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: 7781435
• 负责人: STEVEN C. BORKAN
• 金额: $ 25.35万
• 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/7781435
• 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 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（由申请人提供）：尽管大量证据表明Bax和Bcl 2（BCL 2家族的两个拮抗成员）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BCL 2蛋白的细胞内信号尚不清楚。来自我们实验室的初步数据显示，Hsp 70是体外ATP耗竭和体内肾缺血后Bax和Bcl 2的主要调节剂，所述损伤显著地将Bax：Bcl 2平衡向促凋亡方向转移。使用一种新的合成Bax底物，我们表明，ATP耗竭导致Akt和GSK 3b活性的变化，促进Bax激活。压力还导致：核磷蛋白（NPM）的胞质积累，一种新描述的Bax伴侣;己糖激酶II（HK II）从线粒体的置换;以及Bcl 2（一种有效的Bax拮抗剂）的失活和降解。新的证据表明，热休克蛋白70与细胞死亡途径中的每一个关键步骤相互作用，并可能调节这些步骤。在Hsp 70敲除或Hsp 70杂合小鼠中，缺血性肾衰竭远比Hsp 70完全对照严重，肾小管损伤更大，Bax活化和Bcl 2降解更明显。此外，从这些动物中收获并在原代培养物中生长的近端小管细胞对体内缺血后观察到的ATP耗竭具有平行的敏感性。选择性添加Hsp 70可以完全挽救Hsp 70缺陷细胞对ATP耗尽的敏感性。因此，我们假设Hsp 70通过以下方式干扰Bax活化：（1）修饰Bax活化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重新靶向线粒体膜以防止“Bax攻击”;和/或（4）保留Bcl 2响应于应激而抑制Bax的能力。这些假设将在已建立的Hsp 70过表达转基因小鼠、Hsp 70敲除小鼠和Hsp 70杂合小鼠中、在源自这些动物的原代培养物中、在永生化小鼠近端小管细胞中以及在分离的线粒体中进行测试。在这些研究中，我们将利用具有特异性磷酸模拟和磷酸缺失变化的充分表征的Bax突变体、具有缺陷性靶向的核磷蛋白突变体和缺乏伴侣蛋白或ATP酶结构域或不能运输到细胞核或核仁中的Hps 70缺失突变体。将诱导Hsp 70（GGA）或直接抑制Bax通道（Bci 1和Bci 2）的试剂给予小鼠，以证明它们在预防或治疗缺血性肾衰竭中的治疗功效。通过检查细胞损伤途径中Hsp 70调节的步骤，这些研究将表征BCL 2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措施提供新的靶点。 公共卫生关系： 急性肾衰竭是一种常见的，往往是危及生命的后果减少血流量（缺血）的器官。目前，导致肾脏衰竭的机制尚不清楚，因此没有具体的治疗方法。我们建议研究内在的细胞保护蛋白Hsp 70在预防肾细胞死亡和器官衰竭中的作用。这些研究不仅有助于阐明急性缺血性肾损伤的发病机制，而且可能为治疗提供新的靶点。