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Abstract: FR-PO589

Regulation of PKC-Dependent WNK4 Phosphorylation by Extracellular Potassium: Insight into Familial Hyperkalemia and Hypertension Pathophysiology

Session Information

Category: Fluid and Electrolytes

  • 901 Fluid and Electrolytes: Basic

Authors

  • Murillo-de-Ozores, Adrian R., Universidad Nacional Autónoma de México, Mexico City, Mexico
  • Grajeda-Medina, Leoneli I., INCMNSZ, Ciudad de Mexico, Mexico
  • Gonzalez-Behn-Eschenburg, Sebastian, Instituto de Investigaciones Biomedicas, Universidad Nacional Autonoma de Mexico, Mexico City, Mexico
  • Vázquez, Norma Hilda, UNAM, Mexico City, Mexico
  • Gamba, Gerardo, Instituto Nacional de Ciencias Medicas y Nutricion Salvador Zubiran, Mexico City, Mexico
  • Castañeda-Bueno, Maria, Instituto Nacional de Ciencias Medicas y Nutricion Salvador Zubiran, Mexico City, Mexico
Background

Familial Hyperkalemic Hypertension (FHHt) is caused by mutations in genes such as KLHL3 and WNK4, which result in higher protein levels of the kinase WNK4 and thus upregulation of the phosphorylated renal NaCl cotransporter NCC (pNCC), increasing its activity. Because NCC activity is inversely proportional to plasma K+ in physiological conditions, it is puzzling that NCC activity is not suppressed by hyperkalemia in FHHt. We have shown that WNK4 phosphorylation by PKC/PKA at S64 is important for kinase activity and that it is increased by hypokalemia. Here we investigated how this site is regulated in hyperkalemia and FHHt.

Methods

Wild-type (WT) mice were fed with low (LKD-0%), normal (NKD-1.2%) or high (HKD-5%) K+ diets for 7 days. Transgenic KLHL3R528H/+ mice (with FHHt phenotype) were fed with LKD or NKD for 4 days. Blood samples were obtained to measure electrolyte levels. Western Blot assays with kidney lysates were performed to analyze total and phosphorylated WNK4 and NCC levels. WNK4-transfected HEK293 cells were incubated in low (1mM), normal (5mM) or high (10mM) K+ media. Ex vivo kidney slices from WT mice were exposed for 30 min to control solutions followed by 30 min to low, normal or high K+ media.

Results

We observed that phosho-S64 in WNK4 is increased in WT mice by LKD, but not affected by HKD, while in the same samples, we corroborated that pNCC is increased by LKD and decreased by HKD. Accordingly, in KLHL3R528H/+ mice, WNK4-S64 phosphorylation was not suppressed by hyperkalemia as phosphorylation levels increased in a similar amount than WNK4 total levels. In these mice, both pNCC and pWNK4-S64, were further increased by LKD, showing that DCT cells can still respond to low [K+] via de [Cl-]-WNK4 pathway.
In addition, consistent with the observations in vivo, in WNK4-transfected HEK293 cells and in ex vivo kidney slices, we found that pWNK4-S64 was increased by incubation with low K+, but not decreased by high K+.

Conclusion

WNK4 phosphorylation at S64 is increased by low [K+]e. However, we did not observe the opposite process by high [K+]e. Since WNK4 remains phosphorylated at S64 in this setting, this might help to explain why elevated WNK4 levels in FHHt are enough to maintain higher NCC activity in spite of hyperkalemia.

Funding

  • Government Support - Non-U.S.