Autophagy-Lysosome Pathway in Renal Tubular Epithelial Cells Is Disrupted by Advanced Glycation End Products in Diabetic Nephropathy.

It has been suggested that autophagy protects renal tubular epithelial cells (TECs) from injury in diabetic nephropathy (DN). However, the manner in which the autophagy-lysosome pathway is changed in this state remains unclear. In this study of DN, we investigated the autophagic activity and lysosomal alterations in vivo and in vitro. We found that autophagic vacuoles and SQSTM1-positive proteins accumulated in TECs from patients with DN and in human renal tubular epithelial cell line (HK-2 cells) treated with advanced glycation end products (AGEs), the important factors that involved in the pathogenesis of DN. In HK-2 cells, exposure to AGEs caused a significant increase in autophagosomes but a marked decrease in autolysosomes, and the lysosomal turnover of LC3-II was not observed, although LC3-II puncta were co-localized with the irregular lysosomal-associated membrane protein1 granules after AGEs treatment. Furthermore, lysosomal membrane permeabilization was triggered by AGEs, which likely resulted in a decrease in the enzymatic activities of cathepsin B and cathepsin L, the defective acidification of lysosomes, and suppression of the lysosomal degradation of DQ-ovalbumin. Oxidative stress evoked by AGEs-receptor for AGE interaction likely played an important role in the lysosomal dysfunction. Additionally, ubiquitinated proteins were co-localized with SQSTM1-positive puncta and accumulated in HK-2 cells after exposure to AGEs, indicating blocked degradation of SQSTM1-positive and ubiquitinated aggregates. Taken together, the results show that lysosomal membrane permeabilization and lysosomal dysfunction are triggered by AGEs, which induce autophagic inactivation in TECs from patients with DN. Disruption of the autophagy-lysosome pathway should be focused when studying the mechanisms underlying DN.

Effects of interleukin 18 on injury and activation of human proximal tubular epithelial cells.

BACKGROUND/AIMS: Injury and activation of tubular proximal epithelial cells (TEC) play central roles in renal tubulointerstitial fibrosis (TIF), but its mechanisms remain obscure. Interleukin 18 (IL-18) is overproduced during chronic kidney diseases (CKD), but how IL-18 affects the biological behaviour of TEC is not clear. The aim of the present study is to reveal the role of IL-18 in renal TIF. METHODS: The expressions of IL-18 and IL-18 receptor in TEC were detected by immunohistochemical staining in vivo and by reverse transcriptase polymerase chain reaction (RT-PCR) in vitro. TEC line (HK-2 cells) were incubated without or with IL-18. Cell proliferation and cell cycle were evaluated by methyl thiazolyl tetrazolium assay and flow cytometric analysis, respectively. Cell apoptosis was assessed by Hoechst 33258 staining. Expression of alpha-smooth muscle actin was evaluated by RT-PCR, immunocytochemical staining and flow cytometric analysis, respectively. Type I collagen, fibronectin, MCP-1 and RANTES in cultured supernatants were measured by enzyme-linked immunosorbent assay. RESULTS: IL-18 expression in TEC increased significantly in CKD state. IL-18 receptor was constitutively expressed in normal proximal TEC, and its expression increased strongly in CKD state. Proliferation and cell cycle of HK-2 cells were not affected by IL-18. Cell apoptosis, alpha-smooth muscle actin expression, type I collagen and fibronectin production as well as MCP-1 secretion were promoted by IL-18 in dosage- and/or time-dependent manners, but RANTES secretion was not affected. CONCLUSION: IL-18 may play a crucial role in the process of TIF by promoting TEC injury and activation, and could be a target of the therapeutic approaches against TIF.