Emodin Protects Against Renal Ischemia Reperfusion Injury In Mice Via Mitochondrial Fission By Drp1 Phosphorylation

ObjectiveAcute kidney injury(AKI)is a serious threat to human health,mainly manifested as an.abrupt and acute deterioration of kidney function,resulting in the consequence that nitrogen waste(creatinine and urea nitrogen)cannot be excreted in the body,can only be accumulated in the body,and it is a common clinical emergency illness.Clinically,ischemia reperfusion injury is considered one of the most common contributors to the development of acute kidney injury,which due to kidney transplantation,heart surgery,renal artery stenosis and partial nephrectomy.However,renal tubular epithelial cells in renal tissue are more sensitive to ischemia hypoxia than other harmful factors,which are mainly manifested as renal tubular dilatation,loss of brush edge,tubular formation and cell degeneration and necrosis.Recent studies have shown that decreased mitochondrial fusion and increased mitochondrial fission in renal tubular epithelial cells lead to the destruction of mitochondrial homeostasis,the increased release of reactive oxygen species(ros),the caspase enzyme cascade reaction,and the cell apoptosis are the key causes of acute kidney injury.However,the underlying mechanism involved in mitochondrial fission,a key contributor to renal tubular cell death during AKI,remains largely obscured.It has been reported that emodin can alleviate the ischemia reperfusion injury of heart,brain and small intestine in rats and mice through anti-inflammatory.The aim of this study was to investigate whether emodin can interfere with acute renal injury induced by ischemia reperfusion and to elucidate the exact molecular mechanism of its regulation of mitochondrial dynamics.Methods and Results:In vivo,C57BL/6j male mice were used to establish a model of acute kidney ischemia reperfusion in mice,and Emodin was given and pretreated to detect its effects on renal tubular function and renal function.In vitro,hk-2 cells were cultured to simulate ischemia-reperfusion injury by hypoxia/reoxyge-nation,and changes in mitochondrial dynamic were observed.Experiment results showed that Emodin can significantly improve renal function after ischemia reperfusion injury and decrease serum creatinine and blood urea nitrogen levels.Renal injury score was obtained by H.E.staining of kidney tissue sections,indicating that Emodin can significantly reduce the score of renal injury after ischemia and reperfusion.Western blot analysis of Kiml,a marker of early renal injury,showed that emodin could reduce the degree of renal injury.Further test In vivo showed that in the model group,the generation of mitochondrial reactive oxygen species(mtROS)increased,leading to the release of pro-apoptotic protein cytochrome C,the activation of caspase3,the increase of Bax/Bcl2 ratio,and the increase of apoptosis.Emodin administration can reduce the production of mtROS,inhibit the release of pro-apoptotic protein cytochrome C,down-regulate the expression level of caspase3 protein,and reduce the ratio of Bax/Bcl2,thereby inhibiting apoptosis.We further found that after ischemia reperfusion treatment,mitochondrial fission increased and fusion decreased.Mitochondrial biogenesis decreased and autophagy increased.The expression of mitochondrial dynamic related protein(Drpl)and the phosphorylation level of Drpl Ser616 were significantly increased and mitochondrial homeostasis was destroyed.Emodin can reverse these changes.Consistent with the in vivo results above,emodin treatment can reduce apoptosis,reduce mitochondrial fragmentation,reduce the production of mtROS,inhibit mitochondrial fission,reduce the level of Drpl phosphorylation,mainly reduce the activation of Drpl Ser616 to reduce the damage of hk-2 cells.Further,we gave different kinase inhibitors to HK-2 cells,such as KN93,U0126,Licl,R03306,which were inhibitors of CaMK II kinase,U0126,GSK3 beta kinase and CDK1 kinase.Western blot experiments showed that Emodin could inhibit CaMK ? kinase alone,and inhibit CaMKII kinase together with KN93,which affected the phosphorylation of drpl ser616 siteConclusionOur present study demonstrated that Emodin exerted renoprotective function via down-regulating the expression of the phosphorylation of drpl ser616 caused by acute kidney injury,it can restore the dynamic balance of mitochondrial fusion and fission,maintain mitochondrial homeostasis,reduce mtROS generation and reduce apoptosis.