MECHANISMS OF PESTICIDE INDUCED DISTAL NEPHRON INJURY

本研究的目的是阐明毒性物质对特定离子通道的抑制诱导肾小管上皮细胞凋亡的一些细胞事件和分子机制。这些研究将验证某些农药通过与基底外侧膜g-氨基丁酸/苯二氮卓受体CI-通道（GABA/ bzd -CI-通道）相互作用损害髓质厚升肢（mTAL）和远端肾元其他部分的假设；它们与GABA/BZD CI通道的结合导致细胞超极化，改变细胞间离子通量，并以凋亡的形式导致细胞死亡。本研究将探讨农药相关的GABA/BZD-CI通道离子通量变化与远端肾元细胞凋亡之间的一些分子事件。这些研究将在分离的灌注小鼠mTAL段、ST-1细胞（已建立的小鼠mTAL细胞系）和培养的MDCK细胞中进行。离子通量将用电和荧光光谱法测定；细胞的完整性将通过酶的释放和组织学检查来测定。通过TUNEL和基于酶联免疫吸附试验（ELISA）的方法，对暴露于农药的小鼠肾脏组织切片和培养细胞进行检测，并通过DNA片段的直接分析来证实mTAL的凋亡程度。将通过测量氧气消耗、细胞内[Ca}++]和细胞体积的变化来探索可能将CI-通量抑制与凋亡联系起来的细胞事件。进一步的研究将直接研究GABA/BZD-CI通道激动剂和拮抗剂对农药诱导的tal细胞凋亡的急性和慢性作用。本提案中的研究应明确阐明农药引起的急性和慢性肾毒性的一些机制，表现为细胞凋亡增加。这些研究应提供直接证据，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.