Biochemical functions of kidneys Seminar No. 12 Q. 2 Q.3 The volume of blood plasma that is completely cleared of creatinine in one second Express: • Molar amount of plasma creatinine filtered per 1 s: n[p] = [] • Molar amount of urine creatinine excreted by urine per 1 s: n[u] = [• ]Clearance of creatinine: [] • Molar amount of plasma creatinine filtered per 1 s: n[p] = c[p] × V[p] (mmol/s)[] • Molar amount of urine creatinine excreted by urine per 1 s: n[u] = c[u] × V[u] (mmol/s) [• ]Clearance of creatinine: V[p] = (c[u] × V[u]) / c[p] (ml/s)[] Q. 4 Q. 6 Creatinine formation Creatinine formation Filtration of plasma • glomerular membrane is a filter system • structural barrier (collagen IV) • electrostatic barrier (negative charges of sialic acid in glycoproteins repulse anionic proteins) • the basement membrane allows free movement of electrolytes, water and small molecules (urea, glucose, AA, creatinine ...) The filtration of proteins strongly depends on their molecular mass • Proteins with M[r] < 60 000 (microproteins) pass easily into urine regardless of their charge • Proteins with M[r] 60 000 – 150 000 only very small amount is filtered into urine • Proteins with M[r] > 150 000 do not pass into urine regardless of their charge Q. 8 A. 8 total proteins: < 150 mg/day albumin: < 20 mg/day Q. 10 • Test strips based on protein error of acid-base indicators primarily specific for albumin (albuPhan, Pliva-Lachema, Brno) • Precipitation tests proteins are precipitated (denaturated) by 5-sulfosalicylic acid as fine particles (turbidity) Microalbuminuria • immunochemical tests using antibody against human albumin • antibody can be gold-labelled Þ red coloured zone on a strip corresponds with albumin concentration in urine sample (test strips Micral) • antibody freely soluble  nephelometry Q. 11 (see chapter 1) SDS-PAGE • sodium dodecyl sulfate polyacrylamide gel electrophoresis • separation is carried out in the presence of SDS, oligoproteins are separated into their subunits, SDS binds to proteins and gives them a large uniform negative charge, thus proteins are separated according to their M[r]. Glomerular proteinuria • consequence of the loss of glomerular membrane integrity • typical protein: albumin • selective glomerular proteinuria: proteins with M[r] 60 000 – 100 000 pass into urine • non selective glomerular proteinuria: more severe glomerular lessions proteins of all sizes M[r] > 60 000 pass into urine Tubular proteinuria • tubules are unable to reabsorb proteins • small proteins molecules (microproteins) M[r] < 60 000 appear in the urine • typical protein: β[2]-microglobulin Q. 14 A. 14 Resorption of proteins • microproteins (M[r] < 60 000) are resorbed from the primary urine by receptor-mediated endocytosis • after hydrolysis in secondary lysosome in tubular cells are returned into AA pool Q. 15 Distinguish transcellular vs. paracellular transport 15a) Sodium transport into tubular cells 15b) Q. 16 16a) Potassium transport 16b) Potassium secretion Potassium secretion Q. 17 17a) Chloride transport 17b) Q. 18 Symport of glucose with Na^+ is secondary active transport^ Q. 19 Uric acid is the most abundant plasma antioxidant Q. 20 + 21 Q. 22 Q. 23 Osmolarity = Osmolality = Q. 24 • Plasma osmolality: • Urine osmolality: Q. 27 Endocrine functions of kidney Q. 29 • ammonogenesis • gluconeogenesis Q. 30 Q. 33 Q. 34 – see chapter 10, p. 7 Q. 35 Next seminar: 3^rd revision test (15 Q / 20 min) • Seminar chapters 8 – 12 • Practical chapters 8 – 10