Cellular Pathophysiology of Acute Renal Failure

描述（由申请人提供）：缺血性和中毒性急性肾衰竭仍然是住院患者发病和死亡的重要原因，并大大增加了护理费用。肾近端小管是急性肾功能衰竭的主要损伤部位，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，近端小管细胞的糖酵解不存在或很少。当离体近端小管在体内缺血/再灌注相关条件下遭受缺氧/再氧化时，它们会出现严重的能量缺陷，其特征是持续的ATP消耗和再氧化过程中线粒体膜电位的恢复受损，这对小管细胞的存活和从损伤中恢复起关键作用。能量不足可以通过补充特定的柠檬酸循环代谢物来改善。当前资助期的工作表明，线粒体电子传递、腺嘌呤核苷酸转位酶或f1fo - atp酶的异常不能轻易解释这种缺陷。相反，它似乎主要归因于非酯化脂肪酸（NEFA）产生的线粒体失能。降低NEFA可用性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设，探讨其机制及其对小管细胞损伤的影响，我们计划：1)阐明NEFA穿梭于线粒体内膜阴离子载体在NEFA诱导的线粒体膜电位耗散中的作用。2)确定UCP2是否参与NEFA效应。3)评价恢复线粒体膜电位的药物降低NEFA的效果。4)量化介导线粒体膜电位消散的游离脂肪酸水平。5)评估NEFA诱导的质子泄漏的程度以及NEFA在呼吸抑制中的作用。6)验证nefa诱导的线粒体膜电位耗散是否能解释能量亏缺的基质凝聚特征，以及凝聚本身是否进一步损害线粒体功能。7)进一步研究线粒体活性氧的产生及其被NEFA修饰在能量赤字中的作用。8)表征小管中线粒体通透性转变的表达，NEFA对其的修饰，及其对线粒体功能障碍进展的贡献。这些研究与理解和治疗缺血性急性肾功能衰竭，保存肾脏和其他器官用于移植，以及对线粒体在所有细胞类型的坏死和凋亡细胞死亡中所起的关键作用的基本理解有关。

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.