Manganese Superoxide Dismutase and Renal Ischemia/Reperfusion

锰超氧化物歧化酶与肾缺血/再灌注

• 批准号: 7886068
• 负责人: LEE A MACMILLAN-CROW
• 金额: $ 2.1万
• 依托单位: UNIV OF ARKANSAS FOR MED SCIS
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-01-16 至 2013-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7886068
• 关键词: Aging; Animals; Antioxidants; Atherosclerosis; Biological Assay; Blood Vessels; Cell Death; Cell Survival; Cells; Chronic rejection of renal transplant; Complex; Complication; Data; Down-Regulation; Electron Transport; Electron Transport Complex III; Electrons; Estradiol; Event; Excision; Functional disorder; Gene Delivery; Generations; Genetic; Goals; Heterozygote; Human; In Vitro; Injury; Ischemia; Kidney; Kidney Transplantation; Knockout Mice; Laboratories; Lead; Maintenance; Manganese; Manganese Superoxide Dismutase; Measurement; Mediating; Mitochondria; Mitochondrial Proteins; Modeling; Modification; Molecular; Mus; Mutant Strains Mice; Neurodegenerative Disorders; Operative Surgical Procedures; Organ; Organ Donor; Organ Transplantation; Oxidants; Pathologic; Pathway interactions; Pattern; Play; Porphyrins; Post-Translational Protein Processing; Preservation Technique; Prevention; Process; Production; Proteins; Proteomics; Proximal Kidney Tubules; Publications; Publishing; Rattus; Reagent; Renal function; Reperfusion Injury; Reperfusion Therapy; Research; Rodent; Role; Sepsis; Small Interfering RNA; Solid; Stroke; Superoxides; Techniques; Technology; Testing; Text; Therapeutic; Time; Transgenic Mice; Transplantation; Tubular formation; Tyrosine; Up-Regulation; Work; base; cell injury; delayed graft function; design; feeding; gel electrophoresis; graft function; implantation; improved; improved functioning; in vitro Model; in vivo; insight; kidney cell; mouse model; nitration; novel; oligomycin sensitivity-conferring protein; overexpression; oxidant stress; oxidative damage; prevent; protein complex; public health relevance; renal ischemia; research study; response; success

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.

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一个主要问题。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）和肾缺血再灌注过程中失活。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，这也得到了初步数据的支持。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加剧。 MnSOD 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，与这种损伤相关的机制途径仍然未知。令人兴奋的新研究集中于五个线粒体电子传递复合物，揭示了肾 I/R 后复合物 III、IV 和 V 的变化，这也将有助于线粒体氧化剂的产生。因此，我们假设：电子传输复合物是 I/R 过程中线粒体氧化损伤的目标，并且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旨在双向调节 MnSOD 表达的肾细胞，以及尖端的蛋白质组学分析，这将导致鉴定在肾缺血再灌注后损伤中发挥重要作用的关键线粒体靶标。假设 1：即使 MnSOD 活性适度降低（部分敲低），也会因肾缺血再灌注后氧化剂产生增加而导致线粒体复合物损伤。为了检验这一假设，将 MnSOD 敲低（使用 siRNA 技术和突变小鼠）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分析的测量相结合，以确定 MnSOD 敲低和 I/R 后与线粒体复合物损伤相关的精确靶标（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物功能，并减轻 I/R 后的肾损伤。为了检验这一假设，将 MnSOD 过表达（使用基因递送、转基因小鼠和雌二醇介导的诱导）与氧化剂生成、细胞活力、肾功能和线粒体蛋白质组分析的测量相结合，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 期间的肾损伤和 MnSOD 失活。我们最近发表的研究表明，对大鼠进行长期（24 小时）MnP 预处理可显着改善 I/R 期间的 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。维持足够的线粒体电子复合体功能对于正常的 ATP 产生至关重要。拟议的研究将首次确定肾缺血再灌注期间关键线粒体复合蛋白的修饰，并大力表征提供保护的 MnSOD 依赖性机制。最后，将评估两种增加肾脏 MnSOD 活性的试剂（雌二醇和锰卟啉）的治疗潜力，为肾移植相关的转化工作奠定基础。总之，这些发现可能有助于了解涉及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。