细胞外钾对小鼠远端肾小管离子通道活性的影响

Effect of extracellular potassium on the activity of distal renal tubule in mice

目的·探索细胞外钾在短时间内浓度的变化对小鼠远端肾小管离子通道钠氯共转运体(sodium chloride co-transporter,NCC)、大电导钙激活钾通道(largeconductance Ca^2+-activated K^+channel,BK)活性的影响。方法·6只8~10周龄的无特定病原体动物(specific pathogen free,SPF)级C57BL/6小鼠取肾后获得肾片,并随机放入正常钾溶液、高钾溶液、氯化钡溶液和氯化铷溶液中进行孵育。通过蛋白质印迹法观察在不同浓度钾离子溶液及不同时间下,肾片NCC蛋白的表达丰度及磷酸化水平变化,以及经2种钾离子溶液孵育2h后肾片BK中总蛋白和膜蛋白的表达变化。结果·与正常钾溶液相比,经高钾溶液孵育5、15、30min后NCC磷酸化水平显著下降(均P<0.05);而经氯化钡或氯化铷干预后,NCC磷酸化水平亦受到显著抑制(均P<0.05)。经2种钾离子溶液处理2h后,BK核心亚基α和β4的总蛋白表达间无明显变化,且BKα亚基的膜蛋白表达间亦无显著差异。结论·细胞外高浓度的钾离子可直接下调NCC的磷酸化水平,且该下调可能是为下游离子通道的迅速排钾做准备。

Objective · To investigate the effect of extracellular potassium (K+) concentration on the activities of sodium chloride co-transporter (NCC) and large conductance Ca^2+-activated K^+ channel (BK) in distal renal tubule of mice. Methods · Six specific pathogen free (SPF) C57BL/6 mice aged 8 to 10 weeks were sacrificed, and the kidney slices were made with previously reported method. Then, these slices were incubated randomly in normal K^+, high K^+, BaCl2 and RbCl solutions, respectively. The abundance and phosphorylation level of NCC in kidney slices at different K^+ concentrations and different time courses were detected by Western blotting. The overall and membrane expressions of BK in kidney slices were also detected after incubation with different K^+ solutions for 2 h. Results · Compared with normal K^+ solution, NCC phosphorylation level was significantly decreased after incubation with high K^+ solution for 5, 15, 30 min (all P<0.05), and NCC phosphorylation level was also decreased after intervention with K^+ channel inhibitor Ba^2+ or Rb^+(both P<0.05). After the treatment with high K+ solution for 2 h, neither the overall cell expression of BKα subunit and β4 subunit, nor membrane expression of BKα subunit was found significant changes compared with normal K+ incubation. Conclusion · High K+ can directly down-regulate NCC phosphorylation level, which may be preparation for kaliuresis of the downstream tubule of distal convoluted tubule.