Abstract
The identification of several germline and somatic ion channel mutations in aldosterone-producing adenomas (APAs) and detection of cell clusters that can be responsible for excess aldosterone production, as well as the isolation of autoantibodies activating the angiotensin II type 1 receptor, have rapidly advanced the understanding of the biology of primary aldosteronism (PA), particularly that of APA. Hence, the main purpose of this review is to discuss how discoveries of the last decade could affect histopathology analysis and clinical practice. The structural remodeling through development and aging of the human adrenal cortex, particularly of the zona glomerulosa, and the complex regulation of aldosterone, with emphasis on the concepts of zonation and channelopathies, will be addressed. Finally, the diagnostic workup for PA and its subtyping to optimize treatment are reviewed.
Essential Points
In this review we examine the biology of the normal zona glomerulosa through development and with aging and then analyze the functional role of the major regulators of aldosterone secretion
We discuss the theories that explain the transition of the normal zona glomerulosa into a multinodular disorder, eventually leading to formation of an aldosterone-producing adenoma (APA), with particular focus on the activation of signaling pathways such as wingless-related integration site/β-catenin and somatic mutations of ion channels leading to zona glomerulosa cell depolarization and aldosterone production
Clinical aspects, such as epidemiology and diagnosis of primary aldosteronism (PA), are briefly reviewed, and the potential role of circulating and tissue biomarkers of APA in subtyping PA is discussed
Aldosterone, the main mineralocorticoid hormone, is vital for maintaining body fluid and electrolyte homeostasis, vascular resistance, and, thereby, blood pressure under conditions of salt/water (volume) depletion. However, aldosterone levels are inappropriately high in 50% to 80% of all hypertensive disorders, not only primary and secondary aldosteronism but also overweight and obesity (1, 2) and the most severe (stage II to III) or drug-resistant forms of hypertension (3). This indicates that aldosterone produced in excess with respect to sodium status is a main determinant of high blood pressure, but it is clear from clinical studies from around the world that inappropriately high aldosterone concentrations produce prominent target organ damage, thus contributing to an ominous prognosis (4–8).
Primary aldosteronism (PA) occurs in 5% to 20% of hypertensive patients (9, 10) but is often overlooked because patients are not screened for it. PA can mimic primary (essential) hypertension, and is still perceived as an exceptionally rare condition necessitating a complex diagnostic workup, given the lack of known mechanisms and therefore of specific biomarkers. In fact, since its discovery in 1954 the term primary has been used to emphasize our ignorance of its causes.
In the last decade, multiple seminal discoveries have made PA the paradigm of improved mechanistic knowledge in hypertension. Multiple animal models of hyperaldosteronism, including the knockout of TWIK acid–sensitive potassium (TASK) channels types 1 and 3, replicate the PA phenotype in mice (11). Aldosterone-producing adenomas (APAs) are responsible for about half of PA. Study of APAs has been instrumental in recent advances in our understanding of the regulation of normal and pathological aldosterone synthesis. Whole transcriptome analysis has demonstrated consistent underexpression of the TASK-2 channels in APAs, and in vitro molecular blunting of TASK-2 enhances aldosterone production (12). This TASK-2 underexpression could be attributed to elevated miRNA 23 and 34 levels in ~25% of the APAs and to functional genetic variants in the TASK-2 promoter in another quarter of the cases (12), leaving the mechanism for nearly half of the cases unexplained to date.
Elevated serum parathyroid hormone levels have been noted in patients with PA, and type 1 parathyroid hormone receptor has been found in APAs (13). The demonstration that parathyroid hormone stimulates aldosterone secretion and potentiates the effect of angiotensin II (Ang II) and K+ (14, 15) highlighted hitherto unknown interactions between the parathyroid gland and the adrenocortical zona glomerulosa. Agonistic autoantibodies for the type 1 angiotensin receptor (16) were also found to increase aldosterone production (17, 18).
In 2011 the results of exome sequencing of 24 APAs led Choi et al. (19) to uncover mutations in the selectivity filter of the Kir3.4 (KCNJ5) K+ channel. Subsequent studies demonstrated that KCNJ5 mutations were present in approximately one-third of APAs in a large European cohort (20) and between 18% and 70% in a much larger meta-analysis of studies carried out worldwide (21). Germline KCNJ5 mutations were also found in rare familial forms of PA featuring drug-resistant hypertension and massive bilateral adrenal hyperplasia necessitating bilateral adrenalectomy (19). This finding was soon followed by the discovery of mutations in other genes affecting ion channel function, including that of ATP1A1, ATP2B3, CACNA1D, CACNA1H, CTNNB1 (β-catenin), and CLCN2 chloride channel (22–25), thus providing compelling evidence that channelopathies and malfunctioning signaling pathways are involved in the pathophysiology of many cases of PA (26, 27).
Aldosterone plays a key role in causing not only arterial hypertension but also heart failure, where it contributes substantively to cardiovascular and renal damage and events (28–32). However, our knowledge of the fundamental biology of the normal zona glomerulosa remains limited. The purpose of this review is to update information about the complex regulation of aldosterone, with a particular emphasis on the clinical and pathological implications of discoveries in the last decade.
The Adrenal Cortex: Historical Perspective
The earliest detailed description of the paired human suprarenal or adrenal glands in European literature is ascribed to Bartolomeo Eustachi (Eustachius) in 1563. His detailed drawings of the glands were hidden during the Inquisition and published 150 years later by Giovanni Maria Lancisi as part of the Tabulae Anatomicae (Anatomical Engravings) de Bartolomeo Eustachii (33, 34). Description of the structure of the adrenal gland progressed little for the next three centuries except for the recognition that it comprised a distinct medulla and cortex (Cuvier in 1805) and description of the zonation of the cortex by Gottschau et al. in 1883 (33). By the late 1930s, most of the glucocorticoids produced by the adrenal cortex were isolated and their structures defined, but all bioassays developed to characterize adrenal cortical extracts failed to isolate the fraction containing mineralocorticoid activity (35), generating a 20-year debate over whether biologically relevant mineralocorticoids existed, with the majority of researchers contending that glucocorticoids were the major source of mineralocorticoid activity (35). It was only in 1953 that Simpson and Tait in London, in collaboration with Reichstein from the University of Basel and the then Ciba Pharmaceutica Company, isolated an active mineralocorticoid from an amorphous fraction of beef adrenal extracts (35), which they initially named electrocortin for its activity and, later, aldosterone, once it was recognized that the molecule exists as a tautomer between an aldehyde at position 18 and forms a hemiacetal with the 11β hydroxyl (36). Within months, groups in America and Switzerland confirmed these findings (35). In 1954, Lityńsky (37) in Poland and Jerome Conn, an endocrinologist at the University of Michigan, independently reported the first two clinical cases of APA causing the syndrome of primary hyperaldosteronism, featuring hypertension, hypokalemia due to abnormal potassium excretion, and hypomagnesemia, which were cured by adrenalectomy (35).
Zonation of the Adrenal Cortex
Anatomical and histological features
The adrenal gland may be roughly triangular or crescent-shaped to conform to the contour of the dorsal pole of the kidneys, as in humans, or spherical and held slightly apart from the kidney by fat and fibrous tissue, as in rats and mice. From the capsule encasing the adrenal gland, moving centripetally toward the medulla, the cortex comprises the zona glomerulosa, made of densely packed cells with comparatively little cytoplasm forming glomeruli (or balls); the zona fasciculata, composed of cells with copious cytoplasm, containing numerous lipid droplets arranged in fascicles or columns; and, in some species, notably humans, the zona reticularis, next to the medulla and composed of cells slightly smaller than those of the zona fasciculata, arranged to form a net (38, 39). The distinct morphology, arrangement, and steroid production of the adrenocortical cells within each zone are phylogenetically conserved (40, 41).
Steroidogenesis and steroidogenic enzymes
Cloning of the genes for the enzymes in the steroidogenic cascades for the synthesis of the adrenal steroids confirmed the arduous enzymology tour de force necessary to isolate the multiple enzymes and their steroid substrates specific to each zona of the adrenal cortex. Enzymes for the conversion of cholesterol to deoxycorticosterone (DOC), cytochrome P450 side-chain cleavage (CYP11A1), 3β-hydroxysteroid dehydrogenase-2 (HSD3B2), and 21-hydroxylase (CYP21A2) are expressed in the zona glomerulosa and zona fasciculata. The human has two highly homologous HSD3B enzymes; HSD3B2 is expressed primarily in the adrenal, and hydroxy-delta-5-steroid dehydrogenase (HSD3B1) is expressed in extra-adrenal tissues. The mouse has six different homologous isoenzymes. A recent study of mice with the clock genes cry1 and cry2 deleted showed that development of hyperaldosteronism correlates with increased expression of the hsd3b6 isozyme (corresponding to HSD3B1 in the human) in the zona glomerulosa (42). One recent study indicated that HSD3B1 was also expressed in the zona glomerulosa, whereas HSD3B2 was expressed in the zonae fasciculata and glomerulosa (43). However, another study using highly specific antibodies against both isozymes demonstrated that HSD3B1 was expressed at either very low or negligible levels in the zona glomerulosa and confirmed that HSD3B2 was highly expressed in the zonae glomerulosa and fasciculata (44).
Aldosterone synthase (CYP11B2), which converts DOC to aldosterone, is limited to the zona glomerulosa; 11β-hydroxylase (CYP11B1), which converts DOC to corticosterone, is in the zona fasciculata (Fig. 1). In species, including the human, that express 17α-hydroxylase/17,20-lyase (CYP17A1), necessary for the synthesis of 17α-hydroxypregnenolone and 17α-hydroxyprogesterone, CYP11B1 converts 11-deoxycortisol to cortisol in the zona fasciculata. The 17α-20-lyase activity of CYP17A1 produces androstenedione in the zona fasciculata and dehydroepiandrosterone (DHEA) in the zona reticularis, where HSD3B is low. By volume, the primary corticosteroids in the human are cortisol, DHEA, and aldosterone synthesized in adrenal zonae fasciculata, reticularis, and glomerulosa, respectively. In species, including mouse and rat, that do not express an adrenal 17-hydroxylase, corticosterone is the primary glucocorticoid, and no androgens are produced in the smaller innermost zona fasciculata cells next to the medulla. Thus, these species do not have a true zona reticularis, although their zona fasciculata cells may have a reticular or netlike appearance close to the medulla. The mouse fetal X-zone, which expresses 20α-hydroxysteroid dehydrogenase that catabolizes progesterone, regresses in the male at about the time it is weaned but persists in the female until first pregnancy (45, 46), and it should not be confused with a zona reticularis.
Figure 1.
Steroidogenic pathways in the human adrenal cortex. Enzymes localized in the mitochondria are in yellow boxes. [© 2018 Illustration Presentation ENDOCRINE SOCIETY]
Once antibodies were made that distinguished between highly homologous CYP11B1 and CYP11B2 enzymes of the rat and, relatively recently, the human, it became clear that a narrow zona intermedia between the zona glomerulosa and zona fasciculata, in which cells express neither CYP11B2 nor CYP11B1, is evident in the rat but is not clearly defined in humans (47–50). Whether this is due in part to high chronic sodium (Na+) intake of humans or is a species difference is not clear. Though morphologically similar, many zona glomerulosa cells of the adult human do not express substantial amounts of CYP11B2 protein. Some animals, notably cattle, pigs, and sheep, express 17α-hydroxylase (CYP17) in the zona fasciculata and zona reticularis but have a single cytochrome P450–11β hydroxylase, CYP11B1, in both the zona fasciculata and zona glomerulosa. In these species, the CYP11B1 acts as a sequential hydroxylase to synthesize only aldosterone in the zona glomerulosa, a function provided by CYP11B2 in the human and rat (51).
“A narrow zona intermedia…is evident in the rat but is not clearly defined in humans.”
Ontogeny
The adrenal gland comprises tissues of two different embryological origins with very different functions. The adrenal medulla develops from neural crest cells, progenitors of chromaffin cells, and the adrenal cortex from the intermediate mesoderm (52). The adrenal cortex, ovary, and testis arise from a common progenitor, the adrenogonadal primordium, a specialized region of the celomic epithelium called the urogenital ridge (53, 54) (Fig. 2). The urogenital ridge is also the origin for the kidneys and the progenitors of the definitive hematopoietic cells (53, 55, 56).
Figure 2.
Ontogeny of the human adrenal and time during gestation at which steroidogenic genes are first expressed and steroids synthesized. CYB5R3, cytochrome b5 reductase; HSD3B, 3β-hydroxysteroid dehydrogenase; SULT1A, sulfotransferase. [© 2018 Illustration Presentation ENDOCRINE SOCIETY]
During the fourth to sixth week of gestation in the human, and around day 9.5 in the mouse, there is a thickening of the celomic epithelium in the urogenital ridge to form the adrenogonadal primordium. Shortly after at the eighth week in the human and 10.5 days in the mouse, the primordium divides into the dorsomedial portion to form the adrenal primordia and the ventrolateral portion for the gonadal primordia (57–60). At this stage, use of in situ hybridization demonstrates that the adrenal gland expresses the transcription factors, steroidogenic factor-1 (SF1; also called AD4BP or NR5A1) and dosage-sensitive sex reversal–adrenal hypoplasia congenital critical region on the X chromosome (DAX1; also known as NR0B1) (58). Expression of SF1 exceeds that of DAX1 at all embryonic stages studied (61). The cells from the neural crest invade the adrenal primordium around weeks 8 to 9 of human gestation or day 13 in the mouse, before the adrenal primordium is surrounded by mesenchymal cells to form the capsule (62). The human and primate fetal adrenal cortex exhibits two structurally defined areas, an inner “fetal zone” comprising large polyhedral eosinophilic cells, which begins to regress during the fifth month of gestation and disappears totally 1 year after birth, and a peripheral zone adjacent to the capsule comprised of small basophilic cells that persists after birth and is therefore called the definitive zone (58, 63). By the end of gestation, the human fetal adrenal is nearly the size of the kidney, ~80% of which is fetal zone (64). The fetal zone has large steroidogenic cells that express the CYP17A1 enzyme, which has two activities—17α-hydroxylase and 17,20-lyase activities—that convert pregnenolone into 17α-hydroxypregnenolone and then DHEA. A substantial portion of the latter is sulfated to DHEA sulfate (DHEA-S), which in the placenta is converted to estrogens necessary for the maintenance of a normal pregnancy. The smaller definitive zone that becomes obvious at around the eighth week of gestation between the capsule and the fetal zone is composed of densely packed basophilic cells expressing SF1. Definitive zone cells are lipid-poor during midgestation and appear to be the reservoir of typical proliferative cells, many of which are mitotically active. Midkine, a heparin-binding growth factor, selectively stimulates proliferation of the cells from the definitive zone of the human fetal adrenal cortex (65).
As gestation advances, these cells form fingerlike columns of cells close to the outer rim of the fetal zone. Before week 24, a third zone, named the transitional zone, appears between the definitive zone and the fetal zone in humans (66–68); this transitional zone is not as evident in mice. During the last trimester the transitional zone, which eventually becomes the zona fasciculata of the adult human adrenal cortex, expands and begins to produce cortisol under the regulation of ACTH (63). By the 30th week of gestation, the fetal zone becomes rudimentary, and the definitive and transitional zones become the zona glomerulosa and zona fasciculata, respectively.
CYP11B1 expression is apparent by embryonic day 16 (E16) in the rat adrenal gland and remains nearly constant until postpartum day 1. CYP11B2 expression is also observed at this time in single cells or small cell clusters distributed throughout the adrenal (69). CYP11B2-positive cells scattered throughout the cortex decrease at E17, and by E18 to E18.5 there is an increase in the number of CYP11B2 immunoreactive cells adjacent to the capsule, with only a few remaining in the area stained by the CYP11B1 antibody. At E19 there is a clear distinction between outer and inner cortex, with a small region in between where neither enzyme is expressed in the rat embryological adrenal cortex. The latter has been called the undifferentiated zone, where progenitor stem cells are located (47, 69, 70). Measurable aldosterone and corticosterone production by rat fetal adrenals begins at about E17 (69). In the human immunoreactivity for steroidogenic enzymes including steroidogenic acute regulatory (StAR), CYP11A1, CYP21A2, P450 oxidoreductase, and cytochrome b5 were detected in the fetal and transitional zones between 14 and 22 weeks’ gestation. The HSD3B enzyme necessary for the synthesis of progesterone from pregnenolone was not detected before the 22nd week of gestation and was detected after the 23rd week only in the transitional and definitive zones (71). CYP17A1 and DHEA sulfotransferase were detected in the transitional and definitive zones throughout gestation.
Immunohistochemical and steroid measurements show that the human adrenal produces DHEA in the transitional and fetal zone, but not in the definitive zone, as early as 14 weeks’ gestation (68). Cortisol is produced in the transitional zone after the 23rd week of gestation (71). Mast cells and their secretory products appear to play a role in the maturation of zona glomerulosa cells and their synthesis of aldosterone (72). Studies performed between the 16th and 41st weeks of gestation with quantitative reverse transcription PCR and immunohistochemistry demonstrated that by the 18th week tryptase-positive cells, typically mast cells, are present in the subcapsular area of the adrenal close to cells expressing mRNA for the HSD3B and the CYP11B2 enzymes, which appear by 18 and 23 weeks, respectively (72). Tryptophan hydroxylase and serotonin receptor type 4 expression occurred in the developing cortex by week 18, became limited to the definitive zone by week 28, and peaked between weeks 30 and 33, coinciding with the surge in CYP11B2 expression (72). This finding suggests a paracrine role for mast cell–derived serotonin in aldosterone secretion in the fetus, as has been demonstrated in the adult zona glomerulosa (72).
The development of adrenal cortex zonation has also been addressed via cell fate mapping and gene deletion studies using a variety of techniques, including specific zona glomerulosa Cre expression (39, 73, 74). Two models were classically proposed to explain postnatal adrenal zonation: the zonal model of lineage development and centripetal migration of cells from the subcapsular region. According to the zonal model of lineage development, each zone develops and is maintained independently by progenitor cells embedded in the zone (75). In the centripetal model, undifferentiated progenitor cells in the capsule or subscapular region differentiate into zona glomerulosa cells capable of producing aldosterone when stimulated. These cells migrate centripetally and undergo lineage conversion to cortisol- or corticosterone-producing cells, depending on the species, and eventually undergo apoptosis in the innermost part of the cortex, next to the adrenal medulla (53). Lineage tracing using Shh and Gli1-expressing progenitor cells to map the fate of cells in the undifferentiated zone demonstrated radial stripes of cells that seemed to migrate from the zona glomerulosa into the zona fasciculata, thus supporting the centripetal migration model (76). Cell fate mapping and gene deletion studies using zona glomerulosa–specific Cre expression were used to test this model and localize progenitors of zona glomerulosa and fasciculata cells (39, 73). In a Cyp11b2-Cre mouse model, Cyp11b2-expressing cells were permanently marked with green fluorescent protein (GFP). Lineage tracing showed that the entire zona glomerulosa and the majority of the zona fasciculata were labeled within 3 months, indicating that all or most cortical cells descend from the Cyp11b2+ cell lineage during normal tissue replacement (73). Costaining with Sf1 and GFP also shows an extensive overlap between the GFP-labeled population and the cells of the steroidogenic cortex (73). Mice undergoing Cyp11b2-Cre mediated Sf1 deletion, which can no longer give rise to lineage-marked zona fasciculata cells, still have an entirely normal zona fasciculata. This finding suggests that Cyp11b2-Cre–mediated Sf1 deletion occurs late enough to allow some progenitor cells to escape deletion of the Sf1 to differentiate into fasciculata cells (39, 73). Signaling molecules involved in adrenal cell renewal and zonation are expressed from E12.5 onward in mesenchymal cells surrounding the developing adrenal. Members of the R-spondin gene family, Rspo1 and Rspo3, signaling molecules that bind leucine-rich repeat–containing receptors and modulate the β-catenin pathway, are maintained throughout development and adulthood (74). β-catenin is highly expressed in the zona glomerulosa, and Rspo3 is expressed throughout the capsule. Its role in adrenal development is supported by the finding that tamoxifen-induced deletion of Rspo3 at E11.5 resulted in smaller adrenals (74). Two different pools of β-catenin exist: a cytoskeletal pool, which controls interaction with adherens junctions, and a cytosolic pool, which participates in canonical wingless-related integration site (Wnt) signaling and acts as a coactivator of the TCF/LEF transcription factors (54). Activated β-catenin is expressed in the adrenogonadal primordium, adrenal primordium, and subcapsular cells. Deficiency of Wnt4 alters adrenal function with a decrease in zona glomerulosa function and very low aldosterone levels (77). Constitutive activation of β-catenin in the mouse zona fasciculata causes increased Cyp11b2 expression in this zone with hyperaldosteronism and development of adrenal tumors (78, 79); furthermore, activated β-catenin is expressed in APAs and adrenal carcinomas (80, 81).
Transplantation of rat zona glomerulosa and zona fasciculata cells isolated by Percoll gradient indicate that zona glomerulosa cells completely restore production of aldosterone and corticosterone 30 days after adrenalectomy and transplantation, whereas transplanted zona fasciculata cells only restore corticosterone production (82). This finding suggests that zona glomerulosa cells comprise progenitor cells that can give rise to cells with the function of either zone, whereas zona fasciculata cells are able only to proliferate and maintain their differentiated function, again lending support to the centripetal model. Results of cell fate mapping and gene deletion studies (39, 73) suggest one of two possibilities. The most likely is that a single cell line developed from the adrenogonadal primordium to generate all zones of the adrenal cortex. Temporal expression of transcriptional factors produces the development of the fetal, definitive, and transitional zones from these cells, and some of the progenitor cells of the zona glomerulosa that differentiate into zona fasciculata cells retain the ability to regenerate zona fasciculata cells but cannot redifferentiate into zona glomerulosa cells. However, cell fate mapping techniques and gene deletion studies (39, 73, 74) do not exclude the possibility of development of a second cell line very early in differentiation that generates only zona fasciculata cells, because zona glomerulosa deletion of SF1 prevents lineage conversion of zona glomerulosa into zona fasciculata, but the zona fasciculata develops normally from the other potential cell line. An unusual clinical case supports this second developmental hypothesis: superselective adrenal vein sampling in a patient with bilateral hyperaldosteronism demonstrated an area of the adrenal with much lower production of aldosterone that was spared during subtotal bilateral adrenalectomy. Study of the excised tissue revealed that the both adrenals harbored the same somatic KCNJ5 mutation (G151R) throughout the cortex, with one area of much lower representation of the mutation. This finding suggested that the mutation occurred early in the development of the zona glomerulosa cell line (which followed the lineal conversion to zona fasciculata) and that a second cell line lacking the mutation was also present (83). This intriguing hypothesis clearly deserves further research.
The normal human neonate has a transient physiological renal resistance to aldosterone and higher levels of plasma aldosterone than its mother (84, 85), associated with a low renal mineralocorticoid receptor expression (86). Low aldosterone secretion in very preterm infants compounds the problem of low receptor expression in the kidney (87). Aldosterone secretion rates per body surface area are higher in younger infants than in older infants, who are similar to adults (88, 89).
The fundamental enzyme for aldosterone synthesis is aldosterone synthase, or the CYP11B2 enzyme. Its expression identifies cells that synthesize aldosterone, but its expression is regulated by aldosterone secretagogues, so not all zona glomerulosa cells express CYP11B2. Immunohistochemistry of the adrenal of a very young child shows that cells expressing CYP11B2 form a continuous band within the zona glomerulosa. With increasing age, expression of CYP11B2 becomes less continuous, so in the adult human CYP11B2-expressing cells are scattered throughout the subcapsular cortex among typical zona glomerulosa cells that do not express the enzyme. These CYP11B2-expressing cells in some cases may be difficult to find (5, 49, 90, 91) except as clusters that extend from the subcapsular area and penetrate into the outer zona fasciculata. The terms aldosterone-producing cell clusters (APCCs) (90), aldosterone-producing foci, and mega foci (81, 92), or just aldosterone-producing foci (5, 93), have been coined to identify these clusters; we will use APCCs herein. The number of APCCs increases with age (5). Renin activity decreases in older adults, with no concomitant decline in plasma or urinary aldosterone (5), suggesting an age-related autonomous aldosteronism that might be related to the higher number and abnormal aldosterone physiology of APCCs.
Functional Regulation of Aldosterone Production
Under physiological conditions aldosterone secretion is regulated mainly by Ang II and K+, and to a lesser extent by other secretagogues such as ACTH, endothelin 1 (ET-1), estrogens, and urotensin II. Aldosterone production can be upregulated acutely (within minutes) through increased expression and phosphorylation of the StAR protein or chronically (over hours to days) by augmented expression of the steroidogenic enzymes, mostly CYP11B2.
Ang II
Ang II is a potent activator of Ca2+ influx, which regulates all biosynthetic steps leading to aldosterone secretion (94). Upon binding to the angiotensin II type 1 (AT1) receptor subtype in cells of zona glomerulosa, Ang II activates phosphoinositide-specific phospholipase C, which hydrolyzes phosphatidylinositol 4,5-bisphosphate, which generates the secondary messengers inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (95). IP3 triggers aldosterone secretion by eliciting a transient increase in cytosolic calcium (Ca2+) concentration and activating Ca2+/calmodulin-dependent protein kinases (CaMKs), whereas diacylglycerol stimulates protein kinase C, which regulates StAR levels via cAMP response element binding (CREB) and activator protein-1 (95). Thus, activation of the AT1 receptor stimulates both the early and late regulatory steps of aldosterone synthesis, which include phosphorylation of StAR and expression of the steroidogenic enzymes, respectively.
Ang II regulates both Ca2+ influx into the zona glomerulosa cell via voltage-transient T- and long-lasting l-type Ca2+ channels and intracytoplasmic Ca2+ movement, thus increasing cytosolic Ca2+ concentrations, leading to activation of CaMK II complexes (96, 97). CaMK II complexes mediate the transcription of nuclear receptor–related 1 protein (also known as NR4A2), activating transcription factors 1 and 2, and CREB, which initiate transcription of CYP11B2. The major role for Ca2+ in the regulation of aldosterone biosynthesis is unambiguously supported by the blunted Ang II–induced aldosterone secretion after inhibition of Ca2+ influx with Ca2+ channel antagonists.
Chronic Ang II stimulation maintains aldosterone synthesis not only through increased CYP11B2 transcription but also by increasing cholesterol uptake by glomerulosa cells from high-, low- and very low-density lipoproteins and increasing StAR protein, necessary for cholesterol transport into the mitochondria (98, 99). Activation of the renin-angiotensin-aldosterone system by low Na+ intake induces expression of CYP11B2 without affecting CYP11B1, probably because of the greater expression of AT1 receptors in zona glomerulosa than in zona fasciculata cells (100, 101). Chronic (7- to 14-day) Ang II administration in rats was also found to increase expression of Ang II receptors subtypes 1A and 1B, an effect that was blocked by the AT1 receptor antagonist losartan, suggesting that during chronic stimulation Ang II increases aldosterone production also by upregulating its own receptors (99). As might be expected from its trophic effects in other cell types, chronic Ang II stimulation also increases zona glomerulosa cell size (102).
“The two-pore domain channels TASK-1 and TASK-2 are highly expressed in the normal human adrenal cortex and generate background K+ currents.”
Potassium
Extracellular K+ is a potent regulator of aldosterone synthesis. K+ channels, mostly the leak K+ channels TASK-1, TASK-2, and TASK-3 (coded by Kcnk3, KcnK5, and Kcnk9 genes), the TWIK-related potassium channel 1, and the G protein inward rectifying potassium channel Kir3.4 (KCNJ5), maintain adrenocortical cells hyperpolarized under resting conditions. The two-pore domain channels TASK-1 and TASK-2 are highly expressed in the normal human adrenal cortex and generate background K+ currents, blunting aldosterone production. Inhibition of TASK channels by Ang II results in depolarization, and mice with deletions of TASK-1, TASK-3, or both genes have hyperaldosteronism (103–107). Increases in extracellular K+ depolarize the plasma membrane and activate the voltage-dependent calcium channels, leading to Ca2+ influx and the signaling mechanisms described above. Inhibition of Ca2+ influx abolishes K+-stimulated aldosterone secretion. A role of K+ in zona glomerulosa cell growth is also suggested by the increased thickness of zona glomerulosa found in rats with a chronically high K+ intake.
ACTH
ACTH binds to the transmembrane receptor melanocortin receptor 2 in the cells of zona glomerulosa and zona fasciculata and exerts its downstream effects by activating adenylate cyclase, thus generating cAMP, which in turn activates protein kinase A (PKA). PKA phosphorylates and activates StAR to increase cholesterol delivery to the inner mitochondrial membrane and activates members of the CREB family transcription factors. ACTH also promotes Ca2+ influx through PKA-mediated phosphorylation of l-type calcium channels, further enhancing aldosterone secretion.
Acute ACTH administration stimulates aldosterone secretion transiently, followed by decreased aldosterone synthesis within few days. The escape from ACTH stimulation is restricted to aldosterone, because cortisol synthesis continues during chronic ACTH stimulation. Putative mechanisms for the loss of aldosterone response to ACTH include melanocortin receptor 2 desensitization and internalization through a PKA-dependent mechanism; increased 17α-hydroxylase (P450c17) and 11β-hydroxylase (P450c11) activity, resulting in enhanced production of DOC and precursor steroids; blunting of the late (CYP11B2-mediated) step of aldosterone synthesis; transformation of zona glomerulosa cells into zona fasciculata–like cells featuring round mitochondria with ovoid cristae; suppression of the renin-angiotensin-aldosterone system; desensitization of zona glomerulosa cells to Ang II via downregulation of the AT1 receptor; and a rapid increase in zona fasciculata thickness alongside a decrease in zona glomerulosa width [for a review see Gallo-Paynet (108)]. Moreover, ACTH stimulation leads to an increase in 18-oxocortisol formation. The activities of CYP11B2 and CYP17 are necessary for the synthesis of 18-oxocortisol, either by coexpression in the same cells or by a paracrine mechanism in which cortisol, secreted by cells expressing CYP11B1 and CYP17, is taken up by cells expressing CYP11B2 (109–111). The underlying mechanism is not clear because CYP11B2 and CYP17 are not expressed in the same cells in the normal adrenal but are coexpressed in many adenomas. A paracrine mechanism is also doubtful because blood flow is centripetal in the adrenal, moving substrate from the zona fasciculata away from CYP11B2, and adrenal cells express a high level of the p-glycoprotein that pumps out polar steroids such as cortisol (112).
ET-1
ET-1 is a paracrine hormone that activates ETA and ETB receptors mediating contraction, cell proliferation, and hypertrophy in vascular smooth muscle, and ETB receptors, which stimulates prostacyclin and nitric oxide production in endothelial cells, causing vasorelaxation and inhibition of sodium transport in renal tubules. In freshly isolated human adrenocortical cells ET-1 acts as an aldosterone secretagogue through both ETA and ETB receptors, with a potency similar to Ang II on an equimolar basis, and synergizes with Ang II and ACTH as an aldosterone secretagogue on zona glomerulosa cells (113, 114). ET-1 acts via ETB in rat and via both ETA and ETB in human zona glomerulosa cells by downstream activation of phospholipase C and protein kinase C pathways, rising intracellular Ca2+, and extracellular signal-regulated kinase/ribosomal S6 kinase/CREB phosphorylation activation (113, 114).
ET-1(1-31), which is formed in the adrenal cortex by a chymase acting at the Tyr31-Gly32, acts through ETA receptors to stimulate aldosterone secretion. ET-1(1-31) is a weaker aldosterone secretagogue than ET-1(1-21) in dispersed human adrenocortical cells, but it has a much stronger effect on DNA synthesis and cell proliferation (113). Therefore, it has been suggested that in the human adrenal cortex, depending on local needs, the alternative cleavage of bigET-1 to ET-1(1-21) by endothelin-converting enzyme-1 , or to ET-1(1-31) by chymase, may lead to the production of either peptide, the former mainly stimulating steroidogenesis via both ETA and ETB receptors, the latter mainly exerting growth-promoting effects via ETA receptors (113). How this process is regulated deserves further research.
Serotonin
Serotonin released by perivascular mast cells in the subcapsular region of the adrenal cortex activates the 5-hydroxytryptamine receptor 4 (5-HT4) in zona glomerulosa cells, thus stimulating aldosterone production. Some APAs overexpress 5-HT4 receptors, and others have been described as having a high density of infiltrating mast cells (115–117), suggesting that overexpression of 5-HT4 receptors or overproduction of serotonin contributes to the aldosteronism in subsets of APA.
Urotensin II
The potent vasoactive peptide urotensin II is ubiquitously expressed in the vascular tissue and is involved in blood pressure regulation. It is also expressed in both the medulla and cortex of normal human adrenal glands. Pheochromocytomas express higher urotensin II levels, whereas APAs have lower levels than the normal adrenal gland. In contrast, the opposite was found for the urotensin II receptor, suggesting a urotensin II–mediated paracrine interaction between the cortex and the medulla (102). Infusion of urotensin II in normal rats for 1 week potently increased both CYP11B2 expression and plasma aldosterone levels (102) through activation of a specific urotensin receptor, and these effects were abrogated by the urotensin receptor antagonist palosuran (102). Because the mild increase in CYP11B1 in these adrenals, possibly due to stress, was unaffected by palosuran, the secretagogue effect of urotensin II on aldosterone seems to be specific and thus can be physiologically relevant. Of further note, rats chronically infused with urotensin II and exposed to different Na+ intakes were recently described to show an increase not only of aldosterone but also of renin release, suggesting that under conditions of salt and volume overload the peptide can mediate the inappropriately high secretion of both hormones (118).
17β-Estradiol
In human zona glomerulosa cells, estradiol (E2) can act as secretagogue or antisecretagogue, depending on the estrogen receptor subtype activated (119). In the normal human adult adrenal cortex and APA tissues, ERβ is far more abundantly expressed than ERα and its variant ERα36, whereas the G protein–coupled receptor-1 (GPER-1) is highly expressed in the normal human adrenocortical zona glomerulosa and faintly detectable in zonae fasciculata and reticularis (119). E2 tonically inhibits aldosterone synthesis via ERβ, as demonstrated by the increased aldosterone synthesis after ERβ blockade or silencing, whereas the secretagogue effect of E2 on aldosterone involves GPER-1 and protein kinase A and cAMP signaling (119) and is unmasked when ERβ is blocked. In APAs, GPER-1 is the predominant ER subtype, which suggests that it can be relevant for the pathophysiology of aldosteronism (119, 120). Given the broad use of selective estrogen receptor modulators, some of which act as GPER-1 modulators, in women with hormone-dependent forms of cancer, better knowledge of the effect of selective estrogen receptor modulator treatment on aldosterone secretion would be an important goal. However, this is an area where investigative efforts are clearly necessary.
Dopamine
Dopamine is thought to tonically inhibit aldosterone secretion because the selective D2 receptor antagonist metoclopramide induces aldosterone release in humans (121, 122). Dopamine blunts Ca2+ influx and protein kinase C phosphorylation (122), suggesting that its inhibitory effect involves the early steps of steroidogenesis. However, dopamine or dopamine agonists, such as bromocriptine, did not modify basal plasma aldosterone levels (121), indicating maximal tonic dopaminergic inhibition of endogenous aldosterone production.
Dopamine, Ang II, and salt intake interact in a complex way in regulating aldosterone secretion. Dopamine inhibits Ang II-stimulated aldosterone secretion in Na+-depleted subjects (123), but it is ineffective in preventing basal or ACTH- or Ang II-stimulated aldosterone production in Na+-repleted subjects (124, 125). Of the five known dopamine receptor subtypes, four (D1, D2, D4, and D5) are expressed in the normal human adrenal gland, suggesting their role in the modulation of steroidogenesis (126). Activation of the D4 subtype, the predominant D2-like receptor, tonically inhibits aldosterone secretion, whereas activation of D1 stimulates aldosterone secretion and thus counteracts D2-mediated inhibition (126). A microarray comparison of genes from normal human zona fasciculata and zona glomerulosa and from zona fasciculata–like and zona glomerulosa–like APAs led researchers to identify the expression of the neurofilament medium polypeptide (NEFM) in normal zona glomerulosa cells, small zona glomerulosa–like APAs, and the human adrenocortical cell H295R, but not in normal zona fasciculata and in large APAs, including those that express KCNJ5 mutations (127). Silencing of NEFM in H295R cells increased basal aldosterone production and cell proliferation, as well as aldosterone production in response to D1 agonist fenoldopam; moreover, expression of a mutant KCNJ5 gene in these cells decreased NEFM expression and raised basal aldosterone production, suggesting that NEFM is a negative modulator of aldosterone acting via the D1 receptor (127).
“A decrease in serum ionized Ca2+ levels is the main stimulus for PTH secretion by the chief cells of the parathyroid gland.”
Atrial natriuretic peptide
The blunting of aldosterone secretion was described as one of the multiple actions of the natriuretic peptide (128). In line with this effect, binding studies have revealed a single class of high-affinity saturable sites in H295R cell membranes, which were thereafter characterized as NPRA receptors coupled to the particulate guanylate cyclase (129). These receptors antagonize Ang II–stimulated aldosterone secretion in H295R cells and murine adrenals (129, 130) and also inhibit ACTH-stimulated aldosterone secretion via cyclic guanosine monophosphate–dependent activation of phosphodiesterase 2 and hydrolysis of cAMP in mice (131).
Parathyroid hormone
Although other factors, such as calcitriol, phosphate, magnesium, and the FGF23/klotho system, intervene in a complex manner, a decrease in serum ionized Ca2+ levels is the main stimulus for PTH secretion by the chief cells of the parathyroid gland. PTH is a single-chain 84–amino acid peptide sharing sequence homology with ACTH (108). Like ACTH, PTH also activates cellular adenylate cyclase/cAMP-dependent protein kinase, phospholipase C/protein kinase C–dependent, and cAMP-dependent signaling cascades. PTH also promotes cytosolic Ca2+ entry into the mitochondrial matrix, an essential step for triggering steroidogenesis, and particularly for its late step involving CYP11B2, an enzyme that is Ca2+-activated and cAMP-activated, as discussed in the next section.
A bidirectional link between aldosterone and PTH secretion in humans has been proposed based on the findings that PTH (and also the cancer-derived PTH-related peptide) stimulates aldosterone secretion in a concentration-dependent fashion; APA cells express the type 1 PTH receptor; parathyroid cells express the mineralocorticoid receptor; and patients with PA (132), and particularly those with the florid form of PA usually due to APA, have elevated serum PTH levels, which are corrected by adrenalectomy (13). Thus, the enhanced cardiovascular damage observed in both primary hyperparathyroidism and PA can derive from an interplay between PTH and aldosterone (133, 134).
Calcium ions
Ca2+ affects adrenocortical steroidogenesis in multiple ways: by increasing the activity of cholesterol ester hydrolase, the enzyme that de-esterifies cholesterol, allowing its release from cytoplasmic storage and use in aldosterone synthesis; by promoting cytoskeletal delivery of cholesterol to the outer mitochondrial membrane and the transcription and translation of StAR, which increases transfer to the inner membrane; by augmenting the mitochondrial oxidative metabolism and Ca2+-dependent formation of reduced nicotinamide adenine dinucleotide phosphate, a cofactor necessary for the activity of P450 cytochromes including CYP11A1 and CYP11B2; and by increasing the binding of Ca2+ to calmodulin-activated Ca2+/calmodulin-dependent kinases, which target steroidogenic factors, including CYP11B2, which catalyzes the conversion of 11-DOC to aldosterone.
Under resting conditions Ca2+ concentration is 10,000-fold lower [about 100 to 200 nM (135)] in the cytosol of zona glomerulosa cells than in the extracellular space (1 to 2 mM). Thus, upon depolarization-induced opening of voltage-gated Ca2+ channels there is a large concentration gradient for Ca2+ influx (136). Sustained Ca2+ entry into zona glomerulosa cells is possible because voltage-operated calcium channels have a permissive voltage window through which they conduct steady-state current (137).
Voltage-dependent activation and inactivation curves have been constructed for calcium channels in zona glomerulosa cells. Curves for activation predict the proportion of channels that open at each voltage, whereas those for inactivation predict the proportion of channels that are available to open that have not inactivated. When considered together, these relationships define a permissive voltage window in which there is steady-state opening of calcium channels, enabling a sustained flux of calcium into zona glomerulosa cells [Fig. 3(a)].
Figure 3.
Activation of wild-type and mutated channels. (a) Under physiological conditions the threshold of voltage-operated T-type calcium channels Cav3.2 (blue) is lower than that of l-type Cav1.3 channels (green), leading to activation (and deactivation) at lower voltages (permissive window of voltage), even though the voltage dependence [i.e., the fractional opening per unit increase in voltage (slope factor)] does not differ greatly between channels. The pink rectangle indicates the range of resting membrane potentials of normal glomerulosa cells. The y-axis indicates the amplitude of currents, expressed as normalized steady-state current. (b) Both somatic and germline KCNJ5 mutations cause loss of ion selectivity with ensuing increased Na+ influx and therefore a shift of the resting potential toward less polarized voltages (striped rectangle) that, in turn, causes opening of the voltage-dependent T-type Ca2+ channels [see Choi et al. (19)]. (c) Germline CLCN2 mutations, by causing efflux of chloride ion, also determine a shift of the resting potential toward less polarized voltages [see Scholl et al. (23) and Fernandes-Rosa et al. (24)]. (d) A similar shift of the resting potential toward less polarized voltages can occur in cells with mutations of ATP1A1 that cause loss of function or with mutations of ATP2B3 associated with impaired clearance of cytoplasmic Ca2+ ions [see Beuschlein et al. (138)]. (e) Ile770Met CACNA1D mutation (brown dotted line) causes a curve shift toward the left, leading to activation of the mutated channel at lower voltages than those needed to activate the wild-type Cav1.3 channel (green) [see Scholl et al. (139)]. (f) Mutations of CACNA, as germline CACNA1H M1549V mutation, cause increased constitutive Ca2+ influx at potentials close to the resting potential of zona glomerulosa cells (more rapid activation) and delayed inactivation, with a resulting larger permissive voltage window [see Scholl et al. (140)]. For details on cell type and species in which these curves were generated, see the original references. [© 2018 Illustration Presentation ENDOCRINE SOCIETY]
Two types of voltage-gated Ca2+ channels have been described in mammalian zona glomerulosa cells: low-voltage activated T-type (Cav3.2), and high-voltage activated l-type (Cav1.3), which differ in the voltage dependency of their opening and inactivation properties. Knowing their features and permissive window is key for understanding the impact of the ion channel mutations that have been identified in PA.
The low-threshold Cav3.2 channels activate and inactivate at lower voltages than high-threshold l-type Ca2+ channels (Cav1.3), which means that their permissive window occurs at lower voltages. Accordingly, they activate upon depolarization from resting hyperpolarized potentials, allowing a surge of Ca2+ influx at the beginning of an action potential (when the electrochemical gradient is highly favorable for cation entry). Because only a small fraction of channels open upon modest membrane depolarizations, the resultant current per unit time is small compared with the maximal current that can be elicited by a strong depolarization or a putative action potential when more channels open. However, because channel activation can be sustained for minutes (because of a lack of complete inactivation at hyperpolarized voltages), Ca2+ accumulation within the cell during this time period can still be large. Therefore, these channels are well suited for responding to the small membrane depolarizations from rest induced by physiological zona glomerulosa cell agonists and conveying Ca2+ into the cell at the right place for the control of steroidogenesis. This explains why physiological concentrations of Ang II and K+ may preferentially activate Cav3.2 and not Cav1.3 channels, to allow Ca2+ influx into the cytoplasm.
In contrast, the high-threshold Cav1.3 (l-type) Ca2+ channels are normally activated by much stronger cell depolarization from rest; thus, they probably play a minor role in the tonic regulation of aldosterone secretion. Accordingly, the dihydropyridine (l-type) calcium antagonists are very weak inhibitors of aldosterone secretion, both in vivo and in vitro, supporting the view that the two types of channels may serve different functions in the regulation of steroidogenesis.
Ca2+ cytosol concentration in the zona glomerulosa cells, as well as ACTH-induced cAMP signaling and PKA activation, are key for induction and activation of StAR (135). They are also crucial for activation of calmodulin-dependent kinases I and IV, which are crucial for StAR transcription (141).
Cytosolic Ca2+ concentration is also controlled by the mitochondria, which represent ~30% of zona glomerulosa cell cytoplasm volume. The high density of these organelles allows them to act as Ca2+ buffers: they accumulate Ca2+ during the rising phase of cytosolic Ca2+ transients and release Ca2+ during the decaying phase (136). Even small increases in mitochondrial Ca2+ levels have measurable effects, including reduction of pyridine nucleotide nicotinamide adenosine dinucleotide phosphate, enhanced steroidogenesis, particularly aldosterone biosynthesis, and generation of ATP essential for maintaining cell homeostasis (135). Reduction of nicotinamide adenine dinucleotide phosphate produced in the mitochondria is necessary for 20α and 22-hydroxylations and subsequent scission of cholesterol and also for 11β-hydroxylation of 11-DOC followed by 18-hydroxylation and 18-methyl oxidation of corticosterone, two crucial steps in aldosterone biosynthesis (135).
Biology of APA Formation
The multiple hypothetical mechanisms that may contribute to the development of APAs are depicted in Fig. 4. For the sake of clarity, these will be itemized (142).
Figure 4.
Mechanisms mostly deemed to contribute to the development of APAs. (a) Somatic mutations in the genes KCNJ5, ATP1A1, and CLCN2 coding for Kir3.4, Na+/K+ ATPase, and ClC-2 channels cause membrane depolarization of zona glomerulosa, with ensuing increased influx of Ca2+ into the cells, whereas mutations in CACNA and ATP2B3 genes, which code for Cav1.3 and plasma membrane calcium-transporting ATPase, directly cause an increase in intracellular Ca2+ levels. In both cases the final result is enhanced CYP11B2 expression and increased aldosterone production. For a list of mutations, see Table 1. (b) Wnts are secreted proteins that control growth and stem cell renewal, acting via canonical and noncanonical Wnt pathways. The canonical Wnt signaling is activated by the binding of Wnt to a serpentine Frizzled receptor (Fzd) and the coreceptor LRP5/6, which lead to recruitment of Dishevelled protein Dsh and disassembly of the β-catenin destruction complex. β-catenin, free to move toward the nucleus, binds to the TCF/LEF1 proteins, triggering transcription of genes involved in growth. When activated by the noncanonical Wnt pathway, Wnt first binds to Fzd and recruits Dsh to form a complex that activates the Rho-associated kinase pathway or phospholipase C, finally releasing Ca2+ from intracellular stores. The increased Ca2+ can drive cell growth or aldosterone synthesis. (c) Hypomethylation of the promoter region of CYP11B2 activates gene transcription, thereby promoting aldosterone secretion. (d) Elevated levels of miRNA 23 (miR23) and miRNA 34 (miR34) can downregulate KCNJ5 gene expression, blunting TASK2 synthesis, and finally enhancing aldosterone production. [© 2018 Illustration Presentation ENDOCRINE SOCIETY]
Mutations in ion channels
Somatic or germline mutations in the genes encoding ion channels and proteins that regulate the membrane potential of zona glomerulosa cells are common in APAs, suggesting that they play a key role in the biology of these tumors. Some mutations, such as KCNJ5 (19) and CACNA1D (139) and CCl2 (23, 24) lead to a gain of function, whereas others, such as ATP1A1 (138) and ATP2B3 (138), cause a loss of function. Overall, they are thought to promote CYP11B2 expression and aldosterone biosynthesis via an increase in intracellular Ca2+ levels, albeit through different mechanisms (143).
Somatic mutations
Very soon after PA-related KCNJ5 gene mutations were first described (19), several others were identified in APAs from patients throughout the world (20, 21, 144) (Table 1). The KCNJ5 gene encodes the G protein–activated inward rectifier potassium channel (GIRK4, also known as Kir 3.4) that, under normal conditions, allows the selective efflux of K+ through the channel pore, thereby maintaining zona glomerulosa cells hyperpolarized (19, 20). These mutations are located in exon 2 and cause amino acid changes near, or within, a highly conserved TXGYGFR motif of the GIRK4 selectivity filter, which result in loss of ion selectivity (19). The ensuing increased Na+ influx determines cell membrane depolarization, opening of voltage-dependent T-type Ca2+ channels [Fig. 3(b)], and activation of the Na+/Ca2+ exchanger in the reverse mode (e.g., Na+ out and Ca2+ in) (145), thus increasing cytosolic Ca2+ levels and ultimately triggering aldosterone synthesis.
Table 1.
Somatic and Germline Mutations Causing Excess Aldosterone Production
| Gene and Mutation | Channel and Protein | Transmission | Effect | Phenotype and Clinical Disorder |
|---|---|---|---|---|
| KCNJ5 | GIRK4 (also known as Kir 3.4) | Somatic | ↑ Na+ conductance | More frequent in women and younger patients; larger APA with zona fasciculata–like cells; higher plasma aldosterone levels and ARR at diagnosis |
| Cell membrane depolarization | ||||
| ↑ Ca2+ influx into the cell | ||||
| ATP1A1 | α1 Subunit of the Na+/K+ ATPase | Somatic | Cell membrane depolarization | More common in older men; smaller APA with zona glomerulosa–like cells; higher plasma aldosterone levels and lower K+ at diagnosis |
| ↑ Ca2+ influx into the cell | ||||
| CACNA1D | α1 Subunit of the Cav1.3 voltage-dependent l-type calcium | Somatic | ↑ Ca2+ influx activation | More common in older men; smaller APA with zona glomerulosa–like cells |
| ATP2B3 | Plasma membrane calcium-transporting ATPase 3 | Somatic | ↑ Ca2+ influx into the cell | More common in older men; smaller APA with zona glomerulosa–like cells; higher plasma aldosterone levels and lower K+ at diagnosis |
| CTNNB1 | β-Catenin | Somatic | Activation of canonical or noncanonical Wnt pathways | More common in women and older patients; larger APA |
| CYP11B2/CYP11B1 chimeric | CYP11B2 | Germline | CYP11B2 under control of CYP11B1 promoter | Early and severe hypertension; higher plasma levels of 18-hydroxycortisol and 18-oxocortisol; bilateral hyperplasia or adrenal nodules |
| FH-I or GRA | ||||
| Gene unknown in chromosome 7p22 | Unknown | Germline | ↑ Aldosterone production | No specific phenotype; diagnosis is made after exclusion of other known familiar forms |
| FH-II | ||||
| KCNJ5 | GIRK4 (also known as Kir 3.4) | Germline | ↑ Na+ conductance | Early and severe hypertension and hypokalemia; higher levels of aldosterone, 18-hydroxycortisol, and 18-oxocortisol; bilateral hyperplasia |
| Cell membrane depolarization | FH-III | |||
| ↑ Ca2+ influx into the cell | ||||
| CACNA1H | ↑ Ca2+ influx activation | Germline | ↑ CYP11B2 expression | Early onset of PA and hypertension, developmental delay, and attention deficit |
| FH-IV | ||||
| CLCN2 | ClC-2 | Germline | ↑ Cl− conductance | Detected in young patients |
| Cell membrane depolarization |
Abbreviations: ARR, aldosterone-to-renin ratio; FH, familial hyperaldosteronism; GRA, glucocorticoid-remediable aldosteronism.
Tumors with KCNJ5 mutations have been described to be larger, with a predominance of zona fasciculata–like cells, higher expression of CYP11B1, and lower expression of CYP11B2 than zona glomerulosa–like APAs that tend to be smaller (146–149). A differential gene expression profile between zona fasciculata–like and smaller zona glomerulosa–like APAs identified a consistent expression of NPNT in zona glomerulosa–like APAs and its lack in zona fasciculata–like APAs (150). NPNT is a Wnt target gene that encodes the extracellular matrix protein nephronectin (150). Accordingly, nephronectin was immunochemically demonstrated in zona glomerulosa–like APAs and normal human zona glomerulosa but not in zona fasciculata–like APAs and in the normal human zona fasciculata (150). Both nephronectin and NEFM, as discussed earlier as regards dopamine, are expressed in the normal zona glomerulosa but not in zona fasciculata cells (131, 153). This leads to two hypotheses to be tested. Might either or both factors be involved in adrenocortical cell proliferation and replenishment? Does their absence allow a zona fasciculata–like phenotype? Although it is not yet clear why KCNJ5 mutations result in loss of NEFM expression, it might at least partially explain the zona fasciculata–like phenotype of APA with KCNJ5 mutations (127).
The CACNA1D gene codes for the α1 subunit of the Cav1.3 voltage-dependent l-type calcium channel, which increases Ca2+ influx in response to depolarization (93, 139). The α1-subunit is composed of four repeated domains, each containing six trans-membrane segments, and forms the channel pore. Mutations in hotspot areas of CACNA1D cause a gain of function of CaV1.3 in zona glomerulosa cells due to opening of the Cav1.3 channel at membrane potentials more hyperpolarized than under physiological conditions, thus increasing Ca2+ influx and CYP11B2 expression (139) [Fig. 3(f)].
ATP1A1 and ATP2B3 are members of the P-type ATPase gene family, which encode the α1 subunit of the Na+/K+ ATPase and the plasma membrane calcium-transporting ATPase 3, respectively (93). At rest, ATP1A1 maintains the negative cell membrane potential by exchanging two extracellular K+ ions for three cytoplasmic Na+ ions with ATP consumption. Somatic mutations in ATP1A1 are found in ~5% of APAs. They involve the transmembrane helices M1 and M4 of the protein and cause loss of pump activity, reduced affinity for K+, and Na+ leak, leading to cell membrane depolarization [Fig. 3(d)] and increased Ca2+ influx and, thereby, increased aldosterone production (138).
Mutations of ATP2B3, found in 1.5% of APAs, involve the M4 transmembrane helix, which is responsible for Ca2+ transport and binding of ATP and phosphate (138). They distort the Ca2+ ion-binding motifs, thus increasing intracellular Ca2+ and priming aldosterone production.
Transfection of different mutant channels into adrenocortical cells resulted in increased transcription of CYP11B2, as well as NR4A2 and NR4A3 genes encoding transcription factors NURR1 and NOR1, respectively (138, 151). Somatic mutations in ATP1A1, ATP2B3, and CACNA1D, but not in KCNJ5 so far, have also been detected in APCCs from normal adrenal glands (152, 153) (Table 2).
Table 2.
Prevalence of Mutations in K+ or Ca2+ Channels in Either APCC or APA
| Mutation |
APCC
|
APA (155, 156) | |
|---|---|---|---|
| Without Hypertension (93, 154) | With Hypertension a (155, 156) | ||
| KCNJ5 | 0% | 0%–8% | 38%–63% |
| CACNA1D | 23%–26% | 0%–65% | 9% |
| ATP1A1 | 3%–9% | 0%–4% | 5% |
| ATP2B3 | 0%–5% | 0%–4% | 1% |
| CACNA1D + ATP2B3 | 0%–3% | 0%° | 0% |
Available data are from hypertensive patients with PA.
The effects of these mutations on cell proliferation remain unclear (157). The KCNJ5 T158A mutation was associated with massive bilateral hyperplasia, suggesting a growth-promoting effect (19). In contrast with this contention, studies performed in the laboratory of some of us (C.E.G.-S. and E.P.G.-S.) showed that cells transfected with this mutant GIRK4 exhibited a lower proliferation rate than cells transfected with wild-type GIRK4 or even apoptosis (151), indicating that other mechanisms are involved in abnormal cell growth.
Of note, despite an explosion of research, the factors leading to somatic mutations in APA are unknown. Because the rate of KCNJ5 mutations varies widely (21), ranging from 34% in Europe (20) to 65% to 70% in Asia and Japan (22, 161), whereas mutations in ATP1A1, ATP2B3, and CACNA1D seem to be uncommon in the Asian cohorts, some of us (G.P.R., T.M.S.) have suggested that the occurrence of these mutations depends on ethnic or environmental factors, including salt intake and screening policies implemented in the different countries (157). Moreover, there are APAs associated with hyperplasia or smaller aldosterone-producing nodules in the adjacent zona glomerulosa, and different somatic mutations have been found in the same adrenal gland (149, 158, 159). The hypothesis that one trigger may induce different mutations has been suggested, although no solid evidence for this contention has been provided to date.
It remains unknown how somatic mutations induce the transformation of normal adrenocortical cells into APA cells. One hypothesis is that somatic mutations induce formation of CYP11B2-expressing APCCs from mutated zona glomerulosa cells, leading to renin-independent aldosterone production and, eventually, to an APA. Lending support to this hypothesis are the findings by Nishimoto et al. (93) that APCCs comprise mainly small subcapsular zona glomerulosa–like cells surrounding large lipid-rich zona fasciculata–like cells and that the transcriptome profiles of APCCs are similar to that of zona glomerulosa cells with high expression of CYP11B2, and finally that some APCCs harbor cells with ATP1A1 and CACNA1D mutations. However, how zona glomerulosa cells are induced to form nests of cells (i.e., APCCs) remains to be established. Moreover, it is unknown why KCNJ5 mutations do not occur in APCCs. The hypothesis that APCCs with KCNJ5 mutations rapidly progress to APA, though conceivable (93), remains to be proven.
Germline mutations
Germline mutations cause <5% of PA cases and have been recently reviewed (20, 160–162), therefore, they will be examined only briefly herein (Table 1). A classification proposed recently by one of us (G.P.R.), which considers not only the mutations but also the clinical and imaging features and the response to drug treatment, will be followed (27).
Familial hyperaldosteronism type I (glucocorticoid-remediable aldosteronism)
Familial hyperaldosteronism (FH) type I is an autosomal dominant disease is caused by an unequal crossing-over of the homologous genes coding for CYP11B2 and 11β-hydroxylase (CYP11B1). This results in a chimeric gene comprising the CYP11B2 coding sequences under control of the CYP11B1 promoter and thus controlled by ACTH (160). This explains why in FH-I the clinical and biochemical signs of PA can be antagonized by suppressing ACTH with low-dose dexamethasone (163). Measurements of 18-hydroxycortisol and 18-oxocortisol levels suggest the diagnosis, but it is confirmed with long PCR, a more widely available technique (164).
FH type II
Clinically heterogeneous, FH-II comprises non–glucocorticoid-remediable forms of familial PA inherited with an autosomal dominant pattern (165–167). Although an association of a locus on the region 7p22 has been found in some FH-II families (168, 169), the genetic basis remains unknown; the diagnosis of FH-II is made after exclusion of the other Mendelian forms. The finding of new mutations in families of patients previously classified as having FH-II separates them from this group. Recently two groups independently reported a family with the chloride channel CLCN2 (23, 24).
FH type III (FH-IIIA and FH-IIIB)
Different mutations in the KCNJ5 gene located in or near the selectivity filter cause FH-III. The molecular mechanisms derived from these mutations resemble those found in somatic KCNJ5 mutations found in APAs (19, 151, 170, 171); however, mutations occurring even in the same codon were described to cause altogether different clinical phenotypes and responsiveness to drug treatment, thus leading to the division of FH-III into subtypes A and B. FH-IIIA features stage III, drug-resistant hypertension necessitating bilateral adrenalectomy. FH-IIIB results in a much milder clinical phenotype best treated with antihypertensive agents (27) (Fig. 5).
Figure 5.
Development of APA or multinodular cortical hyperplasia from zona glomerulosa. In FH-I the hybrid chimeric gene comprising the CYP11B2 coding sequences under control of the CYP11B1 promoter induces hyperproduction of aldosterone controlled by ACTH. Growth and proliferation of stem cells cause multinodular cortical hyperplasia or multinodular lesions with tumors. In the only FH-I pedigree [see Jeunemaitre et al. (163) and Pascoe et al. (172)], two patients had a tumor but did not lateralize on adrenal venous sampling (AVS), suggesting that these tumors were not APAs. In FH-III the excess production of aldosterone is caused by mutations in the KCNJ5 gene, which are located in or near the selectivity filter. Whether growth and proliferation of stem cells cause development of APAs remains to be investigated. [© 2018 Illustration Presentation ENDOCRINE SOCIETY]
FH type IV
Germline mutations in CACNA1H cause different phenotypic presentations of PA [reviewed in (26)]. The T-type calcium channel blocker mibefradil reduces the excess aldosterone production in transfected Cav3.2 1549Val cells, suggesting that inhibitors of this channel could be the treatment of choice for patients with CACNA1H gain-of-function mutations (173). Unfortunately, mibefradil has been withdrawn from the market because of increased risk of death in patients concomitantly receiving other antiarrhythmic drugs, mostly amiodarone.
The finding of somatic or germline mutations in K+ and Ca2+ channels in a large percentage of APAs implicated these mutations in oncogenesis and suggested that they might be driver mutations conferring growth advantages to the neoplastic cells (174). However, the heterogeneity of adrenal morphology found in patients with PA with these germline mutations argues against this theory. If these were driver mutations, they would be expected to invariably lead to neoplastic growth. In contrast, patients with FH-III germline KCNJ5 mutations exhibit adrenal phenotypes ranging from normal adrenal to massive bilateral adrenal hyperplasia or nodules (19, 175, 176), and adrenals of patients with germline CACNA1H mutations were found to have an APA, hyperplasia, or even a normal phenotype (140, 177). Therefore, it seems reasonable to hypothesize that K+ and Ca2+ channel mutations, though favoring inappropriate aldosterone production, have little or no impact on the cell growth. Other abnormalities, such as those involving the Wnt/β-catenin signaling pathway or hypomethylation of CYP11B2, may be involved in triggering cell growth in the zona glomerulosa.
Three gain-of-function missense mutations (G403D, I770M, and V401L) in CACNA1D were found to be associated with extra-adrenal signs, such as seizures, cerebral palsy, and other neurologic abnormalities (PASNA, OMIM: 615474) (139, 178). The mutations cause channel activation at membrane potentials close to the resting potential of the zona glomerulosa cells, leading to an increased Ca2+ influx and stimulation of aldosterone production [Fig. 3(d)].
Very recently, six gain-of-function mutations have been identified in the CLCN2 gene via whole-exome sequencing of germline DNA from young-onset hypertension and PA diagnosed before 25 years of age (23, 24). CLCN2 gene encodes the voltage-gated ClC-2 chloride channel, whose opening causes depolarization of zona glomerulosa cells and enhanced expression of CYP11B2 [Fig. 3(c)]. These mutant channels provide higher probabilities of channel opening at the glomerulosa resting potential and ensuing abnormally high aldosterone production.
Wnt/β-catenin signaling pathway
Wnts are secreted proteins that control growth and stem cell renewal through activation of highly conserved signaling pathways, known as canonical and noncanonical (179). The canonical Wnt signaling is activated by the binding of Wnt to a serpentine Frizzled receptor and the coreceptor LRP5/6, which leads to recruitment of Dishevelled protein Dsh and disassembly of the β-catenin destruction complex (179) Once β-catenin is free to move toward to the nucleus, it binds to the TCF/LEF1 proteins and triggers transcription of genes involved in cell growth (179). The noncanonical Wnt pathways mostly regulate intracellular Ca2+, cell polarity, and migration. By binding to the Frizzled receptor and recruiting Dsh, Wnt activates the Rho-associated kinase pathway or phospholipase C, with release of Ca2+ from intracellular stores (179).
In the adrenocortical tissue only zona glomerulosa cells display active Wnt signaling. As mentioned earlier, disruption of the Wnt pathway results in abnormal adrenal development and blunting of aldosterone synthesis. In mice, mutations in the β-catenin gene (CTNNB1) cause development of PA and tumors, which can become malignant (79, 180). Only 5% of APAs harbor CTNNB1 mutations (25), but ≤70% of the tumors exhibit active Wnt signaling, suggesting a role of active Wnt in the development of APAs. Although the underlying mechanisms remain unclear, one hypothesis is that Wnt activation results from decreased expression of the secreted negative Wnt regulator Frizzled-related protein II (80).
Hypomethylation of CYP11B2 and genes controlling CYP11B2 transcription
DNA methylation or demethylation by methyltransferase and demethyltransferase enzymes are key mechanisms of DNA damage repair, cell survival, and, when dysregulated, tumor progression (181). When methylation affects the promoter region, it blunts gene transcription and hormone secretion, whereas hypomethylation has the opposite effect (181). By using DNA methylation array analysis in 35 APAs, Yoshii et al. (182) showed that seven of eight putative methylation sites of the CYP11B2 gene, four of which are located in the promoter region, were hypomethylated in APAs. However, methylation levels showed no correlation with CYP11B2 transcription levels in APAs (182), suggesting the contribution of other mechanisms to excess aldosterone production in APAs. More recently, Purkinje cell protein 4 (PCP4), a calmodulin-binding factor that drives intracellular calcium from the cytoplasm to the nucleus, has been identified as one of the most hypomethylated genes in APAs (183). The PCP4 gene not only is more highly expressed in APAs than in the normal adrenal cortex, but it also has a site in its promoter region that binds the transcription factor CCAAT/enhancer binding protein β. Thus, it has been suggested that hypomethylation of PCP4 DNA in APAs allows increased PCP4 expression, which in turn leads to CYP11B2 upregulation (183). Of note, APAs with underlying KCNJ5 mutations have not been found to have altered PCP4 methylation (183).
Origin of APA cells from zona glomerulosa or zona fasciculata
Although aldosterone is synthetized by zona glomerulosa cells, paradoxically, most APAs are composed of cells with a high cytoplasm-to-nucleus ratio resembling zona fasciculata cells (e.g.,zona fasciculata–like cells). Despite their morphological appearance, zona fasciculata–like cells of these APAs express CYP11B2, DAB2, and CD56, putative markers of zona glomerulosa cells. Different theories have been provided to explain the occurrence of zona fasciculata–like cells in the APA. One contends that APA cells derive from zona glomerulosa cells and that an injury causes the morphological change (92); another suggests that a somatic mutation in zona fasciculata cells causes CYP11B2 overexpression (146, 184) (Fig. 6). To date there is no clear evidence supporting the superiority of one theory over the other, leaving this question open.
Figure 6.
Steroid synthesis in APA cells and origin of APA cells from zona glomerulosa or zona fasciculata. [(a), left] Staining of an APA with hematoxylin and eosin identifies zona glomerulosa–like cells (visualized as pink cells) and zona fasciculata–like cells (yellow). However, hematoxylin and eosin cannot provide functional information about steroidogenetic capability. Note the discontinuous layer of zona glomerulosa cells under the capsule. [(a), right] Immunohistochemistry with antibodies against CYP11B1 and CYP11B2 allows identification of cortisol-producing (green) and aldosterone-producing (violet) cells in the APA, as well as aldosterone-producing (violet) cells in the subcapsular clusters. Both CYP11B1- and CYP11B2-positive cells can be detected in the APA, suggesting that there is no perfect matching between zona fasciculata–like cells. Moreover, the APA may also contain CYP17 positive cells or even double (CYP17 and CYP11B2 or CYP17 and CYP11B1) or triple (CYP17 and CYP11B2 and CYP11B1) positive cells, intermingled with cells that do not show any immunoreaction. (b) Illustration of the two-hit theory with its variants. First, an injury or mutation (first hit) in a CYP11B2-positive cell of zona glomerulosa causes hyperproduction of aldosterone and transforms the zona glomerulosa cell into a zona fasciculata–like cell; then, activation of a pathway involved in cell growth (e.g., Wnt; second hit) causes abnormal proliferation with the tumor mass (APA). Second, the injury or mutation (first hit) occurs in a CYP11B1-positive cell of zona fasciculata, with transformation of the cell into a cell producing aldosterone; the second hit causes abnormal proliferation with the tumor mass (APA). Finally, the injury or mutation can associate with expression of other enzymes involved in steroidogenesis, as CYP17. To date there are no evidence supporting the superiority of one theory over the other. Note that the colors used to visualize the cells have been chosen arbitrarily. [© 2018 Illustration Presentation ENDOCRINE SOCIETY]
A different opinion is that zona fasciculata–like cells are clear and large because they accumulate cholesterol in their cytoplasm, thus displaying lipid droplets and morphological ballooning, because they are hormonally inactive (136).
The detection of hybrid cell types that coexpress the enzymes needed for cortisol [CYP11B1 and cytochrome P450 17A1 (CYP17)] and aldosterone synthesis (CYP11B2) adds further complexity to the issue (185). Nakamura et al. (185) found different types of hybrid cells in APAs characterized by the coexpression of CYP11B1 and CYP11B2, CYP11B2 and CYP17, or CYP11B1 and CYP17. Because in an in vitro model CYP11B1 was found to convert DOC to corticosterone and corticosterone to 18-OH corticosterone (186), and because immature CYP11B1/CYP11B2 double-positive cells were found in the subcapsular zone of murine adrenal glands (76, 185), it was suggested that CYP11B1 expression in APAs could be involved in mineralocorticoid synthesis and that CYP11B1/CYP11B2 hybrid cells could represent an undifferentiated cell phenotype associated with tumorigenesis (185). The CYP11B1/CYP17 and CYP11B2/CYP17 hybrid cells were suggested to make cortisol, but functional evidence is still lacking. The finding of large cells coexpressing CYP11B1, CYP11B2, and CYP17 in APAs was even more intriguing, because these cells are unlikely to make steroids efficiently given the divergent direction of the pathways mediated by CYP11B2 and CYP11B1/CYP17. Whether the coexpression of all three enzymes is a marker of tumorigenesis remains unknown (185). No association was found between such hybrid cells and somatic KCNJ5 and ATPase mutations, leaving uncertain the role played by these mutations in the genesis of the hybrid cells (185).
Origin of APA cells from APCCs
Development of specific antibodies against CY11B1 and CYP11B2 allowed unequivocal identification of cortisol- and aldosterone-producing cells and unveiled different patterns of CYP11B1 and CYP11B2 expression that underlie lateralized, and thus surgically curable, human PA (49). These findings led us to question the classic view of APA as a single, well-demarcated or encapsulated nodule exclusively constituted by cells producing excess aldosterone (187). The antibodies showed that islets of CYP11B2-positive cells, arranged in cuneiform or trapezoid clusters, named APCCs, can be present in the zona glomerulosa and outer zona fasciculata of normal glands and in hyperproducing aldosterone glands (90). APCCs are often heterogeneous, with zona glomerulosa cells intermingled with zona fasciculata cells expressing CYP11B1 in the APCC adjacent to the zona fasciculata (81, 90). The findings that APCC cells have higher CYP11B2 expression levels than the adjacent zona glomerulosa cells, some APCCs carry APA-associated somatic mutations (50, 94, 157), and APCCs resemble APAs in that they harbor zona glomerulosa and zona fasciculata cells suggested that the APCC may be a source of autonomous aldosterone production that may evolve into an APA. The term APCC-to-APA transitional lesion recapitulates this recent hypothesis (50, 188) that, though supported by some experts, remains to be conclusively proven. Moreover, even though mutations were found in APCCs in in two series of adrenal glands from normotensive subjects or kidney donors (93, 154), they were never detected in APCCs of patients with APAs (155, 156) (Table 2).
The two-hit theory
The two-hit hypothesis was originally conceived in 1971 by Alfred G. Knudson, based on Poisson statistics, to explain onset of nonhereditary retinoblastoma (189). In contrast to inherited cancer that can be caused by one mutation, sporadic tumors would result from two or more accumulated mutations. This theory has been proposed also for APAs: the first hit would be a somatic gene mutation causing excess aldosterone secretion, and the second hit would be a novel mutation, or activation of a system (e.g., Wnt), which induces an abnormal balance between cell proliferation and apoptosis, leading to nodule formation (26) (Fig. 6). An alternative view is that mutations as KCNJ5 are the “second hit,” which follows the “first hit” responsible for proliferation (190).
The two-hit or multiple-hit theory is consistent with the following: In vitro data show that KCNJ5 mutations per se do not increase cell proliferation (151), two somatic mutations were detected in different nodules of the same adrenal gland (149, 158), a germline APC mutation was found to concur with a somatic KCJN5 mutation in one patient with macronodular adrenal PA (190), and unilateral adrenocortical micronodules comprising an outer subcapsular portion of cells expressing only CYP11B2 and an inner portion of cells expressing both CYP11B2 and CYP11B1 and harboring KCNJ5 mutations were reported (188). Collectively, these findings suggest that the onset of somatic or germline mutations, possibly associated with some yet unknown mutations or injuries, can produce a spectrum of clinical and pathological phenotypes ranging from APA to micro and macro nodules.
Abnormal electrical excitability of zona glomerulosa cells
Ca2+ is needed for aldosterone synthesis; therefore, prolonged Ca2+ entry into zona glomerulosa cells through voltage-gated calcium channels must be postulated when abnormal aldosterone production occurs (191–193). Because isolated zona glomerulosa cells are hyperpolarized (−90 mV), aldosterone secretagogues should markedly depolarize the cell to allow Ca2+ entry, which is difficult to reconcile with the high sensitivity of zona glomerulosa cells to small changes (0.1 mM) in extracellular K+. The demonstration that mice zona glomerulosa cells have the intrinsic capacity to behave as electrical oscillators that depend on the Cav3.2 provides an explanation for this sensitivity (193). Spontaneous rhythmic oscillations of low periodicity in the membrane potential could create a platform that generates a recurring Ca2+ signal controlled and amplified by aldosterone secretagogues, including Ang II and extracellular K+ (94, 193). Mutations of the Ca2+ or K+ channels have been hypothesized to amplify these oscillating current signals, thereby triggering aldosterone synthesis, but to date evidence to support this contention is lacking.
Abnormal microenvironment
The microenvironment from which a neoplasm arises may play multiple roles in tumor development, for example by selecting the mutations or by promoting cell growth (174). Moreover, factors released by neighboring cells may prevent or promote the transition from normal to hyperplastic or adenomatous cells. The presence of tiny nodules in the tissue adjacent to APAs (81) supports the view of an altered microenvironment in the entire adrenal gland that harbors the APA. However, whether these nodules in the apparent normal tissue are the precursors of an APA or favor transition from normal to hyperplastic or adenomatous cells remains to be clarified.
Epidemiology of PA
About a decade after the description of PA, Jerome Conn contended that PA was not a rare disease because he detected PA in >20% of the hypertensive patients he had screened, even in the absence of hypokalemia (194). Because others could not confirm this experience, most clinicians continued to believe that PA was exceedingly rare, and available estimates of PA prevalence varied widely, from 1.4% to 32% (median 8.8%), probably because of differences in the selection criteria for retrospective studies (194). This gap of knowledge was eventually filled by a large prospective survey of consecutive newly diagnosed hypertensive patients referred to specialized hypertension centers and studied with a predefined diagnostic protocol, the PA Prevalence in Hypertensives (PAPY) study, in 2006 (10). This study provided compelling evidence of a high prevalence (11.2%) among referred hypertensive patients. Subsequently an even higher prevalence of PA was found in patients with drug-resistant hypertension by Douma et al. (195). Evidence for a high prevalence of PA in patients with hypertension seen in general practice was also provided by Olivieri et al. (196) and more recently confirmed (9). Thus, available data support the idea that rather than being a rare disease, PA is the most common, albeit overlooked, cause of endocrine hypertension.
The following reasons can account for the underdiagnosis of PA. First, hypokalemia, previously considered a hallmark of PA, is lacking in the majority of the cases (10). The incidence of normokalemia in PA may be due to tissue potassium released by the common use of a tourniquet during blood sampling (197, 198). Second, current guidelines for case detection of PA are seldom applied by general practitioners (199). Finally, in most PA, aldosterone is within the normal range but with suppressed renin. PA in these patients remains undiagnosed and often mislabeled as low-renin essential hypertension, as we recently reported (200).
The high prevalence of PA, the severity of cardiovascular and renal damage caused by the excess aldosterone, and a more favorable outcome of adrenalectomized patients with APAs compared with patients treated pharmacologically necessitate the early and accurate screening of hypertensive patients (22, 201–203). For this purpose, a simplified and more cost-effective strategy is briefly discussed below.
Diagnosis of APAs
Patients with PA are characterized by higher cardiovascular morbidity and mortality than age- and sex-matched patients with essential hypertension and a similar degree of blood pressure elevation (9, 28, 204, 205). Among the cardiovascular complications, atrial fibrillation, the most common arrhythmia worldwide, has a significantly higher relative incidence in patients with PA compared with those with essential hypertension (206). This is not unexpected, given the compelling evidence relating aldosterone to atrial fibrillation (207). Thus, early identification of patients with PA, followed by targeted treatment, translates into prevention of morbid events and reversal of cardiovascular damage. Compelling evidence has been recently provided by completion of the long-term longitudinal phase of the PAPY Study, where adrenalectomy was found to lower the risk of atrial fibrillation in patients with PA (203). The Endocrine Society Guidelines suggest screening for PA in patients with a high pretest probability for PA (Table 3) (164), but a broader systematic screening for PA in most or all hypertensive patients is endorsed by other experts, based on the high prevalence rate of PA and the high rate of cardiovascular damage (208). Unfortunately, because of the economic burden, wider screening is not feasible in many municipalities and several developed countries.
Table 3.
Conditions That Should Make the Search for PA Mandatory According to the Endocrine Society Guidelines
| 1. Unexplained hypokalemia (spontaneous or diuretic-induced) |
| 2. Hypertension (BP >140/90 mm Hg) resistant to 3 antihypertensive drugs (including a diuretic) |
| 3. Controlled BP (>140/90 mm Hg) on ≥4 antihypertensive drugs |
| 4. Hypertension and spontaneous or diuretic-induced hypokalemia |
| 5. Hypertension and adrenal incidentaloma |
| 6. Obstructive sleep apnea syndrome |
| 7. Hypertension and a family history of early-onset hypertension or cerebrovascular accident at a young age (<40 y) |
| 8. All hypertensive first-degree relatives of patients with PA |
| 9. Incidentally discovered, apparently nonfunctioning adrenal mass (“incidentaloma”) |
| 10. Evidence of organ damage that is disproportionate for the severity of hypertension |
Data from Funder JW, Carey RM, Mantero F, et al. The management of primary aldosteronism: case detection, diagnosis, and treatment. An Endocrine Society Clinical Practice Guideline. J Clin Endocrinol Metab. 2016;101(5):1889–1916.
The diagnosis of PA requires demonstration of excessive aldosterone secretion in relative autonomy of the renin-angiotensin aldosterone system (164). This is commonly done with concomitant measurements of plasma aldosterone levels and renin activity or concentration and calculation of the aldosterone-to-renin ratio (ARR) (164). Direct renin concentration has replaced plasma renin activity (PRA) in many laboratories, rendering measurements of renin simpler, quicker, and more accurate (209). Different cutoff values of ARR, using either PRA or direct renin concentration, have been proposed; only those obtained from large prospective studies are accepted for the diagnosis of PA (164, 209).
A number of so-called confirmatory tests, including the captopril challenge test, are still used in some centers to confirm the diagnosis of PA. With the prevalence rate of PA between 11% and 30% in patients referred to specialized centers, these tests function as exclusionary rather than confirmatory tests because their negative predictive value largely exceeds their positive predictive value, as clearly shown in a PAPY study post hoc analysis (210). By definition these tests identify only the subset of PA cases that are unresponsive to salt or volume suppression of aldosterone secretion, notably a minority of cases (211). Moreover, most studies supporting use of these tests were affected by a tautology bias in that they attempted to validate the confirmatory tests not against a gold reference standard but against another confirmatory test, based on the presumed but unproven autonomy of aldosterone secretion from the renin-angiotensin system (211). The largest study investigating one such test provided unambiguous evidence that when a solid diagnosis of APA was used as reference index, neither the ARR value nor the fall of plasma aldosterone concentration after captopril administration furnished any diagnostic gain over baseline ARR values in two cohorts of patients (212). This study calls for a simplification of the diagnostic algorithm to include only the determination of the ARR at baseline, a strategy that should extend screening for PA to most, or even all, hypertensive patients, even in municipalities with low levels of access to specialized medical care.
“PA mimicking “low-renin essential” hypertension could be far more common than currently perceived.”
Use of the cutoff values of ARR for diagnosis of PA implies a definition of PA as a categorical disorder. However, the available evidence suggests that the spectrum of autonomous aldosterone secretion extends beyond cases featuring an overt phenotype of hypertension or hypokalemia. By analyzing a cohort of normotensive subjects, Baudrand et al. (7) found a continuous spectrum of autonomous aldosterone secretion, ranging from subtle to overtly dysregulated aldosterone secretion, defined as suppressed PRA (<1.0 ng/mL/h) and elevated aldosterone excretion rate (≥12 μg with a urinary sodium excretion rate >200 mmol/d). Together with data showing a 6% prevalence of PA in normotensive subjects from a general population survey (213, 214) and the demonstration of APAs in normoaldosteronemic patients (200), this evidence suggests that autonomous aldosterone secretion may begin early in normotensive subjects and then may gradually transform into low-renin “essential” hypertension, then to more or less florid PA, finally evolving into stage II to III or drug-resistant hypertension. Therefore, PA mimicking “low-renin essential” hypertension could be far more common than currently perceived. Combined measurement of the ARR and 24-hour urinary sodium excretion would allow early identification of these patients. Although animal studies support a direct interaction between high salt intake and aldosterone-induced increase in blood pressure and related target organ damage (215, 216), whether institution of lifestyle intervention based on low sodium and high potassium intake is a valuable strategy to prevent transition from “low-renin essential” hypertension to overt PA remains to be proven.
Adrenal CT is useful to detect a large mass that may represent adrenocortical carcinoma and to assist the interventional radiologist and surgeon if the patient needs further treatment (164, 208). MRI has no advantage over CT in subtype evaluation of PA; in addition to being more expensive, it has less spatial resolution than CT (164). However, very small APAs (<10 mm) or adrenal zona glomerulosa hyperplasia cannot be visualized with either CT or MRI (164).
The most common forms of PA are unilateral APAs, which can be treated with unilateral adrenalectomy, and bilateral idiopathic hyperplasia [idiopathic hyperaldosteronism (IHA)], which requires lifelong mineralocorticoid receptor antagonists. The distinction between APA and IHA is crucial to identify the appropriate treatment (164, 208).
Adrenal venous sampling (AVS) is currently the only way to reliably discriminate between APA and IHA, because CT and MRI have poor accuracy (164). Unilateral aldosterone excess from a small, CT-undetectable APAs in the adrenal gland contralateral to a CT-detectable nonfunctioning adrenal mass cannot be reliably identified without AVS (208). However, AVS is a minimally invasive but technically difficult and expensive procedure, which is potentially affected by several factors (217, 218). For these reasons it should be performed only in centers endowed with a skilled multidisciplinary team with extensive expertise and in properly selected patients (164). As a preliminary test for adrenalectomy, AVS should be reserved for patients who are seeking long-term cure of PA with surgery and are reasonable candidates for general anesthesia and adrenalectomy (164, 208). AVS should be performed after correction of hypokalemia, if present, and adjustment of antihypertensive medications to allow correct interpretation of the AVS results (164).
The Endocrine Society guidelines suggest that unilateral adrenalectomy should not be performed without prior demonstration of lateralized aldosterone production by AVS. An exception to this rule may be young patients (age <35 years) with a florid PA phenotype, comprising spontaneous hypokalemia, marked aldosterone excess, suppressed plasma renin, a unilateral adrenal lesion with radiologic features consistent with a cortical adenoma, and a contralateral normal gland on CT (164). However, even for young patients, bilateral aldosterone secretion cannot be excluded without AVS (208). An in-depth review of the use of AVS has been recently published (208).
Demonstration of a CYP11B2-positive adenoma at pathology provides a conclusive diagnosis of the PA subtype, which in lateralized forms of PA can be an APA or unilateral multinodular adrenocortical hyperplasia (219, 220). Thus, in the PAPY Study the “four corners” criteria were introduced more than a decade ago for the diagnosis of APA (10). With the availability of monoclonal antibodies for human CYP11B2, these criteria must be amended by the addition of immunohistochemical detection of CYP11B2 in the resected adrenal (Table 4) (187).
Table 4.
“Five Corners” Criteria for the Diagnosis of APA
| 1. Biochemical evidence of PA (e.g., an inappropriately high aldosterone/renin ratio) |
| 2. Lateralized aldosterone secretion by AVS |
| 3. Detection of a nodule by imaging (CT or MRI) and an adenoma at pathology |
| 4. Biochemical correction of PA after adrenalectomy |
| 5. Detection of a CYP11B2-positive adenoma in the resected adrenal cortex at immunohistochemistry with a monoclonal antibody for human CYP11B2 |
The “Five corners” criteria imply that, in a setting of PA, a nodule can be defined as an aldosterone-producing adrenocortical adenoma after evidence of CYP11B2 immunoreactivity at immunohistochemistry.
In patients with PA <20 years of age or with a family history of PA or stroke at a young age (<40 years), the guidelines suggest genetic testing for FH-I [i.e., glucocorticoid-remediable aldosteronism (GRA)] and for germline mutations of KCNJ5 causing FH-III (164).
The Biomarkers of APA
A relentless search for biomarkers for APA has continued for decades, given the limited availability of AVS. Unfortunately, this remains a challenging unresolved issue, as we recently reviewed in depth (142) and update here.
The circulating markers of APA and the steroid profiles
Regarding circulating biomarkers, one approach has been the measurement of 18-oxocortisol in peripheral venous plasma to distinguish patients with bilateral adrenal hyperplasia from those with an APA (221, 222). Another approach has been to use liquid chromatography–tandem mass spectrometry (LC-MS/MS) to determine the steroid profiles in urine and peripheral venous plasma. Although LC-MS/MS showed 18-oxocortisol levels in peripheral plasma to be on average 8.5 times higher in patients with APAs than IHA, a more recent study by Eisenhofer et al. (223) showed a marked overlap of values between PA subtypes, thus questioning the usefulness of this marker for discrimination purposes. This led to the proposal of using principal component or discriminant analysis of the profile of all 15 adrenal steroids identified with LC-MS/MS, which was reported to achieve correct classification of 80% of cases of APAs. Of potential relevance, in one study steroid profiles in peripheral samples identified 77% of the 97 patients who did not benefit from adrenalectomy or in whom AVS indicated IHA, suggesting that LC-MS/MS could be an approach to avoid AVS in such patients. However, because LC-MS/MS and complex statistical analysis entail techniques unavailable in most centers, this proposal to replace AVS with an even less available strategy seems premature.
LC-MS/MS can be useful to detect excess lateralized secretion of aldosterone at AVS: LC-MS/MS-derived lateralization ratios of aldosterone normalized to cortisol and to the hybrid steroids 18-oxocortisol and 18-hydroxycortisol were significantly higher than immunoassay-derived ratios (223), but LC-MS/MS-derived lateralized secretion of the hybrid steroids was found only in 76% and 35%, respectively, of patients with APAs. Nonetheless, by identifying steroids such as 17αOH progesterone and, even more so, androstenedione that have a much larger step difference between the adrenal vein blood and the inferior vena cava blood, LC-MS/MS has been instrumental in improving the indexes for assessing selectivity and lateralization (224).
Regarding the measurement of steroids in the urine, gas chromatography–mass spectrometry has been used to characterize the urine steroid metabolome of patients with PA (225). A small but statistically significant increase in excretion of cortisol and total glucocorticoid metabolites was found in patients with PA who had no sign of glucocorticoid excess, suggesting that a mild glucocorticoid excess could be a feature of some patients with PA. Although of potential relevance, both strategies should be validated in larger samples with the gold reference standard of the diagnosis of APA as defined above (Table 4).
Finally, autoimmune mechanisms have been suggested as a cause of human PA. Autoantibodies against type-1 angiotensin II receptor (AT1AAs) were isolated from the plasma of patients with PA at levels comparable to those found in preeclamptic women (16, 17). Moreover, circulating AT1AA levels discriminated between APAs and IHA, suggesting that they might be useful not only for diagnosing PA but also for differentiating APA from non-APA conditions (16). Whether AT1AAs, because of their agonistic activity, have an etiologic role in the development of APA remains to be clarified (16, 17).
The tissue markers of APA
Not only has the search for the circulating markers been difficult, but reliable cellular biomarkers for APA cells have been a major hurdle. For decades, the expression of CYP11B2 was thought to be the signature of zona glomerulosa and APA cells, but this turned out to be untrue. Different laboratories have identified genes and proteins overexpressed or underexpressed in zona glomerulosa and APA cells that could be involved in steroidogenesis or cell growth, but each candidate marker raised questions that still await answers [for a review, see (142)]. The most promising markers for aldosterone-producing cells are CD56 (NM_000615) and Dab2 (NM_001343), which are both markedly expressed in zona glomerulosa and APA, with the former also expressed in some zona fasciculata cells and the latter missing in some zona glomerulosa cells. Because CD56 is expressed at the cell membrane level, it is successfully being used to isolate aldosterone-producing cells for research purposes (226). We recently proposed that the combination of the two markers would allow identification of all aldosterone-producing cells, but studies designed to investigate this issue are lacking thus far. After the publication of our recent review on the markers of zona glomerulosa, two markers have been proposed. CXC chemokine receptor type 4 (CXCR4), which binds its ligand CXC chemokine CXCL12 (stromal cell-derived factor-1), putatively involved in cell migration, was found to be overexpressed in aldosterone-producing tissue of normal adrenals and in about two-thirds of APAs compared with nonfunctional adenomas (227). Therefore, CXCR4 has been proposed as a marker of APA. Limited ex vivo autoradiography and in vivo positron emission tomography imaging studies, using the CXCR4 ligand 68Ga-pentixafor29-31 (68GaCPCR4.2) as tracer, claimed specificity for aldosterone-producing tissues, pointing to positron emission tomography imaging as a promising approach for noninvasive characterization of adrenal lesions in PA. However, because CXCR4 is underexpressed in about one-third of APAs and results were heterogeneous even in a very small series of highly selected patients with APA, caution is warranted in drawing conclusions at this stage (227).
Transcriptome analysis recently revealed that among the genes encoding Ca2+-binding proteins in APA, the CALN1 gene encoding calneuron 1 showed the strongest correlation with CYP11B2 (228). Calneuron 1 is located in the endoplasmic reticulum, where it binds cytosolic Ca2+ and enhances Ca2+ storage. Upon IP3 receptor signaling it releases Ca2+, thus promoting CYP11B2 transcription. Moreover, aldosterone production in HAC15 cells was potentiated by CALN1 expression, and, conversely, CALN1 silencing decreased Ca2+ in the endoplasmic reticulum and abrogated Ang II- and KCNJ5 T158A–mediated aldosterone production. Thus, elevated CALN1 could be a marker of excess aldosterone production (228). However, because what matters for activation of aldosterone biosynthesis is mitochondrial Ca2+ rather than endoplasmic reticulum Ca2+ (159), how the latter is transferred to the mitochondria and whether the calcium uniporter (229) plays a role in this process are fundamental questions that await an answer.
Conclusions
There is no doubt that the progress made in our understanding of the mechanisms of the pathogenesis of PA in the last decade eclipses that of all other endocrine causes of arterial hypertension. The advancements involve molecular mechanisms and immunophenotyping, as well as the diagnostic workup of patients from screening to subtyping. Nonetheless, little doubt exists that PA is a markedly underdiagnosed condition, despite the fact that its high prevalence was identified by Jerome Conn >60 years ago. Major factors contributing to neglecting PA as the most important cause of secondary hypertension are misconceptions, particularly the belief that PA is an exceptional cause of arterial hypertension and that the diagnosis is complex and out of reach for many doctors. An overwhelming amount of data supports exactly the opposite; PA is the most common curable cause of hypertension, and its diagnosis is simple and should not discourage anyone. Overlooking PA translates into missing the opportunity for a cure or optimal treatment of many patients. Accordingly, in our view opinion leaders have an ethical obligation to spread the message and work to improve the education of all primary care providers. The updated information provided in this review can be a small step to bridge the gap between new knowledge and its implementation in clinical practice.
Acknowledgments
Financial Support: G.P.R. and T.M.S. were supported by grants from the Ministry of Health (RF2011-02352318) and from the University of Padova (DOR1625891/16, DOR1670784/16, BIRD163255/16) and by the Foundation for Advanced Research in Arterial Hypertension and Cardiovascular Disease (FORICA). C.E.G.-S. was supported by National Heart, Lung, and Blood Institute Grant R01 HL27255.
Disclosure Summary: The authors have nothing to disclose.
Glossary
Abbreviations
- 5-HT4
5-hydroxytryptamine receptor 4
- Ang II
angiotensin II
- APA
aldosterone-producing adenoma
- APCC
aldosterone-producing cell cluster
- ARR
aldosterone-to-renin ratio
- AT1
angiotensin II type 1
- AT1AA
autoantibody against type 1 angiotensin II receptor
- AVS
adrenal venous sampling
- CaMK
calmodulin-dependent protein kinase
- CREB
cAMP response element binding
- CXCR4
CXC chemokine receptor type 4
- CYP11A1
cytochrome P450 side-chain cleavage enzyme
- CYP11B1
11β-hydroxylase
- CYP11B2
aldosterone synthase
- CYP17
17α hydroxylase
- CYP17A1
17α-hydroxylase/17,20-lyase
- CYP21A2
21-hydroxylase
- DAX1
dosage-sensitive sex reversal–adrenal hypoplasia congenital critical region on the X chromosome
- DHEA
dehydroepiandrosterone
- DHEA-S
dehydroepiandrosterone-sulfate
- DOC
deoxycorticosterone
- E
embryonic day
- E2
estradiol
- ET-1
endothelin-1
- FH
familial hyperaldosteronism
- GFP
green fluorescent protein
- GIRK4
G protein–activated inward rectifier potassium channel
- GPER-1
G protein–coupled receptor-1
- GRA
glucocorticoid-remediable aldosteronism
- HSD3B1
hydroxy-delta-5-steroid dehydrogenase
- HSD3B2
3β-hydroxysteroid dehydrogenase-2
- IHA
idiopathic hyperaldosteronism
- IP3
inositol 1,4,5-trisphosphate
- LC-MS/MS
liquid chromatography–tandem mass spectrometry
- NEFM
neurofilament medium polypeptide
- PA
primary aldosteronism
- PAPY
Primary Aldosteronism Prevalence in Hypertensives
- PCP4
Purkinje cell protein 4
- PKA
protein kinase A
- PRA
plasma renin activity
- SF1
steroidogenic factor-1
- StAR
steroidogenic acute regulatory
- TASK
TWIK acid–sensitive potassium
- Wnt
wingless-related integration site
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