Steroids 75 (2010) 550–554

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Steroids

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Aldosterone as a renal growth factor

Warren Thomas ∗, Ruth Dooley, Brian J. HarveyDepartment of Molecular Medicine, Royal College of Surgeons in Ireland, Education and Research Centre, Beaumont Hospital, Dublin 9, Ireland

a r t i c l e i n f o

Article history:Available online 24 September 2009

Keywords:AldosteroneKidneyProtein kinase D

a b s t r a c t

Aldosterone regulates blood pressure through its effects on the cardiovascular system and kidney. Aldos-terone can also contribute to the development of hypertension that leads to chronic pathologies such asnephropathy and renal fibrosis. Aldosterone directly modulates renal cell proliferation and differentiationas part of normal kidney development. The stimulation of rapidly activated protein kinase cascades is onefacet of how aldosterone regulates renal cell growth. These cascades may also contribute to myofibrob-lastic transformation and cell proliferation observed in pathological conditions of the kidney. Polycystickidney disease is a genetic disorder that is accelerated by hypertension. EGFR-dependent proliferation

EGFRPolycystic kidney diseaseMitogen activated protein kinase

of the renal epithelium is a factor in cyst development and trans-activation of EGFR is a key feature ininitiating aldosterone-induced signalling cascades. Delineating the components of aldosterone-induced

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. Introduction

The renin–angiotensin–aldosterone system (RAAS) is a key reg-lator of systemic blood pressure through its direct action on theenal and cardiovascular systems [1,2]. The release of aldosteroney the adrenal glands contributes to the homeostatic regulationf blood pressure under normal physiological conditions via aumber of interlinked mechanisms including the modulation ofascular tone and the rate of electrolyte transport across tar-et epithelia such as the distal nephron. Defective RAAS activitys a contributory factor in congenital and acquired hypertension3]; consequently the individual receptors, enzymes and effec-ors of the RAAS cascade are important therapeutic targets in thereatment of this condition. The kidney is a key target organ forldosterone action and the regulation of the rate of Na+ absorptiony the distal nephron is an important factor influencing systemiclood pressure. The biological effects of aldosterone are initi-ted through the binding of the hormone to the mineralocorticoideceptor (MR), which is expressed by all aldosterone responsiveissues including the distal nephron [4] and the renin secreting

esangial cells of the juxtaglomerular apparatus [5]. In the distalephron, the aldosterone-induced modulation of ion transporterctivity is mediated through the transcriptional effects of MR on

ransporter subunit and regulatory protein expression or throughhe phosphorylation of transporters and regulatory molecules byapidly activated protein kinases. The combined transcriptionalnd signalling effects of aldosterone on the renal epithelium lead

∗ Corresponding author. Tel.: +353 1 8093825; fax: +353 1 8093778.E-mail address: wthomas@rcsi.ie (W. Thomas).

039-128X/$ – see front matter © 2009 Elsevier Inc. All rights reserved.oi:10.1016/j.steroids.2009.09.008

fy novel therapeutic targets for proliferative diseases of the kidney.© 2009 Elsevier Inc. All rights reserved.

to the modulation of ion fluxes that impact upon whole bodyelectrolyte homeostasis. The regulation of Na+ and K+ transportare held to be the most important functions for aldosterone inthe kidney. However, recent evidence points to aldosterone hav-ing a more complex role in supporting renal cell differentiationand also in contributing to pathological processes in the kidney.The direct effects of aldosterone on renal pathology through itseffects on MR-expressing cells in the renal tubule are adjunct to thehistological damage inflicted on renal tissue by systemic hyperten-sion.

Aldosterone modulates the growth of renal cells both in cultureand in vivo. Evidence points to a contribution from aldosteroneto normal renal tubule cell proliferation and differentiation fromisolated renal stem cells [6] and also from studies in vivo usingjuvenile animal models [7]. Aldosterone may also contribute tothe development of pathological conditions in the kidney throughdysregulation of renal cell growth in the adult resulting in fibro-sis or epithelial hyperplasia. The growth stimulatory effects ofaldosterone, either hypertrophy or hyperplasia depending on thecell type investigated; result from the synergism between thetranscriptional and rapid signalling responses stimulated by thehormone. The cross-talk between these two facets of aldosteroneaction is a prominent aspect of current research and blurs the dis-tinction between genomic and non-genomic effects of steroids.Essentially aldosterone not only promotes the activation of pro-tein kinase signalling cascades which contribute to cell growth but

also up-regulates the expression of key signalling intermediates toamplify the sensitivity and magnitude of responses to aldosteroneitself and other growth factors. The mechanisms by which aldos-terone modulates the growth of renal cells will be the subject of thisreview, in particular the roles of the rapidly activated signalling cas-

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Fig. 1. Suppression of PKD1 expression inhibits aldosterone-induced CCD cell pro-liferation. M1-CCD cells and cells stably suppressed in PKD1 expression using aplasmid expressing a PKD1 specific siRNA were seeded at a density of 1 × 104 cellsper microtitre plate well, in DMEM/F12 containing 5 �M dexamethasone and 10%FBS. The cells were left to adhere overnight at 37 ◦C in a humidified atmosphere of5% CO2. The medium was then exchanged for serum-free DMEM/F12, containing1 �M dexamethasone for 24 h. Cells were treated with 10 nM aldosterone or vehi-cle control for 72 h. Following treatment, the medium was carefully removed. The

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ades in potentiating and sustaining the transcriptional response toldosterone in proliferating renal cells.

. Aldosterone and renal stem cell differentiation

The development of each renal tubule from progenitor cells is aomplex process reliant upon the co-operative actions of nephro-enic mesenchymal stem cells which give rise to the proximalubule, loop of Henle and distal tubule, and other stem cells locatedn the collecting duct ampullae that form the collecting duct tubuletself. Cell proliferation following stem cell stimulation leading tohe formation of S-shaped tubule bodies marks the earliest stagef nephron development in the mesenchymal parenchyma. Theignalling processes which co-ordinate the two groups of stemells to form a single nephron are poorly understood. Isolatedenal stem cells from neonatal animals can be cultured using an initro serum-free perfusion system with the embryonic cells seededetween two layers of porous polyester matrix [8]. However, var-

ous combinations of typically used cell culture supplements suchs epidermal growth factor (EGF), insulin, transferrin, selenium,etinoic acid, bovine pituitary extract or cholecalciferol do nottimulate tubule formation in this model [6]. Under these cultureonditions the progenitor cells will develop hollow, tubular struc-ures only in the presence of 100 nM aldosterone. These tubulartructures display at least partial differentiation with the expres-ion of N-acetylgalactosamine on surface glycoproteins as a markeror the collecting duct and also some degree of apical to basolat-ral epithelial polarity indicated by tight junction formation. Theldosterone biochemical precursors: cholesterol and pregnenoloneid not mimic this effect on renal stem cells while progesteronend 11-deoxycorticosterone treatment resulted in only partialubule development. The MR antagonist spironolactone inhibitedhe aldosterone-induced tubule formation while the GR agonistexamethasone stimulated disorganized cell proliferation but noubule formation [9]. Aldosterone thus acts as a potent differen-iation factor for renal stem cells in neonatal animals. However,imilar tubulogenic effects have not been observed in cells isolatedrom adult animals and the molecular basis of aldosterone-inducedubule differentiation has not been established to date.

. Aldosterone-induced signalling and renal cell growth

The field of aldosterone-induced rapid signalling has developedreatly in recent years with the identification of the physiologi-al roles of these signalling cascades in the cell and in the wholerganism. The regulation of ion fluxes has always been seen as theey function of aldosterone in the kidney [10] and consequentlyttention has been focused on coupling aldosterone-induced sig-alling cascades to Na+ and K+ transport. It has also been known

or some time that aldosterone can modulate the proliferationf MR-expressing cells in vitro [11]. Modulation of cell growthy aldosterone; whether it is a metabolic stimulation (hypertro-hy) or the stimulation of cell proliferation (hyperplasia) requireso-ordination of rapid signalling and transcriptional changes. Thectivation of several signalling cascades by aldosterone has beendentified in cells derived from the distal nephron including theKC� [12], PKD1 [13] and ERK1/2 mitogen activated protein (MAP)inase [14] cascades. A number of the signalling cascades rapidlyctivated by aldosterone such as the Ca2+-dependent PKC isoformsnd ERK1/2 also promote cell proliferation in response to mitogenic

gonists. This begs the question of whether aldosterone modulatesenal cell growth through the activation of these cascades.

Aldosterone treatment of isolated renal cortical collecting ductells stimulates the biphasic activation of the ERK1/2 cascade withn early phase of transient activation followed by a more sus-

cells were detached using trypsin and re-suspended in a final volume of 100 �l freshmedium. Cells were counted using a Neubauer chamber, 4 wells per treatment foreach cell line. Aldosterone stimulated a twofold increase in cell number above basalproliferation rates that was blocked by suppression of PKD1 expression.

tained period of activation. Recent work has investigated the roleof aldosterone on ERK1/2 signalling in vivo. Dosing new born ratswith the MR antagonist spironolactone for 7 days resulted in anincrease in apoptosis detected in kidney sections [15]. This obser-vation suggests that at least in juvenile animals, aldosterone playsa role in inhibiting apoptosis. Spironolactone-induced apoptosiswas most prominent in the tubule cells of the renal cortex whereMR is highly expressed. MR antagonism resulted in a significantreduction in ERK1/2 and p38 MAP kinase protein expression buta slight increase in the abundance of the mRNA of these twokinases. Conversely JNK-2 expression was induced by spironolac-tone in the cells of the glomeruli and in the cortical tubules [15].A role for aldosterone in regulating apoptosis in the adult kidneyhas also been described. Aldosterone stimulated both apoptoticand mitogenic effects in human mesangial cells, which correlatedwith de-phosphorylation of the Bcl-2 family protein Bad and therelease of cytochrome c into the cytoplasm. In vivo, aldosteronehad a similar pro-apoptotic effect on rat mesangial cells whichwas eplerenone-sensitive and correlated with an increase in sys-tolic blood pressure and albuminuria [16]. Aldosterone stimulatedERK1/2 activation within 10 min of treatment in mesangial cellswhich was also eplerenone-sensitive and led to ERK1/2-dependentcell proliferation [5]. Aldosterone is therefore involved in regulatingthe delicate balance between proliferation and apoptosis in matureand immature renal tissues.

The sustained activation of the ERK1/2 signalling cascadepromotes growth factor-stimulated cell cycle progression in fibrob-lasts. Transient activation of ERK1/2 is insufficient to commitstimulated cells to complete cell division [17]. The protein kinaseD (PKD) family of serine threonine protein kinases are involvedin modulating critical cellular processes including sub-cellulartrafficking, hypertrophy, apoptosis and proliferation and are acti-vated in response to diverse agonists [18]. The expression andactivation of PKD family proteins is pre-requisite for sustained

activation of ERK1/2 in response to non-steroid growth factorsthat act through G-protein coupled receptors [17,19]. PKD fam-ily isoforms are in some way responsible for stabilizing theinitial growth factor-stimulated ERK1/2 activation [20]. The MR-dependent autophosphorylation of protein kinase D occurs within

552 W. Thomas et al. / Steroids 75 (2010) 550–554

Fig. 2. ERK1/2 nuclear localization following aldosterone treatment is EGFR-dependent. Murine M1-CCD cells were maintained in serum-free medium for 24 h then treatedw inased ecameo specifia

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ith aldosterone (10 nM) for 2 min. The sub-cellular distribution of ERK1/2 MAP ketected using an anti-rabbit secondary antibody conjugated to Alexa488. ERK1/2 bf ERK1/2 in response to aldosterone was blocked by pre-incubation with the EGFR-ldosterone treatment is dependent on trans-activation of EGFR.

min of treating CCD cells with aldosterone [13]. The rapid PKDctivation coincides temporally with initial ERK1/2 activation inhese cells. The activation of both PKD and ERK1/2 by aldosteronere dependent on the trans-activation and phosphorylation of theGF receptor (EGFR) by c-Src tyrosine kinase [13,21,22]. In wild-ype CCD cells aldosterone stimulates ERK1/2 activation whichemains detectable 2 h after treatment. In CCD cells suppressed inKD1 expression, aldosterone stimulates only a transient activationf ERK1/2 [42]. Aldosterone promotes the growth of sub-confluentCD cells but not of the PKD1 suppressed cells (Fig. 1), consequentlyhe behaviour of aldosterone as a renal growth factor is at leastartially dependent on PKD1 activation.

. Aldosterone-induced signalling and nephropathy

Abnormal cell growth is a factor in the progression of pathogenicenal conditions such as chronic kidney disease, polycystic kid-ey disease and diabetes associated nephropathy. The growthromoting effects of aldosterone on renal tubule cells has beenroposed as a factor contributing to the rate of progression ofhese conditions. However, the pathological effects of circulatingldosterone can only be one component of a multi-factorial pro-ess which displays chronic progression leading to renal damage.rogressive tissue deterioration results from cell growth dysreg-lation and extracellular matrix deposition leading to fibrosisnd inflammation. Animal experiments and human clinical trialsave demonstrated an additive effect of aldosterone antagonismith RAAS blockade in attenuating renal damage. MR antago-

ism in combination with angiotensin-converting enzyme (ACE)

nhibitors attenuates the progression of diabetic and non-diabeticephropathies [23]. A diabetic rat model revealed that the MRntagonist eplerenone in combination with the ACE inhibitor,nalapril enhanced glomerular filtration and suppressed glomeru-

was determined by immuno-fluorescence using an ERK1/2-specific antibody andlocalized to the nucleus following aldosterone treatment. The nuclear localization

c inhibitor tyrphostin AG1478 (1 �M). The nuclear localization of ERK1/2 following

losclerosis with a concurrent reduction in TGF-�1, Collagen type-IVand plasminogen activator factor-1 expression [24]. Aldosteronealso stimulates NADPH oxidase-dependent production of reactiveoxygen species in renal cells that accelerates fibrosis that can beattenuated using eplerenone [25]. The NF�B transcriptional path-way is also activated in response to aldosterone in renal principalcollecting duct cells however this was mediated by SGK activ-ity rather than the stimulation of MAP kinases. The transcriptionof pro-inflammatory cytokine genes such as IL-1� and IL-6 areup-regulated by NF�B [26]. The activation of NF�B-dependent tran-scription in this experimental system was MR-dependent and wasantagonized by concurrent activation of the glucocorticoid recep-tor. Aldosterone treatment of mesangial cells results in a rapidphosphorylation of myosin phosphatase target subunit-1, a sub-strate for Rho kinase within 20 min of hormone treatment andalso results in an increase in actin polymerization [27]. The stateof hypertrophy stimulated by aldosterone in these cells correlateswith the subsequent up-regulation in the expression of colla-gen types I, III and IV and of �-smooth muscle actin, a markerfor myofibroblastic transformation. These changes, stimulated byaldosterone could be blocked by eplerenone and the Rho kinaseinhibitor Y27632 demonstrating that this cascade plays a criticalrole in the differentiation effects of aldosterone.

5. Aldosterone, EGFR and polycystic kidney disease

The aberrant stimulation of cell proliferation in the epitheliumof the distal nephron is one factor in the development of poly-

cystic kidney disease [28]. Susceptible individuals have a geneticpredisposition to developing autosomal recessive polycystic kid-ney disease (ARPKD) and autosomal dominant polycystic kidneydisease (ADPKD) through mutations in the genes encoding thefibrocystin or polycystin 1 and 2 genes, respectively [29,30]. The

W. Thomas et al. / Steroids

Fig. 3. Aldosterone and growth dysregulation in renal cortical collecting duct cells.Chronic renal injury occurs as a result of multiple synergistic processes. Aldosteroneaction can be one of these processes. The interaction between aldosterone and themineralocorticoid receptor (MR) promotes the nuclear localization of MR where itinteracts with other transcription factors (TFs) to modulate gene expression. Sig-nalling events coupled to MR include the Src-dependent trans-activation of EGFRleading to activation of the ERK1/2 and PKD1 signalling cascades. PKD1 activationstabilizes the activation of ERK1/2, while ERK1/2 phosphorylates MR and other tran-scription factors to modulate their activity and contribute to cell growth responsesincluding proliferation. Serum and glucocorticoid regulated kinase-1 (SGK) is oneopc

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[15] Yim HE, Yoo KH, Bae IS, Jang GY, Hong YS, Lee JW. Aldosterone regulates cel-

f the earliest detected proteins to be up-regulated by aldosterone. SGK activationromotes NF�B-dependent transcription leading to the release of pro-inflammatoryytokines IL-1� and IL-6 to promote renal fibrosis.

roducts of these genes are multi-functional structural membraneroteins that have roles in cell polarization, tight junction integrity,

on transport and signal transduction. Chronic hypertension is anxacerbating factor in the progression of polycystic kidney disease31] and controlling hypertension through the administration of

R antagonists is one approach to therapeutic intervention [32].enal cyst formation and enlargement is a consequence of epithelialroliferation [33] and ion transport dysregulation. The forma-ion of cysts in ARPKD is mainly localized to the MR-expressingpithelium of the distal nephron [33] and this points to a roleor aldosterone in promoting aberrant proliferation in the dis-al nephron. Silencing of the ARPKD-associated fibrocystin genen HEK293 cells produced a hyper-proliferative response to EGFnd resulted in over-activation of the ERK1/2 cascade followingGF treatment [34]. Over-expression of Heparin-binding EGF isetected in ARPKD and EGF antagonistic antibodies suppress theitogenic activity of cleared cystic fluid [35]. The abundance of

GFR in cells from the renal tubule is responsive to aldosterone-ependent transcription [36] and expression of EGFR is necessaryo permit the stimulation of rapid signalling responses by aldos-erone in CHO cells [37]. The activation of ERK1/2 signalling byldosterone in cells derived from the distal nephron has beenescribed by several groups including our own [38,39] and this

esponse has been linked to trans-activation of EGFR [40]. Theuclear localization of ERK1/2 occurs subsequent to its activationnd is consistent with its role as a regulator of transcription factors.he nuclear translocation of ERK1/2 in response to the aldosterone

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treatment of CCD cells was blocked by EGFR inhibition (Fig. 2).Antagonism of the RAAS to control hypertension as a means of slow-ing cyst formation in ADPKD is the subject of an on-going clinicalinvestigation [41].

6. Conclusion

Aldosterone is emerging as an important factor in regulatingthe growth and differentiation of renal cells both in vivo andin vitro. Aldosterone has an indirect effect on the pathology ofrenal diseases such as renal fibrosis and polycystic kidney dis-ease through the detrimental effects of systemic hypertension.Aldosterone also has direct effects on renal pathology through therapid activation of protein kinase signalling cascades in cells of therenal epithelium to stimulate cell proliferation and hypertrophy(Fig. 3). Pharmacological intervention in the activation of thesealdosterone-induced signalling cascades, particularly those cou-pled to EGFR trans-activation may provide novel avenues for thetreatment of chronic kidney diseases in combination with existingtherapies.

Funding support

Science Foundation of Ireland Research Frontiers ProgrammeAward (WT). Higher Education Authority, Programme for Researchin Third Level Institutions Cycle 4 (BJH).

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