Epithelial sodium channel regulated by aldosterone-induced protein sgk

S Chen

A Bhargava

L Mastroberardino

O Meijer

^{Division of Nephrology, Departments of Medicine and Cellular and Molecular Pharmacology, Box 0532, University of California, San Francisco, CA 94143;}^{Institute of Physiology, University of Zurich, Zurich, Switzerland; and}^{Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720}

^{S.y.C. and A.B. contributed equally to this work.}

^{To whom reprint requests should be addressed. e-mail:}ude.fscu.lgc@ecraep.

Edited by Maurice B. Burg, National Heart, Lung, and Blood Institute, Bethesda, MD, and approved December 28, 1998

Received 1998 Sep 10

Abstract

Sodium homeostasis in terrestrial and freshwater vertebrates is controlled by the corticosteroid hormones, principally aldosterone, which stimulate electrogenic Na^{absorption in tight epithelia. Although aldosterone is known to increase apical membrane Na}^{permeability in target cells through changes in gene transcription, the mechanistic basis of this effect remains poorly understood. The predominant early effect of aldosterone is to increase the activity of the epithelial sodium channel (ENaC), although ENaC mRNA and protein levels do not change initially. Rather, the open probability and/or number of channels in the apical membrane are greatly increased by unknown modulators. To identify hormone-stimulated gene products that modulate ENaC activity, a subtracted cDNA library was generated from A6 cells, a stable cell line of renal distal nephron origin, and the effect of candidates on ENaC activity was tested in a coexpression assay. We report here the identification of sgk (serum and glucocorticoid-regulated kinase), a member of the serine–threonine kinase family, as an aldosterone-induced regulator of ENaC activity. sgk mRNA and protein were strongly and rapidly hormone stimulated both in A6 cells and in rat kidney. Furthermore, sgk stimulated ENaC activity approximately 7-fold when they were coexpressed in}Xenopus laevis oocytes. These data suggest that sgk plays a central role in aldosterone regulation of Na^{absorption and thus in the control of extracellular fluid volume, blood pressure, and sodium homeostasis.}

Abstract

Unlike marine animals, terrestrial and freshwater vertebrates must separately control salt and water excretion to maintain extracellular fluid volume, blood pressure, and ion concentrations (1). Adaptation to wide variations in dietary Na^{is controlled largely by the adrenal corticosteroid hormone aldosterone, which acts in tight epithelia such as the colon, parotid gland, and renal collecting duct (CD) to stimulate Na}^{absorption (}2). The principal early action of aldosterone (starting after a latent period of 45 min) is to increase apical membrane Na^{transport through the epithelial sodium channel (ENaC; for review, see ref.}3). Like all members of the nuclear receptor superfamily, the steroid receptors that mediate this effect modulate the rate of transcription of specific target genes. Interestingly, in spite of its genomic site of regulation, aldosterone appears to regulate primarily ENaC activity, not abundance (3–6). In renal CD and colon, for example, the bulk of increase in Na^{transport has already occurred before ENaC α-, β-, and γ-subunit mRNA levels begin to increase (}7–10). Furthermore, in kidney, the late increase in ENaC subunit mRNAs is modest, although in colon, ultimately, there is a substantial increase in β- and γ-subunit mRNAs (9). Thus, although it is still uncertain whether aldosterone primarily increases ENaC open probability, number, or both (11), it is clear that the early transcriptional effects of aldosterone are not on ENaC gene transcription.

The above observations suggest that aldosterone rapidly induces the transcription of gene(s) encoding regulatory protein(s) that increase ENaC activity. We, therefore, wanted to identify aldosterone-induced genes whose products might stimulate ENaC activity. Toward this end, we used a PCR-based technique to generate a cDNA library representing rapidly induced genes in A6 cells, a CD-like cell line derived from Xenopus laevis kidney (12). We then characterized the effect of selected clones on ENaC activity in a coexpression assay. One of these, sgk (serum and glucocorticoid-regulated kinase), a member of the serine-threonine kinase family, was rapidly induced by dexamethasone in A6 cells and by aldosterone in rat distal nephron. Furthermore, sgk strongly stimulated ENaC activity in a coexpression assay. These observations suggest that sgk is an important mediator of the early aldosterone effect on sodium transport in distal nephron and possibly in other tight epithelia.

Acknowledgments

M. Dallman and her laboratory are gratefully acknowledged for extensive help with performing in situ hybridizations. We are grateful to B. C. Rossier for providing XENaC cDNAs, J. Xu and S. Hebert for providing mouse ROMK2 cDNA, I. Forster for providing the setup and assistance with the electrophysiological experiments, and R. Pfeiffer for performing electrophysiological experiments. B. Yen is gratefully acknowledged for assistance with interpreting histological data. This work was supported by National Institutes of Health Grant R29-DK51151-03 and Swiss National Science Foundation Grant 31.49727.96. O.C.M. was supported by a TALENT stipend of the Dutch Organization for Scientific Research.

Acknowledgments

ABBREVIATIONS

ENaC	epithelial sodium channel
CD	collecting duct
GR	glucocorticoid receptor
sgk	serum and glucocorticoid-regulated kinase
xsgk	Xenopus sgk
MR	mineralocorticoid receptor
adx	adrenalectomized

ABBREVIATIONS

Footnotes

This paper was submitted directly (Track II) to the Proceedings Office.

Data deposition: The Xenopus laevis sgk (xsgk) cDNA sequence reported in this paper has been deposited in the GenBank database (accession no. {"type":"entrez-nucleotide","attrs":{"text":"AF057138","term_id":"3688802"}}AF057138).

Footnotes

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