MECHANISMS OF PESTICIDE INDUCED DISTAL NEPHRON INJURY

农药所致远端肾单位损伤的机制

• 批准号: 6382293
• 负责人: DONALD A MOLONY
• 金额: $ 23.02万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCI CTR HOUSTON
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-04-01 至 2003-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6382293
• 关键词: MDCK cell; adenosine triphosphate; apoptosis; basolateral membrane; benzodiazepine receptor; calcium ion; cell morphology; chloride channels; chloride ion; chlorohydrocarbon insecticide; epithelium; gamma aminobutyrate; inhibitor /antagonist; laboratory mouse; lindane; membrane potentials; oxidative stress; oxygen consumption; pesticide biological effect; renal ischemia /hypoxia; renal toxin; renal tubule; stimulant /agonist; toxicology

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.

本研究的目的是阐明毒物抑制特定离子通道诱导肾小管上皮细胞凋亡的细胞事件和分子机制。这些研究将检验以下假设：某些杀虫剂通过与基底外侧膜g-氨基丁酸/苯二氮卓受体Cl-通道（GABA/BZD-Cl-通道）的相互作用损伤髓质粗升支（mTAL）和远端肾单位的其他节段;它们与GABA/BZD Cl-通道的结合导致细胞超极化、跨细胞离子通量的改变以及表现为细胞凋亡的细胞死亡。本提案中的研究将检查通过GABA/BZD-CI通道连接农药相关离子通量变化和远端肾单位细胞凋亡的一些分子事件。这些研究将在分离的灌注小鼠mTAL节段、ST-1细胞（一种已建立的小鼠mTAL细胞系）和培养的MDCK细胞中进行。将通过电学和荧光分光光度法测定离子通量;将通过酶的释放和组织学检查测定细胞完整性。将通过TUNEL和基于ELISA的测定方法，在从农药暴露小鼠获得的肾组织切片和培养细胞中测定mTAL中的细胞凋亡程度，并通过DNA片段化的直接分析进行确认。将通过测量氧消耗、细胞内[Ca}++和细胞体积的变化来探测可能将Cl-通量的抑制与细胞凋亡联系起来的细胞事件。进一步的研究将直接检查GABA/BZD-Cl-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作用。本提案中的研究应明确阐明农药引起急性和慢性mTAL肾毒性的一些机制，表现为细胞凋亡增加。这些研究应提供直接证据，支持Cl-离子通量的毒物相关变化与细胞损伤和凋亡诱导之间的联系。