Aldosterone mediates activation of the thiazide-sensitive Na-Cl cotransporter through an SGK1 and WNK4 signaling pathway
David J. Rozansky, … , Chao-Ling Yang, David H. Ellison
David J. Rozansky, … , Chao-Ling Yang, David H. Ellison
Published August 17, 2009
Citation Information: J Clin Invest. 2009;119(9):2601-2612. https://doi.org/10.1172/JCI38323.
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Research Article Nephrology

Aldosterone mediates activation of the thiazide-sensitive Na-Cl cotransporter through an SGK1 and WNK4 signaling pathway

Abstract

Aldosterone regulates volume homeostasis and blood pressure by enhancing sodium reabsorption in the kidney’s distal nephron (DN). On the apical surface of these renal epithelia, aldosterone increases expression and activity of the thiazide-sensitive Na-Cl cotransporter (NCC) and the epithelial sodium channel (ENaC). While the cellular mechanisms by which aldosterone regulates ENaC have been well characterized, the molecular mechanisms that link aldosterone to NCC-mediated Na+/Cl– reabsorption remain elusive. The serine/threonine kinase with-no-lysine 4 (WNK4) has previously been shown to reduce cell surface expression of NCC. Here we measured sodium uptake in a Xenopus oocyte expression system and found that serum and glucocorticoid–induced kinase 1 (SGK1), an aldosterone-responsive gene expressed in the DN, attenuated the inhibitory effect of WNK4 on NCC activity. In addition, we showed — both in vitro and in a human kidney cell line — that SGK1 bound and phosphorylated WNK4. We found one serine located within an established SGK1 consensus target sequence, and the other within a motif that was, to our knowledge, previously uncharacterized. Mutation of these target serines to aspartate, in order to mimic phosphorylation, attenuated the effect of WNK4 on NCC activity in the Xenopus oocyte system. These data thus delineate what we believe to be a novel mechanism for aldosterone activation of NCC through SGK1 signaling of WNK4 kinase.

Authors

David J. Rozansky, Tonya Cornwall, Arohan R. Subramanya, Shaunessy Rogers, Yong-Feng Yang, Larry L. David, Xiaoman Zhu, Chao-Ling Yang, David H. Ellison

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Figure 5

Within the 45–amino acid C-terminal NCC negative regulatory region of WNK4/1178–1222, S1196 acts as a single target of SGK1 phosphorylation.

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Within the 45–amino acid C-terminal NCC negative regulatory region of WN...
(A) Amino acid sequence of this WNK4 region contains 10 serines and threonines. (B) MS/MS spectrum of peptide 246–260 from a tryptic digest of GST-WNK4/1178–1222, localizing phosphorylation to residue S248 in the construct (corresponding to residue S1196 of mouse WNK4) after incubation with SGK1 kinase. An uninterrupted y and b ion series was observed in the fragment ion spectra, as well as a characteristic major doubly charged neutral loss ion at m/z 788.5 resulting from the loss of phosphate. Because of the intensity of this neutral loss ion, the intensities of the other areas of the spectrum increased 10-fold. The inset sequence of the peptide is aligned with the corresponding fragment ions of the y and b ion series. (C) In vitro SGK1 kinase assay confirmed that S1196 was the only SGK1 phosphorylation site in the region 1178–1222 from bacterially derived fusion fragments. The single mutation of WNK4/1178–1222 S1196A was not phosphorylated by SGK1, whereas wild-type and fragment 1112–1177 yielded robust and weak signals, respectively. Shown below is Coomassie Blue staining of the 4 protein preparations used for the SGK kinase assay. n = 3. The schematic diagram denotes the C-terminal WNK4 region and the approximate size of the GST-fusion proteins.