Objective: To determine whether hyperglycemia activates NFAT2 in cultured podocytes to cause podocyte apoptosis and explore the role of NFAT2 in high glucose-induced podocyte apoptosis.
Methods: Immortalized mouse podocytes were cultured in the presence of normal (5.3 mmol/L) or high glucose (10, 20, 30, and 40 mmol/L) or pretreated with 11R-vivit (100 nmol/L) or cyclosporine A (500 nmol/L) before exposure to 20 mmol/L glucose for different durations (0.5-48 h). The activation of NFAT2 in the podocytes was detected using Western blotting and immunofluorescence assay. The role of NFAT2 in hyperglycemia-induced podocyte apoptosis was explored by observing the inhibition of NFAT2 activation by 11R-vivit using flow cytometry. Intracellular Ca2+ was monitored in high glucose-treated podocytes using Fluo-3/AM. The mRNA and protein expressions of the apoptosis gene Bax were detected using real time-qPCR and Western blotting.
Results: Exposure to high glucose in the medium time- and dose-dependently activated NFAT2 in cultured podocytes. Pretreatment with cyclosporine A or 11R- VIVIT completely blocked nuclear accumulation of NFAT2. Treatment with 11R- vivit also inhibited high glucoseinduced apoptosis in cultured podocytes. Exposure to high glucose obviously increased [Ca2 +]I in the podocytes to cause activation of calcineurin and the subsequent increment of nuclear accumulation of NFAT2 and Bax expression.
Conclusions: High glucose-induced apoptosis in podocytes is mediated by calcineurin/NFAT2/Bax signaling pathway, which may serve as a potential target for therapeutic intervention.
目的: 探讨高糖在体外培养足细胞中是否激活NFAT2并导致足细胞凋亡，并探讨其发生机制。
方法: 大鼠足细胞分别在不同的培养液中（葡萄糖5.3、10、20、30和40 mmol/L，葡萄糖5.3 mmol/L+Mannitol 14.7 mmol/L，葡萄糖20 mmol/L+11R-vivit 100 nmol/L，葡萄糖20 mmol/L+CsA 500 nmol/L）培养不同的时间（0.5、1、2、4、12、24和48 h）；分别用免疫荧光、Western blot检测NFAT 2的活化；流式细胞术检测足细胞的凋亡，实时定量PCR和Western blot检测Bax表达；用激光共聚焦动态观察足细胞内Ca2+水平的变化。
结果: 不同葡萄糖浓度培养的足细胞，足细胞核中NFAT2/Histone3的比值分别为0.999±0.001；1.652± 0.188；2.405±0.281；1.850±0.397及2.000±0.381，葡萄糖20 mmol/L（HG）分别作用足细胞0.5、1、2和4 h，Western blot显示NFAT2/Histone 3结果分别为：1.253±0.332、1.656±0.464、2.080±0.319及1.601±0.431，培养2 h时NFAT2的活化达到高峰；经过CsA或者11R-VIVIT预处理后可见完全阻断NFAT2的核积累，也阻断细胞凋亡；同时，我们发现高糖可增加体外培养足细胞的钙离子内流，导致NFAT2的核积累和Bax表达。
结论: 高糖可能通过CaN/NFAT2/Bax信号通路介导足细胞的凋亡，阻断CaN/ NFAT2/Bax信号通路可以减少高糖诱导的足细胞凋亡。
Keywords: NFAT2; apoptosis; high glucose; podocyte.