Biology of Mercapturic Acid Mediated Nephrotoxicity

硫醇酸介导的肾毒性的生物学

• 批准号: 6918100
• 负责人: ALEXANDER PUSHKIN
• 金额: $ 36.69万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-07-25 至 2010-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6918100
• 关键词: amidohydrolases; binding proteins; cell line; confocal scanning microscopy; disease /disorder model; environmental exposure; enzyme activity; immunofluorescence technique; laboratory mouse; protein binding; protein localization; protein structure function; renal toxin; renal tubular transport; sulfur aminoacid; toxin metabolism; trichloroethylene; water pollution; western blottings

DESCRIPTION (provided by applicant): 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.

描述（由申请人提供）：通过诱导近端小管（S2-和S3-PT）的S2和S3段的选择性坏死，导致哺乳动物的急性肾衰竭，导致哺乳动物的急性肾衰竭，而受影响的S1段则显着较大PT（S1-PT）的。这些溶剂引起急性肾衰竭的机制尚不完全理解。众所周知，TCE和其他溶剂的代谢会导致胃酸的产生（N-乙酰基甲基半胱氨酸S-偶联物），这些被PT吸收。在PT中，苏联酸要么分泌或脱乙酰基，并进一步代谢为含有肾毒性的含硫的反应性片段。负责从TCE衍生而成的脱乙酰化苏联的关键肾脏酶，从而介导其肾毒性仅是从大鼠肾脏中部分纯化的，尚未完全表征。我们现在已经克隆并描述了这种酶，我们将其命名为氨基酰基III（AAIII）。 AAIII脱乙酰化的N-乙酰基 - （1,2-二氯环烯基）和N-乙酰基 - （2,2-二氯环烯基）-L-甲基（N-乙酰基-2,2-DCVC），以及其他一些有毒的用毒剂， 。在小鼠肾脏中，AAIII定位于S1-PT的顶膜以及S2-和S3-PT的细胞质。我们假设AAIII的细胞质定位是导致TCE衍生的S2-和S3-PT的更大毒性，而不是S1-PT。 S1-，S2-和S3-PT之间AAIII定位的差异归因于一种新型蛋白质，该蛋白被称为AAIII结合蛋白（AAIIIBP），该蛋白在S1-PT的顶端膜上特别表达，并且在S1-PT的顶端没有表达，并且在S2-，S3-PT和其他肾单位段。在用作模型系统的小鼠PT细胞系（MPCT）中，AAII被定位在细胞质中，如S2-和S3-PT中。当AAIIIBP在这些细胞中外源性表达时，AAIII被靶向顶端膜，这与S1-PT一样，导致硫酸介导的肾毒性显着降低。此外，初步实验表明，AAIII和AAIIIBP的复合物与多药抗性相关蛋白2（MRP2）形成了转运代谢物，从而增加了MRP2的抗胃酸盐的运输。在此提案中，将研究AAIII的假设参与TCE衍生的抗毒性介导的毒性。该项目的成功完成将增强我们对硫酸介导的肾毒性机制的理解。