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: 9275417
• 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/9275417
• 关键词: Acute Renal Failure with Renal Papillary Necrosis; Address; Ammonium Sulfate; Antioxidants; Appearance; Automobile Driving; CASP1 gene; Caring; Caspase; Cell Death; Cell membrane; Cells; Cessation of life; Cisplatin; Clinical; Closure by clamp; Complex; Data; Development; Digestion; Event; Functional disorder; Glycerol; Glycine; Hypoxia; Individual; Injury; Intervention; Ion Channel; Iron; Ischemia; Kidney Diseases; Laboratories; Lipid Peroxidation; Lipid Peroxides; 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; Pharmacology; 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; cell injury; cell killing; cell type; clinically relevant; collagenase; cost; cyclophilin D; immunogenic; improved; in vivo; in vivo Model; inhibitor/antagonist; insight; lipophilicity; mitochondrial membrane; mitochondrial permeability transition pore; mouse model; novel; public health relevance; screening; small molecule inhibitor; 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）： 人们越来越认识到，多种途径以高度调控的方式导致坏死性细胞死亡，并且适合特定的干预措施。所涉及的过程包括与亲环蛋白 D (CypD) 调节的线粒体通透性转变 (MPT) 发育相关的坏死性细胞死亡、受体相互作用蛋白激酶 1 和 3（RIPK1 和 RIPK3）介导的坏死性凋亡并对 necrostatin-1 的抑制敏感，以及半胱天冬酶 1 和 caspase 激活导致的细胞焦亡。 11. 几种坏死性免疫原性过程所需的最终下游事件是甘氨酸敏感的质膜通道打开。该提案的初步研究表明，铁介导的细胞死亡的另一种途径称为“铁死亡”，可能是几种常见和临床相关形式的急性肾损伤（AKI）中涉及的铁相关细胞损伤的原因。铁死亡受到化学文库筛选中出现的新型小分子抑制剂、铁他汀以及其他亲脂性抗氧化剂和 NADPH 氧化酶抑制的调节。该提案的目的是进一步调查和澄清 铁死亡的机制及其在离体新鲜分离的肾近曲小管中的表达，其对体内 AKI 的影响，及其与坏死性凋亡和亲环蛋白 D 调节性坏死途径的相互作用，以解决铁死亡的累加效应、CypD 介导的 MPT 诱导和坏死性凋亡导致的调节性坏死的假设 在 AKI 期间有助于杀死肾小管细胞，是药物干预的目标。研究将利用来自遭受离体损伤的兔子和小鼠的肾小管以及小鼠 AKI 的体内模型以及药理调节剂和缺乏 CypD 或 RIPK3 的突变小鼠、缺乏 CypD+RIPK3 或 caspase+RIPK3 的新开发的小鼠以及缺乏 NADPH 氧化酶 4 的小鼠。 遭受叔丁基过氧化氢或羟基喹啉加硫酸亚铁铵引起的氧化剂和铁诱导的损伤，或缺氧/复氧。致命的膜损伤将被定量作为最终终点，并且在损伤之前的时间点，将测量 ATP、线粒体​​膜电位、活性氧产生和脂质过氧化，以评估这种损伤的机制及其由铁他汀、MPT 和坏死性凋亡的药理学抑制剂以及途径蛋白的缺失引起的修饰。 AKI 将通过甘油诱导的横纹肌溶解、钳夹缺血或顺铂在体内产生，然后评估肾功能和结构变化。这些研究将进一步阐明 AKI 期间新表征的调节性坏死形式的作用，并深入了解改善这些坏死的方法以及由此导致的器官衰竭。