On the other hand, WNK4 hypomorphic mice showed decreased phosphorylation of NCC and lower blood pressure, and other WNKs could not compensate (Ohta et al., 2009). However, lower blood pressure was observed in WNK3 knockout mice fed low-salt diet. WNK4 and WNK1 expression was slightly elevated in the knockout mice under low-salt diet, suggesting compensation for WNK3 knockout by these WNKs. Thus, WNK3 may have some role in the WNK-OSR1/SPAK-NCC/NKCC2 transmission cascade in the kidney, but its contribution to total WNK kinase activity may be minimal. Keywords:Na-K-Cl cotransporter, CCT241736 Na-Cl cotransporter, WNK3, mouse kidney, WNK == CCT241736 Introduction == Pseudohypoaldosteronism type II (PHAII) is an autosomal-dominant disease characterized by hypertension due to increased renal salt reabsorption, hyperkalemia and metabolic acidosis (Gordon, 1986;Schambelan et al., 1981;Achard et al., 2001). Mutations in with-no-lysine kinase 1 (WNK1) and with-no-lysine kinase 4 (WNK4) have been reported to cause PHAII (Wilson et al., 2001). We generated WNK4D561A/+knock-in mice, an ideal mouse model of PHAII, and observed increased phosphorylation of oxidative stress-responsive kinase-1 (OSR1), STE20/SPS1-related proline/alanine-rich kinase (SPAK) and thiazide-sensitive Na-Cl co-transporter (NCC) CCT241736 (Yang, S. S. et al., 2007). We previously exhibited in in vitro experiments that WNK1 and WNK4 phosphorylated and activated OSR1 and SPAK kinases, and that OSR1 and SPAK could phosphorylate NCC (Moriguchi et al., 2005;Vitari et al., 2005;Vitari et al., 2006;Richardson et al., 2008). Furthermore, Pacheco-Alvarez et al. reported that phosphorylation of NCC at Thr 53 and 58, and at Ser 71 was important for NCC function inXenopusoocytes (Pacheco-Alvarez et al., 2006), and we showed that phosphorylated NCC is concentrated around the apical membranes of distal convoluted tubules in the WNK4D561A/+knock-in mice, which suggests that phosphorylation may also be important for intracellular localization of NCC (Yang, S. S. et al., 2007). Based on the above evidence, we postulated that WNK, OSR1/SPAK and NCC constitute a signal cascade in the in vivo kidney, which is usually important for NaCl homeostasis and blood pressure regulation. Recently, we mated WNK4D561A/+knock-in mice with SPAK and OSR1 kinase-dead knock-in mice, in which the T-loop Thr residues in SPAK (Thr 243) and OSR1 (Thr 185) were mutated to Ala to prevent activation by WNK kinases (Rafiqi et al., 2010). In these triple knock-in mice, PHAII phenotypes and increased phosphorylation of NCC were completely corrected (Chiga et al., 2011). Based on the definitive genetic data, we clearly established the presence of the WNK-OSR1/SPAK-NCC kinase cascade in the in vivo kidney. Even though transmission cascade was established, it remains unclear which WNK kinase is usually responsible in the kidney. It is also uncertain whether a single dominant WNK kinase is present in each different type of cell, or whether multiple WNKs are present in the same cells and function as a WNK kinase complex, as postulated by Yang, C. L. et al. (Yang, C. Rabbit Polyclonal to SNIP L. et al., 2007). In fact, in addition to WNK1 and WNK4, whose mutations cause PHAII, WNK3 mRNA expression was reported to be present in the kidney (Holden et al., 2004). Therefore, although WNK3 mutation has not been observed in PHAII, WNK3 could be an important component of WNK kinase-mediated transmission cascade in kidney. Previous in vitro data found that WNK3 regulates SLC12A cotransporters. WNK3 was shown to be an activator of Na-K-Cl cotransporter (NKCC1 and 2) and NCC (Kahle et al., 2005;Rinehart et al., 2005;Yang, C. L. et al., 2007;San-Cristobal et al., 2008;Ponce-Coria et al., 2008;Glover et al., 2009;Cruz-Rangel et al., 2011), and a repressor of K-Cl cotransporters (KCC 1-4) (Kahle et al., 2005;de Los Heros et al., 2006), when co-expressed inXenopus laevisoocytes. Much like WNK1 and WNK4, WNK3 was found to phosphorylate SPAK.
