CYTOPROTECTIVE ROLE OF HSP72 IN RENAL CELL INJURY

描述（由申请人提供）：体内肾缺血和体外ATP消耗导致肾上皮细胞凋亡。细胞凋亡是由线粒体膜损伤引起的，这是由BCL2蛋白家族的成员调控的。我们已经证实，在肾细胞中ATP耗竭会增加bax（促凋亡），降低bcl2（抗凋亡）并导致细胞凋亡。伴随bcl2:bax比值的这种促凋亡变化，细胞色素c和凋亡诱导因子（AIF）从线粒体转移到细胞质中，III态线粒体呼吸减少。胞浆细胞色素c激活半胱天冬酶，而AIF进入细胞核，激活内切酶并引起DNA降解。前期热应激足以诱导分子伴侣hsp72，稳定bcl2:bax比，减少细胞色素c和AIF泄漏，减少caspase激活和DNA降解，完全保留器官功能，提高ATP耗尽后的长期存活。hsp72的选择性过表达模拟了热应激对细胞凋亡和肾细胞存活的保护作用。hsp72结合bcl2（而不是bax）、细胞色素c和AIF的事实表明，hsp72本身是一种抗凋亡蛋白。我们假设hsp72降低了bax对线粒体膜的原发性损伤，并分别抑制了泄漏的细胞色素c和AIF对caspase激活和DNA降解的继发性促凋亡作用。在分离细胞器和完整细胞中，本研究将：(1)确定hsp72对bax介导的线粒体膜损伤的细胞保护是否需要bcl2；(2)评估hsp72挽救bcl2功能，阻止caspase激活和/或抑制aif介导的DNA降解的能力；(3)确定hsp72特定结构域(s)负责其抗凋亡作用。为了实现这些目标，将利用腺病毒感染已建立的载体，包括野生型人hsp72、反义和已知的具有特定功能缺陷的hsp72缺失突变体，来操纵肾上皮细胞中hsp72、bax和bcl2的含量。这些研究将确定hsp72的抗凋亡机制。这些见解可以促进预防性策略的发展，以防止缺血损伤期间肾细胞损伤以及其他形式的肾损伤，其中细胞凋亡导致器官衰竭。

DESCRIPTION (provided by applicant): Renal ischemia in vivo and ATP depletion in vitro cause renal epithelial cells to die by apoptosis. Apoptosis is caused by mitochondrial membrane injury that is regulated by well-characterized members of the BCL2 protein family. We have established that ATP depletion in renal cells increases bax (pro-apoptotic), decreases bcl2 (anti-apoptotic) and causes apoptosis. Concomitant with this pro-apoptotic change in the bcl2:bax ratio, cytochrome c and apoptosis inducing factor (AIF) translocate from mitochondria into the cytosol and state III mitochondrial respiration is reduced. Cytosolic cytochrome c activates caspases, whereas AIF enters the nucleus, activates endonucleases and causes DNA degradation. Prior heat stress, sufficient to induce hsp72, a molecular chaperone, stabilizes the bcl2:bax ratio, decreases cytochrome c and AIF leak, reduces caspase activation and DNA degradation, completely preserves organeile function and enhances long term survival after ATP depletion. The selective over-expression of hsp72 mimics the protective effects of prior heat stress on apoptosis and renal cell survival. The fact that hsp72 binds bcl2 (but not bax), cytochrome c and AIF suggests that hsp72 itself is an anti-apoptotic protein. We hypothesize that hsp72 decreases primary injury to the mitochondrial membrane caused by bax and inhibits the secondary, pro-apoptotic effects of leaked cytochrome c and AIF on caspase activation and DNA degradation, respectively. In isolated organelles and intact ceils, this study will: (1) determine whether cytoprotection by hsp72 against bax-mediated mitochondrial membrane injury requires bcl2; (2) evaluate the ability of hsp72 to rescue bcl2 function, prevent caspase activation and/or to inhibit AIF-mediated DNA degradation and (3) identify the specific hsp72 domain(s) responsible for its anti-apoptotic actions. To achieve these AIMS, hsp72, bax and bcl2 content will be manipulated in renal epithelial cells using adenoviral infection with established vectors including wild type human hsp72, antisense and known hsp72 deletion mutants with specific functional defects. These studies will identify the anti-apoptotic mechanism(s) of hsp72. These insights can prompt the development of preemptive strategies for preventing renal cell injury during ischemic insults as well as other forms of renal injury in which apoptosis contributes to organ failure.