Effect of cyclosporine A on autophagy-lysosomal pathway in tubular epithelial cells / 中华肾脏病杂志

ABSTRACT

Objective To investigate the effect of cyclosporine A (CsA) on autophagylysosomal pathway in tubular epithelial cells.Methods Human renal tubular epithelial cell line (HK-2 cell) was treated with different concentrations (3,5 and 10 μmol/L) of CsA for 24 h.Then the viability and apoptosis of cells were measured by MTT assay or AnnexinV-PI staining followed by flow cytometry analysis,respectively.Autophagy-related protein LC3-Ⅱ and p62 were detected by immunofluorescence assay.Autophagic flux was analyzed in HK-2 cells transfected with a tandem mRFP-GFP fluorescent-tagged LC3 (ffLC3) plasmid by laser confocal microscope.The lysosomal degradation was evaluated by DQ-ovalbumin staining followed by flow cytometry analysis.Results The viability of HK-2 cells was significantly decreased with CsA stimulation when compared with control group (P ＜ 0.01),but the number of apoptotic cells was markedly increased by CsA treatment (P ＜ 0.05).Compared with the control group,different doses of CsA dramatically increased the expressions of LC3-Ⅱ (P ＜ 0.01) and p62 (P ＜ 0.05) in HK-2 cells.Moreover,HK-2 cells treated with CsA displayed a significant increase in autophagosomes but a marked decrease in autolysosomes.In HK-2 cells,exposured to CsA caused a decrease in lysosomal degradation by DQ-ovalbumin staining when compared with control group (P ＜ 0.01).Conclusion Blockade of autophagy via disrupting lysosome degradation may represent a novel mechanism of CsA-induced tubular epithelial cells injury.