Regulation Of Mesangial Cell Apoptosis And Endoplasmic Reticulum Stress By Adipocyte Fatty Acid Binding Protein

Objective: Diabetic nephropathy (DN) is one of the most common com-plications of diabetes and the major cause of end-stage renal failure in China.DN is characterized by excessive amassing of extracellular matrix (ECM) withthickening of glomerular and tubular basement membranes and increasedamount of mesangial matrix,which ultimately progress to glomerulosclerosisand tubulo-interstitial fibrosis.Recent studies have demonstrated the apoptosisof kidney cells plays an important role in the development of DN.Mesangialcells are the most active cells in the glomeruli and their response topathological stimuli is crucial to the development of DN.Diabetes mellitus ischaracterized by hyperglycemia and dyslipidemia. Recently,elevated levels offree fatty acids have been measured in the plasma of patients suffering fromDM.FABP4,known as adipocyte FABP (AFABP) or adipocyte P2(aP2),is oneof the FABPs protein family involved in active lipid metabolism. FABP4mRNA is transcriptionally controlled by intracellular fatty acids, promotes thetransportation of free fatty acid (FFA) and thus modulates systemic insulinsensitivity and energy metabolism.Recent work has shown that FABP4is notonly involved in lipid metabolism but also take part in inflammatory andimmuno reaction.The endoplasmic reticulum (ER) is an important site for lipid bio-synthesis and protein folding.However diverse pathological conditionsincluding ER-Ca2+depletion,ischemia,hypoxia,exposure to free radicals,elevated protein synthesis and so on interfere with the function of the ERaltering protein folding,a condition known as“ER stress”. Adaptation to ERstress depends on the activation of the unfolded protein response(UPR).Persisit or irreversible UPR induces apoptosis.We have demonstrated that type1diabetic rat exhibits elevated serumFFAs levels elevated in the early stage of type1diabetic rat exhibits. Furthermore,FABP4is upregulated in the renal cortex of type1diabetic rat atthe2thweek.Here we examined the changes of FABP4,ER chaperone proteinsand Bcl-2in biopsies from patients with established DN and MCGN in orderto explored the relationship between FABP4and ERS.This study is divided into two sections: First,the distribution,localizationand content of FABP4,GRP78,Caspase-12,Bcl-2were detected by immuno-histochemistry in biopsies from45patients with established DN.Second,weinvestigated the effects of different concentrations of oleic acids on humanmesangial cell and the involvement of ER stress in this process.Materials and Methods1Distribution,localization and content of FABP4,GRP78,Caspase-12andBcl-2in biopsies from patients with established DN.We studied two groups of patients: Established DN (n=45) and MCGN(n=10).The renal tissues were stained with hematoxylin-eosin(H&E) andperiodic acid-Schiff (PAS),and then examined by light microscopy. FABP4,GRP78,Caspase-12and Bcl-2were determined by the method of immuno-histochemistry.The quantitative analysis of FABP4,GRP78,Caspase-12andBcl-2were tested using Image-Pro-Plus5.0software.All of the glomeruli bothgroup DN and group MCGN were examined (fully sclerous glomerularremoved).2The effects of HG and OA on human mesangial cell(HMC) and theexpression analysis of FABP4,GRP78,GADD153/CHOP and Caspase-12.Differentiated HMC were divided into five groups:control group;HGgroup (30mmol/l);Three of OA groups (100μmol/l,200μmol/l and400μmol/l).Cultured HMC were incubated with HG and OA for up to24h and48h.The ultra-structure was assessed by scanning electron microscopy(SEM)and transmission electron microscope(TEM).The expression of FABP4,GRP78,GADD153/CHOP and Caspase-12were assessed by immunocyto-chemistry and western blot.Result:1Analysis of Renal Biopsies from Patients ①Pathologic morphcology of DN groupHistologic analysis of the kidneys from45patients with established DNrevealed glomerular hypertrophy,excessive amassing of extracellular matrix(ECM),thickening of glomerular and tubular basement membranes andincreased amount of mesangial matrix in the early stage.Moreover,the earliestlesions also include capsular drop lesions in the Bowman’s capsule andgranular or vacuolation degenration of renal tubular epithelial cells.Established DN is characterized by diffuse global glomerulosclerosis,nodularglomerulosclerosis resembling Kimmelstiel-Wilson nodules,interstitialinflammation,interstitial fibrosis,tubular atrophy and arteriolar accumulationof hyaline.②The expression of FABP4,GRP78,Caspase-12and Bcl-2in MCGNand DN group:Immunohistochemistic study showed that slight staining withanti-FABP4antibody was observed in MCGN group,located mainly incytoplasm and nucleus of mesangial cell and little capillary endothelial cell.Incontrast,DN group produced widespread strong staining with anti-FABP4antibody in mesangial cell.MCGN group exhibited weak immunoperoxidasestaining for GRP78,and it was strong expressed in cytoplasm of renal tubularepithelial cell,endothelial cell, mesangial cell and glomerular parietalepithelial cells.Interestingly,it was also expressed in inflammatory cells amongthe renal interstitial.Bcl-2was mainly distributed in cytoplasm of renal tubularepithelial cell, glomerular parietal epithelial cell and a part of mesangial cell,but in DN group,the expression level of Bcl-2was reduced.However,it wasalso expressed in cytoplasm of lymphocyte among the renal interstitial. In DNgroup,Caspase-12was immunolocalized to cytoplasm of renal tubularepithelial cell, smooth muscle cells, mesangial cell and glomerular parietalepithelial cell,significantly higher than those in MCGN group.③The quantitative analysis of FABP4,GRP78,Caspase-12and Bcl-2inMCGN and DN group:Compared with MCGN group,the expression ofFABP4,GRP78and Caspase-12in glomeruli of DN group were markedlyhigher（P＜0.05）.Nevertheless, Bcl-2protein was reduced obviously in DN group（P＜0.05）.2Localization and content of FABP4and ER-chaperone proteins incultured HMC①The anlysis of ultrastructure:Scanning electron microscopy:In controlgroup,HMC contacted with each other by pseudopodia.In contrast,the volumeof cells incubated in media containing high glucose were larger and there weremany excretionvesicles on the surface of cells.The HMC exposed to OAresulted in cell pyknosis, pseudopods disappeared and excretionvesiclesincreased.Transmission electron microscope:Compared with control group,therewere numerous lipid droplets,swelled mitochondria and autophagic vacuolesin HG group.In addition,microvillus were reduced or sheded,endoplasmicreticulum and mitochondria were swelled under OA condition.②The results of immunocytochemistry:We found that the exposure ofHMC to high glucose results in significant increase in the expression ofFABP4compared with control group. Similar results were obtained in OAgroup.③The results of western blot:FABP4was markedly increased at24h andthen decreased at48h both in HG and OA groups,however,GRP78,GADD153/CHOP and Caspase-12protein levels were obviously increased at24h and similar findings were observed with up to48h.There was nosignificant difference among the different concentrations of OA.Conclusion:1In our study,the expression of FABP4,GRP78and Caspase-12in DNwas upregulated while Bcl-2was downregulated.These observations indicatedthat the ERS was induced in DN,chronic ERS suppressed the expression ofBcl-2and cells undergo apoptosis.2It has been previously shown that FABP4was significant increased inHG and OA groups suggesting that OA and HG induced ER stress. Both inHG and OA groups was observed lesion of HMC.What is more,apoptosis wasobserved in OA groups.