Trichloroethylene (TCE) and other industrial solvents cause acute renal failure in mammals by inducing selective necrosis of S2 and S3 segments of the proximal tubule (S2- and S3-PT) in contrast to significantly less affected S1 segment of the PT (S1-PT). The mechanism(s) by which these solvents cause acute renal failure is incompletely understood. It is known that the metabolism of TCE and other solvents results in the generation of mercapturic acids (N-acetyl-cysteine S-conjugates), which are absorbed by the PT. In the PT, mercapturic acids are either secreted, or deacetylated and further metabolized into nephrotoxic sulfur-containing reactive fragments. The key renal enzyme responsible for deacetylating mercapturic acids derived from TCE thereby mediating their nephrotoxicity was only partially purified from rat kidney and was not completely characterized. We have now cloned and characterized this enzyme, which we named aminoacylase III (AAIII). AAIII deacetylated N-acetyl-(1,2-dichlorovinyl)- and N-acetyl-(2,2-dichlorovinyl)-L-cysteine (N-acetyl-2,2-DCVC) derived from TCE, and some other toxic mercapturates. In mouse kidney, AAIII was localized to the apical membrane of the S1-PT and to the cytoplasm of the S2- and S3-PT. We hypothesized that the cytoplasmic localization of AAIII is responsible for the greater toxicity of the TCE-derived mercapturates for S2- and S3-PT than for S1-PT. The difference in the localization of AAIII between the S1-, S2- and S3-PT was due to a novel protein that was named AAIII binding protein (AAIIIBP) which was specifically expressed on the apical membrane of S1-PT and was not expressed in S2-, S3-PT and other nephron segments. In the mouse PT cell line (mPCT) used as a model system, AAIII was localized to the cytoplasm as in the S2- and S3-PT. When AAIIIBP was expressed exogenously in these cells, AAIII was targeted to the apical membrane, which resulted in significantly reduced mercapturic acid-mediated nephrotoxicity as in the S1-PT. Furthermore, the preliminary experiments indicated that the complex of AAIII and AAIIIBP formed a transport metabolon with the multi-drug resistance associated protein 2 (Mrp2) thereby increasing the transport of mercapturates via Mrp2. In this proposal, the hypothesis of AAIII is involved in TCE-derived mercapturate-mediated toxicity will be studied. Successful completion of this project will enhance our understanding of the mechanism of mercapturic acid-mediated nephrotoxicity. ? ? ?
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