SEMI-QUANTITATIVE IMMUNOHISTOCHEMICAL ANALYSIS OF MALE RAT-SPECIFIC α2u-GLOBULIN ACCUMULATION FOR CHEMICAL TOXICITY EVALUATION

We purified male rat urinary α2u-globulin, prepared the antibody in rabbits, and improved an immunohistochemical detection method using this antibody for male rat-specific α2u-globulin accumulation appearing as hyaline droplets in the kidneys. Our prepared antibody reacted specifically with α2u-globulin in both immunohistochemical and Western blotting analyses, furthermore, and the graded immuno-reactivities on the slide were well associated with computational image analyzing results. Using this method, we retrospectively analyzed the renal sections from the toxicity studies of 12 nephrotoxic chemicals, which had already been conducted under the Japanese Existing Chemicals Survey Program. We demonstrated that the hyaline droplets induced by treatment with 10 chemicals (1,4-dibromobenzene, dicyclopentadiene, 3,4-dimethylaniline, 1,4-dicyanobenzene, tetrahydrothiophene-1,1-dioxide, 1,3-dicyanobenzene, acenaphthene, 3,4-dichloro-1-butene, 3a,4,7,7a-tetrahydro-1H-indene and 3,5,5-trimethylhexan-1-ol) were directly associated with α2u-globulin accumulation. This immunohistochemical method is convenient for applying, even retrospectively, paraffin sections from general toxicity studies and could be useful for qualifying male rat-specific hyaline droplets consisting of α2u-globulin and renal risk in humans.