Preliminary Study Of Necroptosis Mechanism In Renal Tubular Epithelial Cell

BACKGROUNDThe morbidity of Acute kidney injury(acute kidney injury,AKI)is extremely high.Mortality,the chance to encounter long-term cardiovascular events and the chance to develop end-stage renal disease(ESRD)increased significantly once a patients suffered from AKI.Among all the causes,ischemia-reperfusion is a main cause of acute renal injury,which is the primary cause of hospital-acquired acute renal injury in our country.Acute tubular necrosis(ATN)is an important pathophysiological feature of ischemia and reperfusion.Previous studies suggest that renal tubular epithelial cell necrosis cannot be regulated,but recent studies have revealed variety types of regulatory cell necrosis,one of them is necroptosis.Necroptosis is a kind of cell death that can be regulate.Studies have shown that Necroptosis is involved in the development of ischemia-reperfusion AKI.Necroatin-1,an inhibitor of necroptosis,can reduce renal injury caused by ischemia-reperfusion.RIP-1 and RIP-3 are important proteins that mediate necroptosis.RIP-1 and RIP-3 activation can lead to MLKL phosphorylation and cause cell membrane rupture,eventually lead to necrosis;at the same time it can promote mitochondrial division and increase ROS and causes cell necrosis.However,the role of mitochondrial cleavage and ROS in renal tubular epithelial cell necroptosis is still unknown.We proposed to establish the necroptosis model of renal tubular epithelial cells under hypoxia and observe the mitotic effect of mitochondrial division and ROS in renal tubular epithelial necroptosis.OBJECTIVETo establish the necroptosis model of renal tubular epithelial cells under hypoxia and observe the damage of mitochondrial division and ROS in renal tubular epithelial necroptosis.METHODSTo observe the protective effect of necrostatin-1 to cell deaths on NRK-52E modeled by using TNF-a,Z-VAD and Antimycin in the rat proximal tubular epithelial cell NRK-52E.In the case of necroptosis,NRK-52E cells were examined for mitochondrial cleavage of DRP-1,and the division of mitochondria was observed by mitotracker.The damage of mitochondria in necroptosis of renal tubular epithelial cells was observed.The model of necroptosis was established by using TNF-a,Z-VAD and Antimycin in human proximal tubular epithelial cells HK-2.The changes of ROS were detected during necroptosis,and when come to the case of under the protection of necroatin-1 and NADPH oxidase Diphenyliodonium chloride on ROS and observe the protective effect on HK-2 cells.RESULTSNRK-52E cells were treated with TNF-? 10 ng/ml,Z-VAD 50 ?mol/L and Antimycin A 10 ?mol/L.TNF-?(T)and TNF-? + Antimycin A(TA)(P<0.05).It appeared:the TNF-? + Antimycin A + Z-VAD(TZA)group has fewer apoptotic cells than T and TA groups,but necrotic cells increased significantly,and necroptosis key regulatory proteins RIP-1 and RIP-3 raised significantly.When we used a Nec-1 pretreatment appoint TZA group,we found that Nec-1 reduced TZA-induced cell necrosis and reduced RIP-1 and RIP-3 levels.The expression of Drp-1 in TZA group was significantly higher than that in control group,while mitochondrial cleavage increased while Nec-1 could inhibit the increase of Drp-1 and mitochondrial division in TZA group.The use of siRNA silencing Drp-1 could reduce mitochondrial division,And reduce TZA-induced cell necrosis.According to the preliminary results of the study group,necroptosis model was established in HK-2 cells using TNF-?,Z-VAD and Antimycin A.ROS levels were significantly increased in the TZA group,whereas the use of Nec-1 inhibited the increase in ROS induced by TZA.The use of NADPH oxidase inhibitor DPI in TZA cells can reduce ROS production and reduce cell necrosis.CONCLUSIONSIn this study,hypoxia-induced necroptosis was established in rat renal tubular epithelial cells(NRK-52E)for the first time and confirmed that Nec-1 attenuated hypoxia-induced necrosis of renal tubular epithelial cells.It was also found that Drp-1-mediated mitochondrial division was involved in renal tubular epithelial necroptosis.In the necroptosis model of HK-2 cells,ROS is involved in necroptosis of renal tubular epithelial cells and the necroptosis in renal tubular epithelial cells can be reduced by inhibiting the production of ROS.