Department of Nephrology, Graduate School of Medicine, Tokyo Medical and Dental University, Tokyo, Japan.
PLoS One. 2011;6(8):e24277. doi: 10.1371/journal.pone.0024277. Epub 2011 Aug 31.
The NaCl cotransporter (NCC) is essential for sodium reabsorption at the distal convoluted tubules (DCT), and its phosphorylation increases its transport activity and apical membrane localization. Although insulin has been reported to increase sodium reabsorption in the kidney, the linkage between insulin and NCC phosphorylation has not yet been investigated. This study examined whether insulin regulates NCC phosphorylation. In cultured mpkDCT cells, insulin increased phosphorylation of STE20/SPS1-related proline-alanine-rich kinase (SPAK) and NCC in a dose-dependent manner. This insulin-induced phosphorylation of NCC was suppressed in WNK4 and SPAK knockdown cells. In addition, Ly294002, a PI3K inhibitor, decreased the insulin effect on SPAK and NCC phosphorylation, indicating that insulin induces phosphorylation of SPAK and NCC through PI3K and WNK4 in mpkDCT cells. Moreover, acute insulin administration to mice increased phosphorylation of oxidative stress-responsive kinase-1 (OSR1), SPAK and NCC in the kidney. Time-course experiments in mpkDCT cells and mice suggested that SPAK is upstream of NCC in this insulin-induced NCC phosphorylation mechanism, which was confirmed by the lack of insulin-induced NCC phosphorylation in SPAK knockout mice. Moreover, insulin administration to WNK4 hypomorphic mice did not increase phosphorylation of OSR1, SPAK and NCC in the kidney, suggesting that WNK4 is also involved in the insulin-induced OSR1, SPAK and NCC phosphorylation mechanism in vivo. The present results demonstrated that insulin is a potent regulator of NCC phosphorylation in the kidney, and that WNK4 and SPAK are involved in this mechanism of NCC phosphorylation by insulin.
钠氯共转运蛋白（NCC）对于远曲小管（DCT）的钠重吸收至关重要，其磷酸化可增加其转运活性和顶端膜定位。虽然胰岛素已被报道可增加肾脏的钠重吸收，但胰岛素与 NCC 磷酸化之间的联系尚未得到研究。本研究探讨了胰岛素是否调节 NCC 磷酸化。在培养的 mpkDCT 细胞中，胰岛素以剂量依赖性方式增加 STE20/SPS1 相关脯氨酸-丙氨酸丰富激酶（SPAK）和 NCC 的磷酸化。这种胰岛素诱导的 NCC 磷酸化在 WNK4 和 SPAK 敲低细胞中受到抑制。此外，PI3K 抑制剂 Ly294002 降低了胰岛素对 SPAK 和 NCC 磷酸化的作用，表明胰岛素通过 WNK4 和 mpkDCT 细胞中的 PI3K 诱导 SPAK 和 NCC 的磷酸化。此外，急性给予胰岛素可增加小鼠肾脏中氧化应激反应激酶-1（OSR1）、SPAK 和 NCC 的磷酸化。mpkDCT 细胞和小鼠的时程实验表明，在这种胰岛素诱导的 NCC 磷酸化机制中，SPAK 是 NCC 的上游，这在 SPAK 敲除小鼠中缺乏胰岛素诱导的 NCC 磷酸化得到证实。此外，在 WNK4 低表达小鼠中给予胰岛素不会增加肾脏中 OSR1、SPAK 和 NCC 的磷酸化，表明 WNK4 也参与了体内胰岛素诱导的 OSR1、SPAK 和 NCC 磷酸化机制。本研究结果表明，胰岛素是肾脏中 NCC 磷酸化的有效调节剂，WNK4 和 SPAK 参与了胰岛素诱导的 NCC 磷酸化机制。
