CYTOPROTECTIVE ROLE OF HSP 72 IN RENAL CELL INJURY

应激如缺血或ATP耗竭诱导细胞保护性热应激蛋白（HSP）的合成。 在可诱导的细胞保护剂中，HSP 72是最丰富和最好表征的。 HSP 72是一种分子伴侣，可以清除和修复受损的细胞蛋白。 在非肾细胞和组织中，HSP 72的选择性过表达减少了缺血或ATP耗竭引起的后续损伤。 在这些模型中，HSP 72保护的细胞功能尚未确定。 在肾上皮细胞中，体内缺血或体外ATP耗竭诱导HSP，引起肌动蛋白细胞骨架的崩溃，紧密连接完整性的丧失，损害线粒体ATP产生并触发细胞凋亡。 我们以前已经表明，先前的热应激，足以上调HSP 72，保留这些细胞的功能，并提高肾细胞存活后ATP耗竭。该项目的中心假设是HSP 72是一种主要的肾细胞保护剂。 我们认为HSP 72通过与调节以下功能的蛋白质结合，减少细胞损伤并抑制ATP耗竭引起的细胞凋亡：（a）细胞骨架，（B）细胞-细胞接触位点，（c）天然DNA，（d）线粒体功能和（e）凋亡途径。 我们的初步研究支持了这一假设，其中单独选择性诱导（或抑制）HSP 72改变ATP耗竭的肾上皮细胞的存活率。 在一个类似于体内缺血/再灌注的ATP耗竭的体外模型中，我们将：（1）鉴定HSP 72保护的细胞功能;（2）鉴定与HSP 72相互作用的一些细胞内蛋白;（3）确定蛋白质结合与重折叠在介导HSP 72的细胞保护中的作用和（4）评估细胞内信号不需要热应激就能诱导HSP 72。 我们已经开发了分子、免疫学和生物化学工具来操纵肾上皮细胞中HSP 72的含量，并鉴定与HSP 72结合的蛋白质。在蛋白质结合或重折叠中具有良好描述缺陷的人HSP 72缺失突变体将用于确定这种分子伴侣在ATP耗竭细胞中保护靶蛋白的机制。 为了评估一些诱导HSP 72的细胞内信号，我们已经开发了一种新的，透化的细胞系统，其中细胞内的pH值，钙和腺嘌呤核苷酸含量可以单独操纵。 这些研究将检查一些允许HSP 72保护肾上皮细胞的途径，并可能最终提供一种增加细胞对缺血性损伤（急性肾衰竭的常见原因）的抵抗力的方法。

Stresses such as ischemia or ATP depletion induce the synthesis of cytoprotective heat stress proteins (HSP). Of the inducible cytoprotectants, HSP 72 is the most abundant and well-characterized. HSP 72 is a molecular chaperone that removes and repairs damaged cell proteins. In non-renal cells and tissues, selective over-expression of HSP 72 decreases subsequent injury from ischemia or ATP depletion. In these models, the cell functions protected by HSP 72 have not been identified. In the renal epithelial cell, ischemia in vivo or ATP depletion in vitro induce HSPs, cause collapse of the actin cytoskeleton, loss of tight junction integrity, impair mitochondrial ATP production and trigger apoptosis. We have previously shown that prior heat stress, sufficient to upregulate HSP 72, preserves these cell functions and improves renal cell survival after ATP depletion. The central hypothesis of this project is that HSP 72 is a major renal cytoprotectant. We suggest that by binding to proteins that regulate: (a) the cytoskeleton, (b) cell-cell contact sites, (c) native DNA, (d) mitochondrial function and (e) the apoptotic pathway, HSP 72 decreases cell injury and inhibits apoptosis caused by ATP depletion. This hypothesis is supported by our preliminary studies in which the selective induction (or suppression) of HSP 72 alone alters survival in ATP depleted renal epithelial cells. In an in vitro model of ATP depletion that resembles ischemia/reperfusion in vivo, we will: (1) identify cell functions protected by HSP 72; (2) identify some of the intracellular proteins that interact with HSP 72; (3) determine the role of protein binding vs. refolding in mediating cytoprotection by HSP 72 and (4) evaluate intracellular signal(s) that can induce HSP 72 without requiring heat stress. We have developed the molecular, immunologic and biochemical tools to manipulate HSP 72 content in renal epithelial cells and to identify proteins that bind to it. Human HSP 72 deletion mutants with well-described defects in protein binding or re-folding will be used to determine the mechanism by which this molecular chaperone protects target proteins in ATP depleted cells. To evaluate some of the intracellular signals that induce HSP 72, we have developed a novel, permeabilized cell system in which intracellular pH, calcium and adenine nucleotide content can be individually manipulated. These studies will examine some of the pathways that permit HSP 72 to protect the renal epithelial cell and may ultimately provide a means for increasing cellular resistance to ischemic injury, a common cause of acute renal failure.