Cytoprotective Role of HSP72 in Renal Cell Injury

HSP72 在肾细胞损伤中的细胞保护作用

• 批准号: 8078160
• 负责人: STEVEN C. BORKAN
• 金额: $ 25.1万
• 依托单位: BOSTON MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-07-01 至 2013-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8078160
• 关键词: ATP phosphohydrolase; ATPase Domain; Acute; Acute Kidney Failure; Animals; Apoptosis; Apoptotic; BCL-2 Protein; BCL2 gene; Binding; Biochemical; Blood flow; Cell Death; Cell Nucleolus; Cell Nucleus; Cell Survival; Cells; Cessation of life; Cytoprotective Agent; Cytosol; Data; Epithelial Cells; Epitopes; Equilibrium; Event; Family; Functional disorder; Goals; Harvest; Hexokinase 2; In Vitro; Injury; Ischemia; Kidney; Kidney Failure; Knock-out; Knockout Mice; Laboratories; Life; Measures; Mediating; Mitochondria; Modeling; Molecular Chaperones; Molecular Probes; Mus; Necrosis; Nuclear; Nuclear Export; Nuclear Import; Nuclear Localization Signal; Organ; Organ failure; Outer Mitochondrial Membrane; Pathway interactions; Peptides; Phosphorylation; Phosphotransferases; Protein phosphatase; Proteins; Renal function; Role; Serine; Signal Transduction; Site; Specificity; Stress; Testing; Therapeutic Intervention; Transgenic Mice; Transgenic Organisms; Treatment Efficacy; Tubular formation; base; cell injury; genetic regulatory protein; in vivo; inhibitor/antagonist; kidney cell; member; mitochondrial membrane; mutant; novel; nucleophosmin; prevent; public health relevance; receptor; renal ischemia; response; trafficking

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.

描述(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显示，在体外ATP耗竭和体内肾缺血后，Hsp70是Bax和Bcl2的主要调节因子，导致Bax：Bcl2平衡向促凋亡方向急剧变化。使用一种新的合成Bax底物，我们证明了ATP耗尽会导致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)，一种新发现的Bax伴侣蛋白在细胞内积累；己糖激酶II(HK II)从线粒体中移位；以及Bcl2，一种强有力的Bax拮抗剂的失活和降解。新的证据表明，Hsp70与细胞死亡途径中的每一个关键步骤相互作用，并可能调节这些关键步骤。在Hsp70基因敲除或Hsp70杂合子小鼠中，缺血性肾功能衰竭比Hsp70完全对照小鼠严重得多，肾小管损伤更严重，Bax活化和Bcl2降解更明显。此外，从这些动物采集并在原代培养中生长的近端小管细胞对体内缺血后的ATP耗竭具有类似的敏感性。Hsp70基因缺陷细胞对ATP耗竭的敏感性可以通过选择性地加入Hsp70而完全恢复。因此，我们假设Hsp70通过以下方式干扰Bax的激活：(1)改变Bax激活所需的丝氨酸磷酸化事件；(2)干扰NPM介导的Bax激活；(3)将HK II重新靶向线粒体膜以防止Bax攻击；和/或(4)保留Bcl2在应激反应中抑制Bax的能力。这些假说将在已建立的HSP70过表达转基因、HSP70基因敲除和Hsp70杂合子小鼠身上、在这些动物的原代培养中、在永生化的小鼠近端小管细胞中以及在分离的线粒体中进行验证。在这些研究中，我们将利用具有特定模拟磷化和磷酸缺失变化的特征良好的Bax突变体，具有缺陷靶向的核磷蛋白突变体，以及缺乏伴侣或ATPase结构域或无法运输到细胞核或核仁的Hps70缺失突变体。诱导Hsp70(GGA)或直接抑制Bax通道(Bci1和Bci2)的药物将用于小鼠，以证明它们在预防或治疗缺血性肾功能衰竭方面的疗效。通过研究Hsp70在细胞损伤途径中的调控步骤，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功能衰竭的治疗干预措施提供新的靶点。 公共卫生相关性： 急性肾功能衰竭是一种常见的、通常是危及生命的器官血流量减少(缺血)的后果。目前，导致肾脏衰竭的机制尚不清楚，因此，还没有特效的治疗方法。我们建议研究一种内在的细胞保护蛋白Hsp70在防止肾细胞死亡和器官衰竭中的作用。这些研究不仅将确定急性缺血性肾损伤的一些机制，而且可能为治疗提供新的靶点。