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Abstract: PO1081

WNK4 Is a Transductor of V2 Receptor Signaling in the Thick Ascending Limbs and Distal Convoluted Tubules

Session Information

Category: Fluid, Electrolyte, and Acid-Base Disorders

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

Authors

  • Carbajal-Contreras, Hector, Universidad Nacional Autonoma de Mexico, Ciudad de Mexico, Ciudad de México, Mexico
  • Murillo-de-Ozores, Adrian R., Universidad Nacional Autonoma de Mexico, Ciudad de Mexico, Ciudad de México, Mexico
  • Magaña, German R., Universidad Nacional Autonoma de Mexico, Ciudad de Mexico, Ciudad de México, Mexico
  • Vázquez, Norma Hilda, Universidad Nacional Autonoma de Mexico, Ciudad de Mexico, Ciudad de México, Mexico
  • Gamba, Gerardo, Universidad Nacional Autonoma de Mexico, Ciudad de Mexico, Ciudad de México, Mexico
  • Castañeda-Bueno, Maria, Instituto Nacional de Ciencias Medicas y Nutricion Salvador Zubiran, Tlalpan, DF, Mexico

Group or Team Name

  • Molecular Physiology Unit
Background

Vasopressin (AVP) is essential for water and Na+ homeostasis. In the kidney, its actions are mediated by the V2 receptor (V2R), which signals through protein kinase A (PKA). The phosphorylation of the Na+-Cl- cotransporter (NCC) and the Na+-K+-Cl- cotransporter 2 (NKCC2) by the WNK-SPAK signaling pathway upregulates their activity and increases in response to AVP. WNK4 can be regulated by PKA through the phosphorylation of its RRxS motifs in in vitro models. Thus, we hypothesized that WNK4 mediates the activation of NCC and NKCC2 in response to AVP.

Methods

We transfected HEK293 cells with NKCC2 or the V2R with SPAK and either WNK1, WNK3, WT WNK4 or WNK4 with Ala instead of Ser in its 5 RRxS motifs. Cells were stimulated with 30 nM forskolin or 1 nM desmopressin (dDAVP). We cross-bred our WNK4-/- strain (in a C57BL/6 background) with 129Sv mice while selecting for the full-length allele of NKCC2 to evaluate the phosphorylation status of NKCC2. dDAVP (5 ng/h) was administered in miniosmotic pumps for 3 days. Protein extracts were subjected to immunoblot. qRT-PCR of Wnk4, Slc12a3, and 18S was carried out with Taqman probes. 12 h urine collections were conducted and water intake between the groups was equalized using gelled diets.

Results

In HEK293 cells, we found that an increase in phosphorylation of SPAK and of NKCC2 at T100 and T105 (SPAK regulated sites) with forskolin requires of WT WNK4. In contrast, phosphorylation of S130 of NKCC2 was WNK-independent. Only cells with WT WNK4 and the V2R showed an increase in SPAK phosphorylation when stimulated by dDAVP. dDAVP also increased WNK4’s phosphorylation at S1196.
dDAVP-infused WT mice increased their total and phosphorylated WNK4, NCC, and NKCC2, as well as phosphorylated SPAK and Slc12a3 mRNA levels. These effects were absent in WNK4-/- animals. In contrast, WNK4-/- mice did respond to dDAVP by increasing AQP2 protein levels. In addition, WNK4-/- mice had increased water consumption at baseline and an increased urine output when water-restricted, with a tendency towards lower total kidney osmolality.

Conclusion

Our data suggest that WNK4 is a transductor of AVP signaling in the TAL and DCT, modulating NKCC2 and NCC. This might contribute to the anti-natriuretic effects of this hormone and the increase in medullary osmolality that occurs with antidiuresis.

Funding

  • Government Support – Non-U.S.