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脏损伤的主要问题。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的肾功能障碍的一个关键事件，这一点得到了初步数据的支持，即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，下调MnSOD(使用MnSOD杂合子(-/)转基因小鼠)会导致线粒体和肾脏损伤的加剧。MnSOD的失活会导致线粒体产生超氧化物，并可能导致线粒体损伤；然而，参与这种损伤的机制仍不清楚。令人兴奋的新研究集中在五个线粒体电子传递复合体上，揭示了肾脏I/R后复合体III、IV和V的变化，这也有助于线粒体氧化剂的产生。因此，我们假设：电子传递复合体是线粒体在I/R过程中氧化损伤的靶标，而特定复合体的损伤是MnSOD失活所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旨在双向调节MnSOD表达的肾脏细胞，以及尖端的蛋白质组学分析，这将导致识别在肾脏I/R损伤中发挥基础作用的关键线粒体靶点。即使是MnSOD活性的轻微降低(部分敲除)也会导致线粒体复合体的损伤，这是由于肾I/R后氧化剂产生的增加。为了验证这一假设，将结合氧化剂生成、线粒体完整性、细胞活力、肾功能的测量来验证这一假说。和线粒体蛋白质组分析，以确定与MnSOD击倒和I/R后线粒体复合体损伤有关的精确靶点(复合体和/或复合体亚单位)和途径。假说2.增加MnSOD活性可减少氧化剂产生，恢复正常线粒体复合体功能，并钝化I/R后的肾脏损伤。为了验证这一假说，将结合氧化剂生成、细胞存活率、肾功能和线粒体蛋白质组分析的测量结果，结合氧化剂生成、细胞存活率、肾功能和线粒体蛋白质组分析来确定介导MnSOD诱导的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体来钝化I/R期间的肾脏损伤和MnSOD失活。我们最近发表的研究表明，长期(24小时)给予MNP的大鼠在I/R期间显著改善了MnSOD活性和肾功能(附录2)。新的研究将确定，MNP是否通过保护线粒体电子传递复合体的完整性，从而维持正常的线粒体ATP水平，来防止缺血期间线粒体超氧化物歧化的产生。公共卫生相关性：项目叙述/相关性：本项目的重点是确定线粒体氧化剂增加如何导致缺血/再灌流后的肾脏损伤。维持适当的线粒体电子复合体功能对于正常的ATP生产是至关重要的。这项拟议的研究将首次确定肾脏I/R期间关键线粒体复合蛋白的修饰，并有力地表征提供保护的MnSOD依赖机制。最后，将评估两种提高肾脏MnSOD活性的试剂(雌二醇和锰卟啉)的治疗潜力，为肾移植相关的翻译工作奠定基础。综上所述，这些发现可能提供了对其他涉及线粒体氧化剂产生的病理情况的洞察，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.