Succinate Accumulation-related Reactive Oxygen Species Production Was Involved In Renal Ischemic Reperfusion Injury

Background: Acute kidney injury(AKI)is a common clinical syndrome caused by a variety of diseases.Ischemia reperfusion(I/R)is one of the leading causes of acute renal injury,with high morbidity and mortality.Renal I/R cause a series of pathophysiological events,including cell necrosis,apoptosis,inflammatory cell infiltration and oxidative stress,resulting renal damage.Reactive oxygen species(ROS)not only triggered intracellular signal transduction,but interacted with inorganic molecules,proteins,lipids,carbohydrates and nucleic acids and other molecules,which altered the function of the target molecule and ultimately caused tissue damage.Succinate—an intermediate metabolite of the tricarboxylic acid cycle was accumulated during cardiac ischemia.Succinate was believed to play an important role of ROS generation in ischemic organ.ROS is also an important second messenger.It is associated with endoplasmic reticulum stress(ERS).Therefore,we assume that succinate accumulation leads to ROS production and endoplasmic reticulum stress involved in renal I / R injury.Objective:To determine whether succinate dehydrogenase mediated succinate accumulation and ROS production during renal ischemia-reperfusion injury.Methods: BALb/C male mice were divided into three groups.In ischemia/reperfusion group(I/R),mice were occluded bilateral renal arteries and veins for 35 minutes,and then reperfused for 24 hours.In DM group(DM),dimethyl malonate(140mg/kg)was intravenous injected before I/R.The concentration of succinate in renal tissues and cells were detected by mass spectrometry.Biochemical tests(dry chemical method)were used to examine serum creatinine and urea nitrogen.Kidney injury molecule-1(KIM-1),succinate dehydrogenase(SDH),CCAAT / enhancer binding protein(CHOP),glucose-regulated protein-78(GRP78)and 3-nitrotyrosine(3NT)were detected by immunohistochemistry or immunofluorescence staining.The expression of SDH,superoxide dismutase(SOD),cytochrome C(Cyt C),GRP78 and CHOP were also estimated by western blot.ROS was detected by DCFH-DA..HK-2 cells were used for in vitro experiments.Results: After I/R,the kidneys were larger and darker.Serum creatinine was9.40±3.13μmol/L and 88.00±25.98μmol/L in control and I/R group respectively,P=0.0004.Urea nitrogen was 10.19±1.13mmol/L and 52.64±7.01mmol/L,P <0.0001.HE staining showed that vacuolar changes were found in renal tubular epithelial cells,and the changes were the most significant at the junction of cortex and medulla in I/R group.KIM-1 was also increased in I/R group.Succinate accumulated during ischemia stage and decreased after reperfusion by MS analysis.SDH expression increased at junction of cortex and medulla after I/R Immunofluorescence staining showed that 3NT was increased in I/R kidney.DM pretreatment also attenuated renal injury,the serum creatinine and Urea nitrogen were 27.67±12.66μmol/L 和18.95±9.25mmol/L respectively.DM improved renal histology change in I/R mice and inhibited KIM-1 expression,as well as the SDH production in the junction of cortex and medulla.The expression of SOD was 1.17±0.21,0.72±0.13 and 1.09±0.14 in Con,I/R and DM groups resspectively,P=0.002.The expression of CHOP was0.54±0.08,1.08±0.07 和 0.63±0.01 in each group,P=0.017.In vitro,hypoxia-incubated HK-2 cells increased succinate level,which was decreased after reoxygenation incubation.Hypoxia incubation for 24 hours increased SDH expression,which was dreased after reoxygenation incubation..What’s more,ROS production was significantly increased in Hypoxia-reoxygenation incubated HK-2 cells.Hypoxia-incubated HK-2 cells decreased SOD concentration,promoted cytochrome C release and increased CHOP production,which all could be inhibited by DM pretreatment.Conclusion: Ischemia resulted renal tubular epithelial cells injury at the junction of cortex and medulla,which maybe associated with SDH overexpression.Dimethyl malonate inhibited SDH production,reduced ROS generation,cytochrome C release and CHOP upregulation during reperfusion,as well as alleviated I/R-induced renal injury.