Department of Nephrology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Bunkyo, Tokyo, Japan.
Department of pharmacology, Faculty of Medicine, Fukuoka University, Fukuoka, Japan.
PLoS One. 2020 Jun 30;15(6):e0235360. doi: 10.1371/journal.pone.0235360. eCollection 2020.
The sodium (Na+)-chloride cotransporter (NCC) expressed in the distal convoluted tubule (DCT) is a key molecule regulating urinary Na+ and potassium (K+) excretion. We previously reported that high-K+ load rapidly dephosphorylated NCC and promoted urinary K+ excretion in mouse kidneys. This effect was inhibited by calcineurin (CaN) and calmodulin inhibitors. However, the detailed mechanism through which high-K+ signal results in CaN activation remains unknown. We used Flp-In NCC HEK293 cells and mice to evaluate NCC phosphorylation. We analyzed intracellular Ca2+ concentration ([Ca2+]in) using live cell Ca2+ imaging in HEK293 cells. We confirmed that high-K+-induced NCC dephosphorylation was not observed without CaN using Flp-In NCC HEK29 cells. Extracellular Ca2+ reduction with a Ca2+ chelator inhibited high-K+-induced increase in [Ca2+]in and NCC dephosphorylation. We focused on Na+/Ca2+ exchanger (NCX) 1, a bidirectional regulator of cytosolic Ca2+ expressed in DCT. We identified that NCX1 suppression with a specific inhibitor (SEA0400) or siRNA knockdown inhibited K+-induced increase in [Ca2+]in and NCC dephosphorylation. In a mouse study, SEA0400 treatment inhibited K+-induced NCC dephosphorylation. SEA0400 reduced urinary K+ excretion and induced hyperkalemia. Here, we identified NCX1 as a key molecule in urinary K+ excretion promoted by CaN activation and NCC dephosphorylation in response to K+ load.
钠(Na+)-氯共转运体(NCC)在远曲小管(DCT)中表达,是调节尿钠和钾(K+)排泄的关键分子。我们之前报道过高钾(K+)负荷可迅速使 NCC 去磷酸化并促进小鼠肾脏的尿 K+排泄。该效应被钙调神经磷酸酶(CaN)和钙调蛋白抑制剂抑制。然而,高 K+信号导致 CaN 激活的详细机制尚不清楚。我们使用 Flp-In NCC HEK293 细胞和小鼠来评估 NCC 磷酸化。我们使用 HEK293 细胞中的活细胞 Ca2+成像来分析细胞内 Ca2+浓度([Ca2+]in)。我们证实,在没有 CaN 的情况下,高 K+诱导的 NCC 去磷酸化在 Flp-In NCC HEK29 细胞中观察不到。用 Ca2+螯合剂降低细胞外 Ca2+浓度抑制了高 K+诱导的 [Ca2+]in 增加和 NCC 去磷酸化。我们专注于 Na+/Ca2+交换器(NCX)1,它是 DCT 中表达的细胞质 Ca2+的双向调节剂。我们发现,用特异性抑制剂(SEA0400)或 siRNA 敲低 NCX1 抑制了 K+诱导的 [Ca2+]in 增加和 NCC 去磷酸化。在一项小鼠研究中,SEA0400 处理抑制了 K+诱导的 NCC 去磷酸化。SEA0400 减少了尿 K+排泄并导致高钾血症。在这里,我们确定 NCX1 是 CaN 激活和 NCC 去磷酸化响应 K+负荷促进尿 K+排泄的关键分子。