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.

Type 4 Bartter syndrome (BS) is caused by genetic mutations in barttin, which is coded for by BSND. Barttin serves as the β-subunit of the ClC-K chloride (Cl-) channel, which is widely expressed in distal nephrons. Type 4 BS is characterized by severely impaired reabsorption of salt, which may cause polyuria, hypokalemia, and metabolic alkalosis. Calcineurin inhibitors reportedly induce renal salt retention and hyperkalemia by enhancing the phosphorylation of the sodium (Na+)-potassium (K+)-2Cl- cotransporter (NKCC2) and Na+-Cl- cotransporter (NCC). In addition, we have previously reported that tacrolimus, a calcineurin inhibitor, increases the levels of phosphorylated NCC. In this study, we administered tacrolimus to barttin hypomorphic (Bsndneo/neo) mice, a murine model of type 4 BS that exhibits polyuria, hypokalemia, and metabolic alkalosis. Administration of tacrolimus increased the serum K+ level and suppressed urinary K+ excretion. Furthermore, after treatment with tacrolimus, Bsndneo/neo mice increased levels of phosphorylated NCC and NKCC2. We conclude that tacrolimus partially improves clinical phenotypes of Bsndneo/neo mice, and that calcineurin inhibitors might be effective for treating type 4 BS.

Stimulation of the OSR1 (Oxidative stress-responsive kinase-1)/SPAK [STE20 (sterile 20)/SPS1-related proline/alanine-rich kinase]-NCC (Na(+)-Cl(-) cotransporter) signaling cascade plays an important role in the WNK [With-No-Lysine (K)] kinase 4 D561A knock-in mouse model of pseudohypoaldosteronism type II (PHA II) characterized by salt-sensitive hypertension and hyperkalemia. The aim of this study was to investigate the respective roles of Osr1 and Spak in the pathogenesis of PHA II in vivo. Wnk4 (D561A/+) mice were crossed with kidney tubule-specific (KSP) Osr1 knockout (KSP-Osr1 (-/-)) and Spak knockout (Spak (-/-)) mice. Blood pressure, plasma and urine biochemistries, and the relevant protein expression in the kidneys were examined. Wnk4 (D561A/+), KSP-Osr1 (-/-), and Spak (-/-) mice recapitulated the phenotypes of PHA II, Bartter-like syndrome, and Gitelman syndrome, respectively. Wnk4 (D561A/+).KSP-Osr1 (-/-) remained phenotypically PHA II while Wnk4 (D561A/+).Spak (-/-) mice became normotensive and lacked the PHA II phenotype. Phosphorylated Spak and Ncc were similarly increased in both Wnk4 (D561A/+) and Wnk4 (D561A/+).KSP-Osr1 (-/-) mice while phosphorylated Ncc normalized in Wnk4 (D561A/+).Spak (-/-) mice. Furthermore, Wnk4 (D561A/+).KSP-Osr1 (-/-) mice exhibited exaggerated salt excretion in response to thiazide diuretics while Wnk4 (D561A/+).Spak (-/-) mice exhibited normal responses. Wnk4(D561A/+).Spak (-/-).KSP-Osr1 (-/-) triple mutant mice had low blood pressure and diminished phosphorylated Ncc. Both SPAK and OSR1 are important in the maintenance of blood pressure but activation of SPAK-NCC plays the dominant role in PHA II. SPAK may be a therapeutic target for disorders with salt-sensitive hypertension related to WNK4 activation.