The Role And Mechanism Of DGKE In Acute Kidney Injury Induced By Ischemia/Reperfusion

Acute kidney injury(AKI)is a serious clinical complication with high morbidity and mortality worldwide,which is characterized by rapid decline of renal function and accumulation of metabolic wastes.AKI has become a global public health problem in now days.Renal ischemia/reperfusion injury(IRI)is one of the major causes of AKI for patients subjected to renal vascular occlusion,renal transplantation,etc.Despite substantial progress in understanding the pathophysiology of ischemic AKI,the mechanisms contributing to ischemic AKI remains unclear.Except for the plasma exchange therapy,there is no satisfactory clinical treatment available to prevent or treat ischemic AKI.Therefore,it is not only essential and but also urgent to investigate novel key molecules related to AKI and to develop innovative clinical strategies.Diacylglycerol kinases(DGKs)are important lipid kinases,which may have important biological functions via phosphorylating one signal molecule,diacylglycerol(DAG)to another,phosphatidic acid(PA).Diacylglycerol kinase E(DGKE)is the only member of type III isozymes,which has distinctive primary structure and DAG preference,showing substrate specificity substrate specificity toward arachidonate-containing DAG.Accumulating clinical researches have shown that recessive loss-of-function mutations in DGKE caused several kidney deseases,suggesting that DGKE is a vital target for unraveling mechanisms of some kidney diseases.However,by far,reports about DGKE mainly focus on detection of genetic polymorphism and there still lacks systematic researches,especially researches about the role and mechanism of DGKE in ischemic AKI.ObjectiveIn this study,we will use DGKE-knockin mice to investigate the role of DGKE in acute kidney injury induced by ischemia/reperfusion and explore the biological mechanisms.Methods and Results1.The expression pattern of DGKE acute kidney injury induced by ischemia/reperfusion and its cell localizationHealthy wild type(WT)C57BL/6J(eight-to-twelve-weeks-old)male mice were randomly divided into two groups-sham-operation group(sham)and renal ischemia/reperfusion injury model group(IRI).Bilateral renal pedicles of mice in experimental ischemia/reperfusion injury model group were clipped for 30 min by using microaneurysm clamps(FST)and then the vascular clamps were removed and the kidneys were allowed to reflow.A successful ischemia/reperfusion injury model was confirmed by increased serum creatinine and urea,exacerbated morphology injury as well as increased expression of KIM-1.RT-PCR and western blot analyses showed that compared to sham,DGKE expression levels were significantly increased in the kidney after renal ischemia/reperfusion injury,which was further confirmed in paraffin-embedded sections of kidney tissues by immunohistochemical staining.The results of double immunofluorescence labeling method showed that DGKE was mainly localized in proximal tubules and there was a small amount of expression in distal tubules,but no evident of DGKE expression in collecting ducts.Besides,DGKE was also expressed in infiltrated inflammatory cells under ischemia/reperfusion condition.Different cell lines derived from kidney were cultured in vitro.The results showed that DGJ.E was expressed in various cells of renal tubules and glomeruli.Three different methods were used to mimic ischemia-reperfusion injury in vivo and we found that under hypoxic conditions,the expression of DGKE in renal tubular epithelial cells(HK-2)was increased,which is in consistent with in vivo studies.2.DGKE protects against acute kidney injury induced by ischemia/reperfusionTo elucidate the role and mechanism of DGKE in acute kidney injury induced by ischemia/reperfusion,we constructed DGKE knockin mice(DGKEkI),and use WT littermates as controls.Experimental ischemia/reperfusion injury models were established as described previously.Compared with their wild-type counterparts,the DGKEKI mice had significantly lower levels of serum creatinine and urea as well as KIM-1.Kidneys from DGKEKI group also showed less morphological injury,characterized by improved forms of brush border and more living tubular cells.Similarly,TUNEL staining also exhibited less apoptotic cells in the kidneys after IRI in DGKEKI mice than that in the controls.DGKE overexpression reduced the levels of proinflammatory mediators by real-time polymerase chain reaction and IHC analyses in the kidneys after I/R.Consistently,neutrophil and macrophage accumulation was further decreased in the kidney from ischemic DG1E}1 mice,1ndicating that overexpression of DGKE alleviates renal inflammation.These results suggest that DGKE plays a protective role in acute kidney injury induced by ischemia/reperfusion.3.The mechanisms of the protective role of DGKE in acute kidney injury indruced by ischemia/reperfusionIn order to explore the further mechanisms,Gene Chip,RT-PCR,Western blot and IHC methods were used for seeking for the downstream gene of DGKE.We found that under ischemia/reperfusion conditions,the expression of klotho in WT mice was significantly decreased,and overexpression of DGKE may partly recover the decreased expression levels of klotho in the ischemia/reperfusion kidney.Hence,our results indicate that the protective role of DGKE in acute kidney injury induced by ischemia/reperfusion might be involved in the upregulation of klotho.ConclusionIn summary,our study firstly found that DGKE plays a protective role in acute kidney injury induced by ischemia/reperfusion,which may be related to the upregulation of klotho.Our findings provide a better understanding of biological activities of DGKE in the kidney and suggest that DGKE may be an innovative therapeutic strategy for treating acute kidney injury induced by ischemia/reperfusion in clinic.