The Effects Of Core Fucosylation On The Autophagy In Renal Tubular Cell

objective: To investigate the effect of ?-1,6 fucosyl trans ferase(FUT8)on the autophagy in renal tubular epithelial cells,during transdifferentiation process in proximal renal tubular epithelial cells(human proximal tubular epithelial cells,HK-2)induced by TGF-?1.methods:(1)The patients diagnosed as IgA by renal biopsy in nephropathy in the first affiliated hospital of dalian medical university,random divided into four groups(the no fibrosis,mild,moderate and severe)according to the degree of renal fibrosis,every group selected 10 patients,a total of 40 patients,using electron microscopy counting the autophagy bodies in each view for every group to analysis of statistical average amount.(2)Then we stainded the immunohistochemical of beclin-1 with the biopsy tissue of four groups and analysised the expression of the beclin-1 of the four groups,to investigate the autophagy levels of every groups.(3)The culture of HK-2 cells in vitro and divided into two groups: the control group,DMEM containing 10% serum culture for 48 h.The TGF-?1 stimulation group,added 10 ng/ml TGF-?1into DMEM cell culture medium without serum and stimulate for 48 h.Autophagy related gene beclin-1 and muscle fibroblasts marker a-SMA were compared respectively by multiple immunofluorescence staining for two groups.HK-2(4)Culture normal HK-2 cells in vitro in five groups: The control group.culture with DMEM containing 10% serum for 48 h.The Fut8-siRNA silencing group,using siRNA-mate transfection reagent the fut8-siRNA transient transfection into HK-2 cells,and added 10 ng/ml TGF-?1 into DMEM cell culture medium without serum and stimulate for 48h;The TGF-?1 stimulation group,added 10 ng/ml TGF-?1 into DMEM cell culture medium without serum and stimulate for 48 h.The TGF-?1 + Fut8-siRNA group,transient transfection Fut8-siRNA into HK-2 cells,inhibit Fut8 expression,with a final concentration of 10 ng/ml TGF-?1 stimulate HK-2 cell for 48 h.The TGF-?1+ Mock group,add the out-of-order siRNA transient transfection into HK-2 cells,and added 10 ng/ml TGF-?1 into DMEM cell culture medium without serum and stimulate for 48 h.western blot to detect the a-SMA,FUT8 and beclin-1 levels of each group and analyzed the datas.Result:(1)The no fibrosis group existence of a small amount of autophagy bodies under the electron microscope.The autophagy bodis increased significantly(P < 0.05)in the other three groups,compared to the no fibrosis group.(2)In mild group,beclin-1 expression was weak by immunohistochemical staining,the moderate group of beclin-1 expression is strong,severe group expression of beclin-1 was weaker than the moderate group and slightly stronger than the mild group.(3)Compared with the control group,the TGF-?1 group,enhanced fluorescence expression of a-SMA,and the intensity of the beclin-1 fluorescence decreased significantly for immunofluorescence staining,(4)The a-SMA expression is weak in the control group,but the beclin-1 expression is moderate intensity.The a-SMA fluorescence in the Fut8-siRNA silence group and the control group are similar,and the expression of beclin-1 is obviously increased(P<0.05).The expression of the a-SMA fluorescence is enhanced(P<0.05),but the beclin-1 is decreased(P<0.05)in the TGF-?1 stimulation group compared with the control group.The expression of the a-SMA is decreased(P<0.05)In the Fut8-siRNA silence + TGF-?1 stimulating group than the TGF-?1 stimulation group,while the expression of the beclin-1 is slightly stronger than the TGF-?1 stimulation group(P<0.05),but decreased(P<0.05)than the Fut8-siRNA silent group.The expression of the a-SMA and the beclin-1 are similar between the TGF-?1 stimulation group + mock and the TGF-?1 stimulation group.Conclusion: 1.In the stage of renal interstitial fibrosis,there were significant differences in the number of autophagic bodies in renal tubular epithelial cells.2.Autophagy is involved in the whole process of transdifferentiation of renal tubular epithelial cells,and TGF-?1 inhibits autophagy in the process of fibrosis.3.CF maybe prevent the autophagy of the cells.