RIP3 translocation into Mitochondria promotes Mitofilin degradation to increase inflammation and kidney injury after renal ischemia/reperfusion

Abstract

Background. Acute kidney injury (AKI) is an independent risk factor for mortality and morbidity. Inflammation is now believed to play a major role in the pathophysiology of AKI. Receptor‐interacting protein kinase 3 (RIP3) is a member of the receptor‐interacting protein (RIP) family of serine/threonine protein kinases. RIP3 is a regulator of both programmed necrosis/necroptosis, an inflammatory form of cell death observed in pathogen‐induced and sterile inflammation, and TNFα‐induced apoptosis. In the induction of necroptosis, RIP3 is a major component of the tumor necrosis factor (TNF) receptor‐I signaling complex which through additional interactions with (RIP1) and pseudokinase mixed lineage kinase domain‐like protein (MLKL) forms the necrosome. More recently, RIP3 activity was found to promote sepsis‐induced AKI via mitochondrial dysfunction. Since I/R injury triggers numerous pathological changes, including apoptosis, oxidative stress, and inflammation, suggesting a role of mitochondrial function in RIP3‐dependent I/R injury.ObjectiveWe investigated whether RIP3 translocates into mitochondria in response to ischemia/reperfusion (I/R) to interact with Mitofilin and promote mitochondria damage that facilitates mtDNA release into the cytosol. We postulated that release of mtDNA activates cGAS/STING pathway leading to increased nuclear transcription of pro‐inflammatory markers that exacerbates renal I/R injury.Material and methods. C57/6N and RIP3‐/‐ mice as well as HK2 cells were used. Monolateral kidneys were subjected to 30 min of ischemia followed by either 12, 24, or 48 h of reperfusion. Protein levels of RIP3, Mitofilin, cGAS, STING, and p‐p65 were measured using Western and immunofluorescence analysis, while IL‐6, TNF‐α, and ICAM‐1 expressions as well as mtDNA release were assessed by qRT‐PCR, ELISA. Kidney function was measured by blood urea nitrogen and creatinine kits, and the interaction between RIP3 and Mitofilin was measured by Co‐Immunoprecipitation. In WT,Results. We found that renal I/R increased RIP3 levels, and its translocation into mitochondria. We observed that RIP3 interacts with Mitofilin likely promoting its degradation. While renal I/R associated with mitochondria damage, increased mtDNA release, activation of cGAS/STING/p65 pathway and increased transcription of pro‐inflammatory markers including IL‐6, TNF‐α and ICAM‐1, all these effects were decreased in RIP3‐/‐ mice. In HK‐2, RIP3 overexpression or Mitofilin knockdown increased cell death by activating the cGAS‐STING/p65 pathway.Conclusion. We demonstrated that kidney I/R increases RIP3 levels in the cytosol and it translocates into mitochondria, where it interacts with and promote Mitofilin degradation leading to increased mitochondrial structures damage and dysfunction. The subsequent increase in ROS production in mitochondria is postulated to facilitate mtDNA damage and release into the cytosol where it activates the cGAS/STING/p‐p65 pathway leading to amplified nuclear transcription of pro‐inflammatory markers that subsequently increase renal I/R injury. Together, this study point to an important role of RIP3 in the initiation and development of renal I/R injury.