Direct radioimmunoassay of rat cystatin C: increased urinary excretion of this cysteine proteases inhibitor during chromate nephropathy.
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 +/- 18 micrograms/24 h) was found by the direct assay. In the chromate-intoxicated rat, urinary excretion increased twenty-fivefold (1017 +/- 391 micrograms/24 h) and returned to normal level one week after intoxication. This RIA will allow the study of rat cystatin C metabolism particularly during renal dysfunction.
Collé
A
A
INSERM U 133, Toulouse, France.
Tavera
C
C
Laurent
P
P
Leung-Tack
J
J
Girolami
J P
JP
D058186:Acute Kidney Injury / Q000139:chemically induced / Q000652:urine*; D000818:Animals; D000917:Antibody Formation; D002840:Chromates; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D003429:Cross Reactions / Q000276:immunology; D055316:Cystatin C; D015891:Cystatins / Q000276:immunology / Q000652:urine*; D007122:Immunoelectrophoresis; D008297:Male; D011863:Radioimmunoassay / Q000379:methods*; D051381:Rats; D011919:Rats, Inbred Strains; D017670:Sodium Compounds
C525030:CST3 protein, human; D002840:Chromates; C525032:Cst3 protein, rat; D055316:Cystatin C; D015891:Cystatins; D017670:Sodium Compounds; C028982:sodium chromate(VI)