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.
