Impaired degradation of WNK1 and WNK4 kinases causes PHAII in mutant KLHL3 knock-in mice.

Koichiro Susa,Motoko Chiga,Shinichi Uchida,Moko Zeniya,Tatemitsu Rai,Kenta Takeishi,Naohiro Nomura,Daiei Takahashi,Yutaro Mori,Hidenori Nishida,Sei Sasaki,Yuichi Inoue,Kiyoshi Isobe,Takayasu Mori,Naoki Takeda,Eisei Sohara

doi:10.1093/hmg/ddu217

Koichiro Susa, Motoko Chiga + Show 14 more

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https://doi.org/10.1093/hmg/ddu217

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Abstract

Pseudohypoaldosteronism type II (PHAII) is a hereditary disease characterized by salt-sensitive hypertension, hyperkalemia and metabolic acidosis, and genes encoding with-no-lysine kinase 1 (WNK1) and WNK4 kinases are known to be responsible. Recently, Kelch-like 3 (KLHL3) and Cullin3, components of KLHL3-Cullin3 E3 ligase, were newly identified as responsible for PHAII. We have reported that WNK4 is the substrate of KLHL3-Cullin3 E3 ligase-mediated ubiquitination. However, WNK1 and Na-Cl cotransporter (NCC) were also reported to be a substrate of KLHL3-Cullin3 E3 ligase by other groups. Therefore, it remains unclear which molecule is the target(s) of KLHL3. To investigate the pathogenesis of PHAII caused by KLHL3 mutation, we generated and analyzed KLHL3(R528H/+) knock-in mice. KLHL3(R528H/+) knock-in mice exhibited salt-sensitive hypertension, hyperkalemia and metabolic acidosis. Moreover, the phosphorylation of NCC was increased in the KLHL3(R528H/+) mouse kidney, indicating that the KLHL3(R528H/+) knock-in mouse is an ideal mouse model of PHAII. Interestingly, the protein expression of both WNK1 and WNK4 was significantly increased in the KLHL3(R528H/+) mouse kidney, confirming that increases in these WNK kinases activated the WNK-OSR1/SPAK-NCC phosphorylation cascade in KLHL3(R528H/+) knock-in mice. To examine whether mutant KLHL3 R528H can interact with WNK kinases, we measured the binding of TAMRA-labeled WNK1 and WNK4 peptides to full-length KLHL3 using fluorescence correlation spectroscopy, and found that neither WNK1 nor WNK4 bound to mutant KLHL3 R528H. Thus, we found that increased protein expression levels of WNK1 and WNK4 kinases cause PHAII by KLHL3 R528H mutation due to impaired KLHL3-Cullin3-mediated ubiquitination.

Highlights

Pseudohypoaldosteronism type II (PHAII) is a hereditary disease characterized by salt-sensitive hypertension, hyperkalemia, and metabolic acidosis [1,2]
Exon 15 of the Klhl3 gene was replaced by a cassette expressing a neomycin selective marker flanked by loxP sites, which was followed by the mutant exon 15 (R528H) (Fig. 1A)
Investigation of the pathophysiology of PHAII is extremely important, to increase knowledge of this rare inherited disease, and for the discovery of novel mechanisms of salt handling in the kidney

Summary

Introduction

Pseudohypoaldosteronism type II (PHAII) is a hereditary disease characterized by salt-sensitive hypertension, hyperkalemia, and metabolic acidosis [1,2]. It was previously demonstrated that the WNK kinase family phosphorylates and activates oxidative stress-responsive kinase 1 (OSR1) and STE20/ SPS1-related proline/alanine-rich kinase (SPAK) [4,5], and that activated OSR1/SPAK kinases could phosphorylate and activate Na–Cl cotransporter (NCC), constituting the WNK-OSR1/SPAK-NCC phosphorylation signaling cascade. This regulation of NCC by WNK-OSR1/ SPAK signaling was confirmed in vivo using various genetically engineered mouse models [6,7,8,9,10,11,12,13,14].

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