CYTOPROTECTIVE ROLE OF HSP 72 IN RENAL CELL INJURY

缺血或ATP耗竭等应激可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，HSP72含量最丰富，特性最好。热休克蛋白72是一种分子伴侣，可以去除和修复受损的细胞蛋白。在非肾脏细胞和组织中，选择性地过表达HSP72可以减少随后因缺血或ATP耗竭而造成的损伤。在这些模型中，热休克蛋白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HSPs，导致肌动蛋白细胞骨架崩溃，失去紧密连接的完整性，损害线粒体ATP的产生，并引发细胞凋亡。我们之前已经证明，先前的热应激足以上调HSP72，保留了这些细胞功能，并改善了ATP耗竭后肾细胞的存活率。该项目的中心假设是HSP72是一种主要的肾脏细胞保护剂。我们认为，HSP72通过与调节(A)细胞骨架、(B)细胞-细胞接触部位、(C)天然DNA、(D)线粒体功能和(E)凋亡途径的蛋白质结合，减少细胞损伤并抑制由ATP耗竭引起的细胞凋亡。这一假设得到了我们的初步研究的支持，在这些研究中，选择性地诱导(或抑制)HSP72单独改变了ATP耗竭的肾上皮细胞的存活率。在一个类似于体内缺血/再灌注的体外ATP耗竭模型中，我们将：(1)鉴定受HSP72保护的细胞功能；(2)鉴定与HSP72相互作用的一些细胞内蛋白；(3)确定蛋白质结合与折叠在介导HSP72介导细胞保护中的作用；以及(4)评估在不需要热应激的情况下即可诱导HSP72的细胞内信号(S)。我们已经开发了分子、免疫学和生化工具来操纵肾上皮细胞中HSP72的含量，并识别与其结合的蛋白质。具有蛋白质结合或重折叠缺陷的人HSP 72缺失突变体将被用来确定这种分子伴侣保护ATP耗竭细胞中的目标蛋白质的机制。为了评估诱导HSP72的一些细胞内信号，我们开发了一种新的渗透性细胞系统，在该系统中细胞内的pH、钙和腺嘌呤核苷酸含量可以单独控制。这些研究将研究允许HSP72保护肾上皮细胞的一些途径，并最终可能提供一种提高细胞对缺血损伤的抵抗力的方法，缺血损伤是急性肾功能衰竭的常见原因。

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.