Aldosterone-induced signalling and cation transport in the distal nephron

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Aldosterone-induced signalling and cation transport in the distal nephron Warren Thomas ∗ , Victoria McEneaney, Brian J. Harvey Department of Molecular Medicine, Royal College of Surgeons in Ireland Education and Research Centre, Beaumont Hospital, Dublin, Ireland

a r t i c l e

i n f o

Published on line 19 January 2008

a b s t r a c t Aldosterone is an important regulator of Na+ and K+ transport in the distal nephron modulating the surface expression of transporters through the action of the mineralocorticoid

Keywords:

receptor as a ligand-dependent transcription factor. Aldosterone stimulates the rapid acti-

Aldosterone

vation of protein kinase-based signalling cascades that modulate the genomic effects of the

Mineralocorticoid

hormone. Evidence is accumulating about the multi-factorial regulation of the epithelial

Rapid responses

sodium channel (ENaC) by aldosterone. Recent published data suggests that the activa-

ENaC

tion of a novel PKC/PKD signalling pathway through the c-Src-dependent trans-activation

Protein kinase D

of epidermal growth factor receptor contributes to early ENaC trafficking in response to aldosterone. © 2008 Elsevier Inc. All rights reserved.

1.

Introduction

Salt conservation through re-absorption is a vital function of the kidney in terrestrial animals. The movement of electrolytes between the renal ultra-filtrate and the blood is subject to precise hormonal regulation that is essential to maintain whole body homeostatic balance. The mineralocorticoid hormone aldosterone is released by the adrenal cortex as the last stage in the rennin–angiotensin cascade in response to decreased blood pressure or directly in response to hyperkalaemia. The net effect of aldosterone release is to promote Na+ conservation though re-absorption from the renal ultrafiltrate, which can be coupled to simultaneous elevation of K+ secretion. The re-absorption of Na+ facilitates the osmotic movement of water from the renal ultra-filtrate back into the blood resulting in increased blood pressure. It has long been proposed that excessive salt conservation through aldosterone action leads to the development of hypertension with

pathophysiological consequences including the development of cardiovascular disease [1]. Clinical trials demonstrating the efficacy of the mineralocorticoid receptor (MR) antagonists spironolactone and eplerenone in reducing blood pressure and enhancing cardiovascular disease outcomes [2,3] did not demonstrate a link between the beneficial cardiovascular effect and reduced Na+ re-absorption. This has been interpreted as evidence that the renal effects of aldosterone are largely homeostatic, and that occupancy of MR by glucocorticoids in the cells of the cardiovascular system such as cardiomyocytes contributes to the distinct effects of MR antagonism on reducing hypertension [4]. Liddel’s syndrome patients present with symptoms resembling hyperaldosteronism: hypertension and hypokalaemia [5]. This phenotype is due to a gain in epithelial sodium channel (ENaC) activity that results in excessive Na+ conservation, strengthening the view that dysregulation of aldosterone sensitive transport in the kidney can have pathophysiological

∗ Corresponding author at: Department of Molecular Medicine, Royal College of Surgeons in Ireland Education and Research Centre, Smurfit Building, Beaumont Hospital, Dublin 9, Ireland. Tel.: +353 1 809 3825; fax: +353 1 809 3778. E-mail address: [email protected] (W. Thomas). 0039-128X/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.steroids.2008.01.013

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consequences. The degree of contribution made by the renal effects of aldosterone to the development of cardiovascular disease is thus still unclear. Aldosterone acts through a specific nuclear receptor, MR that is expressed in the epithelial cells of the distal nephron and other target tissues. MR bound by aldosterone translocates to the nucleus and acts as a ligand-dependent transcription factor that regulates the expression of a large repertoire of responsive genes. Aldosterone responsive genes include the membrane transporters that transport ions across the renal epithelium such as the subunits of the ENaC and the Na+ /K+ ATPase pump [6,7]. In the kidney MR is expressed along the aldosterone sensitive distal nephron (ASDN), which represents the principal regulatory site for salt conservation in the body. The ASDN comprises the thick ascending limb (TAL) of the loop of Henle; the distal convoluted tubule (DCT); the connecting tubule (CNT) and the cortical collecting duct (CCD). It was believed that Na+ re-absorption mainly occurred through ENaC located at the apical surface of the principal epithelial cells of the CCD. The DCT and connecting tubule are now regarded as the major sites of Na+ re-absorption in the kidney with the CCD playing a lesser role [8]. Na+ is subsequently transported across the basolateral membrane of the epithelium by the Na+ /K+ ATPase pump and into the blood, which in turn maintains a gradient for apical Na+ uptake. I addition to its direct effects on transporter expression, aldosterone also stimulates the activation of signalling cascades that in turn modulate the activity of these transporters. Some of the signalling cascades activated by aldosterone are dependent on the transcriptional effects of MR such as the up-regulation of serum and glucocorticoid regulated kinase (SGK-1), while other, more rapid signalling events stimulated by aldosterone are independent of its effects on transcription (reviewed in [9]). Determining the interaction between these genomic and nongenomic events is crucial in understanding the full scope of aldosterone’s effects on renal physiology.

2. Aldosterone and K+ transport in the distal nephron Aldosterone stimulated K+ transport in the epithelial cells of the distal nephron can be a process of either apical secretion or basolateral recycling. The switch over between secretion and recycling is dependent upon the plasma K+ concentration. The Na+ /K+ ATPase pump is located in the basolateral membrane of the epithelial cells and provides the electrochemical driving force for the influx of Na+ from the lumen of the nephron. Na+ is pumped from the cytoplasm of the epithelial cells across the basolateral/blood side of the epithelium in exchange for K+ . Potassium ions are recycled back into the blood through basolateral K+ channels or secreted into the lumen of the nephron by apical K+ channels. Na+ /K+ ATPase activity is regulated by aldosterone at the level of transcription and through the activation of signalling cascades. MR promotes the expression of pump subunits and the recruitment of pre-expressed pump subunits to the cell membrane through the up-regulation of SGK-1 [10]. Na+ /K+ ATPase activity is also sensitive to intracellular pH [11,12], which affects the cation binding specificity of the pump [13]. Since the eleva-

tion of intracellular pH through increased Na+ /H+ exchanger type 1 (NHE1) activity is an early response to aldosterone in the renal epithelium, this may contribute to the earliest phase in aldosterone-induced Na+ /K+ ATPase activity [14–16]. Aldosterone suppresses the activity of the apical Na+ /H+ exchanger type 3 (NHE3) in the medullary thick ascending limb of the loop of Henle (TAL) through a nongenomic, MR-independent mechanism to block HCO3 − re-absorption in this part of the nephron [17,18]. These observations suggest divergent roles for the NHE isoforms in aldosterone-stimulated ion transport processes. NHE1 stimulation modulates the transcriptional effects of the hormone through pH-sensitive signalling and transporter activity, while NHE3 activity contributes to the aldosterone-sensitive re-absorption of HCO3 − from the renal ultra-filtrate. The stimulation of NHE1 activity by aldosterone to raise intracellular pH also contributes to the activation ATP+ sensitive K+ (K+ ATP ) channels that promote K recycling in the + recycling across channels facilitate K distal nephron [19]. K+ ATP the basolateral membrane to balance the activity of the Na+ /K+ ATPase pump. Aldosterone treatment of frog skin principal cells stimulated a pH-sensitive K+ ATP channel activation within 2 min [19]. Aldosterone also modulates the activity of the renal outer medullary K+ (ROMK) channel to promote K+ secretion. The regulation of ROMK determines whether K+ secretion or recycling occurs in response to aldosterone. Studying the rare condition of pseudohypoaldosteronism type II (PHAII) has elucidated the precise mechanism of this physiological switch [20]. PHAII patients possess a mutation in the protein kinase with no lysine (WNK) types 1 and 4 resulting in excessive K+ secretion [21]. It has now been established that WNK4 suppresses both ROMK and ENaC but phosphorylation of WNK4 by SGK-1 relieves this inhibition to promote Na+ re-absorption and promote K+ secretion. The PHAII mutation mimics the combined effect of aldosterone and angiotensin II on WNK4 to promote ENaC activity while suppressing ROMK activity to conserve salt and raise blood pressure without loosing K+ . Recent research proposes that the aldosterone-induced upregulation of the ROMK channel activity in murine TAL cells relies upon activation of the cystic fibrosis trans-membrane conductance regulator (CFTR) Cl− channel [22] and expression of ENaC [23]. This emphasizes the integrated nature of aldosterone control over responsive membrane transporters. Since CFTR has multiple potential PKA phosphorylation sites it has been proposed that CFTR acts as a PKA-dependent switch for the up-regulation of K+ secretion by the distal nephron through CFTR-dependent coupling to ROMK [22]. A rapid increase in adenylate cyclase activity was detected in isolated inner medullary collecting duct cells treated with aldosterone that could potentiate PKA activity in this model [24].

3.

Aldosterone and ENaC activity

While it is the Na+ /K+ ATPase pump that provides the driving force for Na+ re-absorption, it is the activity of ENaC that is the effective rate-limiting step for the trans-epithelial movement of Na+ [7]. ENaC activity is stimulated by aldosterone and results in the electrogenic transport of Na+ across the apical membrane of the epithelial principal cells of the

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distal nephron. The regulation of ENaC activity by aldosterone has multiple stages. Recent structural analysis of the ENaC-related acid-sensing ion channel [25] suggests that functional ENaC is a heterotrimer complex made up of ␣, ␤ and ␥ subunits. The expression of the ␤- and ␥-subunits is constant in the ASDN, however, ENaC ␣-subunit expression is under the tight transcriptional control of MR and it is the availability of the ␣-subunit that dictates the level of ENaC activity in the distal nephron [8]. Expression of ENaC␣ subunit doubled in the micro-dissected CCDs of kidneys from aldosterone treated rats within 2 h of hormone administration [8]. Increased expression of the ENaC subunits is part of the direct genomic response to aldosterone. ENaC activity is also subjected to indirect genomic regulation which accounts for the observation that aldosterone stimulates only a twoto threefold increase in ENaC expression but a much higher stimulation of ENaC activity. Mature ENaC is expressed at the cell surface and under goes a cycle of endocytosis and exocytosis. ENaC localization within endocytic vesicles may be subjected to ubiquitylation by the Nedd4-2 E3 ubiquitin–protein ligase at a specific PPPXYXXL motif (reviewed in [26]). Ubiquitylation of ENaC targets the channel for proteolytic degradation by the proteasome. The targeted degradation of ENaC balances ENaC expression resulting in a sustained, low level of ENaC activity. The expression of SGK-1 is up-regulated by aldosterone and SGK-1 phosphorylates Nedd4-2, rendering it inactive. Similar inactivation of Nedd4-2 through phosphorylation at three residues (Ser-327, Ser-221 and Thr-246) by PKA has also been described [27]. The suppression of the ENaC ubiquitylation pathway shifts the balance between degradation and expression in favour of expression such that ENaC activity in the apical membrane increases. Aldosterone also stimulates de-ubiquitinylation of ENaC subunits, up-regulating the expression of the ubiquitin-specific protease (USP)2-45 to further stabilize pre-assembled ENaC [28]. The activation of

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near silent ENaC located at the apical surface through proteolytic cleavage by extracellular proteases such as elastase has been described [29] in addition a mechanism where ENaC is activated indirectly by trypsin through the stimulation of a G-protein-coupled receptor has also been identified [30]. Increased ENaC current has been detected as soon as 2 min after aldosterone treatment in isolated rabbit principal CCD cells [31]. The rapidity of this effect cannot be accounted for by changes in the expression of ENaC subunits or SGK1 activity. Aldosterone-induced rapid signalling events have also been implicated in the stimulation of ENaC activity. It has been proposed that there is crosstalk between aldosterone stimulated ERK1/2 and PI3-K signalling, where PI3-K promotes ENaC activity through SGK-1 activation, while ERK1/2 activation suppresses ENaC activity [32]. The activation of ERK1/2 by aldosterone is coupled to the activity of K-Ras small GTPase [32]. K-RasA becomes methylated following aldosterone treatment of A6 cells and ENaC activity is protein methylation sensitive [33,34]. PKC has been implicated in the phosphorylation of each of the ENaC subunits and subunit phosphorylation leads to increased channel activity in insulintreated A6 amphibian renal principal cells [35].

4.

ENaC trafficking and aldosterone

The regulation of early sub-cellular trafficking events may be another mechanism for aldosterone to regulate ENaC density in the apical membrane and as a consequence channel activity. The ENaC subunits undergo extensive post-translational modification that is necessary to achieve full activity of the channel. The subunits acquire a simple glycosylation profile in the endoplasmic reticulum (ER) before trafficking to the Golgi. Recent work by Blazer-Yost et al. has shown that insulin promotes the very rapid translocation of ENaC subunits to a discrete cluster of membrane bound struc-

Fig. 1 – The rapid translocation of ENaC␣ in response to aldosterone is c-Src tyrosine kinase-dependent. M1-CCD cells were transfected with a plasmid expressing ENaC␣ as a CFP fusion protein then treated with aldosterone (10 nM). After 2 min the cells were paraformaldehyde fixed and analysed using a confocal microscope (Zeiss LSM510 meta, 63× magification at laser excitation 488 nM). The CFP-tagged ENaC␣ had localized to discrete foci within the cytoplasm of aldosterone treated cells (A). Pre-incubation of the M1-CCD cells with the c-Src family kinase antagonist PP2 (100 nM) for 20 min before aldosterone completely inhibited the rapid aldosterone-induced translocation of ENaC␣ (B).

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tures located between the ER and Golgi termed the ER Golgi intermediate compartment (ERGIC) [36]. Assembly of the heterotetrameric ENaC structure occurs in the Golgi where the subunits also acquire a complex glycosylation profile. The ␣and ␥-subunits also undergo proteolytic cleavage by furin in the Golgi. The translocation of ENaC to the cell surface within 6 min and increased amiloride sensitive short-circuit current was observed following removal of blockade in Rho kinase activation [37]. These experiments were conducted in CHO cells over-expressing ENaC subunits with constitutively active RhoA. The rapid translocation event was coupled to the Rho kinase-dependent activation of PI4P5 kinase promoting membrane targeting of pre-expressed ENaC in vesicles rather than the effects of RhoA activation on cytoskeletal re-organization. It has been proposed that repeated challenge of murine CCD cells with cAMP results in the cyclical recruitment of ENaC from an intracellular pool for insertion into the apical membrane [38]. Newly synthesized ENaC contributed to a minor extent as cycloheximide only partially suppressed the amiloride-sensitive current on successive challenge. The existence of an intracellular pool of ENaC in the sub-apical cytoplasm of epithelial cells begs the question why activation and translocation of this pool does not become apparent within minutes of aldosterone treatment. Cell culture models suggest that aldosterone does not induce ENaC activity at least until 1–2 h after hormone treatment coinciding with transcriptional changes in SGK-1 and ENaC␣. The earliest descriptions of rapid responses to aldosterone suggest changes in Na+ transport in kidney within a few minutes of treatment and such rapid effects on ENaC activity have also been reported in isolated renal tubules [31]. Such very rapid increases in ENaC activity have not been detected in cultured cells and a satisfactory explanation for this observation has not been put forward. Numerous laboratories have described the rapid activation of signalling cascades in aldosterone responsive tissues. Activation of the novel PKC isoform PKC␧ by aldosterone contributes to the regulation Na+ /K+ ATPase activity in aldosterone-treated cardiomyocytes [39,40]. Protein kinase D (PKD) is regulated by novel PKC isoforms such as PKC␦, PKC␧ and PKC␩ in different tissues. We have shown that PKD1 is activated within 5 min in response to aldosterone treatment in the M1-CCD cell line [41]. The three PKD family isoforms are emerging as important regulators of sub-cellular trafficking through the maintenance of Golgi structure and regulation of vesicle fission from the Golgi organelle reviewed in [42]. We found that the activation of PKD1 by aldosterone is coupled to the MR-dependent trans-activation of the epidermal growth factor receptor (EGFR) by c-Src tyrosine kinase [41,43]. Aldosterone stimulates the activation of c-Src within 2 min of treatment [44] and aldosterone not only stimulates EGFR expression at the level of transcription [45], but also rapidly promotes its phosphorylation [46,47]. We established that one of the residues on EGFR that becomes phosphorylated following aldosterone treatment is the c-Src targeted site Tyr845 [41]. The stimulation of electro-neutral Na+ reabsorption through NHE activity is suppressed by c-Src/EGFR activation [48], however, the electrogenic reabsorption of Na+ through ENaC may be facilitated by the stimulation of ENaC trafficking. Recent research from our laboratory has shown that aldos-

Fig. 2 – The multi-factorial regulation of ENaC by aldosterone. Aldosterone stimulates the expression of the ENaC␣ subunit in the epithelium of the distal nephron through the transcriptional action of the mineralocorticoid receptor (MR). ENaC␣ together with the constitutively expressed ENaC␤ and ENaC␥ subunits pass through a series of membrane bound organelles before reaching the apical cell surface. The ENaC subunits are transported to the Golgi apparatus through the endoplasmic reticulum intermediate compartment (ERGIC). In the Golgi the subunits undergo modification to their glycosylation pattern and proteolytic cleavage by furin in advance of channel assembly. The channels are transported to the membrane in exocytic vesicles (EX) and in the membrane full channel activity is achieved following proteolysis by extracellular proteases such as elastin. The channels may be targeted for degradation by the proteasome by the Nedd4-2 ubiquitin ligase (N4-2), however, up-regulation of the serum and glucocorticoid regulated kinase (SGK-1) by aldosterone suppresses N4-2 activity and increases ENaC density in the membrane. Aldosterone also up-regulates expression of the ubiquitin-specific protease, USP2-45 that de-ubiquitylates ENaC, further increasing its stability and surface density. Aldosterone regulates the movement of ENaC from a sub-apical pool to the cell membrane through the modulation of vesicle transport. The EGFR-coupled activation of protein kinase D (PKD) by aldosterone may be a further mechanism for aldosterone to modulate the trafficking of pre-expressed ENaC subunits.

terone stimulates the translocation of pre-expressed ENaC subunits to cytoplasmic foci within a few minutes of hormone treatment. The rapid translocation of CFP-tagged ENaC␣ in response to aldosterone can be blocked by pre-incubation with the Src family specific antagonist PP2 (Fig. 1).

5.

Conclusion

The homeostatic role of aldosterone in regulating whole body electrolyte balance through its effects on ion transport in the distal nephron are fully established, however, the contribution of aldosterone to the development of cardiovascular disease

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through mal-absorption of Na+ by the kidney and resulting hypertension is less certain. Apical expression of ENaC and its regulation by aldosterone represents the crucial, rate-limiting component of the Na+ transport process in the kidney (Fig. 2). The expression of the ENaC subunits is regulated by aldosterone through the transcriptional activity of MR and the rate of degradation of pre-expressed ENaC subunits that recycle between the cell membrane and an intracellular pool is regulated by the expression of aldosterone-regulated proteins such as SGK-1 and USP-2-45. Aldosterone also stimulates signalling cascades within a few minutes of hormone treatment that include MAP kinase and Ca2+ -dependent PKC␣ activation which are associated with transient NHE activation. Transporters such as the KATP channels and the Na+ /K+ ATPase are pH-sensitive and so may be regulated by the rise in cytoplasmic pH. The physiological relevance of rapid aldosterone-induced responses in the kidney in the context of the pronounced but delayed effects of the hormone on gene expression and ion transport is the subject of debate. Some of the early protein kinase responses may serve to potentiate the transcriptional effects of aldosterone through the phosphorylation of transcription factors essential for MR-dependent gene expression. The membrane transporters that are regulated by aldosterone also possess specific phosphorylation sites for rapidly activated kinases. A role for the rapid aldosteroneinduced signalling cascades in regulating the earliest phase of electrogenic Na+ transport by the distal nephron may be found in the regulation of sub-cellular ENaC trafficking. The PKD family of protein kinases play important roles in regulating sub-cellular vesicle trafficking and the activation of PKD by aldosterone in a CCD cell line [41] requires further investigation to establish whether aldosterone can stimulate endoplasmic reticulum to Golgi trafficking of ENaC subunits in the manner that has already been described for insulin [36].

Acknowledgements The authors are supported by programme grant 060809/Z/00 from the Wellcome Trust and by the Higher Education Authority of Ireland under the Programme for Research in Third Level Institutions (PRTLI) Cycle 3.

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