Novel Forms of Cell Death During Acute Kidney Injury

描述(由申请人提供)： 越来越多的人认识到，多种途径以高度调控的方式导致坏死性细胞死亡，并受特定干预的影响。已涉及的过程包括与亲环素D(CypD)调控的线粒体通透性转变(MPT)相关的坏死性细胞死亡，由受体相互作用蛋白激酶1和3(RIPK1和RIPK3)介导的、对Necrostatin-1抑制敏感的坏死性下垂，以及由caspase 1和11激活引起的下垂。一些坏死的、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开放。对这一建议的初步研究表明，铁介导的细胞死亡的另一种途径被称为铁性下垂，这可能是几种常见的和临床相关的急性肾损伤(AKI)所涉及的铁相关细胞损伤的原因。铁下垂受到化学文库筛选中出现的新型小分子抑制剂、铁他汀类药物以及其他亲脂性抗氧化剂和NADPH氧化酶抑制的调节。这项提案的目的是进一步调查和澄清 现就铁下垂及其在体外新鲜分离的肾脏近端小管中的表达、对AKI的影响及其与坏死性下垂和亲环素D调节坏死通路的相互作用进行综述，以解决以下假说：铁下垂、CypD介导的MPT和坏死性下垂可导致AKI时肾小管细胞的杀伤，并且是药物干预的靶点。研究将利用兔和遭受损伤的小鼠的体外和体内模型小鼠AKI以及药物调节剂和CypD或RIPK3缺失的突变小鼠，新发育的CypD+RIPK3或Caspase+RIPK3缺失的小鼠，以及NADPH氧化酶4缺失的小鼠。胶原酶消化分离的小鼠肾小管将受到叔丁基氢过氧化氢或羟基喹啉加硫酸亚铁铵氧化和铁诱导的损伤，或缺氧/复氧。致死性膜损伤将被量化为最终终点，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，以评估这种损伤的机制以及铁他汀类药物、MPT和坏死性下垂的药物抑制剂以及途径蛋白的缺失对其影响的机制。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的作用，并为改善它们的方法和导致的器官衰竭提供洞察力。

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.