The Effect Of Autophagy On High Glucose Induced Lipotoxicity In Human Proximal Tubular Epithelial Cells

Background and ObjectiveDiabetic nephropathy (DN) is one of the most devastating complications of diabetes, significantly increases the morbidity and mortality in patients with diabetes. It also accounts for the most common cause of end-stage renal disease (ESRD) worldwide. The clinical characteristics of DN include excessive deposition of extracellular matrix (ECM) caused kidney progressive fibrosis, which ultimately leads to irreversible damage of kidney and decline in kidney function. Renal interstitial fibrosis has been established as the common pathway of end-stage renal failure, and epithelial-mesenchymal transition (EMT) has been considered to be the key process. Our previous study revealed that high glucose induced lipotoxicity participates in epithelial-mesenchymal transition, suggesting a link between the disturbances of glucose/lipid metabolism and cell function maintenance in proximal tubular epithelial cells. Autophagy is a highly conserved "self-eating" pathway, by which cells degrade and recycle macromolecules and damaged organelles to keep the energy homeostasis. Studies have shown that some nutrient signaling pathways which regulate autophagy are also involved in many metabolic diseases. Recently autophagy has attracted intensive attention in the study of diabetes pathogenesis and its complications. It has been proposed as a new potential therapeutic target to prevent or alleviate diabetic nephropathy (DN).Based on the evidences that autophagy and glucose/lipid metabolism are closely related, we investigated HK-2 cells autophagy status under diabetic condition, as well as how it participates the lipotoxicity and EMT progress to evaluate the possible application of autophagy as a new target for prevention and treatment of DN.Methods1. HK-2 cell culture and treatment:The cells were cultured in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum in a 5% CO2 atmosphere at 37℃. Groups were divided as follows:low glucose medium (LG,5.5mmol/L glucose), high glucose medium (HG,30mmol/L glucose), HG+CQ, HG+3-MA, HG+Atg5 siRNA, HG+ scramble siRNA negative control, HG+Rapamycin, HG+DMSO, HG+Atg5 siRNA+Rapamycin, HG+scramble siRNA negative control+Rapamycin.2. The expression of autophagy markers Beclinl, LC3 and p62 under high glucose were detected by western blot.3. Morphological observation of HK-2 under microscope with autophagy inhibitors CQ,3-MA and Atg5 siRNA, as well as rapamycin.4. Intracellular lipid accumulation of HK-2 was obtained by Oil red O staining.5. The expression of EMT markers E-cadherin and Vimentin were detected by western blot.Results1. The expression of autophagy under high glucose:compared to low glucose cultured HK-2 cells, high glucose cultured cells at 6 h,24 h,48 h,96 h showed significantly increased expression of Beclinl and LC3 (P<0.05) and decreased expression of p62 (P<0.05) by western blot.2. Inverted microscope analysis and Oil-Red O staining of HK-2 cells shows accumulation of lipid droplets and morphological changes under high glucose condition: with autophagy inhibitor CQ or 3-MA, the high glucose cultured HK-2 cells started to show a changed morphology at 48h instead of 96h as seen in cells treated by HG alone, with significantly more lipid accumulation. Similar phenomenon was observed in Atg5 knockdown HK-2 cells cultured with high glucose medium. On the contrary, HK-2 cells treated with high glucose with rapamycin did not show lipid accumulation even at 96 h, and the cells still kept the epithelial shape compared to the HG plus DMSO control group,.3. The expression of EMT markers E-cadherin and Vimentin were detected by western blot:compared to HG alone, with autophagy inhibition, either with CQ or 3-MA, or with Atg5 siRNA transfection, the Vimentin expression was significantly upregulated and E-cadherin expression was decreasd starting at 48 h (P<0.05), indicating an enhanced EMT. On the contrary, E-cadherin expression was increased and Vimentin was downregulated by rapamycin treatment 96 h compared to DMSO treatement (P<0.05).ConclusionWe found under diabetic conditions, autophagy activity in HK-2 is elevated. Autophagy deficiency exacerbated high glucose induced lipid accumulation and EMT. Rapamycin ameliorated glucose induced lipid deposition and EMT, so autophagy could have been a new target for prevention and treatment of DN.