Over-expression of ROBO4 reduces permeability of human renal glomerular endothelial cells in high glucose medium

Objective 　To investigate the effect of over-expression of ROBO4 on permeability of human renal glomerular endothelial cells (HRGECs) in high glucose medium. Methods 　HRGECs infected with recombinant lentiviral vector ROBO4 were cultured in high glucose or low glucose medium in vitro. The protein levels of ROBO4 and ARF6 in each group were detected by Western blotting. The endothelial permeability was measured by the efflux of fluorescein isothiocyanate-dextran (FITC-Dextran) permeated through the monolayer endothelial cells using Transwell cell model system. The cell viability after lentivirus transfection was measured by CCK8 assay. Results 　The transfection rate of lentiviruses in HRGECs reached 80% 72h after, and obvious over-expression of ROBO4 protein was in transformed cells compared with the empty vector group (P<0.05). The lentivirus-mediated ROBO4 transfection did not affect cell viability of HRGECs. Compared with the low glucose group, the expression of ROBO4 increased obviously after 12h, but declined after 24h (P<0.05), and reached to minimun after 72h (P<0.05). On the contrary, the expression of ARF6 increased after 12h, and the increase reached to the maximum after 72h (P<0.05). Furthermore, the vascular permeability increased gradually after 24h, and reached to the maximum after 72h (P<0.05) in high glucose group. Compared with the empty vector group, the over-expression of ROBO4 inhibited the expression of ARF6 significantly, and the FITC-Dextran permeability reduced obviously. Conclusion 　Over-expression of ROBO4 may significantly enhance the barrier functions of HRGEC in high glucose medium, and ROBO4 activation may be a potential therapeutic approach in diabetic nephropathy. DOI: 10.11855/j.issn.0577-7402.2017.03.03