Autophagy Protects Human Podocytes From High Glucose-induced Injury By Ameliorating Insulin Resistance

ObjectiveDiabetic nephropathy (DN) is a chronic kidney diseases that ultimately progress to end-stage renal disease (ESRD) with microalbuminuria as the earliest clinical manifestation due to compromised glomerular filtration barrier, which affects at least 40% of patients with diabetes mellitus. The structure alteration of podocyte, namely the glomerular visceral epithelial cell,has been reported to be one of the early feature of DN. Podocytes are insulin sensitive and the normal function of insulin signaling is vital for glomerular function and glucose uptake. Autophagy is an evolutionarily conserved catabolic process degrading aggregated proteins and and recycling damaged organelles for cellular homeostasis, and autophagy deficiency has been involved in the pathogenesis of insulin resistance and diabetes. It was comfirmed that the regulation of autophagy flux play a crucial on insulin signaling in liver, muscle and fat tissue. But the role of autophagy in the regulation of insulin signaling is controversial. In skeletal muscle cells, Atg5 gene silencing or inhibitior administration of autophagy can both reduce insulin sensitivity. The overexpression of Atg5 in mice can activates autophagy and improve insulin sensitivity.In addtion, autophagy deficiency in differentiated mature adipocytes resulted in ER stress and impaired insulin signaling, further supporting the crucial roles of autophagy in insulin resistance. HCV infection disturbs glucose homeostasis or insulin signaling to induce IR and also elicits autophagy. However, adipose tissue-specific disruption of critical autophagic genes such as Atg7 in mice suggested a protective effect from high-fat diet-induced obesity and insulin resistance. However, it remains mystical about the role of autophagy in podocyte malfunction under diabetic conditions and its involvement in the insulin responsiveness in podocyte. In the present study, we aimed to investigate the high g lucose induced insulin resistance and cell injury in human podocytes and the putative role of autophagy in this process.Methods1. Human podocyte cell culture and treatment:Human podocytes, kindly provided by professor Fan Yi (Shandong University, Jinan, China), were cultured as described. The cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum under permissive conditions at 33 ℃ and 5% CO2. For differentiation, cells were transferred to nonpermissive conditions at 37 ℃. Cells were harvested after 7 to 10 days of differentiation. Insulin (200 nmol/1) was added 30 min before the cells were harvested for the insulin signaling experiments. The supplemented medium were as follows:low glucose medium (5.5mmol/Lglucose, LG), high glucose medium (30mmol/L glucose, HG), high osmotic (30 mmol/L mannitol, HO).2. The effects on insulin responsiveness were analyzed by glucose uptake and the phosphorylation of insulin receptor β-subunit with nephrin expression as podocyte injury marker. Glucose uptake in high glucose cultured human podocytes were evaluated by 2-NBDG uptake assay. The cell viability was measured by CCK-8 assay. The phosphorylation of insulin receptor was detected by Western blot analysis and immunofluorescent staining.3. The expression of phosphorylation of insulin receptor β-subunit evaluated by immuno histochemistry on normal and diabetic nephropathy patients.4. The expression of nephrin and autophagy markers, such as LC3B, beclin-1 and p62 were detected by Western blot and RT-qPCR. Total mRNAs were purified from cells and reversely transcribed into cDNA by PrimeScriptTM RT Reagent Kit with gDNA Eraser (Takara). Quantitative real-time PCR was performed using the light cycler 480 (Roche Diagnostics) with SYBR Premix Ex TaqTM Ⅱ (Takara). Gene-specific primers were used for Beclin-1, p62, LC3BII and Nephrin. Samples were normalized to β-actin as the control.5. Atophagy activity as regulated by its inhibitor 3-MA(5 mM) and activator rapamycin (Rapa,100 nM).2-NBDG was used to detect the glucose uptake after regulating the activity of autophagy. Blot western and immunofluorescence were used to detect the p-IR level, and the expression of nephrin was detected by blot western analysis.Results1. Glucose uptake and the phosphorylation of insulin receptor were decreased in high glucose cultured human podocytes. The glucose uptake in HG group was dramatically decreased compared to both LG and HO group(p<0.05). Phosphoryted IR (p-IR) level was clearly decreased compared to low glucose and high osmotic controls(p<0.05), with the total IR amount unchanged. And p-IR immunofluorescent staining was obviously reduced by high glucose stimulation compared to LG and HO groups.2. Expression of nephrin was reduced in high glucose cultured human podocytes. As Nephrin is essential for insulin induced glucose uptake in human glomerular podocytes, the expression of Nephrin was investigated in both mRNA level by qPCR and protein level by western blot. The high glucose exposure led to a reduction of the mRNA level of nephrin about 65% compared to LG group and about 73% compared to HO group. And the protein level was also diminished about 30% upon high glucose stimulation.3. Autophagy was inhibited in high glucose cultured human podocytes. Both LC3B and Beclin-1 were dramatically reduced on the protein level in HG treated cells compared to LG and HO groups(p<0.05), while p62 was significantly increased (p<0.05), indicating a suppressed autophagy flux. The qPCR results had the same tendency.4. Insulin sensitivity was regulated by autophagy in human podocytes. To clarify the role of autophagy in this process and the potential therapeutic intervention targeting autophagy, human podocytes were cultured under 30 mM high glucose environment and were stimulated by 5 mM 3-Methyladenine (3-MA) or 100 nM rapamycin with the absence or presence of insulin for 30 min. The alteration of autophagy level was first validated by both western blot and immunofluorescent staining, there was no obvious difference for LC3BII and Beclin-1 expression under the induction of insulin, however,3-MA significantly reduced the level of LC3BII, which was boosted by rapamycin in high glucose cultured human podocytes. The expression of Beclin-1 had the same tendency. On the contrary, the accumulation of p62 was increased by insulin, which was further enhanced by 3-MA. Rapamycin clearly decreased the accumulation of p62. These results were further convinced by immunostaining.The contribution of the altered autophagy to the HPC insulin resistance was investigated in depth. As shown in the 2-NBDG assay, the glucose uptake was increased by insulin induction, and then reduced to normal level by 3-MA treatment. The phosphorylation of insulin receptor was also decreased after autophagy was inhibited by 3-MA as shown by western blot analysis. Immunofluorescent staining further confirmed this result.Interestingly, when we treated the cells with rapamycin, the glucose uptake was dramatically enhanced in high glucose cultured human podocytes, followed by a significantly enhanced phosphorylation of insulin receptor, with the total insulin receptor level unchanged, indicating a restored insulin sensitivity. Finally, we proved the inhibition of autophagy by 3-MA reduced the Nephrin expression, and the activation of autophagy by rapamycin ameliorated the Nephrin lose.Conclusions1. High glucose exposure for 72 h contributed to human podocyte injury and impaired insulin response.2. Autophagy was inhibited by high glucose in human podocytes.3. The inhibition of autophagy deteriorated podocyte injury induced and the impaired insulin sensitivity by high glucose.4. The activation of autophagy restored the insulin sensitivity and ameliorated cell injury in human podocytes exposed to high glucose.