NFAT2参与高糖诱导的大鼠足细胞凋亡

NFAT2 mediates high glucose-induced apoptosis in glomerular podocytes in vitro

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-vivit100 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。

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 11 R-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 11 R-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 11 R-VIVIT completely blocked nuclear accumulation of NFAT2. Treatment with 11 R-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. Conclusion High glucose-induced apoptosis in podocytes is mediated by calcineurin/NFAT2/Bax signaling pathway, which may serve as a potential target for therapeutic intervention.