Novel Forms of Cell Death During Acute Kidney Injury

急性肾损伤期间细胞死亡的新形式

• 批准号: 8735503
• 负责人: JOEL M. WEINBERG
• 金额: --
• 依托单位: VETERANS HEALTH ADMINISTRATION
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-10-01 至 2018-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8735503
• 关键词: Accounting; Acute Renal Failure with Renal Papillary Necrosis; Address; Antioxidants; Appearance; Automobile Driving; Caring; Caspase; Caspase-1; Cell Death; Cell membrane; Cells; Cessation of life; Cisplatin; Clinical; Complex; Data; Development; Digestion; Event; Functional disorder; Glycerol; Glycine; Hypoxia; Individual; Injury; Intervention; Ion Channel; Iron; Ischemia; Kidney Diseases; Laboratories; Lipid Peroxidation; Measurement; Mediating; Membrane; Membrane Potentials; Methodology; Mitochondria; Modification; Mus; Mutant Strains Mice; NADPH Oxidase; Necrosis; Organ failure; Oryctolagus cuniculus; Oxidants; Pathogenesis; Pathway interactions; Patients; Pattern; Permeability; Play; Prevention; Process; Production; Protective Agents; Protein Kinase; Proteins; Proximal Kidney Tubules; RIPK1 gene; RIPK3 gene; Reactive Oxygen Species; Regulation; Renal function; Renal tubule structure; Reperfusion Therapy; Rhabdomyolysis; Role; Testing; Time; Tissues; Tubular formation; Work; ammonium ferrous sulfate; cell injury; cell killing; clinically relevant; collagenase; cost; cyclophilin D; human RIPK1 protein; immunogenic; improved; in vivo; in vivo Model; inhibitor/antagonist; insight; mitochondrial membrane; mitochondrial permeability transition pore; novel; public health relevance; screening; small molecule; small molecule libraries; tert-Butylhydroperoxide

DESCRIPTION (provided by applicant): It is being increasingly recognized that multiple pathways contribute to necrotic cell death in a highly regulated fashion and are amenable to specific interventions. Processes that have been implicated include necrotic cell death related to development of the mitochondrial permeability transition (MPT) regulated by cyclophilin D (CypD), necroptosis mediated by receptor-interacting protein kinases 1 and 3 (RIPK1 and RIPK3) and sensitive to inhibition by necrostatin-1, and pyroptosis resulting from activation of caspases 1 and 11. The ultimate downstream event required for several of the necrotic, immunogenic processes is glycine-sensitive opening of a plasma membrane channel. Preliminary studies for this proposal show that an additional pathway of iron-mediated cell death termed 'ferroptosis' may account for the iron-related cell injury involved in several common and clinically relevant forms of acute kidney injury (AKI). Ferroptosis is subject to modulation by novel small molecule inhibitors that emerged from chemical library screening, ferrostatins, as well as by other lipophilic antioxidants and by inhibition of NADPH oxidase. The objectives of this proposal are to further investigate and clarify the mechanisms of ferroptosis and its expression in freshly isolated kidney proximal tubules ex vivo, its impact on AKI in vivo, and its interactions with necroptosis and cyclophilin D pathways of regulated necrosis to address the hypothesis that regulated necrosis resulting from additive effects of ferroptosis, induction of the MPT mediated by CypD, and necroptosis contributes to tubule cell killing during AKI and is a target for pharmacological intervention. Studies will utilie tubules from rabbits and mice subjected to injury ex vivo and in vivo models of mouse AKI along with pharmacologic modulators and mutant mice deficient in CypD or RIPK3, newly developed mice deficient in both CypD+RIPK3 or caspase+RIPK3, and mice deficient in NADPH oxidase 4. Tubules isolated by collagenase digestion will be subjected to oxidant and iron-induced injury by tert-butylhydroperoxide or by hydroxyquinoline plus ferrous ammonium sulfate, or to hypoxia/reoxygenation. Lethal membrane damage will be quantitated as the final endpoint, and, at time points preceding that damage, measurements of ATP, mitochondrial membrane potential, reactive oxygen species production, and lipid peroxidation will be made to assess the mechanisms of this injury and its modification by ferrostatins, pharmacologic inhibitors of the MPT and necroptosis, and absence of pathway proteins. AKI will be produced in vivo by glycerol-induced rhabdomyolysis, clamp ischemia, or cisplatin followed by assessment of renal function and structural changes. These studies will further elucidate the role of newly characterized forms of regulated necrosis during AKI and provide insight into approaches for ameliorating them and the organ failure that results.

描述（由申请人提供）： 越来越多的人认识到，多种途径以高度调节的方式促进坏死细胞死亡，并且适合于特定的干预措施。涉及的过程包括与亲环蛋白D（CypD）调节的线粒体通透性转换（MPT）相关的坏死性细胞死亡，受体相互作用蛋白激酶1和3（RIPK 1和RIPK 3）介导的坏死性凋亡，对坏死抑素-1的抑制敏感，以及半胱天冬酶1和11激活引起的细胞凋亡。最终的下游事件所需的几个坏死，免疫原性的过程是甘氨酸敏感的质膜通道的开放。对这一提议的初步研究表明，铁介导的细胞死亡的另一种途径，称为“铁凋亡”，可能是几种常见的和临床相关形式的急性肾损伤（阿基）中涉及的铁相关细胞损伤的原因。铁蛋白缺乏症受到来自化学文库筛选的新型小分子抑制剂铁蛋白抑制素以及其他亲脂性抗氧化剂和NADPH氧化酶抑制剂的调节。本提案的目的是进一步调查和澄清 铁凋亡的机制及其在离体新鲜分离的肾近端小管中的表达，其在体内对阿基的影响，及其与坏死凋亡和亲环素D调节性坏死途径的相互作用，以阐明由铁凋亡，CypD介导的MPT的诱导，并且坏死性凋亡在阿基期间有助于小管细胞杀伤，并且是药物干预的靶点。研究将利用来自遭受损伤的兔和小鼠的肾小管以及小鼠阿基的离体和体内模型，沿着CypD或RIPK 3缺陷的药理学调节剂和突变小鼠、CypD+ RIPK 3或半胱天冬酶+RIPK 3缺陷的新开发小鼠和NADPH氧化酶4缺陷的小鼠。通过胶原酶消化分离的小管将受到氧化剂和铁诱导的损伤，通过叔丁基过氧化氢或羟基喹啉加硫酸亚铁铵，或缺氧/复氧。致死性膜损伤将被定量为最终终点，并且在损伤之前的时间点，将进行ATP、线粒体膜电位、活性氧产生和脂质过氧化的测量，以评估该损伤的机制及其通过铁抑制素、MPT和坏死性凋亡的药理学抑制剂以及途径蛋白的缺乏的修饰。通过甘油诱导的横纹肌溶解、钳夹缺血或顺铂在体内产生阿基，然后评估肾功能和结构变化。这些研究将进一步阐明阿基期间新表征的调节性坏死形式的作用，并提供改善它们的方法和导致的器官衰竭的见解。