How Does Aldosterone Work?

How aldosterone stimulates Na⁺ transport in reabsorbing epithelia has been a question that has intrigued physiologists and nephrologists for more than 50 years.^1,2 There is general agreement that the most important effects of the hormone are mediated through changes in gene expression and that they converge on the upregulation of the epithelial Na channel (ENaC). The next questions, therefore, become which genes are involved and how they affect the channels. In the current issue of Kidney360, Loughlin et al.³ address this problem using the mCCDcl1 cell line⁴ together with RNA-sequencing (RNAseq) techniques. The study was rigorous in several important ways. Na⁺ transport was measured in the same cells used for RNAseq, and conditions were chosen to ensure that responses were mediated by mineralocorticoid receptors (MRs). Candidate genes were confirmed with quantitative PCR measurements and validated using primary cultures of mouse principal cells.

Using the criteria of at least a two-fold change with hormone treatment and a false detection probability of <0.05, the authors identify nine aldosterone-responsive genes (eight induced and one repressed). Many more genes were affected by corticosterone after inhibition of 11-βHSD2, the enzyme responsible for protecting MRs from activation by glucocorticoids. This likely reflects the simultaneous activation of glucocorticoid receptors and their effects on gene expression.

Five genes were considered in detail. The two most strongly induced transcripts were those of the serine-threonine protein kinase Sgk1 and the zinc-finger transcription factor Zbtb16. These had also been identified by previous screens using different approaches. The other three–for the Ras-related protein Rasd1, the sulfotransferase Sultd1, and a noncoding transcript Gm43305—are new candidates for the domain of aldosterone-regulated genes.

Several genes that were previously identified as aldosterone-dependent and suggested to be linked to Na⁺ transport by the kidney did not qualify as induced transcripts in this study. These include the α subunit of ENaC itself,⁵ the leucine-zipper protein GILZ1,⁶ the small G protein Kras,⁷ and the deubiquitinating enzyme Usp2-45.⁸ These proteins may not be essential for upregulation of transport under the conditions studied by Loughlin et al.

However, the effects of steroid hormones, including aldosterone, are complex and highly dependent on context. Even the mechanisms that govern the activity of the target protein ENaC may involve various events such as biosynthesis of the channels themselves⁵ trafficking to the apical membrane,^9,10 increased open probability of membrane-resident channels,¹¹ and protein internalization and degradation^12,13 (Figure 1). These effects depend on the tissue type. In the kidney, aldosterone increases mRNA levels for αENaC but not for β or γENaC. By contrast in the colon, where aldosterone also stimulates ENaC activity, the β and γ subunits are induced while expression of αENaC is constitutive.⁵ Even within the kidney, there are axial differences in ENaC-expressing cells of the distal nephron. Those near the border of the distal convoluted tubule and connecting tubule (CNT) do not respond to aldosterone at all.¹⁴ Principal cells in the late CNT and the cortical, outer medullary, and inner medullary collecting ducts all depend on the hormone, but the detailed responses may differ. The mechanisms underlying ENaC regulation are also time-dependent.^15,16 Early effects of dietary Na restriction (within 1 day) involve trafficking and processing of ENaC protein while later actions (approximately 1 week) may entail activation of channels at the surface.¹⁷ The genes identified by Loughlin et al. belong to the early time frame as they were altered by aldosterone treatment for just 3 hours.

Figure 1: Proposed actions of aldosterone to stimulate ENaC. Aldosterone (A) binds to and activates MRs that control the transcription of genes for AIPs. These proteins may be ENaC itself (1) or might also accelerate trafficking of the channel to the apical membrane (2), decelerate removal from the membrane (3), or increase the amount of time the channels are in the open state (4). AIP, aldosterone-induced protein.

The induction of Sgk1 by aldosterone is a robust response, documented in many different studies of renal cells.^8,18,19 Furthermore, expression of Sgk1 increased ENaC activity and surface expression in Xenopus oocytes,^18–20 suggesting its physiological relevance. Furthermore, lack of the Sgk1 gene in mice gives rise to Na wasting on a low-salt diet^21,22 and hyperkalemia on a high-K diet,^23,24 both explicable by understimulation of ENaC. The kinase may act, at least in part, through inhibition of Nedd4-2, a ubiquitin ligase believed to facilitate ENaC internalization from the plasma membrane.^25,26 Indeed, global knockout of Sgk1 in mice strongly blunted aldosterone-dependent trafficking and surface expression of ENaC.²⁴ However, Sgk1 is clearly not the whole story. Collecting ducts of mice lacking the kinase still showed large increases in ENaC activity after chronic treatment with exogenous aldosterone and diets low in Na or enriched in K.^21,24 Clearly other pathways come into play at least in some contexts.

The roles of the other (non-SGK1) genes identified by Loughlin et al. are not yet clear. Zbtb16, the other gene increased eight-fold by aldosterone, has been associated with decreased rather than increased transport in cultured cortical-collecting duct cells.²⁷ There is no information on the effect of Rasd1 on Na⁺ transport. Saxena et al.²⁸ reported that this gene is mainly expressed in intercalated cells under basal conditions. However, this does not rule out the possibility of increased expression in principal cells when aldosterone levels are elevated, and the related small G protein Kras is induced by the hormone in amphibian cells.⁷ Sultd1 could alter sulfonation in renal cells, but humans lack an orthologue of this gene, casting doubt on its general importance. Gm43305 is a long noncoding RNA. Its role in regulating ENaC is uncertain, although short noncoding RNAs (micro RNAs) do have a role in mediating the aldosterone response.²⁹

A more complete understanding would require knowing the effects of deleting or suppressing each gene, individually and in combinations, on the physiological response to the hormone. The cell line used by Loughlin et al. is an attractive model for carrying out such studies. It may even be possible to modify the mineralocorticoid response elements in the individual genes to eliminate steroid control while maintaining basal expression. The relatively low number of induced genes identified makes the problem tractable, although it remains to be seen if these genes can account for the response or if additional candidates need to be sought. Other protocols and different models may be required to explore all the actions of aldosterone with various cell types, times of exposure, and interacting hormones.

Disclosures

The author has nothing to disclose.

Funding

This article was funded by National Institutes of Health grant R01-DK111380.

Acknowledgments

The content of this article reflects the personal experience and views of the author(s) and should not be considered medical advice or recommendation. The content does not reflect the views or opinions of the American Society of Nephrology (ASN) or Kidney360. Responsibility for the information and views expressed herein lies entirely with the author(s).

Author Contributions

L.G. Palmer wrote the original draft.