Direct Radioimmunoassay of Rat Cystatin C: Increased Urinary Excretion of This Cysteine Proteases Inhibitor During Chromate Nephropathy

摘要

Rat cystatin C was purified to apparent homogeneity from rat urine after induction of a tubular dysfunction with sodium chromate. The two-steps purification procedure included a Carboxymethyl-papain affinity chromatography and anion exchange chromatography. The purified protein was identified as rat cystatin C by the following criteria: firstly retained on a Cm-papain affinity column, secondly an apparent molecular weight of 15 kDa and pI of 10.2. Antisera raised in rabbits against our purified rat cystatin C did not cross-react with other urinary proteins such as rat albumin and rat kallikrein, but partially cross-reacted with human cystatin C. A direct radioimmunoassay was developed and it enabled 8.32 fmol/ml of rat cystatin C to be detected. The detection range was between 0.125 and 62.5 ng/ml, with 10 intra-assay variation and 14 inter-assay variation. Physiological rat cystatin C excretion (40 #xB1; 18 #x3BC;g/24h) was found by the direct assay. In the chromate-intoxicated rat, urinary excretion increased twenty-fivefold (1017 #xB1; 391 #x3BC;g/24h) and returned to normal level one week after intoxication. This RIA will allow the study of rat cystatin C metabolism particularly during renal dysfunction.