The role of mitochondrial dysfunction in ARDS after AKI

急性肾损伤（阿基）发生在30%的危重退伍军人中，6年死亡率更大 50%以上1.22有趣的是，阿基死亡的主要原因是非肾脏并发症和呼吸系统并发症。 衰竭是阿基最严重的非肾脏并发症，其预后比所有其他远端器官更差 失败4.现在认识到，除了容量超负荷，阿基中还有呼吸衰竭的机制， 例如急性呼吸窘迫综合征（ARDS）的发展2-3。调查的机制， 阿基引起的ARDS可能导致新策略的开发，以提高阿基和ARDS的死亡率。 关于机制考虑，肾脏是体内的高度代谢器官， 异常高的线粒体含量，因此线粒体功能障碍是各种形式的关键因素， AKI 5.7在阿基中观察到的线粒体功能障碍导致线粒体片段化， 相关分子模式（DAMP）传播全身炎症和损伤12，13。线粒体 DNA（mtDNA）是最好描述的mtDAMP，并已被证明可以促进先天免疫， 通过激活Toll样受体9（TLR 9）的全身性炎症18。线粒体DNA也会导致肺损伤 当静脉注射给健康动物时18.我假设线粒体功能障碍是 阿基引起的ARDS机制。具体而言，1）阿基中的线粒体功能障碍导致mtDNA 碎片化并释放到流通中。2)从肾脏释放的循环mtDNA导致肺损伤 通过肺细胞和中性粒细胞上的TLR 9活化。阿基患者需要药物治疗的可能性也是其他患者的两倍 与无AKI 6的患者相比，机械通气是一种已知的机制， 呼吸机诱发的肺损伤（VILI）20.我进一步假设阿基增加了对VILI的易感性，并且VILI 在阿基后，通过增强两个器官中的线粒体功能障碍来减轻肺和肾损伤。 我将利用阿基的缺血再灌注（IR）模型来验证这些假设。线粒体 在IR-AKI后，将在肺和肾中评价功能、动力学、自噬和细胞死亡。mtDNA 将在尿液、血浆和支气管肺泡灌洗液（BALF）中评价水平。详细的机制研究 线粒体DNA在肺上皮细胞和内皮细胞、肺泡巨噬细胞和中性粒细胞上的表达， 在体外进行。将在C57 BL/6和C57 BL/6中评价静脉和静脉内mtDNA给药。 体内TLR 9敲除小鼠。Western blot、ELISA、定量PCR、透射电镜、FACS 分析，通过flexiVent®啮齿动物呼吸机进行肺力学评估，XF 96 Seahorse®细胞外流量 分析仪和FITC-菊粉动力学将用于评估肺和肾损伤、线粒体功能和 IR-AKI后mtDNA释放。我还将研究小鼠对机械通气的反应， IR-AKI，以评估VILI对阿基后肺和肾损伤以及线粒体功能的潜在作用。 最后，我将尝试使用mtDNA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作为阿基所致ARDS的机制， 相信这些研究将导致新的疗法，将有利于退伍军人患有严重疾病。我 长期职业目标是发展成为VA独立的物理学家-科学家所需的技能 致力于肺肾相互作用。我在照顾退伍军人方面有很强的临床背景- intensivist，和2年的专门研究培训。在过去的两年里，我制作和出版了 新的数据支持我的假设，并获得了世界级的支持，多学科，指导 在弗吉尼亚州圣地亚哥的团队，将培训我在基础，转化，和临床研究。科研训练 在本申请中提出的结合阿基、ARDS和线粒体生物学的教学培训将 为我提供必要的工具，以发展我作为一个在VA圣地亚哥的物理学家，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.