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 4

SGK1 phosphorylates more than 1 serine/threonine site of the C-terminal region of WNK4.

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SGK1 phosphorylates more than 1 serine/threonine site of the C-terminal ...
GST-WNK4 fusion proteins from 1112 to 1222 were isolated from bacteria and subjected to an SGK1 kinase assay. Shown with each panel is a schematic of the WNK4 region of interest and the approximate size of the GST-fusion protein. (A) SGK1 kinase assay of 0.8 μg GST-WNK/1112–1222 and isoforms conforming to mutants associated with FHHt (R1164C) and putative SGK1 phosphorylation target (S1169A). Autoradiographic signal was equivalent for wild-type and R1164C, with a slight reduction in signal for S1169A. (B) C-terminal GST-WNK4 deletion constructs showed much stronger signal for 1178–1222 compared with 1112–1177 or 1127–1179. Below, Coomassie Blue staining of protein preparations confirmed that equivalent amounts of the 5 substrates were used in the study above. Data are from 2 gels run simultaneously. (C) S1169 was confirmed as the only target of SGK1 phosphorylation in WNK4 fragment 1112–1177. Shown below is Coomassie Blue staining of 4 protein preparations. Blots in A–C are representative samples from 3 experiments.