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Recently, in addition to epithelial sodium channel alpha-subunit (αENaC), the thiazide-sensitive sodium-chloride cotransporter (NCC) and pendrin, also known as sodium-independent chloride/iodide transporter, were reported to be activated by aldosterone. Here, we investigated whether chloride (Cl - ) is responsible for hypertension, inflammation, and renal damage in aldosterone-infused rats. Following left nephrectomy, 8-wk-old male Sprague-Dawley rats were allocated into four groups: 1) drinking 1.0% sodium chloride solution with aldosterone infusion (Aldo/NaCl rats); 2) drinking 1.44% sodium bicarbonate solution with aldosterone infusion (Aldo/NaHCO 3 rats); 3) drinking distilled water with aldosterone infusion (Aldo/water rats); and 4) drinking distilled water without aldosterone infusion (sham rats). Additionally, heminephrectomized rats with aldosterone infusion were fed a 0.26% NaCl diet (control); 8.0% NaCl diet (high-Na/high-Cl); or a 4.0% NaCl 6.67% sodium citrate diet (high-Na/half-Cl). Last, Aldo/NaCl rats were treated with or without hydrochlorothiazide. Blood pressure in the Aldo/NaCl rats was significantly higher than in the Aldo/NaHCO 3 rats, which was associated with the increased expression of NCC. Expression of markers of inflammation (CD3, CD68, interleukin-17A) and fibrosis (α-smooth muscle actin, collagen 1) were also increased in Aldo/NaCl rats. Similarly, aldosterone-infused rats fed a high-Na/half-Cl diet had lower blood pressure than those fed a high-Na/high-Cl diet, with a reduction of phosphorylated NCC, but not αENaC and pendrin. NCC inhibition with hydrochlorothiazide attenuated interleukin-17A protein expression along with the phosphorylation of NCC in Aldo/NaCl rats. These findings suggest that NCC-mediated Cl - uptake plays important roles in the development of aldosterone-induced hypertension and renal injury.

The sodium chloride cotransporter (NCC) has been identified as a key molecule regulating potassium balance. The mechanisms of NCC regulation during low extracellular potassium concentrations have been studied in vitro. These studies have shown that hyperpolarization increased chloride efflux, leading to the activation of chloride-sensitive with-no-lysine kinase (WNK) kinases and their downstream molecules, including STE20/SPS1-related proline/alanine-rich kinase (SPAK) and NCC. However, this mechanism was not studied in vivo Previously, we developed the barttin hypomorphic mouse ( Bsnd neo/neo mice), expressing very low levels of barttin and ClC-K channels, because barttin is an essential β-subunit of ClC-K. In contrast with Bsnd -/- mice, Bsnd neo/neo mice survived to adulthood. In Bsnd neo/neo mice, SPAK and NCC activation after consuming a low-potassium diet was clearly impaired compared with that in wild-type (WT) mice. In ex vivo kidney slice experiment, the increase in pNCC and SPAK in low-potassium medium was also impaired in Bsnd neo/neo mice. Furthermore, increased blood pressure was observed in WT mice fed a high-salt and low-potassium diet, which was not evident in Bsnd neo/neo mice. Thus, our study provides in vivo evidence that, in response to a low-potassium diet, ClC-K and barttin play important roles in the activation of the WNK4-SPAK-NCC cascade and blood pressure regulation., (© 2018 The Author(s).)

Metabolic syndrome patients have insulin resistance, which causes hyperinsulinemia, which in turn causes aberrant increased renal sodium reabsorption. The precise mechanisms underlying this greater salt sensitivity of hyperinsulinemic patients remain unclear. Abnormal activation of the recently identified with-no-lysine kinase (WNK)-oxidative stress-responsive kinase 1 (OSR1)/STE20/SPS1-related proline/alanine-rich kinase (SPAK)-NaCl cotransporter (NCC) phosphorylation cascade results in the salt-sensitive hypertension of pseudohypoaldosteronism type II. Here, we report a study of renal WNK-OSR1/SPAK-NCC cascade activation in the db/db mouse model of hyperinsulinemic metabolic syndrome. Thiazide sensitivity was increased, suggesting greater activity of NCC in db/db mice. In fact, increased phosphorylation of OSR1/SPAK and NCC was observed. In both SpakT243A/+ and Osr1T185A/+ knock-in db/db mice, which carry mutations that disrupt the signal from WNK kinases, increased phosphorylation of NCC and elevated blood pressure were completely corrected, indicating that phosphorylation of SPAK and OSR1 by WNK kinases is required for the increased activation and phosphorylation of NCC in this model. Renal phosphorylated Akt was increased in db/db mice, suggesting that increased NCC phosphorylation is regulated by the phosphatidylinositol 3-kinase/Akt signaling cascade in the kidney in response to hyperinsulinemia. A phosphatidylinositol 3-kinase inhibitor (NVP-BEZ235) corrected the increased OSR1/SPAK-NCC phosphorylation. Another more specific phosphatidylinositol 3-kinase inhibitor (GDC-0941) and an Akt inhibitor (MK-2206) also inhibited increased NCC phosphorylation. These results indicate that the phosphatidylinositol 3-kinase/Akt signaling pathway activates the WNK-OSR1/SPAK-NCC phosphorylation cascade in db/db mice. This mechanism may play a role in the pathogenesis of salt-sensitive hypertension in human hyperinsulinemic conditions, such as the metabolic syndrome.

We recently generated Wnk4(D561A/+) knockin mice and found that a major pathogenesis of pseudohypoaldosteronism type II was the activation of the OSR1/SPAK kinase-NaCl cotransporter (NCC) phosphorylation cascade by the mutant WNK4. However, the physiological roles of wild-type WNK4 on the regulation of Na excretion and blood pressure, and whether wild-type WNK4 functions positively or negatively in this cascade, remained to be determined. In the present study, we generated WNK4 hypomorphic mice by deleting exon 7 of the Wnk4 gene. These mice did not show hypokalemia and metabolic alkalosis, but they did exhibit low blood pressure and increased Na and K excretion under low-salt diet. Phosphorylation of OSR1/SPAK and NCC was significantly reduced in the mutant mice as compared with their wild-type littermates. Protein levels of ROMK and Maxi K were not changed, but epithelial Na channel appeared to be activated as a compensatory mechanism for the reduced NCC function. Thus, wild-type WNK4 is a positive regulator for the WNK-OSR1/SPAK-NCC cascade, and WNK4 is a potential target of anti-hypertensive drugs.

[Figure: see text]. [ABSTRACT FROM AUTHOR]

Background: There have been still few case reports of pseudohypoaldosteronism type II (PHA2), also known as Gordon's syndrome, genetically diagnosed, and this is the first report of familial PHA2 case in Japan with a novel D564N mutation in WNK4. Methods: A 29‐year‐old woman was admitted to our hospital due to hyperkalemia (serum potassium: 6.4 mmol/L). She had mild hypertension (135/91 mm Hg), a bicarbonate level at the lower limit of the normal range (HCO3: 22 mmol/L) with a normal anion gap, low plasma renin activity (0.2 ng ml‐1 hr‐1), and high urinary calcium excretion (505.4 mg/g Cre). A hereditary condition was suspected because her mother also had the same symptoms. We performed a comprehensive genetic analysis for major inherited kidney diseases with next‐generation sequencing including the genes responsible for PHA2 (WNK1, WNK4, KLHL3, and CUL3). Results: Genetic analysis revealed that the patient and her mother had a novel missense mutation (D564N) in the acidic motif in WNK4, which leads to the diagnosis of PHA2. Administration of trichlormethiazide (1 mg/day) effectively ameliorated her blood pressure (114/69 mm Hg), plasma bicarbonate (25 mmol/L), serum potassium (4.3 mmol/L), and urinary calcium excretion (27.2 mg/g Cre). Conclusion: We report the first Japanese familial case of PHA2 with WNK4 mutation. D564N mutation in WNK4 is a novel genetic cause of PHA2 with a relatively mild phenotype. [ABSTRACT FROM AUTHOR]

We recently reported increased phosphorylation of the NaCl cotransporter (NCC) in Wnk4D561A/+ knock-in mice, an ideal model of the human hereditary hypertensive disease pseudohypoaldosteronism type II (PHAII). Although previous in vitro studies had suggested the existence of a phosphorylation cascade involving the WNK, OSR1 and SPAK kinases, whether the WNK-OSR1/SPAK cascade is in fact fully responsible for NCC phosphorylation in vivo and whether the activation of this cascade is the sole mediator of PHAII remained to be determined. To clarify these issues, we mated the Wnk4D561A/+ knock-in mice with Spak and Osr1 knock-in mice in which the T-loop threonine residues in SPAK and OSR1 (243 and 185, respectively) were mutated to alanine to prevent activation by WNK kinases. We found that NCC phosphorylation was almost completely abolished in Wnk4D561A/+SpakT243A/T243AOsr1T185A/+ triple knock-in mice, clearly demonstrating that NCC phosphorylation in vivo is dependent on the WNK-OSR1/SPAK cascade. In addition, the high blood pressure, hyperkalemia and metabolic acidosis observed in Wnk4D561A/+ mice were corrected in the triple knock-in mice. These results clearly establish that PHAII caused by the WNK4 D561A mutation is dependent on the activation of the WNK-OSR1/SPAK-NCC cascade and that the contribution of other mechanisms to PHAII (independent of the WNK-OSR1/SPAK cascade) could be minimal. [ABSTRACT FROM AUTHOR]