Autophagy Downregulation Contributes To Insulin Resistance Mediated Injury In Insulin Receptor Knockout Podocytes In Vitro

Objective Diabetic nephropathy (DN) is the leading cause of end-stage kidney disease (ESRD) worldwide. It is also a major devastating complication of diabetes mellitus (DM), with up to 40％ of diabetic patients experiencing this problem. Molecular mechanisms involved in the etiology and progression of DM and its complications have been studied intensively. Among them, insulin resistance was proved to be a critical one. Previous studies have shown that podocytes are insulin responsive cells in glomeruli, and a loss of podocyte insulin sensitivity in perfused glomerulus results in an albuminuric phenotype even under normal glycemic conditions. Thus, insulin signaling in podocytes is essential for normal glomerular function. Autophagy is an intracellular catabolic process by which aggregates and malfunctioned organelles are degraded to maintain intracellular homeostasis. Accumulating evidence suggests that regulation of autophagy system may become a new therapeutic option for treatment of DN. Whether the process of autophagy in insulin resistant podocytes is altered is still an open question. There are also uncertainties about whether autophagy participates in the insulin resistance mediated podocyte injury. The present study aims at evaluating the role of autophagy in diabetic nephropathy with focus on podocyte insulin resistance.Methods1. Podocytes were transfected by scramble shRNA (Control) or specific shRNA targeting IR(IR shRNA).The knockdown efficiency was evaluated by Western Blot and RT-qPCR.2. The change of podocytes autophagy was tested by Western Blot. (1) The expression of p62 and Beclin was tested by Western Blot after 200 nM insulin stimulation for normal,control and IR knockout group.The expression of LC3 was tested by Western Blot with or without 50 uM chloroquine stimulation for control and IR knockout group.(2) The change of podocytes autophagy was tested by immunofluorescence. The expression of p62 Beclin and LC3 was tested by immunofluorescence after 200 nM insulin stimulation for control and IR knockout group(3) The number of autophagosomes was tested by electronic microscopy.The number of autophagosomes was tested by electronic microscopy after 200 nM insulin stimulation for control and IR knockout group.3. The expression of nephrin was tested after insulin stimulation.The expression of nephrin was tested by Western Blot and immunofluorescence after 200 nM insulin stimulation for normal,control and IR knockout group.4. The change of podocytes autophagy and nephrin expression were tested after rapamycin stimulation. The exppression of nephrin and p62 were tested by Western Blot after 10uM rapamycin stimulation for IR knockout group. The expression of LC3 was tested by Western Blot with or without 50 uM chloroquine stimulation for rapamycin stimulation group.Results1. The expression of IR can be decreased by shRNA transfection.2. Autophagy was downregulated after IR knockdown. (1) Western Blot shows that the expression of Beclin and LC3 were decreased, but P62 was up-regulated in cells transfected with IR shRNA, compared cells with control shRNA. (P﹤0.05)(2) Immunofluorescence shows that the staining of p62 was enhanced but LC3 and Beclinlwere decreased in cells transfected with IR shRNA, compared with cells transfected with control shRNA.(3) Electronic microscopy shows the number of autophagosomes in cells transfected with IR shRNA was decreased compared with control.3. Both Western Blot and immunofluorescence(IF) show that the expression of nephrin in cells transfected with IR shRNA was decreased compared with control.4. Rapamycin (RAPA) activated autophagy in IR-knockdown podocytes and increased the expression of Nephrin.Conclusion Our results show that autophagy is suppressed when podocytes lose insulin sensitivity and that treatment of rapamycin, an mTOR specific inhibitor, could attenuate insulin resistance induced podocytes injury via autophagy activation.