The role of mitochondrial dysfunction in ARDS after AKI

急性肾损伤(AKI)发生在30%的危重退伍军人中，6年死亡率更高 比50%1，22。有趣的是，AKI的主要死亡原因是非肾脏并发症和呼吸系统并发症。 衰竭是AKI最严重的非肾脏并发症，预后比所有其他远隔器官都要差。 失败4.现在人们认识到，除了容量过载之外，AKI还有呼吸衰竭的机制， 如发展为急性呼吸窘迫综合征(ARDS)2-3。探讨其发病机制。 AKI引起的ARDS可能导致新的策略的发展，以提高AKI和ARDS的死亡率。 关于机理方面的考虑，肾脏是体内一个高度新陈代谢的器官，具有 线粒体含量异常高，因此线粒体功能障碍是各种形式的关键因素 AKI5，7.AKI中所见的线粒体功能障碍导致线粒体碎片化，线粒体功能受损 传播全身炎症和损伤的相关分子模式(DAMP)12，13.线粒体 DNA(MtDNA)是最广为人知的mtDAMP，已被证明可促进先天性免疫和 Toll样受体9(TLR9)激活引起的全身性炎症18.线粒体DNA也可引起肺损伤 当静脉注射给健康动物时18。我假设线粒体功能障碍是一种 急性肾损伤致急性呼吸窘迫综合征的发病机制。具体地说，1)AKI的线粒体功能障碍导致mtDNA 碎裂并释放到循环中。2)肾脏释放的循环线粒体DNA会导致肺损伤。 通过TLR9激活肺细胞和中性粒细胞。Aki患者也有两倍的可能需要 与没有AKI6的患者相比，线粒体DNA损伤是一种已知的机制 呼吸机诱导的肺损伤(VILI)20.我进一步假设AKI会增加VILI和VILI的易感性 AKI通过加强肺和肾两个器官的线粒体功能障碍而加重这两个器官的损伤。 我将利用AKI的缺血再灌注(IR)模型来检验这些假说。线粒体 IR-AKI后将对肺和肾脏的功能、动力学、自噬和细胞死亡进行评估。线粒体DNA 将评估尿液、血浆和支气管肺泡灌洗液(BALF)中的浓度。详细的、机械的研究 肺上皮细胞、内皮细胞、肺泡巨噬细胞和中性粒细胞的线粒体DNA 在体外进行。静脉和气管内给予mtDNA将在C57BL/6和 体内TLR9基因敲除小鼠。免疫印迹、酶联免疫吸附试验、定量聚合酶链式反应、透射电子显微镜、流式细胞仪 通过FlexiVent®啮齿动物呼吸机、XF96 SeaHorse®细胞外流量进行分析、肺力学评估 Analyzer和FITC菊粉动力学将用于评估肺和肾脏损伤、线粒体功能和 IR-AKI后线粒体DNA释放。我还将研究有无机械通风对小鼠的反应。 IR-AKI评价VILI对AKI后肺、肾损伤及线粒体功能的影响。 最后，我将尝试使用线粒体DNA修复酶来减轻肺-肾损伤。 我的近期目标是研究线粒体功能障碍是AKI所致ARDS的一个机制。 相信这些研究将导致新的治疗方法，使患有危重疾病的退伍军人受益。我的 长期的职业目标是培养成为退伍军人管理局独立内科医生-科学家所需的技能 致力于肺和肾的相互作用。我在照顾退伍军人方面有很强的临床背景- 集约主义者，以及两年的专职研究培训。在过去的两年里，我制作并出版了 支持我的假设的新数据，并获得了世界级、多学科、指导的支持 在退伍军人事务部圣地亚哥的团队，将在基础、翻译和临床研究方面对我进行培训。研究性培训 建议在此应用程序中结合AKI、ARDS和线粒体生物学方面的教学培训 为我提供必要的工具来发展我作为退伍军人圣地亚哥大学内科科学家的独立性。

Acute kidney injury (AKI) occurs in 30% of critically ill veterans, and the 6-year mortality rate is greater than 50%1,22. Interestingly, the leading causes of death in AKI are non-renal complications, and respiratory failure is the most serious non-renal complication of AKI with a worse prognosis than all other remote organ failures4. It is now recognized that there are mechanisms of respiratory failure in AKI beyond volume overload, such as the development of the acute respiratory distress syndrome (ARDS)2-3. Investigating the mechanisms of ARDS due to AKI may lead to the development of novel strategies to improve the mortality of AKI and ARDS. Regarding mechanistic considerations, the kidney is a highly metabolic organ in the body with an exceptionally high mitochondrial content, therefore mitochondrial dysfunction is a key element in various forms of AKI5,7. Mitochondrial dysfunction as seen in AKI leads to fragmented mitochondria which function as damage associated molecular patterns (DAMPs) that propagate systemic inflammation and injury12,13. Mitochondrial DNA (mtDNA) is the most well-described mtDAMP, and has been shown to promote innate immunity and systemic inflammation via activation of toll-like receptor 9 (TLR9)18. MtDNA is also known to cause lung injury when administered intravenously to healthy animals18. I hypothesize that mitochondrial dysfunction is a mechanism of ARDS due to AKI. Specifically, 1) Mitochondrial dysfunction in AKI leads to mtDNA fragmentation and release into circulation. 2) Circulating mtDNA released from the kidney leads to lung injury via TLR9 activation on pulmonary cells and neutrophils. AKI patients are also twice as likely to require mechanical ventilation compared to patients without AKI6, and mtDNA damage is a known mechanism of ventilator induced lung injury (VILI)20. I further hypothesize that AKI increases susceptibility to VILI, and VILI after AKI worsens lung and kidney injury by potentiating mitochondrial dysfunction in both organs. I will utilize the ischemia-reperfusion (IR) model of AKI to test these hypotheses. Mitochondrial function, dynamics, autophagy, and cell death will be evaluated in the lung and kidney after IR-AKI. MtDNA levels will be evaluated in urine, plasma, and bronchoalveolar lavage fluid (BALF). Detailed, mechanistic studies of mtDNA on pulmonary epithelial and endothelial cells, alveolar macrophages, and neutrophils will be performed in vitro. Intravenous and intratracheal mtDNA administration will be evaluated in C57BL/6 and TLR9 knockout mice in vivo. Western blot, ELISA, quantitative PCR, transmission electron microscopy, FACS analysis, lung mechanics assessments via a flexiVent® rodent ventilator, XF96 Seahorse® extracellular flux analyzer, and FITC-inulin kinetics will be used to assess lung and kidney injury, mitochondrial function, and mtDNA release after IR-AKI. I will also investigate response to mechanical ventilation in mice with and without IR-AKI to evaluate the potential role of VILI on lung and kidney injury and mitochondrial function after AKI. Finally, I will attempt to mitigate lung-kidney injury using a mtDNA repair enzyme. My immediate goal is to investigate mitochondrial dysfunction as a mechanism of ARDS due to AKI as I believe that these studies will lead to novel therapies that will benefit veterans suffering with critical illness. My long term career goal is to develop the skills needed to become an independent physician-scientist at the VA dedicated to lung-kidney interactions. I have a strong clinical background in caring for veterans as a pulmonary- intensivist, and 2 years of dedicated research training. During the past 2 years I have produced and published novel data supporting my hypotheses, and garnered support from a world-class, multidisciplinary, mentoring team at the VA San Diego that will train me in basic, translational, and clinical research. The research training proposed in this application combined with didactic training in AKI, ARDS, and mitochondrial biology will provide me with the tools necessary to develop my independence as a physician-scientist at the VA San Diego.