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Abstract: PO1083

Upregulation of NCC by Hypokalemia Involves Additional Mechanisms to Direct Cl- Sensing by WNK4

Session Information

Category: Fluid, Electrolyte, and Acid-Base Disorders

  • 901 Fluid, Electrolyte, and Acid-Base Disorders: Basic

Authors

  • Murillo-de-Ozores, Adrian R., Universidad Nacional Autonoma de Mexico, CDMX, Mexico
  • Carbajal-Contreras, Hector, Universidad Nacional Autonoma de Mexico, CDMX, Mexico
  • Magaña, German R., Universidad Nacional Autonoma de Mexico, CDMX, Mexico
  • Vázquez, Norma Hilda, Universidad Nacional Autonoma de Mexico, CDMX, Mexico
  • Delpire, Eric J., Vanderbilt University, Nashville, Tennessee, United States
  • Gamba, Gerardo, Instituto Nacional de Ciencias Medicas y Nutricion Salvador Zubiran, CDMX, Mexico
  • Castañeda-Bueno, Maria, Instituto Nacional de Ciencias Medicas y Nutricion Salvador Zubiran, CDMX, Mexico
Background

Cl--sensitive WNK4 kinase plays an important role in the modulation NaCl reabsorption in the distal convoluted tubule (DCT). WNK4 activates the NaCl cotransporter (NCC) in response to hypokalemia, promoting its phosphorylation. Low plasma [K+] decreases intracellular [Cl-] ([Cl-]i) and thus Cl- binding to WNK4’s active site, leading to its activation. We have previously shown that hypokalemia and low [Cl-]i increase WNK4 phosphorylation at S64 and S1196. Hypokalemia also promotes phosphorylation of KLHL3, the substrate adaptor of the Cullin-Ring ligase that regulates WNK degradation, at a site that is similar to S64 and S1196 of WNK4. Thus, we wondered if KLHL3-mediated modulation of WNK abundance in the DCT is also regulated by [Cl-]i.

Methods

Transient transfection of HEK293 cells and incubation with media with varying electrolyte concentrations. Hydrochlorothiazide (HCTZ) administration in the diet to WT mice for 12 hrs. Generation of WNK4-L319F mice with CRISPR/Cas9, which were fed with normal or low K+ diets for 7 days. Immunoblot assays of cell and kidney lysates. Immunofluorescence (IF) of kidney slices.

Results

Co-expression of WNK4 and KLHL3 in HEK293 cells promoted a decrease in WNK4 abundance, which was partially prevented by incubation with a low K+ medium or a hypotonic low Cl- medium. Next, HCTZ-treated mice showed no changes in plasma [K+], but increased pNCC and KS-WNK1 protein levels were observed by immunoblot, possibly due to decreased [Cl-]i. In addition, WNK1- and WNK4- positive cytoplasmatic puncta were observed in the DCTs by immunostaining. In WNK4-KO mice, which might also have decreased [Cl-]i in their DCT, we also observed increased KS-WNK1 levels as well as WNK1-positive cytoplasmic puncta. Finally, WNK4-L319F mice were capable of upregulating pNCC, despite having a Cl--insensitive WNK4. We also observed an increase in WNK4 protein levels and its phosphorylation at Ser64 and Ser1196 in WNK4-L319F mice on low K+ diet.

Conclusion

Our work shows that low K+ mediated upregulation of NCC does not solely depend on the Cl--sensing capability of WNK4. A yet unidentified Cl--regulated mechanism can regulate WNK4 phosphorylation and KS-WNK1 and WNK4 abundance, which converges in the increase of the WNK4-NCC pathway in the DCT.

Funding

  • NIDDK Support