Channels and Pumps in Aldosterone-Producing Adenomas

Primary aldosteronism is the most common cause of secondary hypertension, with a prevalence of about 5% of the general hypertensive population and 10% of patients referred to hypertension specialty clinics. Aldosterone-producing adenomas (APAs) account for approximately 30–50% of cases of primary aldosteronism, and idiopathic hyperaldosteronism accounts for most of the rest of the cases. Aldosterone synthesis is regulated primarily by the renin-angiotensin system, potassium, ACTH, and to a lesser degree other peptides (1). Isolated adrenal zona glomerulosa and fasciculata cells from different species maintain a negative membrane resting potential determined by regulating membrane permeability to potassium (2, 3). Zona glomerulosa cells with hyperpolarized resting membrane voltages are steroidogenically inactive (4). Stimulation with angiotensin II, ACTH, or potassium results in membrane depolarization, opening of plasma membrane calcium channels, and mobilization of intracellular calcium, which increases activation of the calcium-calmodulin pathway, ultimately resulting in an increase in steroid biosynthesis and secretion (1). Several potassium channels have been described in rodent, bovine, and human adrenal cells, including inward rectifying and leak potassium currents that provide background potassium currents (reviewed in Ref. 3).

Until 2011, most human studies of primary aldosteronism addressed the clinical and laboratory differentiation between surgical remediable cases (APA) and idiopathic hyperaldosteronism, which is treated pharmacologically. In 2011, the group of Lifton and colleagues (5) discovered through exome sequencing that approximately 30% of APAs had somatic mutations in the region of the selectivity filter of the G protein-activated inward rectifying potassium channel Kir3.4 (also called KCNJ5). At the time, this channel was not known to be expressed in the adrenal gland. The selectivity filter is a highly conserved region within this family of potassium channels that permits the selective transport of potassium and exclusion of other cations, especially sodium. Kir3.4 is expressed in the zona glomerulosa of the human adrenal where it forms a channel as a homotetramer or heterotetramer, most often with the Kir3.1 subunit. Germ line mutations within the selectivity filter region of the KCNJ5 gene cause familial hyperaldosteronism type 2 (5, 6). Two mutations, G151R and L168R, are the most commonly found in APA. Familial hyperaldosteronism type 3 has a variable phenotype, from relatively mild to severe, depending on the amino acid affected (6, 7). New germline mutations have been found recently in a large cohort of 251 sporadic cases of primary aldosteronism; three heterozygous missense mutations (R52H, E246K, and G247R) were found, and 5% were carriers for a nonsynonymous single nucleotide polymorphism causing a E282Q substitution in the KCNJ5 gene (8). These mutations are outside the selectivity filter of the channel, but functional studies showed that three of the four affected the function of the channel in similar ways to those reported for the other mutations (8). Furthermore, these mutations occurred mostly in patients with idiopathic hyperaldosteronism. Transduction of the human adrenal carcinoma cell line HAC15 with a lentivirus carrying the KCNJ5-T158A mutation (the first mutation described in familial hyperaldosteronism type 3) results in membrane depolarization, increased intracellular sodium, opening of calcium channels, and stimulation of gene expression for several of the enzymes required for aldosterone synthesis (9). Aldosterone secretion is increased under basal conditions and after stimulation with angiotensin II, forskolin, and potassium compared to cells transduced with the empty lentivirus (9). These in vitro studies confirm that mutations in the selectivity filter are responsible for the enhanced ability of the adenoma to produce aldosterone; however, these KCNJ5 mutations decreased cell proliferation (6, 9). The adrenals of patients with APA often exhibit peritumoral hyperplasia, multiple small nodules, and increased expression of stem/proliferative gene markers in the adenoma and peritumoral areas (10). It is likely that expression of some of these genes modulates the antiproliferative effect of KCNJ5 mutations, resulting in adenoma growth.

Mutations in other genes for proteins resulting in an autonomous increase in aldosterone synthesis through an increase in intracellular calcium have been identified by exome sequencing of APAs. Somatic mutations of the α-subunit of the sodium/potassium ATPase (ATP1A1 gene) (11, 12) and the membrane calcium ATPase (ATP2B3 gene), accounting for approximately 7% of cases of APA (11), and the calcium channel Ca_v1.3 (CACNA1D gene), which might account for 11% of cases of APA (12, 13, have been described. Na/K ATPase is expressed throughout the adrenal, with highest concentrations in the zona glomerulosa. This membrane-located enzyme exchanges three cytoplasmic sodium ions for two extracellular potassium ions for each ATP hydrolyzed and drives the ion fluxes that generate resting membrane potential and action potentials (11, 12). Angiotensin II lowers Na/K-ATPase activity, thereby reducing the membrane potential, and leads to the cascade of events promoting aldosterone secretion and cell proliferation (11, 12). The ATP1A1 mutations caused inward leak currents under physiological conditions (12). The ATP2B3, also called PMCA3, is a membrane-associated calcium ATPase that extrudes intracellular calcium to the extracellular space (11). The function of this enzyme in the regulation of aldosterone secretion has not been studied; it may not be directly involved under normal conditions, but a loss of function mutation would result in higher intracellular levels. The CACNA1D gene encodes Ca_v1.3, the α₁ pore-forming subunit of the L-type voltage-gated calcium channel. The mutations identified in APAs cause maximum current amplitudes at less depolarized potentials than the wild-type Ca_v1.3, and thus facilitate channel opening (12, 13).

Adrenal adenoma cell morphology in APAs is variable. A zona fasciculata cell phenotype with larger lipid-filled vacuoles is most often associated with KCNJ5 mutations, whereas a zona glomerulosa cell phenotype is associated with ATP1A1 mutations (12). Somatic mutations of genes that influence membrane potential or intracellular calcium concentrations account for 40–60% of the somatic mutations causing APA; mutations have not been identified for the rest. The study by Lenzini et al (14) tests the hypothesis that blunted expression of leak potassium channel expression was responsible for APAs that did not have mutations described above. Microarray studies of 32 APAs and five normal adrenocortical samples were studied for altered expression of 200 sequences involved in potassium signaling. Although TASK-1 was the most highly expressed leak channel, its expression did not differ between APAs and normal adrenocortical tissue. However, Lenzini et al (14) identified a significant decrease in the expression of TASK-2.

Genetic modification in animal models might provide clues as to the potential mechanism involved in the development of primary aldosteronism. Deletion of the genes for the leak potassium channels (TASK-1, TASK-3, and double deletions) produces mouse models of primary aldosteronism or low renin hypertension (15–17). Female mice with the TASK-1^−/− deletion have hyperaldosteronism with low renin hypertension and aberrant expression aldosterone synthase (CYP11B2) enzyme in the zona fasciculata, whereas males are unaffected (15, 17). The cause of the sexual dimorphism of the phenotype is not clear. This form of hyperaldosteronism is suppressed by inhibition of ACTH with dexamethasone or by administration of T (17). The TASK-3^−/− deletion mice have low-renin, salt-sensitive hypertension, with suppressed plasma renin and aldosterone secretion that is not suppressible by increasing salt intake (15). The TASK-1^−/− and TASK-3^−/− double deletion produces mild hyperaldosteronism with plasma aldosterone levels that are stimulated by a low-sodium diet, but not suppressed by a high salt intake, and are partially responsive to angiotensin II blockade (16). TASK-1^+/− and TASK-3^+/− heterozygote animals have a normal phenotype. The human adrenal and APAs express primarily TASK-1 and very low levels of TASK-3; so far, no cases of a deficiency in the expression of these channels have been encountered in humans (14, 18).

Expression of the TWIK-related acid-sensitive potassium channel 2 (TASK-2) mRNA and protein in the APAs was consistently decreased by approximately 50% regardless of the presence or absence of KCNJ5 mutations (14). Studies of the effects of a dominant-negative TASK-2 transcript in the H295R cells demonstrated an increase in the CYP11B2 enzyme expression and aldosterone production. In an attempt to find the cause for the decreased expression of the TASK-2, Lenzini et al (14) profiled randomly selected APAs for microRNA (miR) expression, and of the 13 miRs that showed a negative correlation with TASK-2 expression, they found three that were predicted to bind the 3′-untranslated of the TASK-2 mRNA. Using a luciferase gene reporter with the 3′untranslated region of TASK-2, they found that miR-23 and to a lesser degree miR-34a decreased the expression of the luciferase signal. These two miRs are expressed in the normal adrenal with a gradient from the zona glomerulosa (highest) to zona reticularis (lowest) and diffuse but lower expression in APAs (14). This study suggests the prominent involvement of TASK-2 in the regulation of potassium homeostasis and setting membrane potentials; however, it remains unclear what role it plays in the presence of 1–2 orders of magnitude greater concentrations of the leak channel TASK-1 in these APAs (14, 18). Animal models of gene deletions of the TASK-1, TASK-3, and the double TASK-1, TASK-3 deletion indicate that the heterozygous states do not have a hyperaldosteronism phenotype (15–17), and it is unclear whether or how a 50% reduction in expression of TASK-2 has pathophysiological consequences.

The study by Lenzini et al (14) also suggests the potential of an important role for microRNAs in the development of adrenal adenomas by regulating other enzymes that might be involved in aldosterone synthesis and/or cell proliferation. miR-21 has been found to increase in the H295R cell after stimulation with angiotensin II, and the transfection of a miR-21 transcript increases aldosterone synthesis and cell proliferation (19). Conditional adrenal deletion of Dicer, a key miRNA processing enzyme, in the mouse results in the failure of the adrenal gland to develop normally (20), whereas knockdown of Dicer in the H295 adrenal cortical carcinoma cell line with a small interfering RNA was reported to up-regulate the expression of the CYP11B1 and CYP11B2 (21), so the effect of down-regulation of Dicer would depend on the stage of development and function of the adrenal. miR-24, which was found to be down-regulated in APAs, has a predicted binding recognition site at the 3′untranslated region of the CYP11B1 and CYP11B2 mRNAs (21). Overexpression of a pre-miR-24 in the H295R cell inhibited aldosterone and cortisol secretion, whereas overexpression of the anti-miR-24 stimulated aldosterone secretion but not cortisol synthesis (21). Thus, a direct regulation of enzymes of steroidogenesis at the mRNA appears to be another potential cause of APA.

APAs have been attributed to several somatic and few germline gene mutations of the KCNJ5, ATP1A1, ATP2B3, and CACNA1D genes. In addition, Lenzini et al (14) demonstrate that a decrease in the expression of TASK-2 leak potassium channel causes an increase in aldosterone synthesis. Figure 1 shows how abnormal function of ion channels and pumps is thought to increase the production of aldosterone. Table 1 is a list of the mutations reported of the various genes found in patients with primary aldosteronism to date. The cause of the remaining cases of APAs and idiopathic hyperaldosteronism remains to be elucidated. Advances in technology promise new answers, some of which may elucidate the cause of idiopathic hyperaldosteronism, which, despite being the most frequent cause of aldosteronism, remains unexplained.

Figure 1.

Proposed mechanism for the increased aldosterone production by various gene mutations described in APAs. A, Normal adrenal cell showing the expression of the various gene products responsible for the maintenance of normal membrane potential through the vectorial movement of potassium, sodium, and calcium across the cell membrane. B, Mutations of the KCNJ5 gene resulting in a leakage of sodium into the cell, membrane depolarization, and opening of the calcium channels increasing intracellular calcium. C, Mutation of the ATP1A1 gene resulting in sodium leakage into the cell, depolarization, and increase in intracellular calcium. D, Mutations of the ATP2B3 resulting in a decrease in calcium disposal and increase in intracellular calcium. E, Mutation of the CACNA1D gene decreases the voltage for activation of the channel, resulting in an increase in calcium transport and intracellular calcium. F, Down-regulation of TASK-2 leak channel results in an increase in intracellular potassium, depolarization, and activation of calcium channels.

Table 1.

Somatic and Germ Line Mutations in APAs or in Familial Hyperaldosteronism Type 3

Gene	Mutation	First Author, Year (Ref.)
KCNJ5	G151Ra	Choi, 2011 (5)
	L168R	Mulatero, 2012 (7)
	T158Aa	Scholl, 2012 (6)
	G151Ea
	I157del	Murthy, 2012 (22)
	E145Q	Åkerström, 2012 (23)
	I157Sa	Charmandari, 2012 (24)
	Y152Ca	Monticone, 2013 (25)
	R52Hb	Murthy, 2014 (8)
	E246Kb
	G247Rb
	E282Qb
ATP1A1	L104R	Beuschlein, 2013 (11)
	V332G	Williams, 2014 (26)
	I580V
	F100_L104 del
	EETA963S	Azizan, 2013 (12)
ATP2B3	L425_V426del	Beuschlein, 2013 (11)
	I232L
	V426_V427del	Williams, 2014 (26)
CACNA1D	V259D	Scholl, 2013 (13)
	G403R	Azizan, 2013 (12)
	F747L
	I750M
	R990H
	M1354I
	P1336R

^aFamilial and sporadic case mutations reported.

^bGermline mutations.