The previous work focused on biochemical properties of glutathione (GSH) transport in isolated renal cells and subcellular organelles. The proposed research will extend those findings by investigating the molecular properties of GSH carrier proteins in kidney mitochondria. The investigators demonstrated previously that two of the known, organic anion carriers of the mitochondrial inner membrane, the dicarboxylate carrier (DCC) and the oxoglutarate carrier (OGC), account for most of the uptake of GSH from the cytoplasm into mitochondria.
Specific Aim 1 will involve cloning, expression, purification, and functional characterization of the role of the DCC and OGC proteins in GSH transport. The DCC and OGC genes will be cloned from total rat kidney RNA by RT-PCR, will be expressed in bacteria, purified, and reconstituted into proteoliposomes. The kinetics and inhibitor and substrate specificity of the two carriers will be studies in detail.
Specific Aim 2 will test the hypothesis that cellular and mitochondrial function differ in cells transfected with wild-type or mutant GSH carriers. DCC and OGC cDNA clones will be manipulated by site-directed mutagenesis using PCR. Wild-type and mutant genes for these carriers will be expressed in bacteria, purified, and reconstituted into proteoliposomes to assess their activity. Clones of wild-types and mutant carriers will be transfected into stable renal cell line, NRK-52E cells, and the effect of different activity levels of GSH transport on mitochondrial function will be assessed. Mitochondrial play a key role in cellular energetics and in the processes of cellular necrosis and apoptosis.
Specific Aim 3 will test the hypothesis that cells transfected with wild-type or mutant mitochondrial GSH carriers have different susceptibilities to oxidant injury, apoptosis and necrosis. Oxidant injury in transfected NRK-52E cells will be induced by tert-butyl hydroperoxide. Cellular and mitochondrial function will be assessed by measurements of respiration, active transport, lipid peroxidation, and GSH status and subcellular distribution. Apoptosis will be quantitated by subdiploid DNA analysis with flow cytometry, measurement of cytochrome c release from mitochondria, activation of caspase-3, and the TUNEL assay for DNA fragmentation. Necrosis will be quantitated by measurements of lactate dehydrogenase release from cells. For each parameter, time and concentration dependent effects will be correlated with GSH transport activity to assess the role of the GSH carriers in the mitochondrial response to toxicants. Achievement of these aims will expand our knowledge of the function of these carriers. This information may have therapeutic applications for prevention of renal cellular injury or for understanding mitochondrial diseases or age-related decreases that occur in mitochondrial function.
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