REVIEW

The Biology of Normal Zona Glomerulosa

and Aldosterone-Producing Adenoma:
Pathological Implications

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Teresa M. Seccia,1 Brasilina Caroccia,1 Elise P. Gomez-Sanchez,2 Celso E. Gomez-Sanchez,3,4
and Gian Paolo Rossi1

1
Department of Medicine-DIMED, University of Padua, 35122 Padua PD, Italy; 2Department of Pharmacology
and Toxicology, G.V. (Sonny) Montgomery VA Medical Center, Jackson, Mississippi 39216; 3Division of
Endocrinology, G.V. (Sonny) Montgomery VA Medical Center, Jackson, Mississippi 39216; and 4University of
Mississippi Medical Center, Jackson, Mississippi 39216

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. (Endocrine Reviews 39: 1029 – 1056, 2018)

A ldosterone, the main mineralocorticoid hor-

mone, 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 in-
appropriately high in % to % of all hypertensive
disorders, not only primary and secondary aldosteronism
but also overweight and obesity (, ) and the most severe
(stage II to III) or drug-resistant forms of hypertension ().
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 dam-
age, thus contributing to an ominous prognosis (–).
Primary aldosteronism (PA) occurs in % to % of
hypertensive patients (, ) 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  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  and , replicate the PA phenotype in mice ().
Aldosterone-producing adenomas (APAs) are re-
sponsible for about half of PA. Study of APAs has been
instrumental in recent advances in our understanding
of the regulation of normal and pathological aldo-
sterone synthesis. Whole transcriptome analysis has
demonstrated consistent underexpression of the TASK-
channels in APAs, and in vitro molecular blunting of
TASK- enhances aldosterone production (). This
TASK- underexpression could be attributed to
elevated miRNA  and  levels in ~% of the
APAs and to functional genetic variants in the TASK-
promoter in another quarter of the cases (), leaving
the mechanism for nearly half of the cases unex-
plained to date.
Elevated serum parathyroid hormone levels have ISSN Print: 0163-769X
been noted in patients with PA, and type  parathyroid ISSN Online: 1945-7189
hormone receptor has been found in APAs (). The Printed: in USA
demonstration that parathyroid hormone stimulates Copyright © 2018
Endocrine Society
aldosterone secretion and potentiates the effect of
Received: 18 February 2018
angiotensin II (Ang II) and K+ (, ) highlighted Accepted: 3 July 2018
hitherto unknown interactions between the para- First Published Online:
thyroid gland and the adrenocortical zona glomerulosa. 10 July 2018

doi: 10.1210/er.2018-00060 https://academic.oup.com/edrv 1029

REVIEW

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/b-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

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role of circulating and tissue biomarkers of APA in subtyping PA is discussed

Agonistic autoantibodies for the type  angiotensin re-
ceptor () were also found to increase aldosterone
production (, ).
In  the results of exome sequencing of 
APAs led Choi et al. () to uncover mutations in the
selectivity filter of the Kir. (KCNJ) K+ channel.
Subsequent studies demonstrated that KCNJ muta-
tions were present in approximately one-third of APAs
in a large European cohort () and between % and
% in a much larger meta-analysis of studies carried
out worldwide (). Germline KCNJ mutations were
also found in rare familial forms of PA featuring drug-
resistant hypertension and massive bilateral adrenal
hyperplasia necessitating bilateral adrenalectomy ().
This finding was soon followed by the discovery of
mutations in other genes affecting ion channel function,
including that of ATPA, ATPB, CACNAD,
CACNAH, CTNNB (b-catenin), and CLCN
chloride channel (–), thus providing compelling
evidence that channelopathies and malfunctioning
signaling pathways are involved in the pathophysiology
of many cases of PA (, ).
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 (–). However, our knowledge
of the fundamental biology of the normal zona glo-
merulosa 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 .
His detailed drawings of the glands were hidden
during the Inquisition and published  years later
by Giovanni Maria Lancisi as part of the Tabulae
Anatomicae (Anatomical Engravings) de Bartolomeo
Eustachii (, ). 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 ) and
description of the zonation of the cortex by Gottschau
et al. in  (). By the late s, 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 mineralocor-
ticoid activity (), generating a -year debate over
whether biologically relevant mineralocorticoids
existed, with the majority of researchers contending
that glucocorticoids were the major source of min-
eralocorticoid activity (). It was only in  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 (), which they initially named
electrocortin for its activity and, later, aldosterone, once
it was recognized that the molecule exists as a tau-
tomer between an aldehyde at position  and forms a
hemiacetal with the b hydroxyl (). Within
months, groups in America and Switzerland con-
firmed these findings (). In , Lityńsky () 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 adre-
nalectomy ().
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 to-
ward the medulla, the cortex comprises the zona

1030 Seccia et al Biology of Zona Glomerulosa and APA Endocrine Reviews, December 2018, 39(6):1029–1056

glomerulosa, made of densely packed cells with com-
paratively 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 (, ). The distinct
morphology, arrangement, and steroid production of the
adrenocortical cells within each zone are phylogenetically
conserved (, ).
Steroidogenesis and steroidogenic enzymes
Cloning of the genes for the enzymes in the ste-
roidogenic 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 cho-
lesterol to deoxycorticosterone (DOC), cytochrome P
side-chain cleavage (CYPA), b-hydroxysteroid
dehydrogenase- (HSDB), and -hydroxylase
(CYPA) are expressed in the zona glomerulosa
and zona fasciculata. The human has two highly
homologous HSDB enzymes; HSDB is expressed
primarily in the adrenal, and hydroxy-delta--steroid
dehydrogenase (HSDB) is expressed in extra-adrenal
tissues. The mouse has six different homologous iso-
enzymes. A recent study of mice with the clock genes
cry and cry deleted showed that development of
hyperaldosteronism correlates with increased expres-
sion of the hsdb isozyme (corresponding to HSDB
in the human) in the zona glomerulosa (). One recent
study indicated that HSDB was also expressed in the
zona glomerulosa, whereas HSDB was expressed in
the zonae fasciculata and glomerulosa (). However,
another study using highly specific antibodies against
both isozymes demonstrated that HSDB was
expressed at either very low or negligible levels in
the zona glomerulosa and confirmed that HSDB
was highly expressed in the zonae glomerulosa and
fasciculata ().
Aldosterone synthase (CYPB), which converts
DOC to aldosterone, is limited to the zona glomerulosa;
b-hydroxylase (CYPB), which converts DOC to
corticosterone, is in the zona fasciculata (Fig. ). In species,
including the human, that express a-hydroxylase/
,-lyase (CYPA), necessary for the synthesis of
a-hydroxypregnenolone and a-hydroxyprogester-
one, CYPB converts -deoxycortisol to cortisol in the
zona fasciculata. The a--lyase activity of CYPA
produces androstenedione in the zona fasciculata and
dehydroepiandrosterone (DHEA) in the zona retic-
ularis, where HSDB 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
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-hydroxylase, corticosterone is the primary glucocor-
ticoid, 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 a-hydroxysteroid dehydrogenase that
catabolizes progesterone, regresses in the male at about
the time it is weaned but persists in the female until first
pregnancy (, ), and it should not be confused with a
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zona reticularis.
Once antibodies were made that distinguished
between highly homologous CYPB and CYPB
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 CYPB nor CYPB, is evi-
dent in the rat but is not clearly defined in humans
(–). Whether this is due in part to high chronic
sodium (Na+) intake of humans or is a species dif-
ference is not clear. Though morphologically similar,
many zona glomerulosa cells of the adult human do not
express substantial amounts of CYPB protein.
Some animals, notably cattle, pigs, and sheep, express
a-hydroxylase (CYP) in the zona fasciculata
and zona reticularis but have a single cytochrome
P–b hydroxylase, CYPB, in both the zona
fasciculata and zona glomerulosa. In these species, the
“A narrow zona intermedia…is
CYPB acts as a sequential hydroxylase to syn- evident in the rat but is not
thesize only aldosterone in the zona glomerulosa, a clearly defined in humans.”
function provided by CYPB in the human and rat
().
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 (). The adrenal
cortex, ovary, and testis arise from a common pro-
genitor, the adrenogonadal primordium, a specialized
region of the celomic epithelium called the urogenital
ridge (, ) (Fig. ). The urogenital ridge is also the
origin for the kidneys and the progenitors of the
definitive hematopoietic cells (, , ).
During the fourth to sixth week of gestation in the
human, and around day . 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 . days in
the mouse, the primordium divides into the dorso-
medial portion to form the adrenal primordia and
the ventrolateral portion for the gonadal primordia
(–). At this stage, use of in situ hybridization
demonstrates that the adrenal gland expresses the
transcription factors, steroidogenic factor- (SF; also
called ADBP or NRA) and dosage-sensitive sex
reversal–adrenal hypoplasia congenital critical region
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Figure 1. Steroidogenic
pathways in the human
adrenal cortex. Enzymes
localized in the mitochondria
are in yellow boxes.
[© 2018 Illustration
Presentation ENDOCRINE
SOCIETY]
1032
on the X chromosome (DAX; also known as NRB)
(). Expression of SF exceeds that of DAX at all
embryonic stages studied (). The cells from the
neural crest invade the adrenal primordium around
weeks  to  of human gestation or day  in the
mouse, before the adrenal primordium is surrounded
by mesenchymal cells to form the capsule (). 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  year after birth, and a pe-
ripheral zone adjacent to the capsule comprised of
small basophilic cells that persists after birth and is
therefore called the definitive zone (, ). By the end
of gestation, the human fetal adrenal is nearly the size
of the kidney, ~% of which is fetal zone (). The
fetal zone has large steroidogenic cells that express the
CYPA enzyme, which has two activities—a-
hydroxylase and ,-lyase activities—that convert
pregnenolone into a-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 SF. Definitive zone cells are lipid-
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poor during midgestation and appear to be the res-
ervoir 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 ().
As gestation advances, these cells form fingerlike
columns of cells close to the outer rim of the fetal zone.
Before week , a third zone, named the transitional
zone, appears between the definitive zone and the fetal
zone in humans (–); 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 (). By the th week of gestation, the fetal
zone becomes rudimentary, and the definitive and
transitional zones become the zona glomerulosa and
zona fasciculata, respectively.
CYPB expression is apparent by embryonic day
 (E) in the rat adrenal gland and remains nearly
constant until postpartum day . CYPB expression
is also observed at this time in single cells or small cell
clusters distributed throughout the adrenal ().
CYPB-positive cells scattered throughout the
cortex decrease at E, and by E to E. there is an
increase in the number of CYPB immunoreactive
cells adjacent to the capsule, with only a few remaining
in the area stained by the CYPB antibody. At E
Endocrine Reviews, December 2018, 39(6):1029–1056

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 (, ,
). Measurable aldosterone and corticosterone pro-
duction by rat fetal adrenals begins at about E ().
In the human immunoreactivity for steroidogenic
enzymes including steroidogenic acute regulatory
(StAR), CYPA, CYPA, P oxidoreductase,
and cytochrome b were detected in the fetal and
transitional zones between  and  weeks’ gestation.
The HSDB enzyme necessary for the synthesis of
progesterone from pregnenolone was not detected
before the nd week of gestation and was detected
after the rd week only in the transitional and de-
finitive zones (). CYPA and DHEA sulfo-
transferase 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  weeks’ gestation (). Cortisol is
produced in the transitional zone after the rd week
of gestation (). Mast cells and their secretory
products appear to play a role in the maturation of
zona glomerulosa cells and their synthesis of aldoste-
rone (). Studies performed between the th and
st weeks of gestation with quantitative reverse
transcription PCR and immunohistochemistry dem-
onstrated that by the th week tryptase-positive cells,
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, 3b-hydroxysteroid dehydrogenase; SULT1A, sulfotransferase. [© 2018 Illustration
Presentation ENDOCRINE SOCIETY]
doi: 10.1210/er.2018-00060
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typically mast cells, are present in the subcapsular area
of the adrenal close to cells expressing mRNA for the
HSDB and the CYPB enzymes, which appear by
 and  weeks, respectively (). Tryptophan hy-
droxylase and serotonin receptor type  expression
occurred in the developing cortex by week , became
limited to the definitive zone by week , and peaked
between weeks  and , coinciding with the surge in
CYPB expression (). This finding suggests a
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paracrine role for mast cell–derived serotonin in al-
dosterone secretion in the fetus, as has been dem-
onstrated in the adult zona glomerulosa ().
The development of adrenal cortex zonation has
also been addressed via cell fate mapping and gene
deletion studies using a variety of techniques, in-
cluding specific zona glomerulosa Cre expression (,
, ). 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 em-
bedded in the zone (). In the centripetal model,
undifferentiated progenitor cells in the capsule or
subscapular region differentiate into zona glomerulosa
cells capable of producing aldosterone when stimu-
lated. These cells migrate centripetally and undergo
lineage conversion to cortisol- or corticosterone-
producing cells, depending on the species, and even-
tually undergo apoptosis in the innermost part of the
cortex, next to the adrenal medulla (). Lineage tracing
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using Shh and Gli-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 (). 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 (, ). In a
Cypb-Cre mouse model, Cypb-expressing cells
were permanently marked with green fluorescent
protein (GFP). Lineage tracing showed that the entire
zona glomerulosa and the majority of the zona fas-
ciculata were labeled within  months, indicating that
all or most cortical cells descend from the Cypb+
cell lineage during normal tissue replacement ().
Costaining with Sf and GFP also shows an exten-
sive overlap between the GFP-labeled population and
the cells of the steroidogenic cortex (). Mice
undergoing Cypb-Cre mediated Sf deletion,
which can no longer give rise to lineage-marked zona
fasciculata cells, still have an entirely normal zona
fasciculata. This finding suggests that Cypb-Cre–
mediated Sf deletion occurs late enough to allow
some progenitor cells to escape deletion of the Sf to
differentiate into fasciculata cells (, ). Signaling
molecules involved in adrenal cell renewal and zo-
nation are expressed from E. onward in mesenchymal
cells surrounding the developing adrenal. Members of
the R-spondin gene family, Rspo and Rspo, signaling
molecules that bind leucine-rich repeat–containing
receptors and modulate the b-catenin pathway, are
maintained throughout development and adulthood
(). b-catenin is highly expressed in the zona glo-
merulosa, and Rspo is expressed throughout the
capsule. Its role in adrenal development is supported
by the finding that tamoxifen-induced deletion of
Rspo at E. resulted in smaller adrenals (). Two
different pools of b-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 (). Activated b-catenin is expressed in the
adrenogonadal primordium, adrenal primordium,
and subcapsular cells. Deficiency of Wnt alters
adrenal function with a decrease in zona glomer-
ulosa function and very low aldosterone levels ().
Constitutive activation of b-catenin in the mouse
zona fasciculata causes increased Cypb expres-
sion in this zone with hyperaldosteronism and
development of adrenal tumors (, ); further-
more, activated b-catenin is expressed in APAs and
adrenal carcinomas (, ).
Transplantation of rat zona glomerulosa and zona
fasciculata cells isolated by Percoll gradient indicate
that zona glomerulosa cells completely restore pro-
duction of aldosterone and corticosterone  days after
1034 Seccia et al Biology of Zona Glomerulosa and APA
adrenalectomy and transplantation, whereas trans-
planted zona fasciculata cells only restore corticoste-
rone production (). 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 pro-
liferate and maintain their differentiated function,
again lending support to the centripetal model. Results
of cell fate mapping and gene deletion studies (, )
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suggest one of two possibilities. The most likely is
that a single cell line developed from the adrenogo-
nadal 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 differ-
entiate into zona fasciculata cells retain the ability to
regenerate zona fasciculata cells but cannot rediffer-
entiate into zona glomerulosa cells. However, cell fate
mapping techniques and gene deletion studies (, ,
) 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 SF prevents lineage conver-
sion 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 ad-
renal 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 KCNJ mutation
(GR) 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 (). This intriguing hypothesis clearly de-
serves further research.
The normal human neonate has a transient
physiological renal resistance to aldosterone and
higher levels of plasma aldosterone than its mother
(, ), associated with a low renal mineralocorticoid
receptor expression (). Low aldosterone secretion in
very preterm infants compounds the problem of low
receptor expression in the kidney (). Aldosterone
secretion rates per body surface area are higher in
younger infants than in older infants, who are similar
to adults (, ).
The fundamental enzyme for aldosterone synthesis
is aldosterone synthase, or the CYPB enzyme. Its
expression identifies cells that synthesize aldosterone,
but its expression is regulated by aldosterone secre-
tagogues, so not all zona glomerulosa cells express
CYPB. Immunohistochemistry of the adrenal of a
Endocrine Reviews, December 2018, 39(6):1029–1056

very young child shows that cells expressing CYPB
form a continuous band within the zona glomerulosa.
With increasing age, expression of CYPB becomes
less continuous, so in the adult human CYPB-
expressing cells are scattered throughout the sub-
capsular cortex among typical zona glomerulosa cells
that do not express the enzyme. These CYPB-
expressing cells in some cases may be difficult to find
(, , , ) except as clusters that extend from the
subcapsular area and penetrate into the outer zona
fasciculata. The terms aldosterone-producing cell clusters
(APCCs) (), aldosterone-producing foci, and mega foci
(, ), or just aldosterone-producing foci (, ), have
been coined to identify these clusters; we will use APCCs
herein. The number of APCCs increases with age ().
Renin activity decreases in older adults, with no con-
comitant decline in plasma or urinary aldosterone (),
suggesting an age-related autonomous aldosteronism
that might be related to the higher number and ab-
normal 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, endo-
thelin  (ET-), estrogens, and urotensin II. Aldo-
sterone production can be upregulated acutely (within
minutes) through increased expression and phos-
phorylation of the StAR protein or chronically (over
hours to days) by augmented expression of the ste-
roidogenic enzymes, mostly CYPB.
Ang II
Ang II is a potent activator of Ca+ influx, which
regulates all biosynthetic steps leading to aldosterone
secretion (). Upon binding to the angiotensin II
type  (AT) receptor subtype in cells of zona glo-
merulosa, Ang II activates phosphoinositide-specific
phospholipase C, which hydrolyzes phosphatidyli-
nositol ,-bisphosphate, which generates the sec-
ondary messengers inositol ,,-trisphosphate (IP)
and diacylglycerol (). IP triggers aldosterone secre-
tion by eliciting a transient increase in cytosolic calcium
(Ca+) concentration and activating Ca+/calmodulin-
dependent protein kinases (CaMKs), whereas diac-
ylglycerol stimulates protein kinase C, which regulates
StAR levels via cAMP response element binding
(CREB) and activator protein- (). Thus, activation
of the AT receptor stimulates both the early and late
regulatory steps of aldosterone synthesis, which in-
clude phosphorylation of StAR and expression of the
steroidogenic enzymes, respectively.
Ang II regulates both Ca+ influx into the zona
glomerulosa cell via voltage-transient T- and long-
lasting L-type Ca+ channels and intracytoplasmic Ca+
doi: 10.1210/er.2018-00060
REVIEW
movement, thus increasing cytosolic Ca+ concentra-
tions, leading to activation of CaMK II complexes (,
). CaMK II complexes mediate the transcription of
nuclear receptor–related  protein (also known as
NRA), activating transcription factors  and , and
CREB, which initiate transcription of CYPB. The
major role for Ca+ in the regulation of aldosterone
biosynthesis is unambiguously supported by the blunted
Ang II–induced aldosterone secretion after inhibition of
Ca+ influx with Ca+ channel antagonists.
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Chronic Ang II stimulation maintains aldosterone
synthesis not only through increased CYPB
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 mito-
chondria (, ). Activation of the renin-angiotensin-
aldosterone system by low Na+ intake induces expression
of CYPB without affecting CYPB, probably be-
cause of the greater expression of AT receptors in zona
glomerulosa than in zona fasciculata cells (, ).
Chronic (- to -day) Ang II administration in rats was
also found to increase expression of Ang II receptors
subtypes A and B, an effect that was blocked by the
AT receptor antagonist losartan, suggesting that during
chronic stimulation Ang II increases aldosterone pro-
duction also by upregulating its own receptors (). As “The two-pore domain
might be expected from its trophic effects in other cell channels TASK-1 and TASK-2
types, chronic Ang II stimulation also increases zona are highly expressed in the
glomerulosa cell size (). normal human adrenal cortex
and generate background K1
currents.”
Potassium
Extracellular K+ is a potent regulator of aldosterone
synthesis. K+ channels, mostly the leak K+ channels
TASK-, TASK-, and TASK- (coded by Kcnk,
KcnK, and Kcnk genes), the TWIK-related potas-
sium channel , and the G protein inward rectifying
potassium channel Kir. (KCNJ), maintain adre-
nocortical cells hyperpolarized under resting condi-
tions. The two-pore domain channels TASK- and
TASK- 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-, TASK-, or both genes have
hyperaldosteronism (–). Increases in extracel-
lular K+ depolarize the plasma membrane and activate
the voltage-dependent calcium channels, leading to
Ca+ influx and the signaling mechanisms described
above. Inhibition of Ca+ 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 mela-
nocortin receptor  in the cells of zona glomerulosa and
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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 cho-
lesterol delivery to the inner mitochondrial membrane
and activates members of the CREB family tran-
scription factors. ACTH also promotes Ca+ influx
through PKA-mediated phosphorylation of L-type
calcium channels, further enhancing aldosterone
secretion.
Acute ACTH administration stimulates aldoste-
rone secretion transiently, followed by decreased al-
dosterone 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 melano-
cortin receptor  desensitization and internalization
through a PKA-dependent mechanism; increased
a-hydroxylase (Pc) and b-hydroxylase
(Pc) activity, resulting in enhanced production
of DOC and precursor steroids; blunting of the late
(CYPB-mediated) step of aldosterone synthesis;
transformation of zona glomerulosa cells into zona
fasciculata–like cells featuring round mitochon-
dria with ovoid cristae; suppression of the renin-
angiotensin-aldosterone system; desensitization of
zona glomerulosa cells to Ang II via downregulation of
the AT receptor; and a rapid increase in zona fas-
ciculata thickness alongside a decrease in zona glo-
merulosa width [for a review see Gallo-Paynet ()].
Moreover, ACTH stimulation leads to an increase in
-oxocortisol formation. The activities of CYPB
and CYP are necessary for the synthesis of
-oxocortisol, either by coexpression in the same cells
or by a paracrine mechanism in which cortisol, se-
creted by cells expressing CYPB and CYP, is
taken up by cells expressing CYPB (–). The
underlying mechanism is not clear because CYPB
and CYP are not expressed in the same cells in the
normal adrenal but are coexpressed in many adeno-
mas. A paracrine mechanism is also doubtful because
blood flow is centripetal in the adrenal, moving
substrate from the zona fasciculata away from
CYPB, and adrenal cells express a high level of the
p-glycoprotein that pumps out polar steroids such as
cortisol ().
ET-1
ET- 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 vaso-
relaxation and inhibition of sodium transport in renal
tubules. In freshly isolated human adrenocortical cells
ET- acts as an aldosterone secretagogue through both
ETA and ETB receptors, with a potency similar to Ang
1036 Seccia et al Biology of Zona Glomerulosa and APA
II on an equimolar basis, and synergizes with Ang II
and ACTH as an aldosterone secretagogue on zona
glomerulosa cells (, ). ET- 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 Ca+,
and extracellular signal-regulated kinase/ribosomal S
kinase/CREB phosphorylation activation (, ).
ET-(-), which is formed in the adrenal cortex
by a chymase acting at the Tyr-Gly, acts through
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ETA receptors to stimulate aldosterone secretion. ET-
(-) is a weaker aldosterone secretagogue than ET-
(-) in dispersed human adrenocortical cells, but it
has a much stronger effect on DNA synthesis and cell
proliferation (). Therefore, it has been suggested
that in the human adrenal cortex, depending on local
needs, the alternative cleavage of bigET- to ET-
(-) by endothelin-converting enzyme- , or to
ET-(-) by chymase, may lead to the production of
either peptide, the former mainly stimulating ste-
roidogenesis via both ETA and ETB receptors, the latter
mainly exerting growth-promoting effects via ETA
receptors (). 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
-hydroxytryptamine receptor  (-HT) in zona
glomerulosa cells, thus stimulating aldosterone pro-
duction. Some APAs overexpress -HT receptors,
and others have been described as having a high
density of infiltrating mast cells (–), suggesting
that overexpression of -HT receptors or over-
production of serotonin contributes to the aldoste-
ronism in subsets of APA.
Urotensin II
The potent vasoactive peptide urotensin II is ubiq-
uitously expressed in the vascular tissue and is in-
volved 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 op-
posite was found for the urotensin II receptor,
suggesting a urotensin II–mediated paracrine in-
teraction between the cortex and the medulla ().
Infusion of urotensin II in normal rats for  week
potently increased both CYPB expression and
plasma aldosterone levels () through activation of a
specific urotensin receptor, and these effects were
abrogated by the urotensin receptor antagonist pal-
osuran (). Because the mild increase in CYPB 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
Endocrine Reviews, December 2018, 39(6):1029–1056

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 ().
17b-Estradiol
In human zona glomerulosa cells, estradiol (E) can act
as secretagogue or antisecretagogue, depending on the
estrogen receptor subtype activated (). In the
normal human adult adrenal cortex and APA tissues,
ERb is far more abundantly expressed than ERa and
its variant ERa, whereas the G protein–coupled
receptor- (GPER-) is highly expressed in the normal
human adrenocortical zona glomerulosa and faintly
detectable in zonae fasciculata and reticularis (). E
tonically inhibits aldosterone synthesis via ERb, as
demonstrated by the increased aldosterone synthesis
after ERb blockade or silencing, whereas the secre-
tagogue effect of E on aldosterone involves GPER-
and protein kinase A and cAMP signaling () and is
unmasked when ERb is blocked. In APAs, GPER- is
the predominant ER subtype, which suggests that it
can be relevant for the pathophysiology of aldoste-
ronism (, ). Given the broad use of selective
estrogen receptor modulators, some of which act as
GPER- modulators, in women with hormone-
dependent forms of cancer, better knowledge of the
effect of selective estrogen receptor modulator treat-
ment on aldosterone secretion would be an important
goal. However, this is an area where investigative ef-
forts are clearly necessary.
Dopamine
Dopamine is thought to tonically inhibit aldosterone
secretion because the selective D receptor antago-
nist metoclopramide induces aldosterone release in
humans (, ). Dopamine blunts Ca+ influx and
protein kinase C phosphorylation (), 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 (), indicating maximal
tonic dopaminergic inhibition of endogenous aldo-
sterone 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 (), but it is
ineffective in preventing basal or ACTH- or Ang II-
stimulated aldosterone production in Na+-repleted
subjects (, ). Of the five known dopamine re-
ceptor subtypes, four (D, D, D, and D) are
expressed in the normal human adrenal gland, sug-
gesting their role in the modulation of steroidogenesis
(). Activation of the D subtype, the predomi-
nant D-like receptor, tonically inhibits aldosterone
doi: 10.1210/er.2018-00060
REVIEW
secretion, whereas activation of D stimulates aldo-
sterone secretion and thus counteracts D-mediated
inhibition (). A microarray comparison of genes
from normal human zona fasciculata and zona glo-
merulosa 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
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cell HR, but not in normal zona fasciculata and in
large APAs, including those that express KCNJ
mutations (). Silencing of NEFM in HR cells
increased basal aldosterone production and cell
proliferation, as well as aldosterone production in
response to D agonist fenoldopam; moreover,
expression of a mutant KCNJ gene in these cells
decreased NEFM expression and raised basal al-
dosterone production, suggesting that NEFM is a
negative modulator of aldosterone acting via the D
receptor ().
Atrial natriuretic peptide
The blunting of aldosterone secretion was described as
one of the multiple actions of the natriuretic peptide
(). In line with this effect, binding studies have
revealed a single class of high-affinity saturable sites in
HR cell membranes, which were thereafter char-
“A decrease in serum ionized
acterized as NPRA receptors coupled to the particulate
Ca21 levels is the main
guanylate cyclase (). These receptors antagonize stimulus for PTH secretion by
Ang II–stimulated aldosterone secretion in HR the chief cells of the
cells and murine adrenals (, ) and also inhibit parathyroid gland.”
ACTH-stimulated aldosterone secretion via cyclic
guanosine monophosphate–dependent activation of
phosphodiesterase  and hydrolysis of cAMP in mice
().
Parathyroid hormone
Although other factors, such as calcitriol, phosphate,
magnesium, and the FGF/klotho system, intervene
in a complex manner, a decrease in serum ionized
Ca+ levels is the main stimulus for PTH secretion by
the chief cells of the parathyroid gland. PTH is a single-
chain –amino acid peptide sharing sequence ho-
mology with ACTH (). 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 Ca+ entry into the
mitochondrial matrix, an essential step for triggering
steroidogenesis, and particularly for its late step in-
volving CYPB, an enzyme that is Ca+-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  PTH receptor; parathyroid cells express the
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REVIEW
mineralocorticoid receptor; and patients with PA
(), and particularly those with the florid form of PA
usually due to APA, have elevated serum PTH levels,
which are corrected by adrenalectomy (). Thus, the
enhanced cardiovascular damage observed in both
primary hyperparathyroidism and PA can derive from
an interplay between PTH and aldosterone (, ).
Calcium ions
Ca+ 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 metab-
olism and Ca+-dependent formation of reduced
nicotinamide adenine dinucleotide phosphate, a co-
factor necessary for the activity of P cytochromes
including CYPA and CYPB; and by increasing
the binding of Ca+ to calmodulin-activated Ca+/
calmodulin-dependent kinases, which target steroido-
genic factors, including CYPB, which catalyzes the
conversion of -DOC to aldosterone.
Under resting conditions Ca+ concentration is
,-fold lower [about  to  nM ()] in the
cytosol of zona glomerulosa cells than in the extra-
cellular space ( to  mM). Thus, upon depolarization-
induced opening of voltage-gated Ca+ channels there
is a large concentration gradient for Ca+ influx ().
Sustained Ca+ entry into zona glomerulosa cells is
possible because voltage-operated calcium channels
have a permissive voltage window through which they
conduct steady-state current ().
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 re-
lationships 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. (a)].
Two types of voltage-gated Ca+ channels have
been described in mammalian zona glomerulosa cells:
low-voltage activated T-type (Cav.), and high-
voltage activated L-type (Cav.), 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 Cav. channels activate and
inactivate at lower voltages than high-threshold L-type
Ca+ channels (Cav.), which means that their
1038 Seccia et al Biology of Zona Glomerulosa and APA
permissive window occurs at lower voltages. Ac-
cordingly, they activate upon depolarization from
resting hyperpolarized potentials, allowing a surge of
Ca+ 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
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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 volt-
ages), Ca+ 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 Ca+ into the
cell at the right place for the control of steroido-
genesis. This explains why physiological concentra-
tions of Ang II and K+ may preferentially activate
Cav. and not Cav. channels, to allow Ca+ influx
into the cytoplasm.
In contrast, the high-threshold Cav. (L-type)
Ca+ 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 dihydropyr-
idine (L-type) calcium antagonists are very weak in-
hibitors of aldosterone secretion, both in vivo and in
vitro, supporting the view that the two types of
channels may serve different functions in the regu-
lation of steroidogenesis.
Ca+ 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 (). They are also crucial for activation of
calmodulin-dependent kinases I and IV, which are
crucial for StAR transcription ().
Cytosolic Ca+ concentration is also controlled by
the mitochondria, which represent ~% of zona
glomerulosa cell cytoplasm volume. The high density of
these organelles allows them to act as Ca+ buffers:
they accumulate Ca+ during the rising phase of cy-
tosolic Ca+ transients and release Ca+ during the
decaying phase (). Even small increases in mito-
chondrial Ca+ levels have measurable effects, in-
cluding reduction of pyridine nucleotide nicotinamide
adenosine dinucleotide phosphate, enhanced steroido-
genesis, particularly aldosterone biosynthesis, and gen-
eration of ATP essential for maintaining cell homeostasis
(). Reduction of nicotinamide adenine dinucleotide
phosphate produced in the mitochondria is necessary for
a and -hydroxylations and subsequent scission of
cholesterol and also for b-hydroxylation of -DOC
followed by -hydroxylation and -methyl oxidation
of corticosterone, two crucial steps in aldosterone
biosynthesis ().
Endocrine Reviews, December 2018, 39(6):1029–1056
 REVIEW

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

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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]

doi: 10.1210/er.2018-00060 https://academic.oup.com/edrv 1039

REVIEW

Biology of APA Formation
The multiple hypothetical mechanisms that may
contribute to the development of APAs are depicted in
Fig. . For the sake of clarity, these will be itemized
().
Mutations in ion channels
Somatic or germline mutations in the genes encoding
ATPA () and ATPB (), cause a loss of
function. Overall, they are thought to promote
CYPB expression and aldosterone biosynthesis via
an increase in intracellular Ca+ levels, albeit through
different mechanisms ().
Somatic mutations
Very soon after PA-related KCNJ gene mutations
were first described (), several others were identified
in APAs from patients throughout the world (, ,

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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
KCNJ () and CACNAD () and CCl (, )
lead to a gain of function, whereas others, such as
) (Table ). The KCNJ gene encodes the G
protein–activated inward rectifier potassium channel
(GIRK, also known as Kir .) that, under normal
conditions, allows the selective efflux of K+ through the
channel pore, thereby maintaining zona glomerulosa

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 b-catenin destruction complex. b-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]

1040 Seccia et al Biology of Zona Glomerulosa and APA Endocrine Reviews, December 2018, 39(6):1029–1056
 REVIEW

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;
Cell membrane depolarization higher plasma aldosterone levels and ARR at
↑ Ca2+ influx into the cell diagnosis

ATP1A1 a1 Subunit of the Na+/K+ ATPase Somatic Cell membrane depolarization More common in older men; smaller APA with

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zona glomerulosa–like cells; higher plasma
↑ Ca 2+
influx into the cell aldosterone levels and lower K+ at diagnosis

CACNA1D a1 Subunit of the Cav1.3 voltage- Somatic ↑ Ca2+ influx activation More common in older men; smaller APA with
dependent L-type calcium zona glomerulosa–like cells

ATP2B3 Plasma membrane calcium- Somatic ↑ Ca2+ influx into the cell More common in older men; smaller APA with
transporting ATPase 3 zona glomerulosa–like cells; higher plasma
aldosterone levels and lower K+ at diagnosis

CTNNB1 b-Catenin Somatic Activation of canonical or More common in women and older patients;
noncanonical Wnt pathways larger APA

CYP11B2/CYP11B1 CYP11B2 Germline CYP11B2 under control of CYP11B1 Early and severe hypertension; higher plasma
chimeric promoter levels of 18-hydroxycortisol and 18-
oxocortisol; bilateral hyperplasia or adrenal
nodules

FH-I or GRA

Gene unknown in Unknown Germline ↑ Aldosterone production No specific phenotype; diagnosis is made after
chromosome 7p22 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
2
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.

cells hyperpolarized (, ). These mutations are
located in exon  and cause amino acid changes near,
or within, a highly conserved TXGYGFR motif of the
GIRK selectivity filter, which result in loss of ion
selectivity (). The ensuing increased Na+ influx
determines cell membrane depolarization, opening of
voltage-dependent T-type Ca+ channels [Fig. (b)],
and activation of the Na+/Ca+ exchanger in the re-
verse mode (e.g., Na+ out and Ca+ in) (), thus
increasing cytosolic Ca+ levels and ultimately trig-
gering aldosterone synthesis.
Tumors with KCNJ mutations have been de-
scribed to be larger, with a predominance of zona
fasciculata–like cells, higher expression of CYPB, and
lower expression of CYPB than zona glomerulosa–like
APAs that tend to be smaller (–). 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
(). NPNT is a Wnt target gene that encodes the
extracellular matrix protein nephronectin (). Ac-
cordingly, nephronectin was immunochemically dem-
onstrated in zona glomerulosa–like APAs and normal
human zona glomerulosa but not in zona fasciculata–like
APAs and in the normal human zona fasciculata ().

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REVIEW
Table 2. Prevalence of Mutations in K+ or Ca2+ Channels in Either APCC or APA
APCC
a Mutation Without Hypertension (93, 154)
Available data are from
hypertensive patients with PA. KCNJ5 0%
CACNA1D 23%–26%
ATP1A1 3%–9%
ATP2B3 0%–5%
CACNA1D + ATP2B3 0%–3%
Both nephronectin and NEFM, as discussed earlier as
regards dopamine, are expressed in the normal zona
glomerulosa but not in zona fasciculata cells (, ).
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 KCNJ mutations result in loss of
NEFM expression, it might at least partially explain the
zona fasciculata–like phenotype of APA with KCNJ
mutations ().
The CACNAD gene codes for the a subunit of
the Cav. voltage-dependent L-type calcium channel,
which increases Ca+ influx in response to de-
polarization (, ). The a-subunit is composed of
four repeated domains, each containing six trans-
membrane segments, and forms the channel pore.
Mutations in hotspot areas of CACNAD cause a gain
of function of CaV. in zona glomerulosa cells due to
opening of the Cav. channel at membrane potentials
more hyperpolarized than under physiological con-
ditions, thus increasing Ca+ influx and CYPB
expression () [Fig. (f)].
ATPA and ATPB are members of the P-type
ATPase gene family, which encode the a subunit of the
Na+/K+ ATPase and the plasma membrane calcium-
transporting ATPase , respectively (). At rest,
ATPA maintains the negative cell membrane potential
by exchanging two extracellular K+ ions for three cy-
toplasmic Na+ ions with ATP consumption. Somatic
mutations in ATPA are found in ~% of APAs. They
involve the transmembrane helices M and M of the
protein and cause loss of pump activity, reduced affinity
for K+, and Na+ leak, leading to cell membrane de-
polarization [Fig. (d)] and increased Ca+ influx and,
thereby, increased aldosterone production ().
Mutations of ATPB, found in .% of APAs,
involve the M transmembrane helix, which is re-
sponsible for Ca+ transport and binding of ATP and
phosphate (). They distort the Ca+ ion-binding
motifs, thus increasing intracellular Ca+ and priming
aldosterone production.
Transfection of different mutant channels into
adrenocortical cells resulted in increased transcription
1042 Seccia et al Biology of Zona Glomerulosa and APA
With Hypertensiona (155, 156) APA (155, 156)
0%–8% 38%–63%
0%–65% 9%
0%–4% 5%
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0%–4% 1%
0%° 0%
of CYPB, as well as NRA and NRA genes
encoding transcription factors NURR and NOR,
respectively (, ). Somatic mutations in ATPA,
ATPB, and CACNAD, but not in KCNJ so far,
have also been detected in APCCs from normal ad-
renal glands (, ) (Table ).
The effects of these mutations on cell proliferation
remain unclear (). The KCNJ TA mutation
was associated with massive bilateral hyperplasia,
suggesting a growth-promoting effect (). In contrast
with this contention, studies performed in the labo-
ratory of some of us (C.E.G.-S. and E.P.G.-S.) showed
that cells transfected with this mutant GIRK
exhibited a lower proliferation rate than cells trans-
fected with wild-type GIRK or even apoptosis (),
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 KCNJ mutations varies
widely (), ranging from % in Europe () to %
to % in Asia and Japan (, ), whereas mutations
in ATPA, ATPB, and CACNAD 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 imple-
mented in the different countries (). 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 (, , ). The
hypothesis that one trigger may induce different mu-
tations has been suggested, although no solid evidence
for this contention has been provided to date.
It remains unknown how somatic mutations in-
duce the transformation of normal adrenocortical cells
into APA cells. One hypothesis is that somatic mu-
tations induce formation of CYPB-expressing
APCCs from mutated zona glomerulosa cells, lead-
ing to renin-independent aldosterone production and,
eventually, to an APA. Lending support to this hy-
pothesis are the findings by Nishimoto et al. () that
APCCs comprise mainly small subcapsular zona
Endocrine Reviews, December 2018, 39(6):1029–1056

glomerulosa–like cells surrounding large lipid-rich
zona fasciculata–like cells and that the transcriptome
profiles of APCCs are similar to that of zona glo-
merulosa cells with high expression of CYPB, and
finally that some APCCs harbor cells with ATPA
and CACNAD mutations. However, how zona glo-
merulosa cells are induced to form nests of cells (i.e.,
APCCs) remains to be established. Moreover, it is
unknown why KCNJ mutations do not occur in
APCCs. The hypothesis that APCCs with KCNJ
mutations rapidly progress to APA, though conceiv-
able (), remains to be proven.
Germline mutations
Germline mutations cause ,% of PA cases and have
been recently reviewed (, –), therefore, they
will be examined only briefly herein (Table ). 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 ().
Familial hyperaldosteronism type I
(glucocorticoid-remediable aldosteronism)
Familial hyperaldosteronism (FH) type I is an auto-
somal dominant disease is caused by an unequal
crossing-over of the homologous genes coding for
CYPB and b-hydroxylase (CYPB). This re-
sults in a chimeric gene comprising the CYPB
coding sequences under control of the CYPB
promoter and thus controlled by ACTH (). This
explains why in FH-I the clinical and biochemical
signs of PA can be antagonized by suppressing ACTH
with low-dose dexamethasone (). Measurements of
-hydroxycortisol and -oxocortisol levels suggest
the diagnosis, but it is confirmed with long PCR, a
more widely available technique ().
FH type II
Clinically heterogeneous, FH-II comprises non–
glucocorticoid-remediable forms of familial PA inherited
with an autosomal dominant pattern (–).
Although an association of a locus on the region
p has been found in some FH-II families (,
), the genetic basis remains unknown; the di-
agnosis 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 CLCN (, ).
FH type III (FH-IIIA and FH-IIIB)
Different mutations in the KCNJ gene located in or
near the selectivity filter cause FH-III. The molecular
mechanisms derived from these mutations resemble
those found in somatic KCNJ mutations found
in APAs (, , , ); however, mutations
doi: 10.1210/er.2018-00060
REVIEW
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 neces-
sitating bilateral adrenalectomy. FH-IIIB results in a
much milder clinical phenotype best treated with
antihypertensive agents () (Fig. ).
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FH type IV
Germline mutations in CACNAH cause different
phenotypic presentations of PA [reviewed in ()]. The
T-type calcium channel blocker mibefradil reduces the
excess aldosterone production in transfected Cav.
Val cells, suggesting that inhibitors of this channel
could be the treatment of choice for patients with
CACNAH gain-of-function mutations (). Un-
fortunately, 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 Ca+ channels in a large percentage of APAs
implicated these mutations in oncogenesis and sug-
gested that they might be driver mutations conferring
growth advantages to the neoplastic cells ().
However, the heterogeneity of adrenal morphology
found in patients with PA with these germline mu-
tations 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 KCNJ mutations exhibit adrenal pheno-
types ranging from normal adrenal to massive bilateral
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]
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REVIEW
adrenal hyperplasia or nodules (, , ), and
adrenals of patients with germline CACNAH mu-
tations were found to have an APA, hyperplasia, or
even a normal phenotype (, ). Therefore, it
seems reasonable to hypothesize that K+ and Ca+
channel mutations, though favoring inappropriate
aldosterone production, have little or no impact on the
cell growth. Other abnormalities, such as those in-
volving the Wnt/b-catenin signaling pathway or
hypomethylation of CYPB, may be involved in
triggering cell growth in the zona glomerulosa.
Three gain-of-function missense mutations
(GD, IM, and VL) in CACNAD were
found to be associated with extra-adrenal signs, such as
seizures, cerebral palsy, and other neurologic abnor-
malities (PASNA, OMIM: ) (, ). The
mutations cause channel activation at membrane
potentials close to the resting potential of the zona
glomerulosa cells, leading to an increased Ca+ influx
and stimulation of aldosterone production [Fig. (d)].
Very recently, six gain-of-function mutations have
been identified in the CLCN gene via whole-exome
sequencing of germline DNA from young-onset hy-
pertension and PA diagnosed before  years of age
(, ). CLCN gene encodes the voltage-gated ClC-
chloride channel, whose opening causes depolarization
of zona glomerulosa cells and enhanced expression of
CYPB [Fig. (c)]. These mutant channels provide
higher probabilities of channel opening at the glo-
merulosa resting potential and ensuing abnormally
high aldosterone production.
Wnt/b-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 non-
canonical (). The canonical Wnt signaling is activated
by the binding of Wnt to a serpentine Frizzled re-
ceptor and the coreceptor LRP/, which leads to
recruitment of Dishevelled protein Dsh and disas-
sembly of the b-catenin destruction complex ()
Once b-catenin is free to move toward to the nucleus,
it binds to the TCF/LEF proteins and triggers tran-
scription of genes involved in cell growth (). The
noncanonical Wnt pathways mostly regulate intracellular
Ca+, 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 Ca+ from intracellular stores ().
In the adrenocortical tissue only zona glomerulosa cells
display active Wnt signaling. As mentioned earlier, dis-
ruption of the Wnt pathway results in abnormal adrenal
development and blunting of aldosterone synthesis. In
mice, mutations in the b-catenin gene (CTNNB) cause
development of PA and tumors, which can become
malignant (, ). Only % of APAs harbor CTNNB
mutations (), but #% of the tumors exhibit active
Wnt signaling, suggesting a role of active Wnt in the
1044 Seccia et al Biology of Zona Glomerulosa and APA
development of APAs. Although the underlying mech-
anisms remain unclear, one hypothesis is that Wnt ac-
tivation results from decreased expression of the secreted
negative Wnt regulator Frizzled-related protein II ().
Hypomethylation of CYP11B2 and genes
controlling CYP11B2 transcription
DNA methylation or demethylation by methyl-
transferase and demethyltransferase enzymes are key
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mechanisms of DNA damage repair, cell survival, and,
when dysregulated, tumor progression (). When
methylation affects the promoter region, it blunts gene
transcription and hormone secretion, whereas hypo-
methylation has the opposite effect (). By using
DNA methylation array analysis in  APAs, Yoshii
et al. () showed that seven of eight putative
methylation sites of the CYPB gene, four of which
are located in the promoter region, were hypo-
methylated in APAs. However, methylation levels
showed no correlation with CYPB transcription
levels in APAs (), suggesting the contribution of
other mechanisms to excess aldosterone production in
APAs. More recently, Purkinje cell protein  (PCP), 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 (). The PCP 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 b. Thus, it has been suggested that hypo-
methylation of PCP DNA in APAs allows increased
PCP expression, which in turn leads to CYPB
upregulation (). Of note, APAs with underlying
KCNJ mutations have not been found to have altered
PCP methylation ().
Origin of APA cells from zona glomerulosa or
zona fasciculata
Although aldosterone is synthetized by zona glomer-
ulosa cells, paradoxically, most APAs are composed of
cells with a high cytoplasm-to-nucleus ratio re-
sembling zona fasciculata cells (e.g., zona fasciculata–
like cells). Despite their morphological appearance,
zona fasciculata–like cells of these APAs express
CYPB, DAB, and CD, putative markers of zona
glomerulosa cells. Different theories have been pro-
vided 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 (); another suggests that a
somatic mutation in zona fasciculata cells causes
CYPB overexpression (, ) (Fig. ). To date
there is no clear evidence supporting the superiority of
one theory over the other, leaving this question open.
A different opinion is that zona fasciculata–like
cells are clear and large because they accumulate
cholesterol in their cytoplasm, thus displaying lipid
Endocrine Reviews, December 2018, 39(6):1029–1056
 REVIEW

droplets and morphological ballooning, because they
are hormonally inactive ().
The detection of hybrid cell types that coexpress
the enzymes needed for cortisol [CYPB and cy-
tochrome P A (CYP)] and aldosterone
synthesis (CYPB) adds further complexity to the
issue (). Nakamura et al. () found different types
of hybrid cells in APAs characterized by the coex-
pression of CYPB and CYPB, CYPB and
source of autonomous aldosterone production that
may evolve into an APA. The term APCC-to-APA
transitional lesion recapitulates this recent hypothesis
(, ) 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 (, ), they were never detected in
APCCs of patients with APAs (, ) (Table ).

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CYP, or CYPB and CYP. Because in an in
vitro model CYPB was found to convert DOC to The two-hit theory
corticosterone and corticosterone to -OH cortico- The two-hit hypothesis was originally conceived in
sterone (), and because immature CYPB/  by Alfred G. Knudson, based on Poisson statistics,
CYPB double-positive cells were found in the to explain onset of nonhereditary retinoblastoma ().
subcapsular zone of murine adrenal glands (, ), it
was suggested that CYPB expression in APAs
could be involved in mineralocorticoid synthesis and
that CYPB/CYPB hybrid cells could represent
an undifferentiated cell phenotype associated with
tumorigenesis (). The CYPB/CYP and
CYPB/CYP hybrid cells were suggested to make
cortisol, but functional evidence is still lacking. The
finding of large cells coexpressing CYPB,
CYPB, and CYP in APAs was even more in-
triguing, because these cells are unlikely to make
steroids efficiently given the divergent direction of the
pathways mediated by CYPB and CYPB/
CYP. Whether the coexpression of all three en-
zymes is a marker of tumorigenesis remains unknown
(). No association was found between such hybrid
cells and somatic KCNJ and ATPase mutations,
leaving uncertain the role played by these mutations in
the genesis of the hybrid cells ().

Origin of APA cells from APCCs

Development of specific antibodies against CYB
and CYPB allowed unequivocal identification of
cortisol- and aldosterone-producing cells and unveiled
different patterns of CYPB and CYPB ex-
pression that underlie lateralized, and thus surgically
curable, human PA (). These findings led us to
question the classic view of APA as a single, well-
demarcated or encapsulated nodule exclusively con-
stituted by cells producing excess aldosterone ().
The antibodies showed that islets of CYPB-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 (). APCCs are
often heterogeneous, with zona glomerulosa cells
intermingled with zona fasciculata cells expressing
CYPB in the APCC adjacent to the zona fasciculata
(, ). The findings that APCC cells have higher
CYPB expression levels than the adjacent zona
glomerulosa cells, some APCCs carry APA-associated
somatic mutations (, , ), and APCCs resemble
APAs in that they harbor zona glomerulosa and zona
fasciculata cells suggested that the APCC may be a
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]

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REVIEW
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 so-
matic gene mutation causing excess aldosterone se-
cretion, 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 ap-
optosis, leading to nodule formation () (Fig. ). An
alternative view is that mutations as KCNJ are the
“second hit,” which follows the “first hit” responsible
for proliferation ().
The two-hit or multiple-hit theory is consistent with
the following: In vitro data show that KCNJ mutations
per se do not increase cell proliferation (), two somatic
mutations were detected in different nodules of the same
adrenal gland (, ), a germline APC mutation was
found to concur with a somatic KCJN mutation in one
patient with macronodular adrenal PA (), and uni-
lateral adrenocortical micronodules comprising an
outer subcapsular portion of cells expressing only
CYPB and an inner portion of cells expressing
both CYPB and CYPB and harboring KCNJ
mutations were reported (). 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
Ca+ is needed for aldosterone synthesis; therefore,
prolonged Ca+ entry into zona glomerulosa cells
through voltage-gated calcium channels must be
postulated when abnormal aldosterone production
occurs (–). Because isolated zona glomerulosa
cells are hyperpolarized (2 mV), aldosterone se-
cretagogues should markedly depolarize the cell to
allow Ca+ entry, which is difficult to reconcile with the
high sensitivity of zona glomerulosa cells to small
changes (. mM) in extracellular K+. The demon-
stration that mice zona glomerulosa cells have the
intrinsic capacity to behave as electrical oscillators that
depend on the Cav. provides an explanation for this
sensitivity (). Spontaneous rhythmic oscillations of
low periodicity in the membrane potential could
create a platform that generates a recurring Ca+ signal
controlled and amplified by aldosterone secretagogues,
including Ang II and extracellular K+ (, ).
Mutations of the Ca+ or K+ channels have been
hypothesized to amplify these oscillating current sig-
nals, 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
1046 Seccia et al Biology of Zona Glomerulosa and APA
example by selecting the mutations or by promoting cell
growth (). 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 () 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
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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 be-
cause he detected PA in .% of the hypertensive
patients he had screened, even in the absence of hy-
pokalemia (). 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 .% to %
(median .%), probably because of differences in the
selection criteria for retrospective studies (). This
gap of knowledge was eventually filled by a large
prospective survey of consecutive newly diagnosed
hypertensive patients referred to specialized hyper-
tension centers and studied with a predefined di-
agnostic protocol, the PA Prevalence in Hypertensives
(PAPY) study, in  (). This study provided
compelling evidence of a high prevalence (.%)
among referred hypertensive patients. Subsequently an
even higher prevalence of PA was found in patients
with drug-resistant hypertension by Douma et al.
(). Evidence for a high prevalence of PA in patients
with hypertension seen in general practice was also
provided by Olivieri et al. () and more recently
confirmed (). 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 ma-
jority of the cases (). The incidence of normoka-
lemia in PA may be due to tissue potassium released
by the common use of a tourniquet during blood
sampling (, ). Second, current guidelines for
case detection of PA are seldom applied by general
practitioners (). 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 ().
The high prevalence of PA, the severity of car-
diovascular and renal damage caused by the excess
aldosterone, and a more favorable outcome of ad-
renalectomized patients with APAs compared with
Endocrine Reviews, December 2018, 39(6):1029–1056

patients treated pharmacologically necessitate the
early and accurate screening of hypertensive pa-
tients (, –). For this purpose, a simplified
and more cost-effective strategy is briefly discussed
below.
Diagnosis of APAs
Patients with PA are characterized by higher cardio-
vascular morbidity and mortality than age- and sex-
matched patients with essential hypertension and a
similar degree of blood pressure elevation (, , ,
). 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 hyper-
tension (). This is not unexpected, given the
compelling evidence relating aldosterone to atrial fi-
brillation (). 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 adre-
nalectomy was found to lower the risk of atrial fi-
brillation in patients with PA (). The Endocrine
Society Guidelines suggest screening for PA in patients
with a high pretest probability for PA (Table ) (),
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 (). Unfortunately,
because of the economic burden, wider screening is
not feasible in many municipalities and several de-
veloped countries.
The diagnosis of PA requires demonstration of
excessive aldosterone secretion in relative autonomy
of the renin-angiotensin aldosterone system ().
This is commonly done with concomitant mea-
surements of plasma aldosterone levels and renin
activity or concentration and calculation of the
aldosterone-to-renin ratio (ARR) (). Direct renin
concentration has replaced plasma renin activity
(PRA) in many laboratories, rendering measure-
ments of renin simpler, quicker, and more accurate
(). Different cutoff values of ARR, using either
PRA or direct renin concentration, have been
proposed; only those obtained from large pro-
spective studies are accepted for the diagnosis of PA
(, ).
A number of so-called confirmatory tests, in-
cluding the captopril challenge test, are still used in
some centers to confirm the diagnosis of PA. With the
prevalence rate of PA between % and % in pa-
tients referred to specialized centers, these tests
function as exclusionary rather than confirmatory tests
because their negative predictive value largely exceeds
doi: 10.1210/er.2018-00060
REVIEW
their positive predictive value, as clearly shown in a
PAPY study post hoc analysis (). By definition these
tests identify only the subset of PA cases that are
unresponsive to salt or volume suppression of aldo-
sterone secretion, notably a minority of cases ().
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
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test, based on the presumed but unproven autonomy
of aldosterone secretion from the renin-angiotensin
system (). 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 (). This study calls for a sim-
plification 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.
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 hyper- “PA mimicking “low-renin
tension or hypokalemia. By analyzing a cohort of essential” hypertension could
normotensive subjects, Baudrand et al. () found a be far more common than
continuous spectrum of autonomous aldosterone se- currently perceived.”
cretion, ranging from subtle to overtly dysregulated
aldosterone secretion, defined as suppressed PRA
(,. ng/mL/h) and elevated aldosterone excretion
rate ($ mg with a urinary sodium excretion
rate . mmol/d). Together with data showing a %
prevalence of PA in normotensive subjects from a
general population survey (, ) and the demon-
stration of APAs in normoaldosteronemic patients
(), this evidence suggests that autonomous aldoste-
rone secretion may begin early in normotensive subjects
and then may gradually transform into low-renin “es-
sential” hypertension, then to more or less florid PA,
finally evolving into stage II to III or drug-resistant
hypertension. Therefore, PA mimicking “low-renin es-
sential” hypertension could be far more common than
currently perceived. Combined measurement of the
ARR and -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 (, ),
whether institution of lifestyle intervention based on low
sodium and high potassium intake is a valuable strategy
to prevent transition from “low-renin essential” hyper-
tension 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
https://academic.oup.com/edrv 1047
REVIEW
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.
1048
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
interventional radiologist and surgeon if the patient
needs further treatment (, ). MRI has no ad-
vantage over CT in subtype evaluation of PA; in addition
to being more expensive, it has less spatial resolution
than CT (). However, very small APAs (, mm) or
adrenal zona glomerulosa hyperplasia cannot be visual-
ized with either CT or MRI ().
The most common forms of PA are unilateral
APAs, which can be treated with unilateral adrenal-
ectomy, and bilateral idiopathic hyperplasia [idio-
pathic hyperaldosteronism (IHA)], which requires
lifelong mineralocorticoid receptor antagonists. The
distinction between APA and IHA is crucial to identify
the appropriate treatment (, ).
Adrenal venous sampling (AVS) is currently the
only way to reliably discriminate between APA and
IHA, because CT and MRI have poor accuracy ().
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 (). However, AVS
is a minimally invasive but technically difficult and
expensive procedure, which is potentially affected by
several factors (, ). For these reasons it should be
performed only in centers endowed with a skilled
multidisciplinary team with extensive expertise and in
properly selected patients (). 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 (, ). AVS should be performed
after correction of hypokalemia, if present, and ad-
justment of antihypertensive medications to allow
correct interpretation of the AVS results ().
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 , years) with a florid PA
Seccia et al Biology of Zona Glomerulosa and APA
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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 (). However, even for young
patients, bilateral aldosterone secretion cannot be
excluded without AVS (). An in-depth review of
the use of AVS has been recently published ().
Demonstration of a CYPB-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 (,
). Thus, in the PAPY Study the “four corners” criteria
were introduced more than a decade ago for the diagnosis
of APA (). With the availability of monoclonal anti-
bodies for human CYPB, these criteria must be
amended by the addition of immunohistochemical de-
tection of CYPB in the resected adrenal (Table ) ().
In patients with PA , years of age or with a
family history of PA or stroke at a young age (,
years), the guidelines suggest genetic testing for FH-I
[i.e., glucocorticoid-remediable aldosteronism (GRA)] and
for germline mutations of KCNJ causing FH-III ().
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 un-
resolved issue, as we recently reviewed in depth ()
and update here.
The circulating markers of APA and the
steroid profiles
Regarding circulating biomarkers, one approach has
been the measurement of -oxocortisol in peripheral
venous plasma to distinguish patients with bilateral
adrenal hyperplasia from those with an APA (,
Endocrine Reviews, December 2018, 39(6):1029–1056

). 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 -oxocortisol levels in peripheral plasma to
be on average . times higher in patients with APAs
than IHA, a more recent study by Eisenhofer et al. ()
showed a marked overlap of values between PA sub-
types, 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  adrenal steroids identified with LC-
MS/MS, which was reported to achieve correct classi-
fication of % of cases of APAs. Of potential relevance,
in one study steroid profiles in peripheral samples
identified % of the  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 cor-
tisol and to the hybrid steroids -oxocortisol and
-hydroxycortisol were significantly higher than
immunoassay-derived ratios (), but LC-MS/MS-
derived lateralized secretion of the hybrid steroids
was found only in % and %, respectively, of patients
with APAs. Nonetheless, by identifying steroids such as
aOH progesterone and, even more so, androstene-
dione 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 ().
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 (). 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. Al-
though of potential relevance, both strategies should be
validated in larger samples with the gold reference stan-
dard of the diagnosis of APA as defined above (Table ).
Finally, autoimmune mechanisms have been suggested
as a cause of human PA. Autoantibodies against type-
angiotensin II receptor (ATAAs) were isolated from the
plasma of patients with PA at levels comparable to those
found in preeclamptic women (, ). Moreover, cir-
culating ATAA 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 (). Whether ATAAs, because of their
agonistic activity, have an etiologic role in the development
of APA remains to be clarified (, ).
doi: 10.1210/er.2018-00060
REVIEW
Table 4. “Five Corners” Criteria for the Diagnosis of APA
The “Five corners” criteria imply
1. Biochemical evidence of PA (e.g., an inappropriately high that, in a setting of PA, a nodule
aldosterone/renin ratio) can be defined as an
aldosterone-producing
2. Lateralized aldosterone secretion by AVS
adrenocortical adenoma after
evidence of CYP11B2
3. Detection of a nodule by imaging (CT or MRI) and an immunoreactivity at
adenoma at pathology immunohistochemistry.
4. Biochemical correction of PA after adrenalectomy
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5. Detection of a CYP11B2-positive adenoma in the resected
adrenal cortex at immunohistochemistry with a
monoclonal antibody for human CYP11B2
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 ex-
pression of CYPB 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 in-
volved in steroidogenesis or cell growth, but each
candidate marker raised questions that still await
answers [for a review, see ()]. The most promising
markers for aldosterone-producing cells are CD
(NM_) and Dab (NM_), 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 CD is expressed at the cell
membrane level, it is successfully being used to isolate
aldosterone-producing cells for research purposes
(). 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  (CXCR), which
binds its ligand CXC chemokine CXCL (stromal
cell-derived factor-), 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 ().
Therefore, CXCR has been proposed as a marker of
APA. Limited ex vivo autoradiography and in vivo
positron emission tomography imaging studies, using
the CXCR ligand Ga-pentixafor- (GaCPCR.)
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 CXCR is underexpressed in about one-third
of APAs and results were heterogeneous even in a very
small series of highly selected patients with APA,
https://academic.oup.com/edrv 1049
REVIEW

caution is warranted in drawing conclusions at this
stage ().
Transcriptome analysis recently revealed that
among the genes encoding Ca+-binding proteins in
APA, the CALN gene encoding calneuron  showed the
strongest correlation with CYPB (). Calneuron  is
located in the endoplasmic reticulum, where it binds
cytosolic Ca+ and enhances Ca+ storage. Upon IP
receptor signaling it releases Ca+, thus promoting
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 . years ago. Major factors con-

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CYPB transcription. Moreover, aldosterone pro-
duction in HAC cells was potentiated by CALN ex-
pression, and, conversely, CALN silencing decreased Ca+
in the endoplasmic reticulum and abrogated Ang II- and
KCNJ TA–mediated aldosterone production. Thus,
elevated CALN could be a marker of excess aldosterone
production (). However, because what matters for
activation of aldosterone biosynthesis is mitochondrial
Ca+ rather than endoplasmic reticulum Ca+ (), how
the latter is transferred to the mitochondria and whether
the calcium uniporter () 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
tributing to neglecting PA as the most important
cause of secondary hypertension are misconcep-
tions, particularly the belief that PA is an excep-
tional cause of arterial hypertension and that the
diagnosis is complex and out of reach for many
doctors. An overwhelming amount of data sup-
ports exactly the opposite; PA is the most common
curable cause of hypertension, and its diagnosis is
simple and should not discourage anyone. Over-
looking 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.

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REVIEW

Wild V, Gomez-Sanchez CE, Reis A-C, Petersenn S,
Wester H-J, Kropf S, Fassnacht M, Lang K, Herrmann
K, Buck AK, Bluemel C, Hahner S. Targeting CXCR4
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and from the University of Padova (DOR1625891/16,
DOR1670784/16, BIRD163255/16) and by the Founda-
tion 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.
Correspondence and Reprint Requests: Gian Paolo
Rossi, MD, FACC, FAHA, Clinica dell’Ipertensione Arteriosa,
Department of Medicine-DIMED, University Hospital, Via
Giustiniani, 2, 35128 Padova PD, Italy. E-mail: gianpaolo.rossi@
unipd.it.
Disclosure Summary: The authors have nothing to
CYP11A1, cytochrome P450 side-chain cleavage enzyme;
CYP11B1, 11b-hydroxylase; CYP11B2, aldosterone synthase;
CYP17, 17a hydroxylase; CYP17A1, 17a-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

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229. Mammucari C, Raffaello A, Vecellio Reane D, Rizzuto
R. Molecular structure and pathophysiological roles
of the Mitochondrial Calcium Uniporter. Biochim
Biophys Acta. 2016;1863(10):2457–2464.
Acknowledgments
Financial Support: G.P.R. and T.M.S. were supported by
grants from the Ministry of Health (RF2011-02352318)
disclose.
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;
dehydrogenase; HSD3B2, 3b-hydroxysteroid dehydrogenase-
2; IHA, idiopathic hyperaldosteronism; IP3, inositol 1,4,5-tri-
sphosphate; LC-MS/MS, liquid chromatography–tandem
mass spectrometry; NEFM, neurofilament medium poly-
peptide; PA, primary aldosteronism; PAPY, Primary Aldo-
steronism Prevalence in Hypertensives; PCP4, Purkinje cell
protein 4; PKA, protein kinase A; PRA, plasma renin activity;
SF1, steroidogenic factor-1; StAR, steroidogenic acute regu-
latory; TASK, TWIK acid–sensitive potassium; Wnt, wingless-
related integration site.

1056 Seccia et al Biology of Zona Glomerulosa and APA Endocrine Reviews, December 2018, 39(6):1029–1056