The Effect of WNK4 on the Na+-Cl- Cotransporter Is Modulated by Intracellular Chloride

Abstract

It is widely recognized that the phenotype of familial hyperkalemic hypertension is mainly a consequence of increased activity of the renal Na(+)-Cl(-) cotransporter (NCC) because of altered regulation by with no-lysine-kinase 1 (WNK1) or WNK4. The effect of WNK4 on NCC, however, has been controversial because both inhibition and activation have been reported. It has been recently shown that the long isoform of WNK1 (L-WNK1) is a chloride-sensitive kinase activated by a low Cl(-) concentration. Therefore, we hypothesized that WNK4 effects on NCC could be modulated by intracellular chloride concentration ([Cl(-)]i), and we tested this hypothesis in oocytes injected with NCC cRNA with or without WNK4 cRNA. At baseline in oocytes, [Cl(-)]i was near 50 mM, autophosphorylation of WNK4 was undetectable, and NCC activity was either decreased or unaffected by WNK4. A reduction of [Cl(-)]i, either by low chloride hypotonic stress or coinjection of oocytes with the solute carrier family 26 (anion exchanger)-member 9 (SLC26A9) cRNA, promoted WNK4 autophosphorylation and increased NCC-dependent Na(+) transport in a WNK4-dependent manner. Substitution of the leucine with phenylalanine at residue 322 of WNK4, homologous to the chloride-binding pocket in L-WNK1, converted WNK4 into a constitutively autophosphorylated kinase that activated NCC, even without chloride depletion. Elimination of the catalytic activity (D321A or D321K-K186D) or the autophosphorylation site (S335A) in mutant WNK4-L322F abrogated the positive effect on NCC. These observations suggest that WNK4 can exert differential effects on NCC, depending on the intracellular chloride concentration.

Keywords: distal tubule; diuretics; hypertension; ion transport.

Figure 1.

WNK4 effect on NCC is modulated by [Cl^-]_i. (A) Functional expression assay shows the thiazide-sensitive Na⁺ uptake in groups of oocytes injected with NCC cRNA alone or together with mouse WNK4 or human WNK4 cRNA, as stated. Uptake was performed in control conditions (white bars) or after 16 hours of LCHS (gray bars) (see Supplemental Material). *P<0.01 versus same group in control conditions. **P<0.05 versus NCC in LCHS. n=13. (B) Uptake in oocytes injected with NCC alone, in control conditions (white bars) or LCHS (gray bars), was arbitrarily set to 100%, and the corresponding groups were normalized accordingly. *P<0.01 versus any group in control conditions and versus NCC in LCHS. (C) The [Cl^-]_i in oocytes was assessed with custom-made glass capillary ion selective microelectrodes 48 hours after injection of water, NCC, or NCC plus WNK4 cRNA, as stated, in control conditions (white bars) or after 16 hours of LCHS (gray bars). *P<0.05 versus water injected in control conditions. n=2 (six oocytes per group, per experiment). (D) Uptake was performed in control conditions (white bars) or in LCHS in 1 hour (light gray bars) and 16 hours (dark gray bars) in groups injected with NCC alone or NCC plus WNK4 group, as stated. *P<0.05 versus own control. n=3. (E) [Cl^-]_i in oocytes injected with water, SLC26A9, or SLC26A9 plus WNK4 cRNA, as stated, after 48 hours of incubation in ND96 or after 30 minutes of LCHS. *P<0.05 versus water in control conditions. n=3. (F) Thiazide-sensitive Na⁺ uptake in oocytes injected with NCC cRNA alone and NCC plus WNK4 cRNA in the absence or presence of SLC26A9 cRNA. *P<0.01 versus NCC or NCC plus WNK4 groups. **P<0.05 versus NCC plus SLC26A9 cRNA injection.

Figure 2.

Elimination of the putative Cl^--binding pocket turns WNK4 into a constitutive activator of NCC. (A) NCC activity in oocytes injected with NCC cRNA (white bars), NCC plus wild-type WNK4 (black bars), or NCC plus catalytically inactive WNK4-D321A (red bars), as stated. Oocytes were incubated in the control solution or LCHS for 16 hours before the measurement of Na⁺ uptake. Uptake in oocytes injected with NCC alone and incubated in control or LCHS was arbitrarily set to 100%, and the NCC plus WNK4 or NCC plus WNK4-D321A groups were normalized accordingly. *P<0.05 versus NCC corresponding control. n=3. (B) Uptake in oocytes injected with NCC alone was arbitrarily set to 100% (white bar), and the rest of the groups were normalized accordingly (as stated, WNK4-LL-FF is the blue bar, WNK4-L322F is the green bar, WNK4-L324F is the orange bar, and WNK4-LL-FF-DA and WNK4-LL-FF-DKKD are the red bars). These series of experiments were performed in control conditions only. *P<0.01 versus NCC alone. n=6. (C) Representative Western blot showing the effect of wild-type or mutant WNK4, as stated, on NCC expression and phosphorylation in control conditions. Blot shows WNK4, pNCC, NCC, and actin expression, as stated. WNK4 is present only in oocytes injected with wild-type or mutant WNK4. (D) Densitometric analysis of data compiled from five different experiments. NCC basal phosphorylation (white bar) was arbitrarily set to 100% and the corresponding groups were normalized accordingly (as stated, WNK4 wild-type (black bar), WNK4-LL-FF (blue bar), WNK4-L322F (green bar), WNK4-L324F (orange bar) and catalytically inactive WNK4-LL-FF-DA (red bar). *P<0.05 versus NCC.

Figure 3.

Modulation of WNK autophosphorylation by decrease of [Cl^-]_i induced by LCHS. (A) Representative Western blot showing the effect of LCHS on wild-type WNK4 autophosphorylation, as stated, in control conditions or LCHS. Blot shows pWNK4, WNK4, and actin expression, as stated. Phosphoantibody recognizing S335 in WNK4 was developed and characterized by Thastrup et al. (B) Densitometric analysis of compiled results from six different experiments. Phosphorylation level of WNK4 in control conditions was arbitrary set to 100% (white bar), and the phosphorylation status in LCHS was normalized accordingly (gray bar). *P<0.05 versus control (Wilcoxon signed-rank test). (C) L-WNK1 and WNK3 autophosphorylation at S382 and S308, respectively, in control and LCHS conditions, as stated. The upper blot depicts phospho-WNK, and the lower blot shows the total amount of L-WNK1 or WNK3 as detected using anti-Myc antibody. Original image was cropped to eliminate empty lanes and show the L-WNK1 and WNK3 lanes only. (D) Representative Western blot of phospho-WNKs (WNK3, WNK4, or mutant WNK4-L322F), total WNKs, phospho-NCC, NCC, and actin in control conditions and after LCHS, as stated. (E) Densitometric analysis of compiled results from six experiments for WNK4: three for WNK3, three for WNK4-L322F, and two for L-WNK1. White bars depict phosphorylation status in control conditions arbitrarily taken as 100%, and gray bars depict the phosphorylation status in LCHS, as normalized for its corresponding white bar. *P<0.05 versus control (Wilcoxon signed-rank test). (F) NCC activity in oocytes injected with mutant WNK4-LL-FF, WNK4-L322F, WNK4-LL-FF-S335A, or WNK4-L322F-S335A, as stated. Uptake in oocytes injected with NCC alone was arbitrarily set to 100%, and the corresponding groups were normalized accordingly. These series of experiments were performed in control conditions only. n=4. *P<0.001 versus corresponding group without S335A substitution.

Figure 4.

A cell scheme of a X. laevis oocyte illustrating the effect of intracellular chloride concentration on wild-type WNK4 and mutant WNK4-L322F effect on NCC. In control conditions, were intracellular chloride concentration fluctuates around 45 mM, wild-type (wt) WNK4 is inactive, resulting in a negative or neutral effect on NCC activity. Disruption of the chloride binding site in mutant WNK4-L322F turns it into a constituitively autophosphorylated actuate kinase that has a positive effect on NCC activity even without chloride depletion. Reduction of intracellular chloride to around 25 mM (after LCHS) is sufficient to promote wt WNK4 autophosphorylation, turning it into an activator of NCC.