Phenotypes of pseudohypoaldosteronism type II caused by the WNK4 D561A missense mutation are dependent on the WNK-OSR1/SPAK kinase cascade

Motoko Chiga,Tatemitsu Rai,Shinichi Uchida,Eisei Sohara,Sei Sasaki,Akihito Ohta,Fatema H Rafiqi,Dario R Alessi

doi:10.1242/jcs.084111

Abstract

We recently reported increased phosphorylation of the NaCl cotransporter (NCC) in Wnk4(D561A/+) 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 Wnk4(D561A/+) 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 Wnk4(D561A/+)Spak(T)(243A/T243A)Osr1(T185A/+) 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 Wnk4(D561A/+) 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.

Highlights

Pseudohypoaldosteronism type II (PHAII) is an autosomal dominant hereditary hypertensive disease characterized by hyperkalemia and metabolic acidosis (Gordon, 1986)
On the basis of these data, we speculated that a WNK-OSR1/SPAKNCC phosphorylation cascade exists in vivo in kidney cells and postulated that the activation of this cascade by the mutant WNK4 is the major cause of PHAII
The purpose of this study was to investigate whether the WNKOSR1/SPAK cascade is fully responsible for NaCl cotransporter (NCC) phosphorylation in vivo and whether the activation of this cascade is the sole mediator of PHAII

Summary

Introduction

Pseudohypoaldosteronism type II (PHAII) is an autosomal dominant hereditary hypertensive disease characterized by hyperkalemia and metabolic acidosis (Gordon, 1986). We had clarified through in vitro experiments that WNK1 and WNK4 phosphorylated and activated OSR1 and SPAK kinases, and that OSR1 and SPAK could phosphorylate threonine and serine residues in the N-terminal domain of NCC (Moriguchi et al, 2005; Vitari et al, 2005; Vitari et al, 2006) Phosphorylation of these residues (threonine 53 and 58, serine 71 in mouse NCC) was previously shown to be necessary for NCC function (Pacheco-Alvarez et al, 2006; Richardson et al, 2008) and possibly important for apical membrane localization of NCC in cells of the distal convoluted tubules We generated Spak knockout mice, further confirming the importance of SPAK in NCC phosphorylation and the involvement of SPAK in the regulation of vascular resistance in the aorta (Yang et al, 2010)

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