Manganese Superoxide Dismutase and Renal Ischemia/Reperfusion

摘要： 肾缺血/再灌注(I/R)是导致肾脏损害的主要问题 在肾移植或大血管手术后。我们的实验室有 证明了线粒体中的主要抗氧化剂，超氧化物锰 在肾移植(人和啮齿动物)过程中被灭活。 肾I/R这些数据表明，MnSOD活性的丧失可能是一个关键事件 这会导致随后的肾功能障碍，初步数据支持这一点 显示MnSOD的诱导(通过基因传递和雌二醇预处理)具有保护作用 I/R损伤所致的肾脏。相反，令人信服的新数据表明，下调监管 (使用MnSOD杂合子(-/+)转基因小鼠)的结果是增加了 线粒体和肾脏损伤。MnSOD失活导致线粒体生成 超氧化物和线粒体损伤；然而，机制 与这种损伤有关的途径尚不清楚。令人兴奋的新研究集中在 在五个线粒体电子传递复合体上，揭示了 肾I/R后的复合体III、IV和V，这也有助于线粒体 氧化剂生产。因此，我们假设：电子传输络合物是靶标。 线粒体氧化剂在I/R期间的损伤以及对特定复合体的损伤是 由于MnSOD失活而导致的关键下游事件(S)。 我们将使用新的转基因小鼠模型和设计成双肾细胞。 定向调节MnSOD的表达，以及前沿的蛋白质组分析 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.即使是轻微的MnSOD活性降低(部分基因敲除) 由于氧化剂产生增加而导致线粒体复合体损伤 肾I/R后，为了验证这一假说，MnSOD基因敲除(使用siRNA 技术和突变小鼠)将与氧化剂的测量相结合 世代、线粒体完整性、细胞存活率、肾功能和线粒体 蛋白质组学分析以确定精确的靶点(复合体和/或亚单位 复合体)和参与线粒体复合体损伤的途径 MnSOD击倒和I/R 假设2.增加的MnSOD活性减少氧化剂的产生，恢复 正常的线粒体复合体功能，并减轻I/R后的肾脏损伤。 测试这一假设，MnSOD过度表达(使用基因传递、转基因小鼠和 雌激素介导的诱导)将与氧化剂的测量相结合 世代、细胞活力、肾功能和线粒体蛋白质组学分析 确定介导对MnSOD所致I/R损伤的保护机制 归纳法。 假设3：新一代催化抗氧化剂锰卟啉 (MNP)通过稳定肾缺血再灌注期肾脏损伤和MnSOD失活 线粒体电子传递复合体。我们最近发表的研究表明 长期(24小时)给予MNP可显著提高大鼠的MnSOD I/R期间的活动和肾功能(附录2)。新的研究将确定 MNP通过保护线粒体超氧化物歧化酶防止线粒体在缺血时产生超氧化物 线粒体电子传递复合体的完整性，从而维持正常 线粒体三磷酸腺苷水平。

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.