MECHANISMS OF PESTICIDE INDUCED DISTAL NEPHRON INJURY

本研究的目的是阐明毒物抑制特异性离子通道诱导肾小管上皮细胞凋亡的一些细胞事件和分子机制。这些研究将检验以下假设：某些农药通过与基侧膜g-氨基丁酸/苯二氮卓类受体CI通道(GABA/BZD-CI通道)相互作用，损伤远端肾单位的延髓粗大上肢(MTAL)和其他节段；它们与GABA/BZD CI通道的结合导致细胞超极化，跨细胞离子通量改变，并导致细胞死亡，表现为细胞凋亡。这项建议中的研究将检验一些分子事件，这些事件通过GABA/BZD-CI-通道将农药相关的离子通量变化与远端肾单位细胞的凋亡联系起来。这些研究将在分离的灌流小鼠mTAL片段、ST-1细胞、已建立的小鼠mTAL细胞系以及培养中的MDCK细胞中进行。离子通量将通过电学和荧光分光光度测定；细胞完整性将通过酶的释放和组织学检查进行分析。MTAL中的凋亡程度将通过TUNEL和基于ELISA法的分析方法在从农药暴露的小鼠获得的肾组织切片和培养细胞中进行检测，并通过直接DNA片段化分析进行确认。可能将CI-通量抑制与细胞凋亡联系起来的细胞事件将通过测量氧耗、细胞内[Ca}++和细胞体积的变化来探讨。其他研究将直接检测GABA/BZD-CI通道激动剂和拮抗剂对农药诱导的mTAL细胞凋亡的急性和慢性影响。这项建议中的研究应该明确地阐明农药引起的急性和慢性mTAL肾毒性的一些机制，表现为细胞凋亡增加。这些研究应该提供直接证据，支持毒物相关的CI-离子通量变化与诱导细胞损伤和细胞凋亡之间的联系。

The purpose of the studies in this proposal is to elucidate some of the cellular events and molecular mechanisms that participate in the induction of apoptosis of renal tubular epithelial cell in response to the inhibition of specific ion channels by toxicants. These studies will test the hypothesis that certain pesticides injure the medullary thick ascending limb (mTAL) and other segments of the distal nephron via their interaction with a basolateral membrane g-aminobutyric acid/Benzodiazepine receptor CI- channel (GABA/BZD-CI-channel); that their binding to the GABA/BZD CI- channel results in cell hyperpolarization, an alteration of transcellular ion fluxes, and in cell death manifest as apoptosis. The studies in this proposal will examine some of the molecular events that link the pesticide associated changes in the ion flux via the GABA/BZD-CI- channel and apoptosis of distal nephron cells. These studies will be performed in isolated perfused mouse mTAL segments, and in ST-1 cells, an established mouse mTAL cell line, and in MDCK cells in culture. Ion fluxes will be determined electrically and spectrofluorometrically; cell integrity will be assayed by released of enzymes and by histologic examination. The degree of apoptosis in the mTAL will be assayed in kidney tissue sections obtained from pesticide exposed mice and in cells in culture by the TUNEL and by an ELISA based assay methods and confirmed by direct analysis for DNA fragmentation. The cellular events that might link the inhibition of CI- flux to apoptosis will be probed via measurement of changes in oxygen consumption, intracellular [Ca}++, and cell volume. Additional studies will examine directly the acute and chronic effects of GABA/BZD-CI- channel agonists and antagonists on pesticide indued apoptosis of mTAL cells. The studies in this proposal should elucidate explicitly some of the mechanisms responsible for acute and chronic mTAL nephrotoxicity from pesticides manifest as increased apoptosis. These studies should provide direct evidence that supports a link between toxicant associated changes in CI- ion flux and induction of cellular injury and apoptosis.