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.

该提案中研究的目的是阐明参与肾小管上皮细胞凋亡的一些细胞事件和分子机制，以响应毒性抑制特定的离子通道。这些研究将测试某些农药通过与基底外侧膜G-氨基丁酸/苯并二氮卓受体CI-CIN通道（GABA/BZD-CI-CHANNEL）的相互作用来测试某些农药损害髓质较厚的升肢（MTAL）和其他远端肾单位的阶段；它们与GABA/BZD CINCH的结合会导致细胞超极化，经跨离子通量的改变，并且在细胞死亡中表现为细胞凋亡。该提案中的研究将检查一些分子事件，这些事件通过GABA/BZD-CI-通道与远端肾单位细胞的凋亡相关的农药相关的变化。这些研究将在孤立的灌注小鼠MTAL段，ST-1细胞，已建立的小鼠MTAL细胞系以及培养物中的MDCK细胞中进行。离子通量将通过电气和光谱测定；细胞完整性将通过酶释放和组织学检查来测定。 MTAL中的凋亡程度将在通过TUNEL和基于ELISA的测定方法中从农药暴露的小鼠和培养物中的细胞中获得的肾脏组织切片进行测定，并通过直接分析DNA碎片分析确认。可能通过测量氧气消耗，细胞内[CA} ++和细胞体积的变化来探测可能将CII通量抑制与凋亡联系起来的细胞事件。其他研究将直接检查GABA/BZD-CI-通道激动剂和拮抗剂对农药的急性和慢性作用。该提案中的研究应明确阐明某些导致农药急性和慢性MTAL肾毒性的机制，这表现为增加的凋亡。这些研究应提供直接的证据，以支持毒性相关的CI-ION通量变化与细胞损伤和凋亡的诱导之间的联系。