Local Control of Aldosterone Production and Primary Aldosteronism.

TEM 1106 No. of Pages 9

[1_TD$IF]Focus on: The Endocrine Pancreas

Opinion

Local Control of Aldosterone Production and Primary Aldosteronism Enzo Lalli,1,2,3,* Jacques Barhanin,4,5 Maria-Christina Zennaro,6,7,8 and Richard Warth9 Primary aldosteronism (PA) is caused by excessive production of aldosterone by the adrenal cortex and is determined by a benign aldosterone-producing adenoma (APA) in a significant proportion of cases. Local mechanisms, as opposed to circulatory ones, that control aldosterone production in the adrenal cortex are particularly relevant in the physiopathological setting and in the pathogenesis of PA. A breakthrough in our understanding of the pathogenetic mechanisms in APA has been the identification of somatic mutations in genes controlling membrane potential and intracellular calcium concentrations. However, recent data show that the processes of nodule formation and aldosterone hypersecretion can be dissociated in pathological adrenals and suggest a model envisaging different molecular events for the pathogenesis of APA. Clinical Relevance of Primary Aldosteronism Primary aldosteronism (PA) is now recognized as the most common form of endocrine hypertension, typically suspected in the presence of hypertension, hypokalemia[1_TD$IF] and low plasma renin levels. The prevalence of PA is estimated at 6–10% in the hypertensive population [1]. Different adrenal diseases are responsible for PA: (i) aldosterone-producing adenoma (APA or Conn's adenoma, 50% of cases) (see Glossary); (ii) idiopathic hyperaldosteronism or bilateral adrenal hyperplasia (BAH, 30–40%); (iii) unilateral primary adrenal hyperplasia (documented unilateral aldosterone secretion without detectable adenoma, 5–10%); and (iv) aldosterone-producing adrenal carcinoma in rare cases. Once the diagnosis of PA has been made, it is important to identify its etiology to distinguish between surgically correctable forms such as APA and unilateral primary adrenal hyperplasia from forms that can be treated pharmacologically such as BAH. This distinction is often difficult to make because of the clinical, hormonal, and pathological continuum that exists between low renin hypertension and the different forms of PA. Diagnostic elements include computed tomography (CT) scan, dynamic testing of aldosterone response to various stimuli, and, most importantly, adrenal venous sampling (reviewed in [2]). PA is currently considered the most common curable form of hypertension [3]. Early detection of PA is important and has an enormous impact on clinical outcome and survival, given the severe adverse effects of aldosterone excess independent of high blood pressure levels. Patients with PA have been reported to exhibit more severe left ventricular hypertrophy and diastolic dysfunction than patients with essential hypertension and a high prevalence of myocardial infarction, stroke, and atrial fibrillation [4,5]. However, surgical treatment of unilateral forms of PA does not cure hypertension in all patients [6]. Even if published guidelines [7] have been instrumental in

Trends in Endocrinology & Metabolism, Month Year, Vol. xx, No. yy

Trends Primary aldosteronism (PA) is the most common form of endocrine hypertension. Patients with PA exhibit more severe cardiovascular damage than patients with essential hypertension. The most common cause of PA is a benign aldosterone-producing adenoma (APA) of the adrenal cortex. Local mechanisms controlling aldosterone production in the adrenal cortex are particularly relevant in the pathogenesis of PA. In APA, somatic mutations are present that induce increased cytosolic calcium activity and consequent activation of aldosterone production. Recent data show that nodule formation and aldosterone hypersecretion can be dissociated in pathological adrenals and suggest a two-hit model for APA formation. Remarkably, somatic mutations in the same genes that are mutated in APA are also found in cell clusters producing aldosterone in normal adrenal.

1 Institut de Pharmacologie Moléculaire et Cellulaire CNRS, 06560 Valbonne, France 2 NEOGENEX CNRS International Associated Laboratory, 06560 [7_TD$IF]Valbonne,[2_TD$IF] France 3 Université de Nice – Sophia Antipolis, 06560 Sophia Antipolis, France 4 Laboratoire de PhysioMédecine Moléculaire CNRS-UNS UMR 7370, 06108 Nice Cedex 2, France

http://dx.doi.org/10.1016/j.tem.2016.01.003 © 2016 Elsevier Ltd. All rights reserved.

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standardizing screening procedures and treatment of PA, important open questions still remain related to its diagnosis, subtype differentiation[1_TD$IF] and management of forms that cannot be treated with surgery. For these reasons, the diagnosis of PA may be missed in a certain number of cases and consequently its treatment delayed, with negative impact on cardiovascular and metabolic complications. Moreover, progress in the understanding of the pathogenic mechanisms underlying PA is essential to allow for the development of new diagnostic tools and biomarkers[1_TD$IF] and for the identification of new therapeutic targets, concerning up to 10% of the hypertensive population.

Local Regulation and Pathophysiology of Aldosterone Secretion by the Adrenal Cortex In the physiological setting, aldosterone secretion in the adrenal zona glomerulosa (ZG) is under the control of the renin–angiotensin system (RAS) and potassium, which promote cell depolarization and activate calcium influx through voltage-gated calcium channels, as well as calcium release from intracellular stores (reviewed in [8]). Activation of calcium signaling is the central element in the regulation of aldosterone biosynthesis as it stimulates, among others, the expression of CYP11B2, the gene coding for aldosterone synthase, the rate-limiting enzyme in aldosterone biosynthesis. By contrast, increasing evidence exists highlighting the importance of local mechanisms, as opposed to circulatory ones, that control aldosterone production in the adrenal cortex and that are particularly relevant in the pathogenesis of PA. Readers are referred to an excellent recent review for extensive coverage of the cell types, anatomical structures[1_TD$IF] and released factors involved in autocrine and paracrine regulation of aldosterone release in normal and pathological conditions [9]. In this opinion article, we evaluate rapidly accumulating and evolving data in the field[1_TD$IF] in the light of a new model for the pathogenesis of APA[1_TD$IF] that integrates novel genomic and functional findings. Adrenal RAS The regulation of the RAS within the adrenal differs from the regulation of cardiovascular RAS. Renin is mainly expressed in ZG cells in rat adrenal glands and its secretion is stimulated by angiotensin II (Ang II) and K+[6_TD$IF] [10]. Nephrectomy stimulates the intra-adrenal RAS, which is probably the cause of the increase in plasma aldosterone measured in this condition [11]. Renin is also expressed in human adrenal glands and in the H295R adrenocortical carcinoma cell line [12]. In mice, adrenal renin expression peaks at embryonic day 16 (E16) during development and is strongly downregulated after birth [13], except in mutant mice lacking the Cyp11b2 gene, where a robust expression of adrenal renin can be detected even in adults [14]. Remarkably, the local adrenal RAS is abnormally activated in newborn Kcnk9 null mice, lacking the potassium channel Task3, which have very high aldosterone plasma levels (see later; [15]). These data suggest that dysregulation of the local RAS in the adrenal may play a role in the pathogenesis of some cases of hyperaldosteronism. Mast Cells, Receptors, and Their Potential Role in APA Perivascular mast cells, which are localized in the subcapsular region of the human adrenal cortex, stimulate aldosterone production through local release of serotonin and activation of the 5-hydroxytryptamine receptor 4 (5-HT4) expressed by ZG cells [16]. Furthermore,[9_TD$IF] some APA overexpress 5-HT4 receptors [17], which were shown to be functional and able to induce an exaggerated aldosterone response when stimulated by a specific agonist [18]. Recent studies have also shown that a subset of APA contains a high density of infiltrating mast cells, which is correlated with CYP11B2 expression and in vivo aldosterone secretory parameters [19]. Genes associated with mast cell function are upregulated in APA. It has then been proposed that local release of serotonin and other mast cell-derived factors may influence aldosterone secretion in APA [19]. In addition to serotonin receptors, other G-protein-coupled receptors are either overexpressed or aberrantly expressed in APA, whose stimulation by circulating or paracrine ligands may affect aldosterone production (reviewed in [20]).

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5 Laboratories of Excellence, Ion Channel Science and Therapeutics, 06107 Nice, France 6 INSERM, UMRS 970, Paris Cardiovascular Research Center, Paris, France 7 Université Paris Descartes, Sorbonne Paris Cité, Paris, France 8 Assistance Publique-Hôpitaux de Paris, Hôpital Européen Georges Pompidou, Service de Génétique, 75015 Paris, France 9 Medical Cell Biology – University of Regensburg, 93053 Regensburg, Germany

*Correspondence: [email protected] (E. Lalli).


Transcription Factors The nuclear receptor transcription factors of the NR4A subfamily NURR1 and NGFIB are expressed in the adrenal and were shown to be able to directly regulate the expression of the CYP11B2 gene [21]. The other nuclear receptor that is a major regulator of adrenocortical development and function is SF-1 (reviewed in [22]). While SF-1 functions as a transcriptional activator of most genes encoding for proteins involved in steroid biosynthesis, it can also act as a repressor: it downregulates basal CYP11B2 promoter activity as well as its activation by Ang II and by NURR1 in adrenocortical H295R cells, thus negatively regulating aldosterone production [23]. Signaling Pathways The following signaling pathways are essential for adrenal development and are also constitutively activated in the adult adrenal, [10_TD$IF]and implicated in the pathogenesis of APA. The Wnt/b-catenin pathway has a key role in the regulation of adrenocortical growth and differentiation (reviewed in [24]). In the adrenal cortex, activated b-catenin, as detected by its nuclear and cytoplasmic localization, is localized selectively in the ZG both in mice and in humans [25,26]. Somatic mutations in exon 3 of the b-catenin gene (CTNNB1), encoding a serine/ threonine-rich domain, which is phosphorylated by the casein kinase 1 (CK1) and GSK-3b kinases[1_TD$IF] and which leads to targeted degradation of b-catenin, are frequently found in tumors. Mutations in this domain make b-catenin less sensitive to degradation with consequent accumulation in the cytoplasm and in the nucleus, where the protein can interact with transcription factors and regulate gene expression. Recent data indicate that CTNNB1 mutations are found at low frequency in APA [26,27], while b-catenin activation, as detected by immunohistochemistry, was reported in approximately two-thirds of APA cases and also in the peritumoral adrenal cortex [26]. Interestingly, a recent study reported the finding of somatic CTNNB1 mutations in APA in two pregnant and one postmenopausal women who expressed the aberrant receptors luteinizing hormone–choriogonadotropin receptor (LHCGR) and gonadotropin-releasing hormone receptor (GNRHR) in their tumors [28]. Studies using a transgenic mouse model expressing an activated Ctnnb1 allele in the adrenal cortex showed that these animals expressed ectopic Cyp11b2 in the inner zone of the cortex and were hyperaldosteronemic [29]. This phenotype was linked to a direct regulation by b-catenin of the expression of transcription factors regulating Cyp11b2 expression and was postulated to represent a specific effect of the Wnt/b-catenin pathway in the activation of ZG cell differentiation, with a direct impact in the pathogenesis of APA [30]. However, CTNNB1 mutations and activation of the Wnt/b-catenin pathway are also found in other benign and malignant adrenocortical neoplasms that do not produce aldosterone [31–33], making it unlikely that b-catenin activation by itself can have an impact on both cell differentiation and aldosterone production in APA. The sonic hedgehog (Shh) signaling pathway also has a major role in adrenal development. In the mouse adrenal cortex, Shh produced by cells located in the outer cortex/ZG signals to mesenchymal cells located predominantly in the overlying capsule expressing the Shh receptor Patched and the Gli1 transcription factor and recruits them to the steroidogenic lineage [34]. Shh signaling is required for adrenocortical growth, but not for cortical zonation or steroidogenic cell differentiation. Lineage analysis in transgenic mice revealed that both cells that produce Shh and those that are responsive to it have properties of adrenocortical stem and/or progenitor cells [34]. While in normal human adrenal Shh expression is barely detectable in a few cells situated in the outer part of the ZG, it is present in the entire area of APA and is dramatically increased in the hyperplastic peritumoral ZG [26]. Transcriptome profiling also revealed differential expression of genes involved in the Shh signaling pathway in a subgroup of APA [26]. These results suggest that upregulated Shh signaling, which in normal conditions is characteristic of stem/progenitor cells, may participate to the processes of tissue remodeling and nodule formation in APA.

Glossary Aldosterone: a steroid hormone produced in the adrenal zona glomerulosa that has a major role in the regulation of plasma volume and ion homeostasis. It activates the mineralocorticoid receptor that regulates target gene expression. Aldosterone-producing adenoma (APA): a benign tumor of the adrenal cortex that produces aldosterone. Aldosterone-producing cell clusters (APCCs): they are groups of cells in the normal adrenal cortex that are present in the zona glomerulosa and extend centripetally in the zona fasciculata and that express aldosterone synthase. Potassium channels: ion channels with selectivity for potassium. They play a pivotal role in regulating membrane potential and intracellular calcium concentration in adrenocortical cells. Protein kinase A (PKA) pathway: cAMP-regulated protein signaling pathway that is essential in regulating growth and steroid hormone production in the adrenal cortex. Renin–angiotensin system (RAS): the proteolytic enzyme renin cleaves the plasma precursor angiotensinogen to angiotensin I, from which angiotensin II (Ang II) is made through subsequent cleavage by angiotensin-converting enzyme (ACE). Ang II has a vasoconstrictor effect and increases aldosterone production from the adrenal cortex. Sonic hedgehog (Shh) signaling pathway: a conserved developmental pathway with an important role in regulating cell differentiation in the adrenal cortex. Wnt/b-catenin pathway: another conserved signaling pathway that has a major role in development and in tissue homeostasis and renewal. It involves stabilization and nuclear translocation of the membrane protein b-catenin, which is translocated into the cell nucleus and regulates gene expression.


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A Tale of Channels Potassium channels have a major role in the regulation of aldosterone secretion by adrenal ZG cells (reviewed in [8]). The resting membrane potential of human ZG cells depends on a number of K+ channels, particularly of the K2P (two-pore domain) family, which are expressed at high levels in the adrenal cortex. In particular, TASK channels have been implicated in regulating the resting membrane potential of adrenal ZG cells and are inhibited by Ang II, with consequent cell depolarization [35]. Genetic inactivation of Kcnk3 (Task1) and/or Kcnk9 (Task3) in the mouse induces hyperaldosteronism of variable severity and phenotypic manifestation [36–39]. Remarkably, Dkk3, which encodes an unusual member of a family of inhibitors of Wnt/b-catenin signaling, acts as a modifier gene of the hyperaldosteronemic phenotype of Kcnk3 null mice [40]. Hyperaldosteronism, which is restricted to the female sex in those animals, is in fact extended to males in Kcnk3–/–[1_TD$IF] mice bred in the Dkk3 null background [40]. Interestingly, in humans a single nucleotide polymorphism (SNP) close to the KCNK3 gene was found to be associated with systolic and mean arterial pressure levels [41], while another genome-wide association study found a correlation between SNPs in the KCNK9 gene with aldosterone levels and hypertension risk [42]. A recent study also showed association between KCNK3 and mean arterial pressure in individuals of East Asian, European[1_TD$IF] and South Asian ancestry [43]. A seminal finding shedding new light on the pathophysiological mechanisms underpinning PA has been the identification of somatic gain-of-function mutations in the KCNJ5 gene, encoding the potassium channel GIRK4, in approximately 40% of APA (frequencies up to 80% have been observed in the Japanese population) and of germline mutations in the same gene in rare forms of familial PA [44–47]. These mutations alter channel selectivity for K+ and produce increased Na+ conductance and cell depolarization. Recent genomic studies have also identified in APA somatic mutations in the genes encoding the ATPases ATP1A1 and ATP2B3 and the L-type calcium channel coding gene CACNA1D [48–50]. Early-onset hyperaldosteronism has also been associated with germline mutations in CACNA1H, coding for the T-type calcium channel Cav3.2 in a new familial form of PA [51]. Altogether, these data indicate that molecular alterations found in APA converge towards a common pathway inducing increased cytosolic calcium activity, upregulation of CYP11B2 expression and consequent activation of aldosterone production. However, a key question remains open about the pathogenic mechanisms of APA: while in the initial study it was postulated that increased calcium entry elicited by GIRK4 gain-offunction mutants also activates proliferation of adrenocortical cells in addition to aldosterone secretion [44], further studies showed that expression of KCNJ5 mutants in human adrenocortical cells decreased, rather than increased, cell proliferation [52] or even led to rapid sodiumdependent cell lethality [53]. These findings suggest that the processes of aldosterone hypersecretion and adenoma formation that characterize APA are distinct entities.

Hypersecretion versus Nodulation: The Two Facets of APA In the peritumoral tissue of APA, important remodeling of the adrenal cortex has been observed with increased nodulation and decreased vascularization that were not correlated with CYP11B2 expression [54]. A recent study examining pathological multinodular adrenals showed that KCNJ5, ATP1A1, and CACNA1D mutations were present in only one of the nodules in each adrenal, which was systematically the one displaying CYP11B2 overexpression [55]. Remarkably, another study showed that somatic mutations in different known genes may be present in different aldosterone-producing nodules belonging to the same pathological multinodular adrenal gland, indicating that those mutations are independent genetic events [56]. These data show that the processes of nodule formation and aldosterone hypersecretion can be dissociated in pathological adrenals, suggesting a two-hit model for APA formation (Figure 1A, Key Figure). One hit, consisting of somatic mutation of one of the known genes in approximately 60% of cases and of other unknown genetic or epigenetic alterations in the remaining patients would cause autonomous aldosterone hypersecretion. The second hit would lead to alterations in the

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Key Figure

Mechanisms of Local Control of Aldosterone Production (A)

Two-hit model for APA pathogenesis

Aldosterone hyperproducon

Nodule formaon

Mutaons in: KCNJ5 CACNA1D ATP1A1 ATP2B3 ...

Acvaon of: Wnt/β-catenin Shh PKA? ...

APA

Adrenal medulla

Adrenal cortex

Ang ll / K + – regulated aldosterone producon

(B)

Constuve aldosterone producon

Foci

APCC

ZG

ZF

CACNA1D ATP1A1 Somac mutaons

Figure 1. (A) A two-hit model for the pathogenesis of APA. Somatic mutations in KCNJ5, CACNA1D, ATP1A1, ATP2B3, and possibly other genetic alterations produce cell depolarization, increased cytoplasmic calcium activity[1_TD$IF] and increased CYP11B2 expression, leading to aldosterone hyperproduction. By contrast, aberrant activation of candidate signaling pathways (Wnt/b-catenin, Shh, PKA[1_TD$IF] etc.) causes imbalances between cell proliferation and death in the adrenal, ultimately leading to adenoma formation. (B) Aldosterone is produced in distinct cell types in the human adrenal cortex. In normal adrenal, CYP11B2-positive cells are present as small clusters (foci) exclusively in the ZG or as larger clusters composed of an outer layer of morphological ZG cells and inner, columnar zona [5_TD$IF]fasciculata (ZF)-like cells (APCCs). Somatic mutations in the ATP1A1 and CACNA1D genes, that are also found mutated in APA, have been described in APCCs [66]. This would render aldosterone production autonomous in APCCs, while ZG foci are sensitive to regulation of their aldosterone output by Ang II and potassium. Abbreviations: Ang II, angiotensin II; APA, aldosterone-producing adenoma; APCCs, aldosteroneproducing cell clusters; PKA, protein kinase A; Shh, sonic hedgehog; ZG, zona glomerulosa.

normal balance between adrenocortical cell proliferation and apoptosis triggering nodule formation. Considering its association with adrenal tumors, activation of the Wnt/b-catenin pathway [24,26] is a good candidate to represent, at least in some cases, the second hit required for adrenal nodule formation. Another candidate pathway to be explored is the protein kinase A (PKA) pathway that has a paramount importance in the regulation of adrenocortical growth and hormone secretion. Somatic gain-of-function mutations in the gene encoding the alpha catalytic subunit of PKA (PRKACA) have been recently found in cortisol-producing adrenocortical adenomas [33,57–60], but the presence of genetic alterations in genes involved in the PKA pathway in APA is currently unknown. However, it is interesting that potentially damaging germline variants in ARMC5 – a gene found mutated in macronodular adrenal hyperplasia


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and whose connection with the PKA pathway has been suggested [61] – have also been described in patients with PA [62]. The sequence of events by which these hypothetical two hits required for APA formation occur is not known, but a very recent study suggests that autonomous aldosterone hypersecretion may, at least in some cases, precede tumor formation (see later).

Autonomous versus Regulated Aldosterone Production in Normal Adrenals Morphological studies of aldosterone production in the human adrenal gland have been made possible by the long-awaited production of antibodies that specifically recognize the very similar enzymes aldosterone synthase (encoded by CYP11B2), which is expressed by the cells that synthesize aldosterone, and steroid 11-beta-hydroxylase (encoded by CYP11B1), which is expressed by the cells that synthesize cortisol [63,64]. By the use of those reagents, as well as by in situ hybridization with a CYP11B2-specific RNA probe [26], a peculiar distribution of CYP11B2-expressing cells in the human adrenal cortex was observed. Alongside with scattered groups of cells in the ZG, clusters of CYP11B2-positive cells (aldosterone-producing cell clusters, APCCs) were present, composed of an outer layer of morphological ZG cells and inner, columnar ZF-like cells [26,63,64]. APCCs are also frequently found in adrenal tissue adjacent to APA, despite the low circulating renin/angiotensin levels in APA patients [26,63–65]. These findings suggest that aldosterone production is independent of the RAS in APCCs. With the purpose of identifying the basis of sustained CYP11B2 expression in APCCs, Nishimoto et al. [66] analyzed the presence of APA-related somatic mutations in APCCs by targeted nextgeneration sequencing. Those authors found that approximately one-third of APCCs from human normal adrenal glands from renal transplantation donors analyzed carried somatic mutations in CACNA1D and ATP1A1 that were not present in the adjacent normal tissue. However, surprisingly, mutations in KCNJ5 were not detected in those APCCs. The frequency of somatic mutations in CACNA1D in APCCs from normal adrenals in Japanese autopsy subjects increases with age [67]. Transcriptome profiles showed that, as expected, APCCs show higher expression of CYP11B2 compared with the adjacent ZG cells. Conversely, genes involved in the Wnt/b-catenin pathway, including the newly identified adrenal b-catenin target gene AFF3 [68] and LGR5, the receptor for R-spondins that enhances Wnt signal strength [69], are more abundantly expressed in ZG cells other than APCCs [66]. Based on those results, we can speculate that in the human adrenal cortex two different pools of cells can produce aldosterone: in ZG cell clusters aldosterone is synthesized in a manner regulated by the RAS and potassium, while in APCC aldosterone secretion is constitutive and may ensure basal production of the hormone even in conditions of salt loading (Figure 1B). Conversely, the Wnt/b-catenin pathway is activated preferentially in ZG cells that do not produce aldosterone and functions to sustain adrenocortical progenitor cell function. In APA, activation of the Wnt/b-catenin pathway may contribute to neoplastic nodule formation, while somatic mutations of genes connected to APA (possibly originating in the APCC population) allow for autonomous aldosterone production. Previous studies on large cohorts of individuals without PA have shown that an inability to appropriately stimulate or suppress aldosterone in response to physiological stimuli is associated with cardiometabolic disease. Using the sodium-modulated aldosterone suppression– stimulation index (SASSI) as a measure of physiological aldosterone responses to dietary sodium manipulation, Vaidya et al. found an association of cardiometabolic risk factors with either impaired suppression of aldosterone on a liberal salt diet or impaired stimulation on a restricted salt diet, or both, even in the ‘normal’ range of aldosterone response [70,71]. On one hand, these data may be interpreted in the sense that failure to fully suppress aldosterone on one side may be related to the degree of autonomous aldosterone secretion from APCCs, while on the other hand APCCs may suppress normal physiological ZG foci of aldosterone secretion such that stimulation of aldosterone is concurrently affected. This type of dysregulation of aldosterone has clinical relevance since it has been shown to robustly predict impaired renal function and future cardiovascular risk [71].

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Concluding Remarks and Future Perspectives

Outstanding Questions

The recent important advances in our understanding of the local mechanisms that regulate aldosterone production in the adrenal cortex have revealed a fascinating diversity of factors whose dysfunction can significantly contribute to the pathogenesis of PA. New molecular findings have changed the classification of PA. Morphological phenotypes (APA versus BAH versus normal morphology on histopathological examination) are now accompanied by the definition of the genetic phenotype (glucocorticoid-remediable aldosteronism, KCNJ5, CACNA1D, ATPase mutations, or no mutations). Under this perspective, it will be very important for future work to elucidate the relative contribution of the different somatic mutations in genes involved in PA both to nodule formation and aldosterone hypersecretion with the help of new animal models presenting gain- or loss-of-function of the relevant genes that are homologous to those involved in human pathology (see Outstanding Questions). However, this task is complicated by the lack of expression of Kcnj5, the gene homologous to human KCNJ5, in the mouse adrenal gland [72]. It will also be important to ascertain whether mutant ion channels found in APA may be specifically targeted by drugs, as preliminary studies indicate that this may be the case for GIRK4 mutants [73], to be able to offer personalized therapy to patients with APA for whom surgery represents a risk. Another field that will undoubtly receive priority attention in the near future is the precise mapping of the occurrence and histological localization of somatic mutations in genes linked to APA in the normal adrenal cortex and their relationship with clinical forms of PA.

What are the mechanisms by which adenoma formation in the adrenal and increased aldosterone production are connected? What genetic and/or epigenetic alterations drive APA development in cases lacking known genetic alterations? Are APCCs in normal adrenal gland potential precursors of APA? In this regard, it is puzzling that currently no KCNJ5 mutations (the most common mutations found in APA) have been identified in APCCs. Is pharmacological therapy of APA bearing mutant ion channels feasible?

Acknowledgments We thank Franck Aguila for artwork. This work was supported by the BeyondTASKs (ANR11-BSV1-005) and LOCALDO (ANR15-CE14-0017) grants from the French Agence Nationale de la Recherche (to E.L., J.B., M-C.Z., R.W.), LabEx Ion Channel Science and Therapeutics (ANR11-LABX-0015-01) (to J.B.), GEA (ANR-13-ISV1-0006-01)[1_TD$IF] and the Fondation pour la Recherche Médicale (number DEQ20140329556) (to M-C.Z.).

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9

Regulation of aldosterone secretion in primary aldosteronism.

Role of ACTH and Other Hormones in the Regulation of Aldosterone Production in Primary Aldosteronism.

Aldosterone-induced oxidative stress: a potential mechanism of aldosterone autonomy in primary aldosteronism.

Aldosterone-induced oxidative stress: a potential mechanism of aldosterone autonomy in primary aldosteronism.

Dopaminergic regulation of aldosterone secretion: assessment in different subtypes of primary aldosteronism and in essential hypertension.

MS aldosterone and automated direct renin in screening for primary aldosteronism.

[Secretory regulation and episodic secretion of aldosterone in patients with primary aldosteronism (author's transl)].

Association between urine aldosterone and diastolic function in patients with primary aldosteronism and essential hypertension.

Minireview: potassium channels and aldosterone dysregulation: is primary aldosteronism a potassium channelopathy?

Plasma aldosterone response to ACTH in primary aldosteronism and in patients with low renin hypertension.

Immunohistochemistry of aldosterone synthase leads the way to the pathogenesis of primary aldosteronism.

Importance of contralateral aldosterone suppression during adrenal vein sampling in the subtype evaluation of primary aldosteronism.

The role of urinary aldosterone for the diagnosis of primary aldosteronism.

Primary aldosteronism and salt.

Primary aldosteronism presenting with an atypical aldosterone-renin ratio in the acute phase of cerebral hemorrhage.

Role of endogenous ACTH on circadian aldosterone rhythm in patients with primary aldosteronism.

ACTH-dependent aldosterone excess due to adrenocortical adenoma: a variant of primary aldosteronism.

Pituitary peptides other than ACTH may not be aldosterone secretagogue in primary aldosteronism.

Genetics of primary aldosteronism.

Progress in primary aldosteronism. Mineralocorticoid antagonist treatment for aldosterone-producing adenoma.

Genetics of primary aldosteronism.

Aldosterone-to-renin ratio acts as the predictor distinguishing the primary aldosteronism from chronic kidney disease.

Twenty-four-hour urinary aldosterone predicts inappropriate left ventricular mass index in patients with primary aldosteronism.

Quantitative Value of Aldosterone-Renin Ratio for Detection of Aldosterone-Producing Adenoma: The Aldosterone-Renin Ratio for Primary Aldosteronism (AQUARR) Study.