Cellular Pathophysiology of Acute Renal Failure

描述(申请人提供)：缺血性和中毒性急性肾功能衰竭仍然是住院患者发病率和死亡率的重要原因，并极大地增加了护理费用。肾近端小管是急性肾功能衰竭时的主要损伤部位，其ATP的产生对线粒体功能障碍特别敏感，因为根据不同的节段，近端小管细胞中没有糖酵解或糖酵解很少。当离体近端小管在与体内缺血/再灌注相关的条件下遭受缺氧/复氧时，它们会出现严重的能量缺陷，其特征是持续的ATP耗竭和复氧过程中线粒体膜电位的恢复，这在肾小管细胞的存活和损伤后的恢复中起着关键作用。能量缺乏可以通过特定的补充柠檬酸循环代谢物来改善。目前资助期的工作表明，这种赤字不能很容易地用线粒体电子传递、腺核苷酸转位酶或F1FO-ATPase的异常来解释。相反，它似乎主要归因于非酯化脂肪酸(NEFA)产生的线粒体去能。降低NEFA的利用率可以恢复线粒体膜电位和ATP的产生，这是补充底物提供益处的主要机制。为了进一步验证这一假说，研究NEFA对肾小管上皮细胞损伤的机制和意义，我们计划：1)阐明NEFA在线粒体内膜阴离子载体上穿梭在NEFA诱导的线粒体膜电位耗散中的作用。2)确定UCP2是否参与了NEFA效应。3)评估恢复线粒体膜电位的药物降低NEFA的效果。4)定量测定参与线粒体膜电位耗散的游离脂肪酸水平。5)评估NEFA引起的质子泄漏的程度以及NEFA在呼吸抑制中的作用。6)测试NEFA引起的线粒体膜电位的耗散是否解释了能量缺失的基质凝聚特征，以及凝结本身是否进一步损害了线粒体的功能。7)进一步研究线粒体活性氧的产生和NEFA的修饰在能量缺乏中的作用。8)研究线粒体通透性转变在肾小管上皮细胞中的表达、NEFA的修饰及其在线粒体功能障碍进展中的作用。这些研究对于了解和治疗缺血性急性肾功能衰竭，保存肾脏和其他器官以供移植，以及对线粒体在所有细胞类型的坏死性和凋亡性细胞死亡过程中所起的关键作用的基本理解是相关的。

DESCRIPTION (provided by applicant): Ischemic and toxic acute renal failure remain important causes of morbidity and mortality in hospitalized patients and greatly increase the expense of care. ATP production in the kidney proximal tubule, a major site of injury during acute renal failure, is especially sensitive to mitochondrial dysfunction because, depending on the segment, glycolysis is absent or minimal in proximal tubule cells. When isolated proximal tubules are subjected to hypoxia/reoxygenation under conditions relevant to ischemia/reperfusion in vivo, they develop a severe energetic deficit characterized by persistent ATP depletion and impaired recovery of mitochondrial membrane potential during reoxygenation, which plays a pivotal role in tubule cell survival and recovery from the insult. The energetic deficit can be ameliorated by specific, supplemental citric acid cycle metabolites. Work during the current funding period has shown that the deficit cannot be readily explained by abnormalities of mitochondrial electron transport, the adenine nucleotide translocase or the F1FO-ATPase. Instead, it appears to be primarily attributable to mitochondrial de-energization produced by nonesterified fatty acids (NEFA). Lowering NEFA availability restores mitochondrial membrane potential and ATP production and is a major mechanism for the benefit provided by the supplemental substrates. To further test this hypothesis and investigate the mechanisms involved and the implications for tubule cell injury, we plan to: 1) Clarify the role of NEFA shuttling on mitochondrial inner membrane anion carriers in the mediation of NEFA-induced dissipation of mitochondrial membrane potential. 2) Determine whether UCP2 is involved in the NEFA effects. 3) Assess the NEFA lowering efficacy of agents that restore mitochondrial membrane potential. 4) Quantify the free fatty acid levels mediating dissipation of mitochondrial membrane potential. 5) Assess the magnitude of the NEFA- induced proton leak and the role of NEFA in respiratory inhibition. 6) Test whether NEFA-induced dissipation of mitochondrial membrane potential accounts for the matrix condensation characteristic of the energetic deficit and whether condensation itself further impairs mitochondrial function. 7) Further investigate the role of mitochondrial reactive oxygen species production and its modification by NEFA in the energetic deficit. 8) Characterize expression of the mitochondrial permeability transition in the tubules, its modification by NEFA, and its contribution to progression of mitochondrial dysfunction. These studies are relevant to understanding and treating ischemic acute renal failure and preserving kidneys and other organs for transplantation and to the basic understanding of the critical role that mitochondria are now recognized to play during both necrotic and apoptotic cell death in all cell types.