Study On Effect Of AGEs On Injuring Of Rat Renal Microvascular Endothelial Cells And Protective Effect Of Probucol

IntroductionDiabetic nephropathy is a leading cause of end-stage renal failure.Glucose and other reducing sugars can react nonenzymatically with the free amino groups of proteins to form reversible Schiff bases followed by Amadori rearrangement.These early glycation products undergo further complex reactons such as dehydtraton,condersation,and cross-linking to yield irreversible fluorescent derivatives termed advanced glycation end products(AGEs).Many studies reveal ed that AGEs have been implicated in the initiation and development of diabetic microvascular complication primarily by inter with their intracellular signal transducing receptor for advanced glycation end products(RAGE).The engagement of RAGE on endothelial cells by AGEs results in the induction of intracellular reactive oxygen species(ROS),which seems to be linked to the activaton of the NADPH-oxidase system and the mitochondrial electron transport system.The increased ROS in turn activates acomplex cascade of signal transduction pathways and sbusequentlly enhances the expression of many genes that are highly relevant for inflammation,and atherosclerosis.Probucol,an old-line anticholesterol medication,has powerful antioxidant properties also.In clinical studies,probucol improved the function of diabetic kidney.In this study we aimed to explore the effect of AGEs on injuring of rat renal microvascular endothelial cells and protective effect of probucol. MethodsMicrovascular endothelial cells isolated and cultured from rat renal were divided into 3 groups:normal control group,AGEs group and probucol group.The levels of MDA and NO were determined by the assay TBA and nitrate reductase method respectively.The expression of eNOS mRNA and NADPH oxidase protein was detected by RT-PCR and Western-blot respectively.The intracellular disposition of NF-κB was observed by immunostaining microscopy.ResultsIn AGEs group,the level of MDA and the expression of NADPH oxidase protein increased but and the expression of eNOS mRNA decreased,as compared with control. The level of NO in culture medium was increased after exposure to AGE-BSA for 30 min and 6h,but after 12h,NO level was decreased.MDA level and the expression of NADPH oxidase protein were decreased but the expression of eNOS mRNA and NO level were upregulated by probucol with a dose-dependent effect in the concentrations of 10,20,50,100μmol/L.The localization of NF-κB shifted from cytoplasm to nucleus after microvascular endothelial cells were exposed to AGE-BSA.After exposed for 30 min,the expression in nucleus reached the peaked and then maintained high level.DiscussionThe classical criteria for identification of microvascuar endothelial cells included cobblestone appearance in vitro culture,synthesis of v Wf by cells,and tubular structure by matrigel in vitro.In the present study,Microvascular endothelial cells isolated and cultured from rat renal were identificated by the classical criteria successfully. Numerous reports have demonstrated that oxidative stress induced by diabetes plays an important role in the development and progression of diabetic vascular complications including nephropathy.Indeed,there is emerging evidence that the formation of reactive oxygen species(ROS)is a direct consequence of high level of advanced glyciation end products.In the present study,we clearly demonstrated that AGEs could cause dysfunction of rat renal microvascular cells.Our result confirmed Oxidative Stress played an important role in Injuring of Rat Renal Microvascular Endothelial Cells.In the present study,we demonstrated in AGEs group the expression of NADPH oxidase protein increased but the expression of eNOSmRNA decreased,resulting in an enhancement of Oxidative Stress in RMEC.The enhancement oxidative stress was further confirmed by normalization of level of MDA,expression of NADPH oxidase protein and in RMEC treated with probucol in parallel with the decrease in oxidative stress.The transcription factors of the NF-kB/Rel family form dimeric complexes that control expression of various genes involved in inflammation and proliferation.In our study,we observed that localization of NF-κB shifted from cytoplasm to nucleus after microvascular endothelial cells were exposed to AGE-BSA and probucol could prevent activation of NF-κB.Protective Effect of Probucol on microvascular entothelial cells probably via antagonisting AGE-induced activating of NF-κB.ConclusionAGEs can cause the injury and dysfunction of renal microvascular endothelial cells via the following possible mechanisms:(1)inducing lipid preoxidation(2)activating NADPH oxidase and increasing the production of ROS;(3)abnormal expression of eNOS mRNA and further affecting NO production;(4)activatin of NF-κB.Probucol can protect microvascular entothelial cells probably via antagonisting the lipid preoxidation,activating of NF-κB and NADPH oxidase,and increasing the AGE-induced abnormal expression of eNOS caused by AGEs.