Manganese Superoxide Dismutase and Renal Ischemia/Reperfusion

摘要： 肾缺血/再灌注（I/R）是导致肾损伤的主要问题 肾移植或大血管手术后。本实验室 证明了线粒体中的主要抗氧化剂超氧化锰 在肾移植过程中（人和啮齿动物）， 肾I/R。这些数据表明MnSOD活性的丧失可能是一个关键事件 导致随后的肾功能障碍，这得到了初步数据的支持， 显示MnSOD的诱导（通过基因递送和雌二醇预处理）保护了 I/R损伤的肾脏相反，令人信服的新数据显示，下调 的MnSOD（使用MnSOD杂合子（-/+）转基因小鼠）导致增加 线粒体和肾损伤。MnSOD失活导致线粒体生成 超氧化物和可能的线粒体损伤;然而， 与这种损伤有关的途径仍然未知。令人兴奋的新研究， 在五个线粒体电子传递复合物上， 肾I/R后的复合物III、IV和V，这也有助于线粒体 氧化剂生产。因此，我们假设：电子传递复合物是靶点 I/R过程中线粒体氧化损伤，对特定复合物的损伤是 由MnSOD失活引起的关键下游事件。 我们将使用新的转基因小鼠模型和肾细胞， 定向调节MnSOD表达，沿着前沿蛋白质组学分析 这将导致识别关键的线粒体靶点，这些靶点在 肾I/R后损伤。 假设1.即使MnSOD活性适度降低（部分敲低） 导致线粒体复合体损伤 肾I/R后。为了验证这一假设，MnSOD敲低（使用siRNA 技术和突变小鼠）将与氧化剂的测量相结合， 生成、线粒体完整性、细胞活力、肾功能和线粒体 蛋白质组学分析，以确定精确的目标（复合物和/或亚基的 复合物）和参与线粒体复合物损伤的途径 MnSOD敲除和I/R。 假设2. MnSOD活性增加可减少氧化剂的产生， 正常的线粒体复合体功能，并减轻I/R后的肾损伤。到 测试这一假设，MnSOD过表达（使用基因传递，转基因小鼠， 雌二醇介导的诱导）将与氧化剂的测量相结合 代，细胞活力，肾功能和线粒体蛋白质组学分析， 确定MnSOD介导I/R损伤保护的机制 诱导 假设3.新一代催化抗氧剂锰卟啉 (MnP)I/R期间，通过稳定 线粒体电子传递复合物。我们最近发表的研究表明， 长期（24小时）预处理可显著提高MnSOD活性 I/R期间的活动和肾功能（附录2）。新的研究将确定是否 MnP通过保护线粒体膜蛋白，防止缺血期间线粒体超氧化物的产生。 线粒体电子传递复合物的完整性，从而维持正常的 线粒体ATP水平。

ABSTRACT: Renal ischemia/reperfusion (I/R) is a major problem leading to kidney damage following renal transplantation or major vascular surgery. Our laboratory has demonstrated that the major antioxidant in the mitochondria, manganese superoxide dismutase (MnSOD), is inactivated during renal transplantation (human and rodent) and renal I/R. These data suggested that the loss of MnSOD activity may be one key event that results in subsequent renal dysfunction, which is supported by preliminary data showing that induction of MnSOD (via gene delivery and estradiol pretreatment) protects the kidney from I/R injury. Conversely, compelling new data show that downregulation of MnSOD (using MnSOD heterozygote (-/+) transgenic mice) results in augmentation of mitochondrial and renal injury. Inactivation of MnSOD results in mitochondrial generation of superoxide and presumably mitochondrial damage; however, the mechanistic pathways involved with this injury remain unknown. Exciting new studies which focused on the five mitochondrial electron transport complexes, revealed alterations in Complexes III, IV, and V following renal I/R, which would also contribute to mitochondrial oxidant production. Thus, we hypothesize that: Electron transport complexes are targets of mitochondrial oxidant damage during I/R and that damage to specific complexes are the critical downstream event(s) that result from inactivation of MnSOD. We will use novel transgenic mouse models and renal cells designed to bi- directionally modulate MnSOD expression, along with cutting-edge proteomic analysis that will lead to identification of key mitochondrial targets that play a fundamental role in injury following renal I/R. Hypothesis 1. Even modest reductions in MnSOD activity (partial knockdown) lead to mitochondrial complex damage due to increased oxidant production following renal I/R. To test this hypothesis, MnSOD knockdown (using siRNA technology and mutant mice) will be combined with measurements of oxidant generation, mitochondrial integrity, cell viability, renal function, and mitochondrial proteomic analyses to determine the precise targets (complexes and/or subunits of complexes) and pathways involved with mitochondrial complex damage following MnSOD knockdown and I/R. Hypothesis 2. Increased MnSOD activity reduces oxidant production, restores normal mitochondrial complex function, and blunts renal injury following I/R. To test this hypothesis, MnSOD overexpression (using gene delivery, transgenic mice, and estradiol-mediated induction) will be combined with measurements of oxidant generation, cell viability, renal function, and mitochondrial proteomic analyses to determine the mechanisms that mediate protection from I/R injury due to MnSOD induction. Hypothesis 3. The new generation catalytic antioxidant manganese porphyrin (MnP) blunts renal injury and MnSOD inactivation during I/R via stabilization of mitochondrial electron transport complexes. Our recent published studies show that the long-term (24 hr) pretreatment of rats with MnP significantly improved MnSOD activity and renal function during I/R (Appendix 2). New studies will determine whether MnP prevents mitochondrial superoxide production during ischemia by preserving the integrity of the mitochondrial electron transport complexes, hence maintaining normal mitochondrial ATP levels.