MOLECULAR MECHANISMS OF RENAL CELL INJURY

该方案研究肾上皮细胞的损伤和修复。 细胞使用一种名为化学缺氧的模型来耗尽细胞内 ATP店。我们已经开发了一个可逆的ATP耗竭模型 氧化磷酸化和糖酵解的化学抑制剂。 氧化磷酸化抑制剂鱼藤酮和2-脱氧葡萄糖， AD糖酵解竞争性抑制剂，被添加到肾上皮细胞 生长在膜过滤器支撑物上的细胞。在应用 抑制药后，细胞内的三磷酸腺苷迅速、可重复地下降。 当向细胞加入灌流介质时，细胞内的三磷酸腺苷 水平迅速上升。在添加或添加后2.5小时 灌流液中，细胞内ATP水平与对照组相当。 这种新的可逆的化学缺氧模型将被用来研究 两种肾上皮细胞系的细胞损伤和修复的性质， MDCK(马丁达比犬肾脏)和JTC(猴近端小管细胞 行)在三个方面。 1.MDCK和JTC细胞株将受到ATP的耗竭 然后恢复其细胞内的ATP储备。这将是 允许我们测试肾细胞重现其正常状态的假设 细胞损伤恢复后的发育步骤(巴卡洛 和Fine，1991)。在这些研究中，细胞的亚细胞组织 肌动蛋白细胞骨架、灶性黏附、紧密连接和黏附连接 将通过共聚焦激光扫描荧光显微镜进行检查。在……里面 此外，我们还将研究紧密结势垒的恢复 功能(Mandel，Bacallao和Zampighi，自然，1993)通过测量 跨上皮单分子层在不同时期的跨上皮阻力 从化学性缺氧中恢复后的次数。 2.我们还将检验这样一种假设，即 化学缺氧症扰乱了蛋白质分选机的准确性 肾上皮细胞。这会导致功能极性的丧失。 我们认为，受损细胞的改变的极性不会 纠正，直到蛋白质分选机修复。这 假说将通过量化动力学和化学计量进行检验。 多种内源性膜蛋白的膜组装。 这些研究可能会发现蛋白质存在显著的不同 归类了，尤其是在伤病恢复期间。例如， 细胞骨架连接蛋白的蛋白质靶向机制可能非常 对化学缺氧敏感，而其他质膜 蛋白质的极化传递可能不会发生变化。这些 研究可能会描绘出蛋白质的不同途径 分类。 3.此外，我们还将研究化学缺氧对 蛋白质挽救途径。这条途径识别错误分类的蛋白质， 将它们从质膜上移除，并将蛋白质输送到 正确的质膜结构域。这一途径将在低pH值下进行检测 水疱性口炎病毒G蛋白在人牙周炎根尖周膜中的融合 细胞及其向基底外侧输送动力学的测定 薄膜。

This proposal studies cellular injury and repair in renal epithelial cells using a model called chemical anoxia to deplete the intracellular ATP stores. We have developed a reversible model of ATP depletion using chemical inhibitors of oxidative phosphorylation and glycolysis. Rotenone, an inhibitor of oxidative phosphorylation, and 2-deoxyglucose, ad competitive inhibitor of glycolysis, are added to renal epithelial cells grown on membrane filer supports. Following the application of the inhibitors, there is a rapid, reproducible fall in intracellular ATP. When a perfusion" media is added to the cells the intracellular ATP levels rapidly rise. At 2.5 hours following the addition or the perfusion media, the intracellular ATP levels are equivalent to controls. This new, reversible, chemical anoxia model will be used to study the nature of cellular injury and repair in two renal epithelial cell lines, MDCK (Madin-Darby canine kidney) and JTC (monkey proximal tubule cell line) in three ways. 1. The MDCK and JTC cell lines will be subjected to ATP depletion followed by restoration of their intracellular ATP reserves. This will allow us to test the hypothesis that renal cell recapitulate their normal developmental steps following the recovery from cellular injury (Bacallao and Fine, 1991). In these studies, the subcellular organization of the actin cytoskeleton, focal adherens, tight junction and adherens junctions will be examined by confocal laser scanning fluorescence microscopy. In addition we will study the recovery of the tight junction barrier function (Mandel, Bacallao and Zampighi, Nature, 1993) by measuring the transepithelial resistance across the epithelial monolayer at various times after recovery from chemical anoxia. 2. We will also test the hypothesis that cellular injury caused by chemical anoxia disrupts the accuracy of the protein sorting machinery of renal epithelial cells. This leads to loss in functional polarity. We suggest that the altered polarity of the injured cells will not be corrected until the protein sorting machinery is repaired. This hypothesis will be tested by quantifying the kinetics and stoichiometry of membrane assembly for a variety of endogenous membrane proteins. These studies may uncover significant different in the way proteins are sorted, especially during the recovery from the injury. For example the protein targeting mechanism of cytoskeletal linked proteins may be very sensitive to the effects of chemical anoxia, while other plasma membrane proteins may exhibit no change in their polarized delivery. These studies could potentially delineate different pathways of protein sorting. 3. Additionally, we will examine the effects of chemical anoxia on the protein salvage pathway. This pathway identifies mis-sorted proteins, removes them from the plasma membrane and delivers the proteins to the correct plasma membrane domain. This pathway will be examined by low pH fusion of vesicular stomatitis virus G protein in the apical membrane of the cells and determining the kinetics of delivery to the basolateral membrane.