Cytoprotective Role of HSP72 in Renal Cell Injury

描述（由申请人提供）：尽管大量证据表明Bcl2家族的两个拮抗成员Bax和Bcl2是缺血后线粒体膜损伤和细胞死亡的主要原因，但调节这些Bcl2蛋白的细胞内信号尚不清楚。我们实验室的初步数据表明，Hsp70是体外ATP耗竭和体内肾缺血后Bax和Bcl2的主要调节因子，这种损伤显著地改变了Bax:Bcl2的平衡，使其向促凋亡方向发展。利用一种新的合成Bax底物，我们发现ATP消耗导致Akt和GSK3b活性的变化，从而促进Bax的激活。应激还导致：核磷蛋白（NPM）的细胞质积累，这是一种新发现的Bax伴侣；己糖激酶II （HK II）从线粒体中移位；Bcl2的失活和降解，Bcl2是一种有效的Bax拮抗剂。新的证据表明，Hsp70与细胞死亡途径中的这些关键步骤相互作用，并可能调节这些关键步骤。在Hsp70敲除或Hsp70杂合小鼠中，缺血性肾功能衰竭远比Hsp70完全对照组严重，肾小管损伤更大，Bax激活和Bcl2降解更为明显。此外，从这些动物身上获得的近端小管细胞和在原代培养中培养的细胞在体内缺血后对ATP消耗具有相似的敏感性。Hsp70缺陷细胞对ATP耗竭的敏感性可以通过选择性添加Hsp70完全恢复。因此，我们假设Hsp70通过以下两种方式干扰Bax的激活：(1)修改Bax激活所需的丝氨酸磷酸化事件；(2)干扰npm介导的Bax活化；(3)将HK II重新靶向线粒体膜，防止“Bax攻击”；和/或(4)保持Bcl2在应激反应中抑制Bax的能力。这些假设将在已建立的Hsp70过表达转基因小鼠、Hsp70敲除小鼠和Hsp70杂合小鼠、来源于这些动物的原代培养物、永活小鼠近端小管细胞和分离的线粒体中进行验证。在这些研究中，我们将利用具有特异性拟磷和磷酸化缺失变化的Bax突变体，靶向缺陷的核磷蛋白突变体，以及缺乏伴侣或atp酶结构域或无法进入细胞核或核仁的Hps70缺失突变体。将诱导Hsp70 （GGA）或直接抑制Bax通道（Bci1和Bci2）的药物给予小鼠，以证明其预防或治疗缺血肾衰的治疗效果。通过研究hsp70调控的细胞损伤通路中的步骤，这些研究将表征BCL2蛋白诱导细胞死亡的机制，并为开发预防或治疗急性缺血性肾衰竭的治疗干预提供新的靶点。

DESCRIPTION (provided by applicant): Although substantial evidence implicates Bax and Bcl2, two antagonistic members of the BCL2 family, as a primary cause of mitochondrial membrane injury and cell death after ischemia, the intracellular signals that regulate these BCL2 proteins are not known. Preliminary data from our laboratory show that Hsp70 is a major regulator of Bax and Bcl2 after ATP depletion in vitro and renal ischemia in vivo, insults that dramatically shift the Bax:Bcl2 balance in a pro-apoptotic direction. Using a novel synthetic Bax substrate, we show that ATP depletion causes changes in Akt and GSK3b activity that promote Bax activation. Stress also results in: cytosolic accumulation of nucleophosmin, (NPM) a newly described Bax chaperone; displacement of hexokinase II (HK II) from mitochondria; and inactivation and degradation of Bcl2, a potent Bax antagonist. New evidence shows that Hsp70 interacts with, and potentially regulates each of these key steps in the cell death pathway. In Hsp70 knockout or Hsp70 heterozygous mice, ischemic renal failure is far more severe, tubular injury greater, and Bax activation and Bcl2 degradation are more pronounced than in Hsp70 replete control. Furthermore, proximal tubule cells harvested from these animals and grown in primary culture have parallel sensitivity to ATP depletion as observed after ischemia in vivo. The sensitivity of Hsp70 deficient cells to ATP depletion can be completely rescued by the selective addition of Hsp70. We therefore hypothesize that Hsp70 interferes with Bax activation either by: (1) modifying serine phosphorylation events required for Bax activation; (2) interfering with NPM-mediated Bax activation; (3) re-targeting HK II to the mitochondrial membrane to prevent "Bax attack"; and/or (4) preserving the ability of Bcl2 to inhibit Bax in response to stress. These hypotheses will be tested in established Hsp70 over-expressing transgenic, Hsp 70 knockout and Hsp70 heterozygous mice, in primary culture derived from these animals, in immortalized murine proximal tubule cells, and in isolated mitochondria. In these studies, we will utilize well- characterized Bax mutants with specific phosphomimetic and phospho-deletion changes, nucleophosmin mutants with defective targeting, and Hps70 deletion mutants that either lack the chaperone or ATPase domains or are unable to traffic into nuclei or nucleoli. Agents that either induce Hsp70 (GGA) or directly inhibit the Bax channel (Bci1 and Bci2) will be administered to mice to demonstrate their therapeutic efficacy in preventing or treating ischemia renal failure. By examining Hsp70-regulated steps in the cell injury pathway, these studies will characterize the mechanisms of BCL2 protein-induced cell death and provide new targets for developing therapeutic interventions to prevent or treat acute ischemic renal failure. PUBLIC HEALTH RELEVANCE: Acute kidney failure is a common and often a life-threatening consequence of reduced blood flow (ischemia) to the organ. At present, the mechanisms that cause the kidney to fail are unclear and as a result, there are no specific treatments. We propose to study the role of an intrinsic, cytoprotective protein, Hsp70, in preventing kidney cell death and organ failure. These studies will not only identify some of the mechanisms of acute ischemic kidney injury, but also may provide new targets for therapy.