The Effects Of Core Fucosylation On Podocyte Injury Caused By Puromycin Aminonucleoside

Objective:To investigate the effects of ?1,6-fucosyltransferase 8(FUT8)on murine podocyte injury caused by puromycin aminonucleoside(PAN).Methods:A conditionally immortalized mouse podocyte clonal cell line(mouse podocyte clone 5,MPC5)was cultured in medium containing mouse y-interferon at 33? in vitro,then cells were induced to differentiate by transfer to 37? and removal of y-interferon from the medium for 10-14 days.1.The fully differentiated podocytes were randomly divided into six groups:(1)normal control(termed as Normal):cells were cultured in RPMI 1640 medium containing 10%fetal bovine serum;(2)mock group(termed as Mock):transfection with 33 nM scrambled siRNA for 24 h;(3)PAN group(termed as PAN):cells were treated with PAN at 5 ug/ml for 2 h;(4)PANF group(termed as PANF):transfection with 33 nM Fut8siRNA for 24 h and then with 5 ug/ml PAN for 2 h;(5)PANM group(termed as PANM):transfection with 33 nM scrambled siRNA for 24 h and then with 5 ug/ml PAN for 2 h;and(6)Fut8siRNA group(termed as Fut8siRNA):transfection with 33 nM Fut8siRNA for 24 h.2.The cell morphology was detected by phase contrast microscope,the expression of FUT8mRNA was detected by RT-PCR,the expression of core fucose and podocyte marker-Nephrin was detected by immunofluorescent analysis,the expression of FUT8 and Nephrin was detected by western-blotting,and the apoptosis was examined by flow cytometry.Statistical analysis was performed by one-way ANOVA.Results:1.It was confirmed by immunofluorescence that core fucose existed in MPC5.2.Comparing with the Control group,incubation with 5 ug/ml PAN for 2 h led to shrinkage of podocytes,together with foot process retraction,and some cells detached from the bottom of the culture dish;immunofluorescent analysis showed that the expression of core fucose was significantly increased,while Nephrin was decreased;western-blotting showed that the expression of FUT8 was significantly increased,while Nephrin was decreased;the apoptosis of cells was increased.Preincubation with FUT8siRNA could inhibit cells detachment from the bottom of the culture dish;the expression of core fucose and FUT8 was decreased,while Nephrin was increased;the apoptosis of cells was decreased.Conclusion:1.The core fucosylation involved in the apoptosis and the change of structural protein in podocyte injury caused by puromycin aminonucleoside.2.Utilizing RNAi technology to silence the expression of FUT8 gene,blocking of the core fucosylation,led to inhibition of podocyte injury induced by puromycin aminonucleoside.