Effect and mechanism of insulin-like growth factor binding protein 2 on hyperglycemia-induced apoptosis in human podocyte
Background The number of diabetic nephropathy patients is increasing,and there are still patients who progress to end-stage renal disease with existing treatments.Thus,new effective therapeutic targets are urgent to be discovered.Objective To investigate the effect and mechanism of insulin-like growth factor binding protein 2(IGFBP2)on apoptosis of human podocyte under hyperglycemia stimulation.Methods The in vitro cultured human podocyte were randomly divided into four groups:the normal glucose group(NG=5 mM)and the high glucose groups(HG=30 mM)treated with different durations(24,48,72 h).IGFBP2,TNFα and ICAM-1 mRNA were detected by RT-qPCR,as well as IGFBP2 and cleaved caspase3 protein levels were detected by Western blotting.The best HG treatment time was determined to use in subsequent research.Podocyte transfected with IGFBP2 small interfering RNA(IGFBP2-siRNA)was divided into NG group,negative control intervention group and IGFBP2 knockdown siRNA intervention group(NG-IGFBP2-siRNA1,NG-IGFBP2-siRNA2,NG-IGFBP2-siRNA3).RT-qPCR was used to detect IGFBP2 mRNA expression level,and the group with the highest IGFBP2-siRNA knockdown efficiency was selected for subsequent transfection experiment.Podocyte were randomly divided into NG and HG group,NG and NG+125 ng/mL rhIGFBP2 group,HG and HG-IGFBP2-siRNA group.In the three groups,RT-qPCR was used to detect TNFα and ICAM-1 mRNA levels,JC-1 staining was used to detect mitochondrial membrane potential,confocal microscopy to detect fluorescence intensity of mitochondrial superoxide(Mito SOX)and reactive oxygen species(ROS),and flow cytometry to detect the apoptosis rate.Results RT-qPCR showed IGFBP2,TNFα and ICAM-1 mRNA levels,as well as Western blotting results showed IGFBP2 and cleaved caspase3 protein levels increased with HG treatment time.Compared with NG group,RT-qPCR showed that IGFBP2,TNFα and ICAM-1 mRNA levels in human podocyte all peaked at HG 72 h(P＜0.05),while Western blotting results showed that IGFBP2 level peaked at HG 72 h(P＜0.05)and cleaved caspase3 protein level peaked at HG 48 h(P＜0.05).Therefore,72 h was selected as the time point to induce in further studies.RT-qPCR result showed that compared with NG group,the mRNA expression of negative control intervention group had no significant difference(P＞0.05).The mRNA expression level of NG-IGFBP2-siRNA2 group was lowest with the highest knockdown efficiency(P＜0.05).Therefore,IGFBP2-siRNA2 was selected for follow-up experiments.Compared with NG group,green/red fluorescence intensity ratio of mitochondrial membrane potential,Mito SOX and ROS,and apoptosis rate in HG group were all increased(P＜0.05).Compared with NG group,TNFα and ICAM-1 mRNA levels,green/red fluorescence intensity ratio of mitochondrial membrane potential,Mito SOX and ROS,and apoptosis rate in NG+125 ng/mL rhIGFBP2 group were also all increased(P＜0.05).Compared with HG group,TNFα and ICAM-1 mRNA levels,green/red fluorescence intensity ratio of mitochondrial membrane potential,Mito SOX and ROS,apoptosis rate in HG-IGFBP2-siRNA group were all decreased(P＜0.05).Conclusion IGFBP2 knockdown decrease human podocyte apoptosis by alleviating mitochondrial dysfunction and oxidative stress under hyperglycemia.Therefore,inhibition of IGFBP2 may be a potential therapeutic target for diabetic nephropathy.
insulin like growth factor binding protein 2mitochondrial damageoxidative stressapoptosisdiabetic nephropathy
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