The Apoptosis And Transformation Of HK-2Cells Induced By Oleic Acids Via Endoplasmic Reticulum Stress

Objective: It is the common pathological process that lipid accumulatingin renal parenchymal cells, and following hypo-function cells called“lipotoxicity”. In proteinuric kidney disease, lipotoxicity has been proposed asa pathogenic mechanism of progressive renal tubulointerstitial damage withrenal interstitial fibrosis(RIF), which correlates with renal prognosis. Freefatty acid (FFA)-bound albumin, which is filtrated through the glomeruli andreabsorbed into proximal tubular cells, is one of the crucial mediators oftubular damage in proteinuric kidney disease. L-FABP, known as liver-typefatty acid binding protein (L-FABP, FABP1), is one of the fatty acid bindingprotein protein family involved in lipid metabolism, and expresses in liver,kidney, intestinum tenueand and pancreas. In chronic kidney disease(CKD)study, urinary L-FABP reflected the progression of CKD and the degree oftubulointerstitial damage. Several experimental studies have shown that excessFFA-load in proteinuria induces severe tubulointerstitial injury consisting ofinflammatory cell infiltration and renal tubular cell apoptosis. But the possiblemechanisms related to the induction of injure by FFAs are not fullyunderstood.It is reported that proteinuria can cause serious endoplasmicreticulum stress(ERS) in tubular cells.The endoplasmic reticulum (ER) is an important site for protein folding,synthesis and transfer dealing with stress. Adaptation to ER stress depends onthe activation of the unfolded protein response(UPR). Persisit or irreversibleUPR induces apoptosis.FFAs induce pancreatic β cell and hepatic cellapoptosis throuhg ERS.But the reports concerning kidney inherent cell ofapoptosis via ERS were seldom.Here, we hypothesize that L-FABP could involve in the tubulointerstitialdamage by FFAs and investigate the mechanisms which FFAs induce kidney tubulointerstitial injury through culturing people proximal convoluted tublesepithelial cells(HK-2). In this study, we take oleic acids(OA), unsturated fattyacids containing18carbon to present FFAs.Method：Human proximal convoluted tubles epithelial cells (HK-2) wereobtained from cells institution of Chinese Academy of Sciences locatedshanghai. They were cultured in DMEM-F12medium (3:1) supplementedwith5%fetal bovine serum,2mmol/L-glutamine,100U/ml penicillin, and100μg/ml streptomycin in a95%air,5%CO2atmosphere. HK-2were grownto80%-90%confluence and growth-arrested in serum-free DMEM-F12medium for24h to synchronize the cell growth. Differentiated HMC weredivided into three groups: control group; OA group; albumin FFA-free group.1The MTT reduction assay to detect diffrent OA concentration (0,50,100,200,400μmo/l) and albumin FFA-free concentration (0,5,10,20,40mg/ml) for HK-2cell proliferation;2Ultrastructure of HK-2cells from three groups were observed byscanning electron microscopy (SEM) and transmission electron microscope(TEM).3Apoptosis rate of HK-2cells from three groups were analyzed by situterminal deoxynucleotidyl transferase labeling technique (terminaldeoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay,TUNEL).4The expression of L-FABP HK-2cells from three groups assessed bywestern blot in1h,3h,6h,12h and24h, and the expression of GRP78,GADD153/CHOP, α-SMA, Bcl-2, Bax, Caspase-3and Cleaved Caspase-3were assessed by western blot in different time.Result:1When HK-2cells were cultured in100μmol/L for12h, the survivalrate of HK-2was50%, and10mg/ml for12h in albumin FFA-free group.2The anlysis ultrastructure of cultured HK-2in different groups(1) Scanning electron microscopy: In control group, HK-2cells contactedwith each other by microvilli. The nucleus lay in the center of cells and the nucleolus were obvious.In contrast, the cytomembrane of HK-2cellsincubated in media containing albumin FFA-free were shrinkage andappearanced patellate or spoking; in OA group, the volume of cells weresmaller and membrane damage were mesh shape, and meanwhile, there werenuclear hiatus.(2) Transmission electron microscope:Compared with control group,inthe albumin FFA-free group, the volume of cells were larger and full oforganelles. While, in OA group, the cell membrane damage disintegrationwith microvilli dropping. In addition, endoplasmic reticulum andmitochondria were swelled under OA condition, and numerous lipid dropletsand glycogen granules accumulated in cytoplasmic.3OA induced the expression of L-FABP of HK-2cells in different timeThe expression of L-FABP begin to rise at6h in HK-2cells exposed inOA, and reach the peak at12h.4OA induced HK-2cells endoplasmic reticulum stressAfter stimulation with different groups for6h,12h and24h, theexpression of GRP78and GADD153/CHOP were markedly higher incubatingin media containing OA compared with control group and the albuminFFA-free group.5OA induced HK-2cells apoptosis(1) TUNEL assay showed compared with control group, OA significantlypromoted cell apoptosis.(2) The results of western blot: The expression of Caspase-3, CleavedCaspase-3and ratio of Bax/Bcl-2significantly increased in OA group.6Effect of OA on transformation in HK-2cellsThe expression of α-SMA significantly increased in OA group at12hcompared with the other two groups.Conclusions:1OA lead to the expression of L-FABP up-regulation before the upregulation of GRP78and GADD/CHOP. We hypothesize that OA play a keyrole in apoptosis involved in ERS. 2OA induced Caspase-3, Cleaved Caspase-3and ratio of Bax/Bcl-2increasing. We demonstrated apoptosis appearing in OA group by TUNELassay.3HK-2cells gradually show the character of myofibroblast in morphousand function occuring transformation.In summary, not only can OA induce apoptosis but also transformation,and play an important role in progress of renal interstitial fibrosis.