Hypertension Editors' Picks: Hyperaldosteronism.

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Context:Aldosterone synthase (CYP11B2) immunohistochemistry and next-generation sequencing (NGS) have revealed the frequent presence of aldosterone-producing cell clusters (APCCs) harboring somatic mutations in aldosterone-regulating genes in adrenals from Americans without defined hypertension status.Objective:Determine the frequency and somatic mutation status of APCCs in a Japanese nonhypertensive cohort.Design, Setting, Patients, and Interventions:Adrenals from 837 consecutive autopsies at a Japanese institution, Tohoku University Hospital, were screened to select 107 unilateral adrenal glands from nonhypertensive patients. APCC score (APCC number/adrenal cortex area per case) was assessed by CYP11B2 immunohistochemistry. DNA from all APCCs and adjacent adrenal cortex was subjected to NGS using two panels targeting aldosterone-regulating genes.Primary Outcome Measure:APCC frequency and somatic mutation spectrum.Results:In 107 adrenals, 61 APCCs were detected (average of 0.6 APCCs per gland). APCC score was positively correlated with age (r = 0.50, P < 0.0001). NGS demonstrated high confidence somatic mutations in 21 of 61 APCCs (34%). Notably, 16 of 21 APCCs (76%) harbored somatic mutations in CACNA1D, the most frequently mutated gene in our previous studies of APCCs in Americans and CYP11B2-positive micronodules in cross-sectional imaging (computed tomography) negative primary aldosteronism (PA), whereas no APCCs harbored mutations in KCNJ5, the most frequently mutated gene in aldosterone-producing adenoma. APCC score was significantly lower than our previous cohort of unilateral computed tomography–negative PA.Conclusions:APCCs are frequent in nonhypertensive Japanese adrenals, accumulate with age, and frequently harbor somatic mutations (most commonly in CACNA1D). The role of APCCs in PA pathobiology and non-PA hypertension warrants further investigation.

Aldosterone-producing adenoma (APA) cause primary aldosteronism-the most frequent form of secondary hypertension. Somatic mutations in genes coding for ion channels and ATPases are found in APA and in aldosterone-producing cell clusters. We investigated the genetic, cellular, and molecular heterogeneity of different aldosterone-producing structures in adrenals with APA, to get insight into the mechanisms driving their development and to investigate their clinical and biochemical correlates. Genetic analysis of APA, aldosterone-producing cell clusters, and secondary nodules was performed in adrenal tissues from 49 patients by next-generation sequencing following CYP11B2 immunohistochemistry. Results were correlated with clinical and biochemical characteristics of patients, steroid profiles, and histological features of the tumor and adjacent adrenal cortex. Somatic mutations were identified in 93.75% of APAs. Adenoma carrying KCNJ5 mutations had more clear cells and cells expressing CYP11B1, and fewer cells expressing CYP11B2 or activated β-catenin, compared with other mutational groups. 18-hydroxycortisol and 18-oxocortisol were higher in patients carrying KCNJ5 mutations and correlated with histological features of adenoma; however, mutational status could not be predicted using steroid profiling. Heterogeneous CYP11B2 expression in KCNJ5-mutated adenoma was not associated with genetic heterogeneity. Different mutations were identified in secondary nodules expressing aldosterone synthase and in independent aldosterone-producing cell clusters from adrenals with adenoma; known KCNJ5 mutations were identified in 5 aldosterone-producing cell clusters. Genetic heterogeneity in different aldosterone-producing structures in the same adrenal suggests complex mechanisms underlying APA development.

Aldosterone and Primary Aldosteronism: Star Performers in Hypertension Research

Primary aldosteronism (PA) is the most frequent form of secondary arterial hypertension and is caused in the majority of cases by an aldosterone producing adenoma (APA) or bilateral adrenal hyperplasia. Different somatic mutations have been identified in APA and in other aldosterone producing structures, which can be distinct within the same adrenal, suggesting multiple mechanisms underlying APA development. Also, APA show important cellular and molecular heterogeneity which may be due to interaction of different signaling pathways involved in adrenal cortex cell differentiation and function. The aim of this study was to investigate the role of Wnt/β-catenin and ACTH signaling as well as elements of paracrine regulation of aldosterone biosynthesis and vascularization in the development of APA and aldosterone producing cell clusters (APCC) and their relationship with intratumoral heterogeneity and mutational status. We performed immunohistochemistry and multiplex immunofluorescence (CYP11B2, CYP17A1, β-catenin, MC2R, pCREB, Tryptase, S100, CD34) multispectral image analysis on 11 adrenals with APA and one with micronodular hyperplasia from patients with PA. CYP11B2 (aldosterone synthase) IHC guided RT-qPCR was performed on RNA extracted from formalin-fixed paraffin-embedded tissues in 7 adrenals. Multiplex immunofluorescence revealed high abundance of tryptase positive mast cells and a dense vascular component in APA, which were independent of the mutational status. Within APA, mast cells were mainly localized in zones expressing CYP11B2, but not in areas expressing CYP17A1, and were rarely colocalized with nerve fibers, suggesting that their activity is not controlled by innervation. In cells expressing aldosterone synthase, β-catenin was activated, i.e. shows nuclear and/or cytoplasmic staining, features suggestive of a zona glomerulosa cell identity; MC2R was found at the cell membrane. Expression of MC2R mRNA was observed at different levels in APA, similar to expression of MRAP and VEGFA; MRAP2 was not detected. Within heterogeneous APA carrying KCNJ5 mutations, both MC2R and VEGFA expression was higher in areas expressing CYP11B2. Remarkably, this pattern was maintained in APCC, where cells show high CYP11B2 expression, together with activated β-catenin, independently of the mutation status. In addition, a high number of mast cells was detected around APCC, with a reorganization of the capillaries around the CYP11B2 positive cells. Our results suggest that aldosterone producing structures in adrenals with APA share common molecular characteristics and cellular environment, despite different mutation status. Mast cells appear to be closely associated with cells expressing aldosterone synthase, both in APA and APCC, and their role in regulating aldosterone biosynthesis in the context of somatic mutations in PA remains to be established.

Objective: Primary aldosteronism (PA) is the most common form of secondary hypertension. Somatic mutations in the KCNJ5, ATP1A1, ATP2B3 and CACNA1D genes have recently been described in aldosterone-producing adenoma (APA). In addition, adrenals with APA show increased nodulation and cortical remodeling with zona glomerulosa hyperplasia. Although the functional link between somatic mutations and aldosterone production is clearly established, it is still not clear whether and how mutations lead to increased proliferation and nodulation. To verify if the somatic mutations are responsible for the entire adrenal phenotype (nodulation and aldosterone production) or isolated events occurring in a previously altered adrenal gland, the aim of this study was to investigate the presence of somatic KCNJ5, ATP1A1, ATP2B3 and CACNA1D mutations in secondary functional nodules of adrenals with APA. Design and method: We performed aldosterone synthase immunohistochemistry in multinodular adrenals to assess the functional characteristics of the secondary nodules for aldosterone secretion, followed by DNA extraction from functional secondary nodules from FFPE. Results: KCNJ5, ATP1A1, ATP2B3 and CACNA1D sequencing was performed on 33 functional secondary nodules from 26 multinodular adrenals of PA patients, collected through the COMETE network. In 12 adrenals, 4 harboring a somatic mutation in the APA and 8 without mutation in known genes, we identified the same mutation status between the APA and the secondary nodule. In 10 adrenals with the APA harboring a mutation, no mutations were identified in the secondary nodule. In 2 adrenals without mutation in APA, we identified a KCNJ5 mutation in the secondary nodule. In 1 adrenal harboring a KCNJ5 mutation in the APA, we identified the same mutation in two secondary nodules but no mutation was identified in a third secondary nodule. Finally, in 1 adrenal a CACNA1D mutation was identified in the APA and a KCNJ5 mutation in the secondary nodule. Conclusions: The identification of a different mutation status between APA and secondary nodules in the same adrenal suggests that somatic mutations found in APA are independent events leading to increased aldosterone production. The sequence of events underlying formation of aldosterone producing nodules remains to be elucidated.

Aldosterone-producing adenomas (APAs) are one of the main causes of primary aldosteronism and the most prevalent surgically correctable form of hypertension. Aldosterone-producing cell clusters (APCCs) comprise tight nests of zona glomerulosa cells, strongly positive for CYP11B2 (aldosterone synthase) in immunohistochemistry. APCCs have been suggested as possible precursors of APAs because they frequently carry driver mutations for constitutive aldosterone production, and a few adrenal lesions with histopathologic features of both APCCs and APAs have been identified. Our objective was to investigate the metabolic phenotypes of APCCs (n=27) compared with APAs (n=6) using in situ matrix-assisted laser desorption/ionization mass spectrometry imaging of formalin-fixed paraffin-embedded adrenals from patients with unilateral primary aldosteronism. Specific distribution patterns of metabolites were associated with APCCs and classified 2 separate APCC subgroups (subgroups 1 and 2) indistinguishable by CYP11B2 immunohistochemistry. Metabolic profiles of APCCs in subgroup 1 were tightly clustered and distinct from subgroup 2 and APAs. Multiple APCCs from the same adrenal displayed metabolic profiles of the same subgroup. Metabolites of APCC subgroup 2 were highly similar to the APA group and indicated enhanced metabolic pathways favoring cell proliferation compared with APCC subgroup 1. In conclusion, we demonstrate specific subgroups of APCCs with strikingly divergent distribution patterns of metabolites. One subgroup displays a metabolic phenotype convergent with APAs and may represent the progression of APCCs to APAs.

In primary aldosteronism, the adrenal gland produces excessive amounts of the steroid hormone aldosterone, which causes hypertension. Common causes are aldosterone‐producing adenomas (benign tumours) and bilateral adrenal hyperplasia. The majority of aldosterone‐producing adenomas carry somatic mutations in known disease genes. These include KCNJ5 (a potassium channel), CACNA1D (a calcium channel) and ATP1A1 and AT2B3 (ATPase subunits). These mutations either directly or indirectly cause increased intracellular calcium levels, which lead to increased aldosterone production and proliferation. Mutations in CTNNB1 (beta‐catenin) are rare. The molecular mechanisms underlying bilateral adrenal hyperplasia are largely unknown, with the exception of rare forms of familial hyperaldosteronism (FH). FH‐I, ‐III and ‐IV are caused by germ line mutations in CYP11B2 (aldosterone synthase), KCNJ5 and CACNA1H (another calcium channel), respectively, and a syndrome that includes neurologic abnormalities is due to germ line mutations in CACNA1D . These observations suggest pathways for the development of novel diagnostic and therapeutic strategies in primary aldosteronism. Key Concepts Primary aldosteronism is the most common cause of secondary hypertension, caused by aldosterone‐producing adenoma or bilateral adrenal hyperplasia. Recent exome sequencing studies have identified the genetic basis of the majority of aldosterone‐producing adenomas. Somatic mutations in the potassium channel KCNJ5 explain about 40% of aldosterone‐producing adenomas. Mutations in KCNJ5 are associated with female gender, early onset and larger tumour size. Other somatic mutations in aldosterone‐producing adenomas affect the CACNA1D , ATP1A1 , ATP2B3 and CTNNB1 genes. The shared common final pathway of somatic mutations in aldosterone‐producing adenomas is increased calcium signalling, which causes excessive aldosterone production and proliferation. The pathophysiology of sporadic bilateral adrenal hyperplasia is largely unknown. Rare forms of familial hyperaldosteronism (FH) include FH‐I, ‐III and ‐IV, caused by germ line mutations in CYP11B2 , KCNJ5 and CACNA1H , respectively, and FH‐II, whose genetic basis remains unknown. The identification of the molecular mechanisms of primary aldosteronism suggests pathways for the development of novel diagnostic and therapeutic strategies.

Primary aldosteronism affects ≈5% to 10% of hypertensive patients and has unilateral and bilateral forms. Most unilateral primary aldosteronism is caused by computed tomography-detectable aldosterone-producing adenomas, which express CYP11B2 (aldosterone synthase) and frequently harbor somatic mutations in aldosterone-regulating genes. The cause of the most common bilateral form of primary aldosteronism, idiopathic hyperaldosteronism (IHA), is believed to be diffuse hyperplasia of aldosterone-producing cells within the adrenal cortex. Herein, a multi-institution cohort of 15 IHA adrenals was examined with CYP11B2 immunohistochemistry and next-generation sequencing. CYP11B2 immunoreactivity in adrenal glomerulosa harboring non-nodular hyperplasia was only observed in 4/15 IHA adrenals suggesting that hyperplasia of CYP11B2-expressing cells may not be the major cause of IHA. However, the adrenal cortex of all IHA adrenals harbored at least 1 CYP11B2-positive aldosterone-producing cell cluster (APCC) or micro-aldosterone-producing adenomas. The number of APCCs per case (and individual APCC area) in IHA adrenals was significantly larger than in normotensive controls. Next-generation sequencing of DNA from 99 IHA APCCs demonstrated somatic mutations in genes encoding the L-type calcium voltage-gated channel subunit α 1-D ( CACNA1D, n=57; 58%) and potassium voltage-gated channel subfamily J-5 ( KCNJ5, n=1; 1%). These data suggest that IHA may result from not only hyperplasia but also the accumulation or enlargement of computed tomography-undetectable APCC harboring somatic aldosterone-driver gene mutations. The high prevalence of mutations in the CACNA1D L-type calcium channel provides a potential actionable therapeutic target that could complement mineralocorticoid blockade and inhibit aldosterone overproduction in some IHA patients.

Due to its rare incidence, molecular features of primary aldosteronism (PA) in young adults are largely unknown. Recently developed targeted mutational analysis identified aldosterone-driver somatic mutations in aldosterone-producing lesions, including aldosterone-producing adenomas (APAs), aldosterone-producing nodules (APNs), and aldosterone-producing micronodules, formerly known as aldosterone-producing cell clusters. To investigate histologic and genetic characteristics of lateralized PA in young adults. Formalin-fixed, paraffin-embedded adrenal tissue sections from 74 young patients with lateralized PA (<35 years old) were used for this study. Immunohistochemistry (IHC) for aldosterone synthase (CYP11B2) was performed to define the histopathologic diagnosis. Somatic mutations in aldosterone-producing lesions were further determined by CYP11B2 IHC-guided DNA sequencing. Based on the CYP11B2 IHC results, histopathologic classification was made as follows: 48 APAs, 20 APNs, 2 multiple aldosterone-producing nodules (MAPN), 1 double APN, 1 APA with MAPN, and 2 nonfunctioning adenomas (NFAs). Of 45 APAs with successful sequencing, 43 (96%) had somatic mutations, with KCNJ5 mutations being the most common genetic cause of young-onset APA (35/45, 78%). Of 18 APNs with successful sequencing, all of them harbored somatic mutations, with CACNA1D mutations being the most frequent genetic alteration in young-onset APN (8/18, 44%). Multiple CYP11B2-expressing lesions in patients with MAPN showed several aldosterone-driver mutations. No somatic mutations were identified in NFAs. APA is the most common histologic feature of lateralized PA in young adults. Somatic KCNJ5 mutations are common in APAs, whereas CACNA1D mutations are often seen in APNs in this young PA population.

Primary aldosteronism (PA) is the most common cause of secondary hypertension. The hallmark of PA is adrenal production of aldosterone under suppressed renin conditions. PA subtypes include adrenal unilateral and bilateral hyperaldosteronism. Considerable progress has been made in defining the role for somatic gene mutations in aldosterone-producing adenomas (APA) as the primary cause of unilateral PA. This includes the use of next-generation sequencing (NGS) to define recurrent somatic mutations in APA that disrupt calcium signaling, increase aldosterone synthase (CYP11B2) expression, and aldosterone production. The use of CYP11B2 immunohistochemistry on adrenal glands from normal subjects, patients with unilateral and bilateral PA has allowed the identification of CYP11B2-positive cell foci, termed aldosterone-producing cell clusters (APCC). APCC lie beneath the adrenal capsule and like APA, many APCC harbor somatic gene mutations known to increase aldosterone production. These findings suggest that APCC may play a role in pathologic progression of PA. Herein, we provide an update on recent research directed at characterizing APCC and also discuss the unanswered questions related to the role of APCC in PA.

Primary aldosteronism (PA) represents the most common cause of secondary hypertension, but little is known regarding its adrenal cellular origins. Recently, aldosterone-producing cell clusters (APCCs) with high expression of aldosterone synthase (CYP11B2) were found in both normal and PA adrenal tissue. PA-causing aldosterone-producing adenomas (APAs) harbor mutations in genes encoding ion channels/pumps that alter intracellular calcium homeostasis and cause renin-independent aldosterone production through increased CYP11B2 expression. Herein, we hypothesized that APCCs have APA-related aldosterone-stimulating somatic gene mutations. APCCs were studied in 42 normal adrenals from kidney donors. To clarify APCC molecular characteristics, we used microarrays to compare the APCC transcriptome with conventional adrenocortical zones [zona glomerulosa (ZG), zona fasciculata, and zona reticularis]. The APCC transcriptome was most similar to ZG but with an enhanced capacity to produce aldosterone. To determine if APCCs harbored APA-related mutations, we performed targeted next generation sequencing of DNA from 23 APCCs and adjacent normal adrenal tissue isolated from both formalin-fixed, paraffin-embedded, and frozen tissues. Known aldosterone driver mutations were identified in 8 of 23 (35%) APCCs, including mutations in calcium channel, voltage-dependent, L-type, α1D-subunit (CACNA1D; 6 of 23 APCCs) and ATPase, Na(+)/(K+) transporting, α1-polypeptide (ATP1A1; 2 of 23 APCCs), which were not observed in the adjacent normal adrenal tissue. Overall, we show three major findings: (i) APCCs are common in normal adrenals, (ii) APCCs harbor somatic mutations known to cause excess aldosterone production, and (iii) the mutation spectrum of aldosterone-driving mutations is different in APCCs from that seen in APA. These results provide molecular support for APCC as a precursor of PA.

Primary aldosteronism (PA) significantly increases the risk of cardiovascular complications, and early diagnosis and targeted treatment based on its pathophysiology is warranted. Next-generation sequencing (NGS) has revealed recurrent somatic mutations in aldosterone-driving genes in aldosterone-producing adenoma (APA). By applying CYP11B2 (aldosterone synthase) immunohistochemistry and NGS to adrenal glands from normal subjects and PA patients, we and others have shown that CYP11B2-positive cells make small clusters, termed aldosterone-producing cell clusters (APCC), beneath the adrenal capsule, and that APCC harbor somatic mutations in genes mutated in APA. We have shown that APCC are increased in CT-negative PA adrenals, while others showed potential progression from APCC to micro APA through mutations. These results suggest that APCC are a key factor for understanding the origin of PA, and further investigation on the relation between APCC and PA is highly needed.

Recently somatic mutations of KCNJ5, ATP1A1, ATP2B3, and CACNA1D have been identified in patients with aldosterone-producing adenoma (APA). The present study sequenced the DNA in the tissues and blood samples from Chinese patients with APA for KCNJ5, ATP1A1, ATP2B3, and CACNA1D gene mutations.Among the 114 patients, 86 (75.4%) were identified with KCNJ5 somatic mutations, including 3 previously reported (G151R, L168R, T158A) and 2 other unreported mutations. One patient presented with both a point mutation (E147) and an insertion mutation, whereas another had a 36-base duplication, G153_G164dup. No mutation of ATP1A1 and ATP2B3 in the known hotspots was identified and only 1 male patient was detected with a novel CACNA1D mutation, V748I. Unlike other studies, male and female patients had similar KCNJ5 mutation rates (76.9% vs 74.2%). Mutation carriers were younger and had lower preoperative potassium level, whereas male (but not female) mutation carriers had higher preoperative plasma aldosterone concentration and preoperative blood pressures. Mutation carriers also had higher LV mass index (LVMI) than nonmutation carriers. After surgery, LVMI improved significantly in the KCNJ5 mutation group but not in the nonmutation group. The mRNA expression of KCNJ5, CYP11B2, and ATP2B3 was higher in the KCNJ5-mutated APA tissues. Functional characterization of the 2 novel KCNJ5 mutations showed that they were associated with decreased proliferation, membrane depolarization, elevated secretion of aldosterone, and increased expression of CYP11B1 and CYP11B2.In conclusion, Chinese APA patients appear to have a high frequency of somatic KCNJ5 mutation. Mutation prevalence rates are similar among men and women and 2 novel mutations are identified. KCNJ5-mutated patients benefit more from surgical resection of APA than nonmutated patients.

Aldosterone-producing adenomas with somatic mutations in the KCNJ5 G-protein-coupled inwardly rectifying potassium channel are a cause of primary aldosteronism. These mutations drive aldosterone excess, but their role in cell growth is undefined. Our objective was to determine the role of KCNJ5 mutations in adrenal cell proliferation and apoptosis. The Ki67 proliferative index was positively correlated with adenoma diameter in aldosterone-producing adenomas with a KCNJ5 mutation (r=0.435, P=0.007), a negative correlation was noted in adenomas with no mutation detected (r=-0.548, P=0.023). Human adrenocortical cell lines were established with stable expression of cumate-inducible wild-type or mutated KCNJ5. Increased cell proliferation was induced by low-level induction of KCNJ5-T158A expression compared with control cells (P=0.009), but increased induction ablated this difference. KCNJ5-G151R displayed no apparent proliferative effect, but KCNJ5-G151E and L168R mutations each resulted in decreased cell proliferation (difference P<0.0001 from control cells, both comparisons). Under conditions tested, T158A had no effect on apoptosis, but apoptosis increased with expression of G151R (P<0.0001), G151E (P=0.008), and L168R (P<0.0001). We generated a specific KCNJ5 monoclonal antibody which was used in immunohistochemistry to demonstrate strong KCNJ5 expression in adenomas without a KCNJ5 mutation and in the zona glomerulosa adjacent to adenomas irrespective of genotype as well as in aldosterone-producing cell clusters. Double immunofluorescence staining for KCNJ5 and CYP11B2 (aldosterone synthase) showed markedly decreased KCNJ5 immunostaining in CYP11B2-positive cells compared with CYP11B2-negative cells in aldosterone-producing adenomas with a KCNJ5 mutation. Together, these findings support the concept that cell growth effects of KCNJ5 mutations are determined by the expression level of the mutated channel.