasma sample enables the estimation of GFR • Creatinine Creatinine (CrCl) approximates to GFR, and is commonly used in most clinical settings in lieu of other more accurate but cumbersome assessments of GFR, e.g., inulin clearance, 99Tc-DTPA scintigraphy, 51Cr-EDTA clearance. Clearance Clearance as an estimation of GFR: Creatinine Clearance (Cr Cl) is calculated as follows: Ucr x V Cr Cl = ---------------Pcr Ucr = Urine concentration of Creatinine Pcr = Plasma concentration Creatinine V = Volume of urine produced over a fixed period (usually a 24-hour collection) Creatinine Clearance is usually expressed in mL/min CREATININE CLEARANCE (Cr Cl) (Cockcroft & Gault, 1976) (140 - Age) x Body Weight Cr Cl ( mL / min ) = ----------------------------------------- x 0.85 (for female) 814 x Plasma [Creatinine] Age: Body Weight: Plasma Creatinine conc: Year Kg mmol / L Correlates with measured values of GFR provided that: i. ii. iii. Plasma [creatinine] is not within normal range Renal impairment is not severe No inhibition of tubular secretion of creatinine by medications Plasma CREATININE and UREA: • Plasma concentrations of Creatinine and Urea are used as convenient but rather insensitive measures of glomerular function - their levels may remain within reference ranges in the presence of a significant reduction of GFR CREATININE • Creatinine is an end product of muscle metabolism that is released into circulation at a relatively constant rate • Creatinine is freely filtered by glomeruli and not reabsorbed by renal tubules, but there is some tubular secretion the degree of which increases with rising UREA Waste product of Amino Acid metabolism Excretory Load dependent on amino acid and protein Intake as well as Net Body Protein Metabolism Filtered freely by the Glomeruli and Diffuse back into the Renal Tubules by a Passive process Clearance dependent on Urine Flow Rate Plasma [UREA] is a poor indicator of GFR because: 1. production (low protein intake) can lower the plasma [urea] sufficiently to enable a normal plasma level to be associated with significant renal insufficiency 2. GFR has to drop ~ 40% before the plasma [urea] begins to rise 3. production (eg. high protein intake) in the face of minor degrees of renal impairment can result in disproportionately high plasma [urea] Conditions Affecting [UREA] Independent of GFR UREA UREA * High Protein Diet * Liver Disease * Gastrointestinal Bleeding * Malnutrition * Tissue Trauma * Glucocorticoids * Tetracycline Conditions Affecting [CREATININE] Independent of GFR Condition Mechanism Spurious or True Elevation: * * * * Ketoacidosis Cefoxitin, Cephalothin Ingested cooked meat Drugs (aspirin, cimetidine Non creatinine chromogen Non creatinine chromogen Gastrointestinal absorption of creatinine Inhibition of tubular creatinine secretion trimethoprim, amiloride triamterene, spironolactone) Decrease: * Increasing age * Cachexia Physiological in muscle mass Pathological in muscle mass Although Plasma [CREATININE] directly reflects GFR, it is not always a good indicator of this parameter because: 1. Plasma level is dependent on muscle mass 2. Creatinine secretion by the proximal tubule increases as GFR decreases; some drugs (eg. cimetidine) interfere with this secretion 3. Various substances interfere with the Jaffe Reaction (commonly employed) assay causing positive and negative bias (eg. acetoacetate, cephalothin, bilirubin) 4. Dietary factors e.g., roasted meat contain significant amount of creatinine and ingestion of these can raise the plasma level temporarily Assessment of Renal Tubular Function: Renal tubules are involved in the formation of a concentrated urine and handling of acid load that is normally produced from the intermediary metabolism of the body Abnormality of the renal tubules can be detected by: • Osmolality measurements in plasma and urine, water deprivation test • Inability to handle an acid load (acid-loading test) • Specific proteinuria and tubular enzymes • Presence of abnormal aminoaciduria and other compounds normally reabsorbed by the renal tubules (e.g., Fanconi syndrome) Urine and Plasma Osmolality: • Normal kidney can produce urine at a wide range of concentrations with osmolality between 50 - 1400 mmol/kg • As renal tubules lose their ability to absorb water and retain or secrete other substances, urine begins to resemble the plasma ultrafiltrate • Because of the variation in urinary concentration with hydration and volume, a random Urine Osmolality (Uosm) has little diagnostic value unless correlated with the clinical state • In patient suspected of diabetes insipidus, an overnight urine osmolality > 600 mmol/kg or > 2 x concomitant plasma Osmolality practically excludes the diagnosis Renal Tubular Acidosis (RTA): A group of diverse disorders in which the kidney is unable to acidify the urine normally The tubular defects may be acquired or hereditary Biochemically characterized hyperchloraemic metabolic acidosis with a normal anion gap and often a low plasma potassium level; a urine pH > 5.3 in the presence of metabolic acidosis Type 1: defective H+ ion secretion in the distal tubule Type 2: reduced capacity of the proximal tubule to conserve bicarbonate An Acid Loading Test may be required to establish the diagnosis Acute Renal Failure (ARF) Definition: Acute renal failure (ARF) is a syndrome characterized by rapid (hours to weeks) decline in glomerular filtration rate (GFR) and retention of nitrogenous waste products such as blood urea nitrogen and creatinine. Brenner & Rector, The Kidney, 6th ed Acute Renal Failure (ARF) These may be classified according to aetiology as: Pre-renal (impaired renal perfusion), and resulting in pre-renal azotaemia - a rapidly reversible form of ARF. Post-renal (obstruction to urinary flow), and resulting in post-renal azotaemia - also a rapidly reversible form of ARF. Intrinsic renal (structural damage to the kidney), and resulting in acute intrinsic renal failure - a form of ARF which is not rapidly reversible and may even be progressive. Both pre- and post-renal causes of ARF have the potential for causing structural damage to the kidney if left untreated. CAUSES OF ACUTE INTRINSIC RENAL FAILURE 1. Acute Tubular Necrosis: Post-ischaemic, Nephrotoxic 2. Acute Interstitial Nephritis Drug Hypersensitivity Infection 3. Gram -ve Sepsis 4. Postpartum Haemorrhage 5. Renal Artery Occlusion (bilateral) 6. Acute Glomerulonephritis * ~ 70% of ATN have more than one underlying causes * Some ATN patients, particularly those with nephrotoxic injury, are non-oliguric initially Acute tubular necrosis (ATN): a potentially (but not rapidly) reversible form of acute renal failure involving structural damage to the tubules and a consequential reduction in glomerular filtration rate (GFR). Recovery usually takes several weeks even when the cause is removed. ARF: Biochemical Investigations P - Creatinine (muscle) P - Urea (protein catabolism) P - Osmolality P - K+ (life threatening if substantially increased) U - Na+ U - Creatinine U - Urea U - Osmolality Creatinine Clearance (CrCl) U - Sediment Also tests for decreased effective circulating volume, e.g. haematocrit Differentiation between PRA and ATN Laboratory Test Pre-Renal Azotaemia Acute Tubular Necrosis P- Urea / Creatinine ratio > 60 Ur Sodium (mmol/L) < 20 Ur Osmolality (mmol/kg) > 500 Ur / P Creatinine ratio > 40 Fractional excretion of < 1% filtered Sodium % (%FENa) < 40 > 40 < 350 < 20 > 2% Urine Sediment Brown granular casts, cellular debris Normal or occasional granular casts FRACTIONAL EXCRETION Fractional Excretion of a substance “X” (Fex) is that portion of the total amount filtered by the glomeruli which is finally excreted in the urine Ux . Pcr FEx % = -------------- x 100% Px . Ucr Fractional Excretion of Sodium FENa = Na excreted / Na filtered = clearence for Na / clearence for creatinine [ U-Na x U-volume / P-Na x time ] or -------------------------------------------------[ U-Creat x U-volume / P-Creat x time ] or U-Na x P-Creatinine -----------------------------------------P-Na x U-Creatinine % FENa = 100% x FENa ACUTE RENAL FAILURE BIOCHEMICAL CHANGES IN PLASMA Increased Decreased POTASSIUM (K+) SODIUM (Na+) HYDROGEN ION (H+) BICARBONATE (HCO3-) PHOSPHATE (PO42-) CALCIUM (Ca2+) UREA CREATININE (Cr) MAGNESIUM (Mg2+) URATE PLASMA BIOCHEMICAL CHANGES IN PROGRESSIVE RENAL FAILURE GFR (mL/min) Analyte Increased 60 – 120 Nil 30 – 60 Creatinine Urea 20 – 30 K+, H+, ( HCO3-) 10 – 20 Urate PO42- Clinical Example A 71-year-old man presented with dizziness and melaena from a bleeding peptic ulcer. Plasma : Na+ K+ Urea Creatinine HCO3- Reference range 140 5.1 23.9 156 19 mmol/L mmol/L mmol/L umol/L mmol/L 135 - 145 3.5 - 4.8 3.0 - 8.0 60 - 120 22 - 32 Urine : Na+ Creatinine Osmolality 17 mmol/L 9350 umol/L 785 mmol/kg 10000 - 20000 50 - 1200 Clinical Example P-Urea / Creatinine ratio = 153 Urine Osmolality = 785 U / P-Creatinine ratio = 60 % FENa = 0.23 Clinical Example Diagnosis: Mild pre-renal azotaemia due to acute blood loss. PROTEINUIRA (1) • Filtration through the glomerular membrane is dependent on molecular size with a cutoff between 20 - 40 A, corresponding to a protein molecular mass about 30 - 70 kDa • Negatively charged molecules have lower permeability • Small proteins like 2-microglobulin (13.5 kDa) and Lyzozyme (11.5 kDa) are freely filtered but they are almost completely reabsorbed in the proximal tubules • Normal daily excretion < 150 mg; about 40 - 50% is Albumin (67 kDa) PROTEINURIA (2) Measurement of Urine Protein: Specimen: • Timed collection: 24-hour, 12-hour overnight, 4-hour • Urine Protein / Creatinine ratio with random sample Dipstick methods for Urine Protein: • Most sensitive to albumin • Poor method for detecting tubular proteinuria PROTEINURIA: Classification 1. Overload Proteinuria: • Bence Jones (multiple myeloma) • Myoglobin (crush injury, rhabdomylosis) • Haemoglobin 2. Tubular Proteinuria: • Mostly low MW proteins (not albumin) • e.g., Fanconi’s, Wilson, pyelonephritis, cystinosis, heavy metal toxicity (Cd, Pb, Hg), galactosaemia 3. Glomerular Proteinuria: • Mostly albumin at first, but larger proteins appear as glomerular membrane selectivity is lost as disease progresses Other Causes of Proteinuria: • Orthostatic proteinuria: Protein excretion varies with posture, increasing on standing. Orthostatic proteinuria present in about 10 - 20% healthy subjects at prolonged upright posture; remits if the subject remains recumbent • Transient Proteinuria: Mild to moderate proteinuria may be found in systemic illnesses apparently not related to the kidneys, e.g., high fever, congestive heart failure, and seizures. Transient proteinuria may also be found in healthy athletes after strenuous exercise and often in urinary tract infection. Nephrotic Syndrome Proteinuria > 3.5 gm/day/1.73 m2 Associated with hypoalbuminaemia, hyperlipoproteinaemia and oedema. Where protein loss is relatively selective for small molecules, larger proteins such as 2-macroglobulin are increased in the plasma Glomerular diseases due to e.g., diabetes, systemic lupus erythematosis, glomerulonephritis RENAL CALCULI (1) Investigation of patients with renal calculus formation is of value because the majority of these patients will have further episodes of stone formation, which is often the final common path for several disorders Biochemical analysis of renal calculi is thus important in detecting the underlying causes of stone formation RENAL CALCULI (2) Types of Stones: • Calcium phosphate/carbonate: may be a consequence of primary hyperparathyroidism or renal tubular acidosis • Magnesium, ammonium and phosphate: often associated with urinary tract infections • Calcium oxalate: commonest, aetiology often obscure; often associated with idiopathic hypercalciuria, intermittent hyperoxaluria, low urinary citrate, or rarely primary hyperoxaluria • Uric acid: may be a consequence of hyperuricaemia • Cystine: very rare, associated with cystinuria RENAL CALCULI: Investigation • Chemical analysis of the calculus • Blood biochemical profile looking in particular at calcium, phosphate, bicarbonate, creatinine and alkaline phosphatase • Determination of pH and amino acids, microscopy, culture on a random early morning urine specimen • 2 or more 24-hour urine collected while patients are on their usual diet, looking at volume, creatinine, calcium, phosphate, oxalate, urate and citrate • PTH level and acid loading test for selected patients (e.g., suspected primary hyperparathyroidism, RTA) Prostate Specific Antigen (PSA) - (1) • Approved by FDA as a tumour marker for carcinoma of prostate • A protease • Localised to prostatic ductal cells • Barely detectable in female • Liquefies coagulum of seminal fluid • Enzymatically active PSA forms stable complexes in blood with antichymotrypsin (PSA-ACT) and 2macroglobulin (PSA-A2M) • Enzymatically inactive PSA remains as free form (fPSA) Prostate Specific Antigen (PSA) - (2) Free PSA (fPSA): • 30 kDa, 10 - 40% of total in plasma • % of fPSA decreases in patients with CA prostate as total PSA level increases Total PSA: • Upper cutoff usually taken at 4 ng/mL • Probability of carcinoma generally parallel the blood levels • Probability of carcinoma increases further with lower “Free / Total PSA ratio” • Total PSA serial measurement is adequate for monitoring of disease progression / recurrence after treatment Thank You! Dr. Sidney Tam