GLUTATHIONE AND MITOCHONDRIA IN TOXIC RENAL INJURY

哺乳动物的肾单位由几个细胞群组成， 独特的生化和生理特性， 人群对化学毒物或病理性毒物的反应不同， 例如缺氧或缺血的病症。为了理解什么是属性 负责细胞类型特异性的化学敏感性， 病理损伤，我们集中在两个突出的细胞因子， 谷胱甘肽（GSH）状态和线粒体功能。GSH是关键 细胞防御反应性亲电体和氧化剂的组成部分， 两种最常见的细胞损伤产生者， 疾病肾脏中的GSH含量是不寻常的， 合成和运输能力。细胞类型特异性差异 在调节谷胱甘肽状态可能是重要的，在确定细胞 易受伤。正常的线粒体功能对于 维持肾功能的恒定和高能量输入。 因此，肾细胞容易受到能量供应中断的影响 是由于线粒体功能障碍由于线粒体是常见的靶点， 毒性或病理损伤的部位， 线粒体的特性也可能有助于细胞易感性 受伤。这些研究中提出的研究将特别侧重于 GSH跨细胞质膜和跨膜转运的机制 线粒体内膜新鲜分离的大鼠细胞 肾近端小管（PT）和远端小管（DT），线粒体来源 从肾皮质，和线粒体纯化的肾PT和DT细胞， 一种新的分离技术将是所使用的生物系统。的 分馏技术是新颖的，因为它是唯一可用的方法 从单个细胞中分离功能上可行的线粒体， 足以进行生物化学研究。具体目标是 （1）确定GSH跨膜转运的机制 不同肾细胞群的质膜，并研究 这些过程在调节细胞内GSH状态中的作用;（2） 探讨谷胱甘肽和谷胱甘肽二硫化物（GSSG）的作用机制 肾皮质线粒体中的转运;和（3）表征 从特定肾细胞中纯化的线粒体的生化特性 人群，并确定线粒体GSH转运的作用， 对毒物敏感性差异的能量学。通过完成 上述目标，我们将描绘谷胱甘肽运输机制， 细胞质膜和线粒体内膜，我们将 确定负责线粒体GSH转运的蛋白质，我们将 描述了线粒体GSH转运活性与 线粒体代谢和细胞能量学，我们将利用这些 结果，以获得线粒体和细胞的作用的信息， 肾PT和DT细胞对化学药物和 病理性损伤这些成果最终将有助于 维持或改善肾功能的方法。

The mammalian nephron is composed of several cell populations that possess distinctive biochemical and physiologic properties and each cell population responds differently to chemical toxicants or to pathologic conditions such as hypoxia or ischemia. To understand what properties are responsible for cell type-specific susceptibility to chemical and pathologic injury, we have focussed on two salient cellular factors, glutathione (GSH) status and mitochondrial function. GSH is a key component of cellular defense against reactive electrophiles and oxidants, two of the most common producers of cellular injury in both toxicity and disease. The kidney is unusual with respect to GSH in that it possesses both synthetic and transport capabilities. Cell type-specific differences in regulation of GSH status may be important in determining cellular susceptibility to injury. Proper mitochondrial function is critical to maintain a constant and high energy input for renal function. Consequently, renal cells are vulnerable to interruptions in energy supply due to mitochondrial dysfunction. Since mitochondria are frequent target sites in toxic or pathologic injury, cell type-specific differences in properties of mitochondria may also contribute to cellular susceptibility to injury. Research proposed in these studies will focus specifically on mechanisms of GSH transport across cellular plasma membranes and across mitochondrial inner membranes. Freshly isolated cells derived from rat kidney proximal tubule (PT) and distal tubule (DT), mitochondria derived from renal cortex, and mitochondria purified from renal PT and DT cells by a novel fractionation technique will be the biological systems used. The fractionation technique is novel because it is the only method available to isolate functionally viable mitochondria from single cells in quantities sufficient for biochemical studies. The specific aims of this research will be: (l) To determine the mechanism of GSH transport across plasma membranes in different renal cell populations and to investigate the role of these processes in regulation of intracellular GSH status; (2) to determine the mechanisms of GSH and glutathione disulfide (GSSG) transport in renal cortical mitochondria; and (3) to characterize biochemical properties of mitochondria purified from specific renal cell populations and to determine the role of mitochondrial GSH transport and energetics in differential susceptibility to toxicants. By completion of the above aims, we will delineate mechanisms of GSH transport across cellular plasma membranes and mitochondrial inner membranes, we will identify the protein responsible for mitochondrial GSH transport, we will describe the interaction of mitochondrial GSH transport activity with mitochondrial metabolism and cellular energetics, and we will use these results to gain information on the role of mitochondrial and cellular heterogeneity in susceptibility of renal PT and DT cells to chemical and pathologic injury. These results will ultimately help in development of procedures to maintain or improve kidney function.