Kidney Week

Abstract: SA-PO1033

Intracellular Chloride Depletion Promotes WNK4-RRXS Phosphorylation by a PKC/PKA Dependent Mechanism

Session Information

• Fluid and Electrolytes: Basic - II
  October 27, 2018 | Location: Exhibit Hall, San Diego Convention Center
  Abstract Time: 10:00 AM - 12:00 PM

Category: Fluid and Electrolytes

• 901 Fluid and Electrolytes: Basic

Authors

• Murillo-de-Ozores, Adrian R., INCMNSZ, Mexico City, Mexico

Adrian R. Murillo-de-Ozores,

• Grajeda-Medina, Leoneli I., INCMNSZ, Mexico City, Mexico

Leoneli I. Grajeda-Medina,

• Gamba, Gerardo, INCMNSZ, Mexico City, Mexico

Gerardo Gamba,

• Castañeda-Bueno, Maria, INCMNSZ, Mexico City, Mexico

Maria Castañeda-Bueno,

Background

Mutations in WNK4 gene cause Familial Hyperkalemic Hypertension (FHHt) due to increased salt reabsorption by the Na⁺:Cl^- cotransporter (NCC). While WNK4 activity is inhibited by chloride, this kinase can be activated by angiotensin II or CaSR-induced PKC or PKA-mediated phosphorylation in different WNK4-RRXS motifs. This is essential for full activation of WNK4.

NCC activity is inversely proportional to plasma K⁺ concentration and evidence suggests that modulation of WNK4 by chloride underlies this phenomenon. Here we analyzed if WNK4 phosphorylation in its RRXS motifs is also modulated by intracellular chloride concentration ([Cl^-]_i).

Methods

We used a mouse model of hypokalemia generated by feeding WT mice with low K⁺ diet for 7 days. WNK4-RRXS phosphorylation was analyzed by Western Blot and immunofluorescence in hypokalemic mice and in WNK4-transfected HEK293 cells incubated with different media and pharmacological inhibitors. ELISA was used to determine PKC activity.

Results

WNK4 phosphorylation at RRXS motifs S64 and S1196 increased in mice fed with low K⁺ diet compared to controls. Interestingly, this was only observed in NCC positive tubules.

Incubation of transfected HEK293 cells with low K⁺ medium also resulted in WNK4-RRXS phosphorylation. Furthermore, two different maneuvers that decrease [Cl^-]_I (low chloride hypotonic stress or NEM-incubation) in HEK293 cells also caused an increase in WNK4-RRXS phosphorylation. This was prevented by PKC inhibitor BIM and PKA inhibitor H89, as well as by intracellular Ca²⁺ chelation by BAPTA-AM. Accordingly, we found that PKC activity was increased upon [Cl^-]_I depletion.

Given that WNKs are the only kinases known to be regulated by [Cl^-]_I, we hypothesized that WNK activity was involved in the PKC/A-induced WNK4-RRXS phosphorylation. However, the specific WNK inhibitor WNK463 did not ablate low K⁺-induced WNK4-RRXS phosphorylation. Instead, WNK463 increased WNK4-RRXS phosphorylation, probably due to the expected [Cl^-]_I decrease.

Conclusion

WNK4 is phosphorylated in its RRXS motifs during reduction of [Cl^-]_I by a mechanism that involves PKC or PKA activation. This process seems to be independent of WNK activity and was also observed in vivo since low potassium diet resulted in WNK4-RRXS phosphorylation in mouse DCT.

Funding

• Government Support - Non-U.S.