Mutation affecting the conserved acidic WNK1 motif causes inherited hyperkalemic hyperchloremic acidosis
Hélène Louis-Dit-Picard, … , Juliette Hadchouel, Xavier Jeunemaitre
Hélène Louis-Dit-Picard, … , Juliette Hadchouel, Xavier Jeunemaitre
Published August 13, 2020
Citation Information: J Clin Invest. 2020;130(12):6379-6394. https://doi.org/10.1172/JCI94171.
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Research Article Genetics Nephrology

Mutation affecting the conserved acidic WNK1 motif causes inherited hyperkalemic hyperchloremic acidosis

Abstract

Gain-of-function mutations in with no lysine (K) 1 (WNK1) and WNK4 genes are responsible for familial hyperkalemic hypertension (FHHt), a rare, inherited disorder characterized by arterial hypertension and hyperkalemia with metabolic acidosis. More recently, FHHt-causing mutations in the Kelch-like 3–Cullin 3 (KLHL3-CUL3) E3 ubiquitin ligase complex have shed light on the importance of WNK’s cellular degradation on renal ion transport. Using full exome sequencing for a 4-generation family and then targeted sequencing in other suspected cases, we have identified new missense variants in the WNK1 gene clustering in the short conserved acidic motif known to interact with the KLHL3-CUL3 ubiquitin complex. Affected subjects had an early onset of a hyperkalemic hyperchloremic phenotype, but normal blood pressure values”Functional experiments in Xenopus laevis oocytes and HEK293T cells demonstrated that these mutations strongly decrease the ubiquitination of the kidney-specific isoform KS-WNK1 by the KLHL3-CUL3 complex rather than the long ubiquitous catalytically active L-WNK1 isoform. A corresponding CRISPR/Cas9 engineered mouse model recapitulated both the clinical and biological phenotypes. Renal investigations showed increased activation of the Ste20 proline alanine–rich kinase–Na+-Cl– cotransporter (SPAK-NCC) phosphorylation cascade, associated with impaired ROMK apical expression in the distal part of the renal tubule. Together, these new WNK1 genetic variants highlight the importance of the KS-WNK1 isoform abundance on potassium homeostasis.

Authors

Hélène Louis-Dit-Picard, Ilektra Kouranti, Chloé Rafael, Irmine Loisel-Ferreira, Maria Chavez-Canales, Waed Abdel-Khalek, Eduardo R. Argaiz, Stéphanie Baron, Sarah Vacle, Tiffany Migeon, Richard Coleman, Marcio Do Cruzeiro, Marguerite Hureaux, Nirubiah Thurairajasingam, Stéphane Decramer, Xavier Girerd, Kevin O’Shaugnessy, Paolo Mulatero, Gwenaëlle Roussey, Ivan Tack, Robert Unwin, Rosa Vargas-Poussou, Olivier Staub, Richard Grimm, Paul A. Welling, Gerardo Gamba, Eric Clauser, Juliette Hadchouel, Xavier Jeunemaitre

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Figure 4

KLHL3 interaction with WNK1 isoforms in HEK293T cells: KLHL3 ubiquitinates KS-WNK1 and significantly reduces its protein levels.

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KLHL3 interaction with WNK1 isoforms in HEK293T cells: KLHL3 ubiquitinat...
(A) Flp-In T-Rex 293 cells stably and inducibly expressing (His)6-protein C-Flag-hKLHL3 were transfected with myc-tagged L-WNK1 (WT or D635N mutant) or KS-WNK1 (WT or D228N mutant), as indicated. At 34 hours after transfection, cells were induced with tetracycline. Fourteen hours later (48 hours after transfection), cells were harvested and lysed in denaturing conditions. Cell lysates were subjected to immunoblot analysis with the indicated antibodies. Data shown are representative of 3 independent experiments. (B) Flp-In T-Rex 293 cells stably and inducibly expressing (His)6-protein C-Flag-hKLHL3 were transfected with ubiquitin-HA and myc-tagged L-WNK1, L-WNK1 D635N, KS-WNK1 or KS-WNK1 D228N, as indicated. At 34 hours after transfection, cells were induced with tetracycline. Fourteen hours later (48 hours after transfection), cells were harvested and lysed in denaturing conditions. Upper panel: Myc-tagged WNK1 isoforms were immunoprecipitated with anti-myc antibody (9B11, Cell Signaling Technology); immunoprecipitates were analyzed by immunoblotting with anti-HA antibody (3724S; Cell Signaling Technology). Nitrocellulose membranes were stripped and reblotted with anti-myc antibody. Immunoblot of cell lysates is represented in D (input). Data shown are representative of 3 independent experiments. (C) Cells were transfected with myc-tagged L-WNK1 (WT or D635N mutant) and KS-WNK1 (WT or D228N mutant), as indicated and in conditions similar to those in A. Cell lysates were subjected to immunoblot analysis with the indicated antibodies. Densitometric analysis was performed using FUJI FILM Multi-Gauge software. Results are shown as mean ± SEM. *P < 0.05 compared with control, unpaired Student’s t test. n = 3. (D) Flp-In T-Rex 293 cells stably and inducibly expressing (His)6-protein C-Flag-hKLHL3 were transfected with myc-tagged L-WNK1, L-WNK1 D635N, KS-WNK1, or KS-WNK1 D228N, as indicated. At 43 hours after transfection, cells were induced with tetracycline and simultaneously treated with MG132 for 5 hours. At 48 hours after transfection, cells were harvested and lysed in native conditions. Left panel: cell lysates were immunoprecipitated with anti-myc antibody, and immunoprecipitates were analyzed by immunoblotting with anti-protein C and anti-myc antibodies. Right panel: cell lysates (input) were subjected to immunoblot analysis with anti-myc and anti–protein C antibodies (HPC4, Roche) to check for even expression of KLHL3. Data shown are representative of 3 independent experiments.