WNK-SPAK-NCC cascade revisited: WNK1 stimulates the activity of the Na-Cl cotransporter via SPAK, an effect antagonized by WNK4 - PubMed (original) (raw)

. 2014 Nov;64(5):1047-53.

doi: 10.1161/HYPERTENSIONAHA.114.04036. Epub 2014 Aug 11.

Chong Zhang 1, Christelle Soukaseum 1, Erika Moreno 1, Diana Pacheco-Alvarez 1, Emmanuelle Vidal-Petiot 1, María Castañeda-Bueno 1, Norma Vázquez 1, Lorena Rojas-Vega 1, Nicholas P Meermeier 1, Shaunessy Rogers 1, Xavier Jeunemaitre 1, Chao-Ling Yang 1, David H Ellison 1, Gerardo Gamba 2, Juliette Hadchouel 2

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WNK-SPAK-NCC cascade revisited: WNK1 stimulates the activity of the Na-Cl cotransporter via SPAK, an effect antagonized by WNK4

María Chávez-Canales et al. Hypertension. 2014 Nov.

Abstract

The with-no-lysine (K) kinases, WNK1 and WNK4, are key regulators of blood pressure. Their mutations lead to familial hyperkalemic hypertension (FHHt), associated with an activation of the Na-Cl cotransporter (NCC). Although it is clear that WNK4 mutants activate NCC via Ste20 proline-alanine-rich kinase, the mechanisms responsible for WNK1-related FHHt and alterations in NCC activity are not as clear. We tested whether WNK1 modulates NCC through WNK4, as predicted by some models, by crossing our recently developed WNK1-FHHt mice (WNK1(+/FHHt)) with WNK4(-/-) mice. Surprisingly, the activated NCC, hypertension, and hyperkalemia of WNK1(+/FHHt) mice remain in the absence of WNK4. We demonstrate that WNK1 powerfully stimulates NCC in a WNK4-independent and Ste20 proline-alanine-rich kinase-dependent manner. Moreover, WNK4 decreases the WNK1 and WNK3-mediated activation of NCC. Finally, the formation of oligomers of WNK kinases through their C-terminal coiled-coil domain is essential for their activity toward NCC. In conclusion, WNK kinases form a network in which WNK4 associates with WNK1 and WNK3 to regulate NCC.

Keywords: Xenopus laevis; familial hypertensive hyperkalemia; hypertension, renal; kidney tubules, distal; mice, knockout; pseudohypoaldosteronism, type II; water-electrolyte balance.

© 2014 American Heart Association, Inc.

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Conflict of interest statement

Conflict(s) of Interest/Disclosure(s). None

Figures

Figure 1

Figure 1. L-WNK1 induces hyperkalemic hypertension and NCC phosphorylation independently of WNK4 in mice

(A) Renin transcripts level was measured by quantitative real-time PCR in the kidney cortex of _WNK4_−/− (n=4), WNK1+/i1lox (n=5), WNK1+/FHHt (n=6) and _WNK1+/FHHt:WNK4_−/− (n=3) males. Results are expressed in arbitrary units relative to the expression of ubc. The control group (WNK1+/i1lox mice) has been arbitrarily set to 1. Data are mean ± s.e.m. *: P<0.05 compared to the control group. (B) Left: Immunoblots performed on membrane-enriched fraction of renal cortex dissected from _WNK4_−/− (n=4), WNK1+/i1lox (n=6), WNK1+/FHHt (n=6) and _WNK1+/FHHt:WNK4_−/− (n=5) males, incubated with antibodies against anti-NCC or its T53 phosphorylated residue (pNCC-T53). Right: Densitometric analysis of the immunoblots. Data, expressed as a percentage of the average signal intensity measured in the control group, set arbitrarily to 100%, are mean ± s.e.m. *: P<0.05, **: P<0.01 and ***: P<0.001 compared to the control group (Student’s unpaired t-test).

Figure 2

Figure 2. L-WNK1 is a powerful activator of NCC activity in Xenopus laevis oocytes

(A) NCC activity was measured in Xenopus oocytes overexpressing NCC alone or in combination with different L-WNK1 splicing variants. All L-WNK1 variants, except the one deleted from exon 9 (L-WNK1-Δ9), stimulated NCC activity. (B) Effect of L-WNK1-Δ11 point mutations affecting its kinase activity (Δ11-DA), SPAK binding (Δ11-F316A), dimer formation (Δ11-HQ) or both (Δ11-F316A-HQ) on NCC activity. For (A) and (B), NCC activity in oocytes injected with NCC cDNA alone (black bar) was arbitrarily set to 100%. Data are mean ± s.e.m. ***: P<0.001 and ****: P<0.0001 vs. NCC alone (Student’s unpaired t-test). (C) Immunoblots from NCC-HEK293 cells transiently transfected with NCC and human L-WNK1-Δ11-myc constructs, and probed with antibodies indicated. Data, indicated below the blots, are mean ± s.e.m. **: P<0.01 and ***: P<0.001 vs. the control (Student’s unpaired t-test). (D) The inhibition of L-WNK1 expression by RNA-interference decreased NCC phosphorylation. Immunoblots from NCC-HEK-293 cells transfected with control and WNK1-specific siRNA, probed with the antibodies indicated.

Figure 3

Figure 3. WNK4 inhibits the activation of NCC by L-WNK1 and WNK3

(A) NCC activity was measured in Xenopus oocytes injected alone or in combination with L-WNK1-Δ11 (WNK1), WNK3 and/or WNK4 as stated. WNK4 not only inhibited NCC activity but also prevented the L-WNK1-Δ11 or WNK3-dependent stimulation of NCC (N=15 independent experiments per combination). (B) Immunoblots from NCC-HEK293 cells transiently overexpressing L-WNK1-Δ11 and WNK4. WNK4 overexpression prevented the activation of NCC by L-WNK1-Δ11. (C-D) Immunoblots from NCC HEK293 cells transiently transfected with myc-tagged L-WNK1-Δ11 and WNK4 constructs (C) or from Xenopus oocytes injected with myc-tagged L-WNK1-Δ11 alone or in combination with WNK4 (D). WNK4 overexpression significantly decreased abundance of L-WNK1-Δ11 in both systems. *: P<0.05 and **: P<0.01 for the comparison between L-WNK1-Δ11 alone and L-WNK1-Δ11 + WNK4 (Student’s unpaired t-test). (E) NCC activity alone or in combination of wild type or mutant-HQ WNKs as stated. The inhibition of the formation of WNKs dimers by mutating the C-terminal HQ motif relieved L-WNK1-Δ11 or WNK3 from WNK4-mediated inhibition. For oocytes experiments, the activity measured in oocytes injected with NCC alone was arbitrarily set to 100% and data are mean ± s.e.m. **: P<0.01 for the comparison between wild type WNK4 and WNK4-HQ (Student’s unpaired t-test).

Figure 4

Figure 4. Proposed new model for the regulation of NCC by WNKs

Note that WNK3 is not shown and that L-WNK1 is indicated instead of L-WNK1-Δ11 to simplify the Figure. At baseline, L-WNK1-Δ11 forms homomers that activate NCC in a SPAK-dependent manner. WNK4 could antagonize the effect of L-WNK11-Δ11 on NCC by either forming inactive WNK4-WNK1 heteromers or inhibitory WNK4 homomers.

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