Autophagy-lysosome System In Renal Tubular Epithelial Cells Is Disrupted By Advanced Glyction End Products Via RAGE/ROS Pathway

Objective It has been suggested that autophagy protects renal tubular epithelial cells(TECs) from injury in diabetic nephropathy. This study was designed to investigate the effects of advanced glycation end products(AGEs) on the autophagy-lysosome pathway in renal tubular epithelial cells, and Whether AGEs interact with RAGE to evoke oxidative stress, so to induce lysosomal dysfunction in TECs.Methods The autophagic activity and lysosomal alterations in HK-2 exposed with AGE-BSA(100μg/ml) for 12 h were investigated. Expression of LC3-II was studied after exposing HK-2cells to AGE-BSA and LC3-II turnover was examined after exposure to AGE-BSA in presence of Lysosomal inhibitors by western blot assay and Immunofluorescence experiments.Immunofluorescence was also used to detect lysosome-associated membrane protein-1(LAMP1)and LC3-II co-localization, to determine whether AGE-BSA blocked the fusion of autophagosomes and lysosomes. HK-2 cells were treated with AGE-BSA(100μg/ml) for 6,12 and 24h, the activity of CB, CD or CL were measured by fluorescence-based assay kits, then the mean fluorescent intensities of LTR and DQ-ovalbumin were examined by fluorescence microscopy and FACS analysis. Immunofluorescence was also used to detect LAMP1 and CB double-staining, so to detect if AGE-BSA triggers LMP in HK-2 cells. HK-2 cells were incubated with AGE-BSA with or without anti-RAGE antibody or antioxidant pre-treatment, we used fluorescence microscopy and FACS analysis to examine the mean fluorescent intensities of LTR and DQ-ovalbumin. Then we used flow cytometry to detect the level of ROS.Results 1. A decrease in autophagic activity of HK-2 cells exposed with AGE-BSA was found.The protein levels of LC3-II were significantly increased after exposure to AGE-BSA for 12 h.However, LC3-II expression was not further elevated by the addition of bafilomycin A1,leupeptin or chloroquine by western blot assay and by immunostaining. That is the lysosomal turnover of LC3-II was not observed although LC3-II puncta were co-localized with the irregular LAMP1 granules after AGEs treatment.2. AGE-BSA could significantly abolish LTR staining and DQ-ovalbumin fluorescence.However, these actions were inhibited partially by anti-RAGE antibody pre-treatment under the microscope or by flow cytometer. Pre-treatment with antioxidant NAC or catalase also improved the lysosomal dysfunction in AGE-BSA-treated cells, characterized by an increase in LTR andDQ-ovalbumin fluorescence signals. Furthermore, we found that the production of reactive oxygen species(ROS) was significantly enhanced after exposure of HK-2 cells to AGE-BSA for12 h. However, similar to the effect of antioxidant NAC or catalase, anti-RAGE antibody notably suppressed the ROS production mediated by AGE-BSA.3. After incubated with AGE-BSA, a decrease in the enzymatic activities of cathepsin B(CB)and CL was found, the defective acidification of lysosomes, and suppression of the lysosomal degradation of DQ-ovalbumin were found in the HK-2 cells. LMP was triggered by AGEs, a diffuse cytoplasmic immunostaining pattern of CB and irregular immunostaining of LAMP1 were also found in AGE-BSA treated HK-2 cells.Conclusion TECs autophagy-lysosome pathway is disrupted by AGEs via RAGE/ROS pathway.The regulation of RAGE/ROS pathway can protect lysosomes and enhance autophagy flux.