GLUTATHIONE AND MITOCHONDRIA IN TOXIC RENAL INJURY

描述：（改编自研究者摘要）：既往工作 专注于谷胱甘肽（GSH）转运的生化特性， 肾细胞和亚细胞器。这项研究将扩大这些 通过研究谷胱甘肽载体蛋白的分子特性， 肾脏线粒体调查人员先前证明， 已知的线粒体内膜的有机阴离子载体， 二羧酸盐载体（DCC）和氧戊二酸盐载体（OGC）占 大部分GSH从细胞质进入线粒体。具体目标1 将涉及克隆、表达、纯化和功能表征 DCC和OGC蛋白在GSH转运中的作用。DCC和OGC基因 将通过RT-PCR从大鼠肾脏总RNA中克隆，将在 细菌，纯化，并重组成脂蛋白体。的动力学和 两种载体的抑制剂和底物特异性将在 详细具体目标2将检验细胞和线粒体 在用野生型或突变型GSH载体转染的细胞中功能不同。DCC 和OGC cDNA克隆将通过使用PCR的定点诱变来操作。 这些载体的野生型和突变基因将在细菌中表达， 纯化并重构成脂蛋白体以评估其活性。 野生型和突变载体的克隆将转染到稳定的肾细胞中。 细胞株NRK-52 E细胞，以及不同活性水平的GSH 将评估线粒体功能的转运。线粒体是关键 在细胞能量学和细胞坏死过程中的作用， 凋亡特异性目的3将检验以下假设： 野生型或突变型线粒体GSH载体具有不同的亲和性， 氧化损伤、凋亡和坏死。氧化损伤 NRK-52 E细胞将被叔丁基过氧化氢诱导。蜂窝和 线粒体功能将通过测量呼吸、活性 转运、脂质过氧化、GSH状态和亚细胞分布。 将通过流式细胞术的亚二倍体DNA分析定量细胞凋亡， 测量细胞色素C从线粒体的释放，半胱天冬酶-3的活化， TUNEL法检测DNA断裂。通过以下方法定量坏死： 测定从细胞释放的乳酸脱氢酶。对于每个参数， 时间和浓度依赖性效应将与GSH转运相关 活性，以评估GSH载体在线粒体反应中的作用 有毒物质。这些目标的实现将扩大我们对 这些运营商的功能。这一信息可能具有治疗应用 预防肾细胞损伤或了解线粒体 疾病或年龄相关的线粒体功能下降。

DESCRIPTION: (Adapted from the investigator's abstract): 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.