Muscle wasting or sarcopenia contributes to morbidity and mortality in patients with cancer, renal failure, or heart failure, and in elderly individuals. Na+-K+-2Cl− cotransporter 1 (NKCC1) is highly expressed in mammalian skeletal muscle, where it contributes to the generation of membrane ion currents and potential. However, the physiologic function of NKCC1 in myogenesis is unclear. We investigated this issue using the NKCC1 inhibitors bumetanide and furosemide, which are commonly used loop diuretics. NKCC1 protein levels increased during C2C12 murine skeletal myoblast differentiation, similarly to those of the myogenic markers myogenin and myosin heavy chain (MHC). NKCC1 inhibitors markedly suppressed myoblast fusion into myotubes and the expression of myogenin and MHC. Furthermore, phosphorylated and total NKCC1 levels were elevated in mouse skeletal muscles after 6 weeks’ voluntary wheel running. Immunofluorescence analyses of myofiber cross-sections revealed more large myofibers after exercise, but this was impaired by daily intraperitoneal bumetanide injections (0.2 or 10 mg/kg/day). NKCC1 plays an essential role in myogenesis and exercise-induced skeletal muscle hypertrophy, and sarcopenia in patients with renal or heart failure may be attributable to treatment with loop diuretics.

Mutations in the with-no-lysine kinase 1 (WNK1), WNK4, kelch-like 3 (KLHL3), and cullin3 (CUL3) genes are known to cause the hereditary disease pseudohypoaldosteronism type II (PHAII). It was recently demonstrated that this results from the defective degradation of WNK1 and WNK4 by the KLHL3/CUL3 ubiquitin ligase complex. However, the other physiological in vivo roles of KLHL3 remain unclear. Therefore, here we generated KLHL3 Ϫ/Ϫ mice that expressed ␤-galactosidase (␤-Gal) under the control of the endogenous KLHL3 promoter. Immunoblots of ␤-Gal and LacZ staining revealed that KLHL3 was expressed in some organs, such as brain. However, the expression levels of WNK kinases were not increased in any of these organs other than the kidney, where WNK1 and WNK4 increased in KLHL3 Ϫ/Ϫ mice but not in KLHL3 ϩ/Ϫ mice. KLHL3 Ϫ/Ϫ mice also showed PHAII-like phenotypes, whereas KLHL3 ϩ/Ϫ mice did not. This clearly demonstrates that the heterozygous deletion of KLHL3 was not sufficient to cause PHAII, indicating that autosomal dominant type PHAII is caused by the dominant negative effect of mutant KLHL3. We further demonstrated that the dimerization of KLHL3 can explain this dominant negative effect. These findings could help us to further understand the physiological roles of KLHL3 and the pathophysiology of PHAII caused by mutant KLHL3.KEYWORDS kelch-like 3 (KLHL3), distal convoluted tubule, hypertension, kidney, NaCl cotransporter, with-no-lysine kinase (WNK) P seudohypoaldosteronism type II (PHAII) is a hereditary disease that is characterized by salt-sensitive hypertension, hyperkalemia, metabolic acidosis, and thiazide sensitivity (1). Mutations in the with-no-lysine kinase 1 (WNK1) and WNK4 genes are known to cause PHAII (2). Furthermore, it has generally been considered that overactivation of the thiazide-sensitive Na-Cl cotransporter (NCC) is the main cause of PHAII (3).Many studies have demonstrated that WNK kinases are at the top of the signaling cascade, along with oxidative stress-responsive gene 1 (OSR1), Ste20-related prolinealanine-rich kinase (SPAK), and the solute carrier family 12a (SLC12a) transporter family, which includes the NCC and Na-K-Cl cotransporter (NKCC). WNK phosphorylates and activates OSR1/SPAK, which in turn phosphorylate and activate the SLC12a transporters (4-6). The regulation of NCC by WNK-OSR1/SPAK signaling was confirmed in vivo using various genetically engineered mouse models (7-14) and overactivation of this WNK-OSR1/SPAK-NCC phosphorylation signal in the kidney causes PHAII (5, 6, 15, 16).

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Recently, the kelch-like protein 3 (KLHL3)-Cullin3 complex was identified as an E3 ubiquitin ligase for with no lysine (WNK) kinases, and the impaired ubiquitination of WNK4 causes pseudohypoaldosteronism type II (PHAII), a hereditary hypertensive disease. However, the involvement of WNK kinase regulation by ubiquitination in situations other than PHAII has not been identified. Previously, we identified the WNK3-STE20/SPS1-related proline/alanine-rich kinase-Na/K/Cl cotransporter isoform 1 phosphorylation cascade in vascular smooth muscle cells and found that it constitutes an important mechanism of vascular constriction by angiotensin II (AngII). In this study, we investigated the involvement of KLHL proteins in AngII-induced WNK3 activation of vascular smooth muscle cells. In the mouse aorta and mouse vascular smooth muscle (MOVAS) cells, KLHL3 was not expressed, but KLHL2, the closest homolog of KLHL3, was expressed. Salt depletion and acute infusion of AngII decreased KLHL2 and increased WNK3 levels in the mouse aorta. Notably, the AngII-induced changes in KLHL2 and WNK3 expression occurred within minutes in MOVAS cells. Results of KLHL2 overexpression and knockdown experiments in MOVAS cells confirmed that KLHL2 is the major regulator of WNK3 protein abundance. The AngII-induced decrease in KLHL2 was not caused by decreased transcription but increased autophagy-mediated degradation. Furthermore, knockdown of sequestosome 1/p62 prevented the decrease in KLHL2, suggesting that the mechanism of KLHL2 autophagy could be selective autophagy mediated by sequestosome 1/p62. Thus, we identified a novel component of signal transduction in AngII-induced vascular contraction that could be a promising drug target. 26: 212926: -213826: , 201526: . doi: 10.1681 Recently, the kelch-like protein 3 (KLHL3) and Cullin3 (Cul3) were identified as the genes responsible for a hereditary hypertensive diseasepseudohypoaldosteronism type II (PHAII). 1 Because the with no lysine (WNK) kinases (WNK1 and WNK4) had already been identified as the responsible genes for PHAII and the KLHL proteins were known to serve as substrate adaptors of Cul3-based E3 ubiquitin ligase, 1-3 we speculated and recently showed that KLHL3 functions as an E3 ligase with Cul3 for WNK4 and that the impaired ubiquitination of WNK4 and its subsequent increase within the cell stimulates the downstream OSR1/STE20/SPS1-related proline/alanine-rich kinase (SPAK)-NaCl cotransporter signaling and causes PHAII. 4 In addition to WNK4, WNK1 and other WNK kinases (WNK2 and WNK3) have been identified as substrates of KLHL3-Cul3 E3 ligase, because KLHL3 can bind to all WNKs in a highly conserved domain (acidic domain). 5 Furthermore, we recently reported that KLHL2 possesses a kelch repeat domain (WNK binding domain) highly similar to that of KLHL3 and that J Am Soc Nephrol