Acute Insulin Stimulation Induces Phosphorylation of the Na-Cl Cotransporter in Cultured Distal mpkDCT Cells and Mouse Kidney

Abstract

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.

Distal Convoluted Tubule

“…Several clinically relevant hormones stimulate salt reabsorption in the distal nephron by increasing NCC activity, and in each of these cases, SPAK and/or OSR1 seem to be involved. These hormones include aldosterone (56,57), angiotensin II (58,59), insulin (60,61), and vasopressin (62,63). All of these hormones have been shown to increase the abundance of phosphorylated active NCC and, in some cases, have also been shown to increase NCC abundance or stimulate the movement of NCC from intracellular vesicles to the luminal membrane (64)(65)(66).…”

Kelch-Like Protein 2 Mediates Angiotensin II–With No Lysine 3 Signaling in the Regulation of Vascular Tonus

“…12,28 The supernatant (120 ml) was then denatured at 60°C for 20 minutes. The primary antibodies used in this study were rabbit anti-KLHL2/KLHL3 antibody (Santa Cruz Biotechnology), anti-KLHL3 antibody (Proteintech), sheep anti-WNK3 antibody (S346C), 29,30 rabbit antiphosphorylated SPAK antibody, 31 rabbit antiphosphorylated NKCC1 (T206) antibody, 32 rabbit antiactin antibody (Cell Signaling Technology), anti-T7 antibody (EMD Millipore), anti-Cul3 antibody (BETHYL), anti-WNK1 antibody (A301-516A; BETHYL), anti-WNK4 antibody, 33 and rabbit anti-p62 antibody (Cell Signaling Technology). Alkaline phosphatase-conjugated anti-IgG antibodies (Promega) were used as secondary antibodies for immunoblotting.…”

The sodium chloride cotransporter SLC12A3: new roles in sodium, potassium, and blood pressure regulation

“…Similarly, chronic metabolic acidosis also increases aldosterone and therefore NCC [26]. Why other hormones such as insulin [13,57,108,109], vasopressin [83,91,104], estrogen [125], and norepinephrine [82] regulate NCC is less clearly defined ( Table 1), but warrants further study given the role of these hormones in normal physiology and human diseases such as diabetes mellitus, obesity, and hypertension.…”