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ldosterone-induced signalling and cation transportn the distal nephron

arren Thomas ∗, Victoria McEneaney, Brian J. Harveyepartment of Molecular Medicine, Royal College of Surgeons in Ireland Education and Research Centre,eaumont Hospital, Dublin, Ireland

r t i c l e i n f o

ublished on line 19 January 2008

eywords:

ldosterone

a b s t r a c t

Aldosterone is an important regulator of Na+ and K+ transport in the distal nephron mod-

ulating the surface expression of transporters through the action of the mineralocorticoid

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

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

ineralocorticoid

apid responses

NaC

rotein kinase D

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

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

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

of epidermal growth factor receptor contributes to early ENaC trafficking in response to

aldosterone.

. Introduction

alt conservation through re-absorption is a vital functionf the kidney in terrestrial animals. The movement of elec-rolytes between the renal ultra-filtrate and the blood isubject to precise hormonal regulation that is essential toaintain whole body homeostatic balance. The mineralocorti-

oid hormone aldosterone is released by the adrenal cortex ashe last stage in the rennin–angiotensin cascade in responseo decreased blood pressure or directly in response to hyper-alaemia. The net effect of aldosterone release is to promotea+ conservation though re-absorption from the renal ultra-ltrate, which can be coupled to simultaneous elevation of+ secretion. The re-absorption of Na+ facilitates the osmotic

ovement of water from the renal ultra-filtrate back into

he blood resulting in increased blood pressure. It has longeen proposed that excessive salt conservation through aldos-erone action leads to the development of hypertension with

∗ Corresponding author at: Department of Molecular Medicine, Royal Coluilding, Beaumont Hospital, Dublin 9, Ireland. Tel.: +353 1 809 3825; fa

E-mail address: wthomas@rcsi.ie (W. Thomas).039-128X/$ – see front matter © 2008 Elsevier Inc. All rights reserved.oi:10.1016/j.steroids.2008.01.013

© 2008 Elsevier Inc. All rights reserved.

pathophysiological consequences including the developmentof cardiovascular disease [1].

Clinical trials demonstrating the efficacy of the miner-alocorticoid receptor (MR) antagonists spironolactone andeplerenone in reducing blood pressure and enhancing cardio-vascular disease outcomes [2,3] did not demonstrate a linkbetween the beneficial cardiovascular effect and reduced Na+

re-absorption. This has been interpreted as evidence that therenal effects of aldosterone are largely homeostatic, and thatoccupancy of MR by glucocorticoids in the cells of the cardio-vascular system such as cardiomyocytes contributes to thedistinct effects of MR antagonism on reducing hypertension[4]. Liddel’s syndrome patients present with symptoms resem-bling hyperaldosteronism: hypertension and hypokalaemia

lege of Surgeons in Ireland Education and Research Centre, Smurfitx: +353 1 809 3778.

[5]. This phenotype is due to a gain in epithelial sodium chan-nel (ENaC) activity that results in excessive Na+ conservation,strengthening the view that dysregulation of aldosterone sen-sitive transport in the kidney can have pathophysiological

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980 s t e r o i d s 7 3

consequences. The degree of contribution made by the renaleffects of aldosterone to the development of cardiovasculardisease is thus still unclear.

Aldosterone acts through a specific nuclear receptor, MRthat is expressed in the epithelial cells of the distal nephronand other target tissues. MR bound by aldosterone trans-locates to the nucleus and acts as a ligand-dependenttranscription factor that regulates the expression of a largerepertoire of responsive genes. Aldosterone responsive genesinclude the membrane transporters that transport ions acrossthe renal epithelium such as the subunits of the ENaC andthe Na+/K+ ATPase pump [6,7]. In the kidney MR is expressedalong the aldosterone sensitive distal nephron (ASDN), whichrepresents the principal regulatory site for salt conservationin 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 occurredthrough ENaC located at the apical surface of the principalepithelial cells of the CCD. The DCT and connecting tubule arenow regarded as the major sites of Na+ re-absorption in thekidney with the CCD playing a lesser role [8]. Na+ is subse-quently transported across the basolateral membrane of theepithelium 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 cas-cades that in turn modulate the activity of these transporters.Some of the signalling cascades activated by aldosterone aredependent on the transcriptional effects of MR such as theup-regulation of serum and glucocorticoid regulated kinase(SGK-1), while other, more rapid signalling events stimulatedby aldosterone are independent of its effects on transcription(reviewed in [9]). Determining the interaction between thesegenomic and nongenomic events is crucial in understandingthe full scope of aldosterone’s effects on renal physiology.

2. Aldosterone and K+ transport in thedistal nephron

Aldosterone stimulated K+ transport in the epithelial cells ofthe distal nephron can be a process of either apical secretionor basolateral recycling. The switch over between secretionand recycling is dependent upon the plasma K+ concentra-tion. The Na+/K+ ATPase pump is located in the basolateralmembrane of the epithelial cells and provides the electro-chemical driving force for the influx of Na+ from the lumenof the nephron. Na+ is pumped from the cytoplasm of theepithelial cells across the basolateral/blood side of the epithe-lium in exchange for K+. Potassium ions are recycled back intothe blood through basolateral K+ channels or secreted into thelumen of the nephron by apical K+ channels. Na+/K+ ATPaseactivity is regulated by aldosterone at the level of transcrip-tion and through the activation of signalling cascades. MRpromotes the expression of pump subunits and the recruit-

ment of pre-expressed pump subunits to the cell membranethrough the up-regulation of SGK-1 [10]. Na+/K+ ATPase activ-ity is also sensitive to intracellular pH [11,12], which affectsthe cation binding specificity of the pump [13]. Since the eleva-

0 8 ) 979–984

tion of intracellular pH through increased Na+/H+ exchangertype 1 (NHE1) activity is an early response to aldosterone inthe renal epithelium, this may contribute to the earliest phasein aldosterone-induced Na+/K+ ATPase activity [14–16]. Aldos-terone suppresses the activity of the apical Na+/H+ exchangertype 3 (NHE3) in the medullary thick ascending limb of theloop of Henle (TAL) through a nongenomic, MR-independentmechanism to block HCO3

− re-absorption in this part of thenephron [17,18]. These observations suggest divergent rolesfor the NHE isoforms in aldosterone-stimulated ion transportprocesses. NHE1 stimulation modulates the transcriptionaleffects of the hormone through pH-sensitive signalling andtransporter activity, while NHE3 activity contributes to thealdosterone-sensitive re-absorption of HCO3

− from the renalultra-filtrate.

The stimulation of NHE1 activity by aldosterone to raiseintracellular pH also contributes to the activation ATP-sensitive K+ (K+

ATP) channels that promote K+ recycling in thedistal nephron [19]. K+

ATP channels facilitate K+ recycling acrossthe basolateral membrane to balance the activity of the Na+/K+

ATPase pump. Aldosterone treatment of frog skin principalcells stimulated a pH-sensitive K+

ATP channel activation within2 min [19]. Aldosterone also modulates the activity of the renalouter medullary K+ (ROMK) channel to promote K+ secretion.The regulation of ROMK determines whether K+ secretion orrecycling occurs in response to aldosterone. Studying the rarecondition of pseudohypoaldosteronism type II (PHAII) has elu-cidated the precise mechanism of this physiological switch[20]. PHAII patients possess a mutation in the protein kinasewith no lysine (WNK) types 1 and 4 resulting in excessive K+

secretion [21]. It has now been established that WNK4 sup-presses both ROMK and ENaC but phosphorylation of WNK4by SGK-1 relieves this inhibition to promote Na+ re-absorptionand promote K+ secretion. The PHAII mutation mimics thecombined effect of aldosterone and angiotensin II on WNK4to promote ENaC activity while suppressing ROMK activityto conserve salt and raise blood pressure without loosing K+.Recent research proposes that the aldosterone-induced up-regulation of the ROMK channel activity in murine TAL cellsrelies upon activation of the cystic fibrosis trans-membraneconductance regulator (CFTR) Cl− channel [22] and expres-sion of ENaC [23]. This emphasizes the integrated nature ofaldosterone control over responsive membrane transporters.Since CFTR has multiple potential PKA phosphorylation sites ithas been proposed that CFTR acts as a PKA-dependent switchfor the up-regulation of K+ secretion by the distal nephronthrough CFTR-dependent coupling to ROMK [22]. A rapidincrease in adenylate cyclase activity was detected in isolatedinner medullary collecting duct cells treated with aldosteronethat could potentiate PKA activity in this model [24].

3. Aldosterone and ENaC activity

While it is the Na+/K+ ATPase pump that provides the driv-ing force for Na+ re-absorption, it is the activity of ENaC

that is the effective rate-limiting step for the trans-epithelialmovement of Na+ [7]. ENaC activity is stimulated by aldos-terone and results in the electrogenic transport of Na+ acrossthe apical membrane of the epithelial principal cells of the

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istal nephron. The regulation of ENaC activity by aldos-erone has multiple stages. Recent structural analysis of theNaC-related acid-sensing ion channel [25] suggests that func-ional ENaC is a heterotrimer complex made up of �, � and

subunits. The expression of the �- and �-subunits is con-tant in the ASDN, however, ENaC �-subunit expression isnder the tight transcriptional control of MR and it is thevailability of the �-subunit that dictates the level of ENaCctivity in the distal nephron [8]. Expression of ENaC� sub-nit doubled in the micro-dissected CCDs of kidneys fromldosterone treated rats within 2 h of hormone administra-ion [8]. Increased expression of the ENaC subunits is part ofhe direct genomic response to aldosterone. ENaC activity islso subjected to indirect genomic regulation which accountsor the observation that aldosterone stimulates only a two-o threefold increase in ENaC expression but a much highertimulation of ENaC activity.

Mature ENaC is expressed at the cell surface and underoes a cycle of endocytosis and exocytosis. ENaC localizationithin endocytic vesicles may be subjected to ubiquityla-

ion by the Nedd4-2 E3 ubiquitin–protein ligase at a specificPPXYXXL motif (reviewed in [26]). Ubiquitylation of ENaCargets the channel for proteolytic degradation by the pro-easome. The targeted degradation of ENaC balances ENaCxpression resulting in a sustained, low level of ENaC activ-ty. The expression of SGK-1 is up-regulated by aldosteronend SGK-1 phosphorylates Nedd4-2, rendering it inactive.imilar inactivation of Nedd4-2 through phosphorylation athree residues (Ser-327, Ser-221 and Thr-246) by PKA has alsoeen described [27]. The suppression of the ENaC ubiquity-

ation pathway shifts the balance between degradation andxpression in favour of expression such that ENaC activity

n the apical membrane increases. Aldosterone also stim-lates de-ubiquitinylation of ENaC subunits, up-regulatinghe expression of the ubiquitin-specific protease (USP)2-45 tourther stabilize pre-assembled ENaC [28]. The activation of

ig. 1 – The rapid translocation of ENaC� in response to aldosterransfected with a plasmid expressing ENaC� as a CFP fusion prells were paraformaldehyde fixed and analysed using a confocaxcitation 488 nM). The CFP-tagged ENaC� had localized to discrA). Pre-incubation of the M1-CCD cells with the c-Src family kinaompletely inhibited the rapid aldosterone-induced translocation

8 ) 979–984 981

near silent ENaC located at the apical surface through pro-teolytic cleavage by extracellular proteases such as elastasehas been described [29] in addition a mechanism where ENaCis activated indirectly by trypsin through the stimulation of aG-protein-coupled receptor has also been identified [30].

Increased ENaC current has been detected as soon as 2 minafter aldosterone treatment in isolated rabbit principal CCDcells [31]. The rapidity of this effect cannot be accountedfor by changes in the expression of ENaC subunits or SGK-1 activity. Aldosterone-induced rapid signalling events havealso been implicated in the stimulation of ENaC activity. Ithas been proposed that there is crosstalk between aldosteronestimulated ERK1/2 and PI3-K signalling, where PI3-K pro-motes ENaC activity through SGK-1 activation, while ERK1/2activation suppresses ENaC activity [32]. The activation ofERK1/2 by aldosterone is coupled to the activity of K-Ras smallGTPase [32]. K-RasA becomes methylated following aldos-terone treatment of A6 cells and ENaC activity is proteinmethylation sensitive [33,34]. PKC has been implicated in thephosphorylation of each of the ENaC subunits and subunitphosphorylation leads to increased channel activity in insulin-treated A6 amphibian renal principal cells [35].

4. ENaC trafficking and aldosterone

The regulation of early sub-cellular trafficking events may beanother mechanism for aldosterone to regulate ENaC densityin the apical membrane and as a consequence channel activ-ity. The ENaC subunits undergo extensive post-translationalmodification that is necessary to achieve full activity ofthe channel. The subunits acquire a simple glycosylation

profile in the endoplasmic reticulum (ER) before traffickingto the Golgi. Recent work by Blazer-Yost et al. has shownthat insulin promotes the very rapid translocation of ENaCsubunits to a discrete cluster of membrane bound struc-

one is c-Src tyrosine kinase-dependent. M1-CCD cells wereotein then treated with aldosterone (10 nM). After 2 min thel microscope (Zeiss LSM510 meta, 63× magification at laserete foci within the cytoplasm of aldosterone treated cellsse antagonist PP2 (100 nM) for 20 min before aldosteroneof ENaC� (B).

( 2 0 0 8 ) 979–984

Fig. 2 – The multi-factorial regulation of ENaC byaldosterone. Aldosterone stimulates the expression of theENaC� subunit in the epithelium of the distal nephronthrough the transcriptional action of the mineralocorticoidreceptor (MR). ENaC� together with the constitutivelyexpressed ENaC� and ENaC� subunits pass through aseries of membrane bound organelles before reaching theapical cell surface. The ENaC subunits are transported tothe Golgi apparatus through the endoplasmic reticulumintermediate compartment (ERGIC). In the Golgi thesubunits undergo modification to their glycosylationpattern and proteolytic cleavage by furin in advance ofchannel assembly. The channels are transported to themembrane in exocytic vesicles (EX) and in the membranefull channel activity is achieved following proteolysis byextracellular proteases such as elastin. The channels maybe targeted for degradation by the proteasome by theNedd4-2 ubiquitin ligase (N4-2), however, up-regulation ofthe serum and glucocorticoid regulated kinase (SGK-1) byaldosterone suppresses N4-2 activity and increases ENaCdensity in the membrane. Aldosterone also up-regulatesexpression of the ubiquitin-specific protease, USP2-45 thatde-ubiquitylates ENaC, further increasing its stability andsurface density. Aldosterone regulates the movement ofENaC from a sub-apical pool to the cell membrane throughthe modulation of vesicle transport. The EGFR-coupledactivation of protein kinase D (PKD) by aldosterone may be

982 s t e r o i d s 7 3

tures located between the ER and Golgi termed the ER Golgiintermediate compartment (ERGIC) [36]. Assembly of the het-erotetrameric ENaC structure occurs in the Golgi where thesubunits also acquire a complex glycosylation profile. The �-and �-subunits also undergo proteolytic cleavage by furin inthe Golgi. The translocation of ENaC to the cell surface within6 min and increased amiloride sensitive short-circuit currentwas observed following removal of blockade in Rho kinaseactivation [37]. These experiments were conducted in CHOcells over-expressing ENaC subunits with constitutively activeRhoA. The rapid translocation event was coupled to the Rhokinase-dependent activation of PI4P5 kinase promoting mem-brane targeting of pre-expressed ENaC in vesicles rather thanthe effects of RhoA activation on cytoskeletal re-organization.

It has been proposed that repeated challenge of murineCCD cells with cAMP results in the cyclical recruitment ofENaC from an intracellular pool for insertion into the api-cal membrane [38]. Newly synthesized ENaC contributed toa minor extent as cycloheximide only partially suppressedthe amiloride-sensitive current on successive challenge. Theexistence of an intracellular pool of ENaC in the sub-apicalcytoplasm of epithelial cells begs the question why activa-tion and translocation of this pool does not become apparentwithin minutes of aldosterone treatment. Cell culture modelssuggest that aldosterone does not induce ENaC activity at leastuntil 1–2 h after hormone treatment coinciding with transcrip-tional changes in SGK-1 and ENaC�. The earliest descriptionsof rapid responses to aldosterone suggest changes in Na+

transport in kidney within a few minutes of treatment andsuch rapid effects on ENaC activity have also been reported inisolated renal tubules [31]. Such very rapid increases in ENaCactivity have not been detected in cultured cells and a sat-isfactory explanation for this observation has not been putforward.

Numerous laboratories have described the rapid activa-tion of signalling cascades in aldosterone responsive tissues.Activation of the novel PKC isoform PKC� by aldosteronecontributes to the regulation Na+/K+ ATPase activity inaldosterone-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 isactivated within 5 min in response to aldosterone treatmentin the M1-CCD cell line [41]. The three PKD family isoformsare emerging as important regulators of sub-cellular traffick-ing through the maintenance of Golgi structure and regulationof vesicle fission from the Golgi organelle reviewed in [42].We found that the activation of PKD1 by aldosterone is cou-pled to the MR-dependent trans-activation of the epidermalgrowth factor receptor (EGFR) by c-Src tyrosine kinase [41,43].Aldosterone stimulates the activation of c-Src within 2 minof treatment [44] and aldosterone not only stimulates EGFRexpression at the level of transcription [45], but also rapidlypromotes its phosphorylation [46,47]. We established that oneof the residues on EGFR that becomes phosphorylated follow-ing 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], how-ever, the electrogenic reabsorption of Na+ through ENaCmay be facilitated by the stimulation of ENaC trafficking.Recent research from our laboratory has shown that aldos-

a further mechanism for aldosterone to modulate thetrafficking of pre-expressed ENaC subunits.

terone stimulates the translocation of pre-expressed ENaCsubunits to cytoplasmic foci within a few minutes of hormonetreatment. The rapid translocation of CFP-tagged ENaC� inresponse to aldosterone can be blocked by pre-incubation withthe Src family specific antagonist PP2 (Fig. 1).

5. Conclusion

The homeostatic role of aldosterone in regulating whole bodyelectrolyte balance through its effects on ion transport in thedistal nephron are fully established, however, the contributionof aldosterone to the development of cardiovascular disease

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hrough mal-absorption of Na+ by the kidney and resultingypertension is less certain. Apical expression of ENaC and itsegulation by aldosterone represents the crucial, rate-limitingomponent of the Na+ transport process in the kidney (Fig. 2).he expression of the ENaC subunits is regulated by aldos-

erone through the transcriptional activity of MR and the ratef degradation of pre-expressed ENaC subunits that recycleetween the cell membrane and an intracellular pool is reg-lated by the expression of aldosterone-regulated proteinsuch as SGK-1 and USP-2-45. Aldosterone also stimulates sig-alling cascades within a few minutes of hormone treatmenthat include MAP kinase and Ca2+-dependent PKC� activationhich are associated with transient NHE activation. Trans-orters such as the KATP channels and the Na+/K+ ATPase areH-sensitive and so may be regulated by the rise in cytoplas-ic pH.The physiological relevance of rapid aldosterone-induced

esponses in the kidney in the context of the pronouncedut delayed effects of the hormone on gene expression and

on transport is the subject of debate. Some of the earlyrotein kinase responses may serve to potentiate the tran-criptional effects of aldosterone through the phosphorylationf transcription factors essential for MR-dependent genexpression. The membrane transporters that are regulatedy aldosterone also possess specific phosphorylation sites forapidly activated kinases. A role for the rapid aldosterone-nduced signalling cascades in regulating the earliest phasef electrogenic Na+ transport by the distal nephron maye found in the regulation of sub-cellular ENaC traffick-

ng. The PKD family of protein kinases play important rolesn regulating sub-cellular vesicle trafficking and the activa-ion of PKD by aldosterone in a CCD cell line [41] requiresurther investigation to establish whether aldosterone cantimulate endoplasmic reticulum to Golgi trafficking of ENaCubunits in the manner that has already been described fornsulin [36].

cknowledgements

he authors are supported by programme grant 060809/Z/00rom the Wellcome Trust and by the Higher Education Author-ty of Ireland under the Programme for Research in Third Levelnstitutions (PRTLI) Cycle 3.

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