OMA1Mediates OPA1Proteolysis And Mitochondrial Fragmentation In Experimental Models Of Ischemic Kidney Injury

Objective:Acute kidney injury (AKI) is associated with mitochondrial fragmentation, which contributes to mitochondrial damage and tubular cell apoptosis. Mitochondrial fragmentation involves the cleavage of both mitochondrial outer and inner membranes. Cleavage of the outer membrane results from Drp-1-mediated fision activation and Bak-promoted fusion arrest, but the molecular mechanism of inner membrane cleavage remains elusive. OMA1-mediated proteolysis of OPA1, a key inner membrane fusion protein, was recently suggested to account for inner membrane cleavage during cell stress. In this study we mainly investigated (1) whether OMA1plays roles in the proteolysis of OPA1, mitochondrial fragmentation, apoptosis and kidney functions in kidneys with ischemia and reperfusion injury.(2) We also investigated how does OMA1release from the prohibitin ring complex by testing the interaction between Bif-1and PHB2.Methods:To mimic the ischemia and reperfusion in vivo, we used buffer with sodium azide to treat rat renal proximal tublar cells (RPTC) that were stablely transfected with OMA1-shRNA or scramble-shRNA for2.5hours. Cells were then incubated with fresh RPTC medium for3hours. Apoptosis was observed with nuclear morphology, caspase3cleavage. The mitochondrial dynamics were evaluated with Cyt-c release, OPA1proteolysis and mitochondrial fragmentation. We used co-IP to detect the interaction of Bif-1and PHB2. Use Native Gel Electrophoresis for separating the prohibitin complex and its degraded proteins. Examine the expression of Bax in cytoplasm and mitochondria to observe its translocation. For in vivo experimental models of ischemic AKI, both wild-type and OMA1knock-out mice were subjected with25minutes of bilateral ischemia. Kidney functions were determined by values of BUN and serum creatinine as well as the expression of Kim-1. Kidney apoptosis and necrosis were measured with TUNEL and H&E staining respectively. Mitochondrial fragmentation was examed by electronic microscope. Cyt-c release and OPA1proteolysis were detected by immunoblot. Results:knockdown of OMA1suppressed OPA1proteolysis, mitochondrial fragmentation, cytochrome c release and consequent apoptosis in renal proximal tubular cells. In mice, OMA1-deficiency prevented ischemic AKI as indicated by better renal function, less tubular damage, and lower apoptosis. OPA1proteolysis and mitochondrial injury during ischemic AKI were ameliorated in OMA1-deficient mice.Co-IP and electronic microscopy results suggested that Bif-1interacts with PHB2when cells have stress. The native gel electrophoresis result confirmed that bif-1disrupted the ring complex assembled by prohibitin-1(PHB1) and prohibitin-2(PHB2). Bif-1regulated cell apoptosis, mitochondrial fragmention and OPA1proteolysis.Conclusions:1. OMA1-mediated OPA1proteolysis plays an important role in the disruption of mitochondrial dynamics in ischemic AKI.2. Bif-1interacts with PHB2to release OMA1, which functionates inducing mitochondrial fragmentation and cell apoptosis.