Manganese Superoxide Dismutase and Renal Ischemia/Reperfusion

描述（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脏损害的主要问题。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）和肾脏I/R过程中失活。这些数据表明，MnSOD活性的丧失可能是导致随后肾功能障碍的一个关键事件，初步数据表明MnSOD的诱导（通过基因传递和雌二醇预处理）可以保护肾脏免受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）会导致线粒体和肾损伤的增加。MnSOD失活导致线粒体产生超氧化物，可能导致线粒体损伤；然而，与这种损伤有关的机制途径尚不清楚。令人兴奋的新研究集中在五种线粒体电子传递复合物上，揭示了肾脏I/R后复合物III， IV和V的改变，这也有助于线粒体氧化剂的产生。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目标，而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，以及尖端的蛋白质组学分析，这将导致鉴定在肾I/R损伤中起基本作用的关键线粒体靶点。假设1。即使是MnSOD活性的适度降低（部分敲低）也会导致肾脏I/R后氧化剂产生增加而导致线粒体复合物损伤。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相结合，以确定MnSOD敲低和I/R后线粒体复合体损伤的精确靶点（复合体和/或复合体亚基）和途径。假设2。MnSOD活性的增加减少了氧化剂的产生，恢复了正常的线粒体复合体功能，并减轻了I/R后的肾损伤。为了验证这一假设，MnSOD过表达（使用基因传递、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相结合，以确定介导MnSOD诱导引起的I/R损伤保护的机制。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过程中肾损伤和MnSOD失活。我们最近发表的研究表明，长期（24小时）预处理MnP大鼠可显著改善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，从而维持正常的线粒体ATP水平，从而在缺血期间阻止线粒体超氧化物的产生。公共卫生相关性：项目描述/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。维持足够的线粒体电子复合物功能对于正常的ATP生成至关重要。拟议的研究将首次确定肾脏I/R过程中关键线粒体复合物蛋白的修饰，并大力表征提供保护的mnsod依赖机制。最后，我们将评估两种增加肾脏MnSOD活性的试剂（雌二醇和卟啉锰）的治疗潜力，为肾移植相关的转化工作奠定基础。总之，这些发现可能为了解线粒体氧化剂产生的其他病理状况，包括动脉粥样硬化、中风、神经退行性疾病、衰老和败血症提供线索。

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.