Manganese Superoxide Dismutase and Renal Ischemia/Reperfusion

描述（由申请人提供）：肾缺血/再灌注（I/R）是肾移植或大血管手术后导致肾损伤的主要问题。我们的实验室已经证明，线粒体中的主要抗氧化剂锰超氧化物歧化酶（MnSOD）在肾移植（人和啮齿动物）和肾I/R期间失活。这些数据表明，MnSOD活性的丧失可能是导致随后肾功能不全的一个关键事件，这得到了初步数据的支持，初步数据显示MnSOD的诱导（通过基因递送和雌二醇预处理）保护肾脏免受I/R损伤。相反，令人信服的新数据表明，MnSOD的下调（使用MnSOD杂合子（-/+）转基因小鼠）导致线粒体和肾损伤的增加。MnSOD的失活导致线粒体产生超氧化物，并可能导致线粒体损伤;然而，与这种损伤有关的机制途径仍然未知。令人兴奋的新的研究，重点是五个线粒体电子传递复合物，揭示了在复杂的III，IV和V肾I/R，这也将有助于线粒体氧化剂的生产后的改变。因此，我们假设：电子传递复合物是I/R过程中线粒体氧化损伤的靶点，对特定复合物的损伤是MnSOD失活导致的关键下游事件。我们将使用新型转基因小鼠模型和设计用于双向调节MnSOD表达的肾细胞，沿着最先进的蛋白质组学分析，以鉴定在肾I/R后损伤中发挥重要作用的关键线粒体靶点。假设1.由于肾I/R后氧化剂产生增加，即使MnSOD活性的适度降低（部分敲低）也会导致线粒体复合体损伤。为了检验这一假设，MnSOD敲除（使用siRNA技术和突变小鼠）将与氧化剂生成、线粒体完整性、细胞活力、肾功能和线粒体蛋白质组学分析的测量相结合，以确定MnSOD敲除和I/R后线粒体复合物损伤的精确靶标（复合物和/或复合物亚基）和途径。假设2. MnSOD活性增加可减少氧化剂的产生，恢复正常的线粒体复合体功能，并减轻I/R后的肾损伤。为了验证这一假设，MnSOD过表达（使用基因递送、转基因小鼠和雌二醇介导的诱导）将与氧化剂生成、细胞活力、肾功能和线粒体蛋白质组学分析的测量相结合，以确定介导MnSOD诱导保护I/R损伤的机制。假设3.新一代催化抗氧化剂锰卟啉（MnP）通过稳定线粒体电子传递复合物来减轻I/R期间的肾损伤和MnSOD失活。我们最近发表的研究表明，大鼠长期（24小时）MnP预处理可显著改善I/R期间MnSOD活性和肾功能（附录2）。新的研究将确定MnP是否通过保持线粒体电子传递复合物的完整性来防止缺血期间线粒体超氧化物的产生，从而维持正常的线粒体ATP水平。公共卫生相关性：项目叙述/相关性：该项目的重点是确定线粒体氧化剂增加如何导致缺血/再灌注后肾损伤。线粒体电子复合体功能的维持对于正常的ATP产生是必不可少的。拟议的研究将首次确定肾I/R期间关键线粒体复合物蛋白的修饰，并有力地表征提供保护的MnSOD依赖性机制。最后，两种试剂（雌二醇和锰卟啉），增加肾脏MnSOD活性的治疗潜力，将进行评估，为相关的肾移植的翻译工作的基础。总之，这些发现可以提供对涉及线粒体氧化剂产生的其他病理条件的深入了解，包括动脉粥样硬化、中风、神经退行性疾病、衰老和败血症。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: PROJECT NARRATIVE/RELEVANCE: The focus of this project is to determine how increased mitochondrial oxidants lead to renal injury after ischemia/reperfusion. Maintenance of adequate mitochondrial electron complex function is essential for normal ATP production. The proposed studies will, for the first time, identify modifications of key mitochondrial complex proteins during renal I/R, and vigorously characterize the MnSOD-dependent mechanisms that offer protection. Finally, the therapeutic potential of two reagents (estradiol and manganese porphyrin), which increase renal MnSOD activity, will be evaluated to set a basis for translational work relevant to renal transplantation. In summary, these findings may provide insight into other pathologic conditions involving mitochondrial oxidant production including atherosclerosis, stroke, neurodegenerative diseases, aging, and sepsis.