Lysosomal degradation of α2u-globulin and α2u-globulin-xenobiotic conjugates

Abstract

A diverse group of chemicals cause a male rat-specific nephrotoxicity in which α2u-globulin accumulates in renal lysosomes. It has been suggested that these chemicals bind to the protein and decrease its degradation by lysosomal proteinases. To test this hypothesis, the lysosomal degradation of native α2u-globulin and that to which d-limonene, d-limonene-1,2-oxide, isophorone, 1,4-dichlorobenzene, and 2,5-dichlorophenol were bound was studied. α2u-Globulin was purified from male rat urine, and male rat renal cortical lysosomes, isolated by differential centrifugation, served as the proteolytic enzyme source. Pepstatin, an inhibitor of aspartic acid proteinases, and leupeptin, an inhibitor of cysteine proteinases, reduced α2u-globulin degradation to 28 ± 8 and 17 ± 5% of control, respectively, whereas addition of both inhibitors decreased α2u-globulin degradation to 8 ± 1% of control values. These results indicate that both classes of endopeptidases are important in the degradation of α2u-globulin. Under the incubation conditions used, 30% of native α2u-globulin was degraded in a 4-hr period. Conjugates of the protein were made for in vitro binding experiments. Binding of d-limonene and 1,4-dichlorobenzene to α2u-globulin did not alter the degradation of the protein, whereas binding of d-limonene-1,2-oxide, 2,5-dichlorophenol, and isophorone decreased α2u-globulin degradation by 33%. These results indicate that not all chemicals which have been shown to bind in vivo to α2u-globulin alter the in vitro lysosomal degradation of the protein. However, in all cases, one metabolite of each hyaline droplet inducer did alter degradation of α2u-globulin, suggesting that a decrease in lysosomal degradation is involved in the accumulation of this protein in male rat kidney lysosomes.