RanBPM对肾小管钠-氯共同转运子的调控作用及对ERK1/2信号通路的影响

目的：观察Ran结合蛋白M（RanBPM）对肾小管钠-氯共同转运子（NCC）蛋白表达及相关ERK1/2信号通路蛋白表达的影响。方法：免疫共沉淀检测RanBPM蛋白与WNK4蛋白是否存在直接相互作用；观察转染RanBPM质粒对EGF诱导细胞ERK1/2磷酸化的影响，通过转染质粒过表达RanBPM蛋白或siRNA抑制RanBPM基因，免疫蛋白印迹法检测细胞内源性NCC蛋白含量和ERK1/2磷酸化水平的变化。结果：免疫共沉淀实验证实WNK4和RanBPM之间存在直接的相互作用。RanBPM可以抑制EGF诱导的ERK1/2磷酸化。RanBPM过表达可使细胞ERK1/2磷酸化减少（1.000±0.074 vs. 0.275±0.041，P＜0.01），而NCC蛋白表达增加（1.000±0.115 vs. 1.470±0.105，P＜0.01）。siRNA沉默RanBPM基因后，ERK1/2磷酸化增加（1.000±0.194 vs. 2.301±0.220，P＜0.01），NCC蛋白表达下降（1.000±0.223 vs. 0.556±0.132，P＜0.01）。转染RanBPM可阻止WNK4对ERK1/2信号通路的影响和对NCC蛋白表达的抑制作用。结论：RanBPM通过与WNK4相互作用，影响ERK1/2信号通路对NCC蛋白表达的调控作用。

The regulatory role of RanBPM in renal tubular sodium chloride cotransporter and its effect on ERK1/2 signaling pathway

Objective: To observe the effect of Ran binding protein M (RanBPM) on the expression of NCC protein in renal tubules and the change of ERK1/2 signaling pathway protein. Methods: Immunocoprecipitation was used to detect whether there was direct interaction between RanBPM and WNK4 protein. The effect of transfection of RanBPM plasmid on ERK1/2 phosphorylation induced by EGF was observed. RanBPM gene was inhibited by overexpression of RanBPM protein or siRNA. The content of endogenous NCC protein and the level of ERK1/2 phosphorylation were detected by Western blot. The effect of RanBPM on WNK4 regulation of NCC by ERK1/2 phosphorylation was explored before and after transfection. Results: Immunocoprecipitation confirmed that there was a direct interaction between WNK4 and RanBPM. RanBPM could inhibit EGF induced ERK1/2 phosphorylation. Overexpression of RanBPM reduced ERK1/2 phosphorylation (1.000±0.074 vs. 0.275± 0.041, P<0.01) and increased NCC protein expression (1.000±0.115 vs. 1.470±0.105, P<0.01). After siRNA silenced RanBPM gene, ERK1/2 phosphorylation increased (1.000±0.194 vs. 2.301±0.220, P<0.01), resulting in decreased NCC protein expression (1.000±0.223 vs. 0.556±0.132, P<0.01). When RanBPM was transfected, WNK4 inhibited NCC protein expression and no longer affected ERK1/2 phosphorylation. Conclusion: RanBPM interacted with WNK4 affects the regulation of ERK1/2 signaling pathway in NCC protein expression.