L-FABP Mediates HK-2 Cell Apoptosis Induced By Free Fatty Acid Via Endoplasmic Reticulum Stress

Objective:Chronic proteinuria is now considered to play an essential role in progression of tubulointerstitial damage which is considered as a final common pathway to end-stage renal disease. Free fatty acids(FFAs) are bound to albumin, therefore, might play a role in the generation of tubulointerstitial disease by lipotoxicity. Palmitic acid, has been identified as a proapoptotic factor in some cell types.Lipotoxicity linked a variety of stress reactions, such as the endoplasmic reticulum stress(ER) and oxidative stress, etc. Disruption of ER homeostasis leads to accumulation of unfolded proteins and successively activates the unfolded protein response(UPR), which is also referred to as the ER stress response, and then triggers apoptosis.Liver-type fatty acid binding protein(L-FABP, also known as FABP1) as one member of fatty acid binding protein family, is expressed in human proximal tubular epithelium. Urine L-FABP was closely related to tubulointerstilitial damage and was used as one monitor of progression of renal diseases.Our previous study had demonstrated that oleic acid induced the up-regulated of L-FABP protein ER stress and apoptosis related proteins in human proximal tubular cells(HK-2). We hypothesized that FABP1 might mediate apoptosis via ER stress in HK-2 induced by PA and OA. To test this hypothesis, we first evaluated the expression of FABP1 and ER stress marker as well as apoptosis-related proteins in HK-2. Then we used RNA interference to further investigate the role of FABP1 in ER stress and apoptosis induced by PA and OA in vitro.Method:1 Grouping:⑴ HK-2 cells were randomly divided into four groups:Control, PA, OAand Alb.⑵ HK-2 cells were treated with PA,OA and Alb with gene silence of sh NC or sh L-FABP, and the control group of sh NC and sh L-FABP. The grouping were as followed.2 The MTT assay was used to determine the concentration of PA in HK-2 cells.3 Western blot was used to determine the intervention time of PA and OA in HK-2 cells.4 Observation the variation of ER related proteins and apoptosis related proteins with treatment of PA,OA and Alb in HK-2 cells.5 Observation the variation of ER related proteins and apoptosis related proteins with treatment of PA, OA and Alb in HK-2 cells with gene silence of sh L-FABP.Result:1 Effect of different PA concentrations in HK-2 cells.Cell survival rate was 40% at the concentration of 200μmol/L of PA, so that 200μmol/L was picked as treatment concentration.2 The time course of L-FABP expression treated in HK-2 cells with PA.Compared with control group of HK-2 cells, the expression of L-FABP at3 h, 6h and 12 h did not increase significantly, while reached the peak at 24 h,and then decreased slightly at 48 h.3 The time course of L-FABP expression treated with OA.Compared with control group of HK-2 cells, the expression of L-FABP at12 h, 24 h and 48 h increased significantly, while reached the peak at 24 h.4 The variation of L-FABP treated with OA,PA and Alb.Compared with control group of HK-2 cells, the expression of L-FABP treated with OA and PA were obviously unregulated, while not changed significantly in Alb group.5 The variation of ERS related proteins and apoptosis proteins in HK-2cells treated with PA,OA and Alb.GRP78、CHOP、Caspase3 and Cleave Caspase3 were higher in PA and OA group than in Alb group; While those proteins in Alb group were not changed greatly.6 L-FABP can be down-regulated by sh L-FABP plasmid in HK-2 cells with.The result of fluorescence showed that HK-2 cells can get sh L-FABP and sh NC and express green fluorescent.L-FABP was significantly less in group of sh L-FABP than in group of sh NC.7 ERS related proteins can be down-regulated by sh L-FABP plasmid in HK-2 cells treated with PA and OA.⑴ The expression of GRP78, PERK and p-PERK were significantly increased in PA+sh NC compared with sh NC. While these proteins was decreased in PA+sh L-FABP.⑵ The expression of GRP78,PERK and p-PERK were significantly increased in OA+sh NC compared with sh NC. While these proteins was decreased in OA+sh L-FABP.⑶ The expression of PERK was up-regulated slightly in Alb, while GRP78 and p-PERK did not change obviously in Alb+sh NC. After transfection with sh L-FABP, the expression of PERK and p-PERK was decreased a little in Alb+sh L-FABP, except GRP78.8 Apoptosis related proteins could be down-regulated by sh L-FABP plasmid in HK-2 cells treated with PA and OA.⑴ The expression of CHOP, Caspase3 and Cleave Caspase3 were significantly increased in PA+sh NC compared with sh NC. While these proteins were decreased in PA+sh L-FABP.⑵ The expression of CHOP, Caspase3 and Cleave Caspase3 were significantly increased in OA+sh NC compared with sh NC. While these proteins was decreased in OA+sh L-FABP.⑶ The expression of CHOP, Caspase3 and Cleave Caspase3 were not changed obviously in Alb+sh NC. After transfection with sh L-FABP, the expression of Cleave Caspase3 was decreased a little in Alb+sh L-FABP,except CHOP and Caspase3.Conclusion:1 PA and OA induced not only high expression L-FABP,but also ER stress related proteins and apoptosis ER stress related in HK-2cells. These results proved that PA and OA up-regulated the expression of L-FABP and ER stress markers.2 Increased expression of ER stress markers and apoptosis related proteins induced by PA and OA significantly decreased with gene L-FABP was knock-down. This showed that L-FABP was involved in ER stress induced by FFAs, and eventually induced ER stress related death FABP4 si RNA significantly suppresses the inductions of GRP78.3 ER stress markers and apoptosis ER stress-related treated with anlbumin, whether with L-FABP gene silence or not, did not change significantly, which confirmed that it was FFAs that induced damage and apoptosis in proximal tubule epithelial cells, instead of albumin.