The role of mitochondrial dysfunction in ARDS after AKI

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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.