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.
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