RESUMO
Recent discoveries of somatic mutations permit the recognition of subtypes of aldosterone-producing adenomas with distinct clinical presentations and pathological features. Here we describe three women with hyperaldosteronism, two who presented in pregnancy and one who presented after menopause. Their aldosterone-producing adenomas harbored activating mutations of CTNNB1, encoding ß-catenin in the Wnt cell-differentiation pathway, and expressed LHCGR and GNRHR, encoding gonadal receptors, at levels that were more than 100 times as high as the levels in other aldosterone-producing adenomas. The mutations stimulate Wnt activation and cause adrenocortical cells to de-differentiate toward their common adrenal-gonadal precursor cell type. (Funded by grants from the National Institute for Health Research Cambridge Biomedical Research Centre and others.).
Assuntos
Adenoma/genética , Neoplasias das Glândulas Suprarrenais/genética , Hiperaldosteronismo/etiologia , Complicações Neoplásicas na Gravidez/genética , beta Catenina/genética , Adenoma/metabolismo , Adenoma/patologia , Neoplasias das Glândulas Suprarrenais/metabolismo , Neoplasias das Glândulas Suprarrenais/patologia , Adulto , Aldosterona/metabolismo , Feminino , Humanos , Hipertensão/etiologia , Hipopotassemia/etiologia , Pessoa de Meia-Idade , Pós-Menopausa , Gravidez , Receptores do LH/metabolismo , Receptores LHRH/metabolismo , Regulação para CimaRESUMO
BACKGROUND: Cell origin of aldosterone-producing adenomas, a major cause of hypertension, is unknown. A less common subtype of these adenomas, composed of cells resembling zona glomerulosa, have mutations in genes ATP1A1 and CACNA1D. To understand whether the adenomas originate from zona glomerulosa, we carried out a microarray analysis comparing transcriptomes of zona glomerulosa, zona fasciculata, and tumour in human adrenal tissue, and investigated the functional role of genes upregulated in the zona glomerulosa. METHODS: Using a microarray analysis (Affymetrix, High Wycombe, UK), we compared transcriptomes of zona glomerulosa, zona fasciculata, and tumour obtained by laser capture microdissection of 14 patients with aldestosterone adenomas and seven with phaeochromocytoma. One of the most zona glomerulosa-selective genes was ANO4, a member of the anoctamin family. Subcellular localisation was observed by immunofluorescence microscopy of transfected HEK293 cells. Yellow fluorescent protein-based assay was performed to detect ANO4 activity as a calcium-activated chloride channel. H295R cells were transfected by ANO4 to measure aldosterone and CYP11B2 expression. FINDINGS: Microarray analysis revealed 28 genes that were at least five times overexpressed in zona glomerulosa compared with zona fasciculata. ANO4 was 19·9 times higher in zona glomerulosa than in zona fasciculata (p=6·6 × 10(-24)). Haemagglutinin-tagged ANO4 was localised to the plasma membrane of transfected HEK293 cells. In response to increased intracellular calcium, ANO4-transfected cells triggered a lower flow of iodide than did other anoctamins. ANO4 overexpression in H295R cells increased aldosterone secretion from mean 0·9 pmol/µg protein (SE 0·2) to 1·1 (0·1), whereas CYP11B2 mRNA expression increased five times. INTERPRETATION: We show that ANO4 is one of the most highly expressed genes in zona glomerulosa of the human adrenal gland. When overexpressed in vitro, it increases aldosterone production. FUNDING: British Heart Foundation.
RESUMO
Primary aldosteronism is the most common single cause of hypertension and is potentially curable when only one adrenal gland is the culprit. The importance of primary aldosteronism to public health derives from its high prevalence but huge under-diagnosis (estimated to be <1% of all affected individuals), despite the consequences of poor blood pressure control by conventional therapy and enhanced cardiovascular risk. This state of affairs is attributable to the fact that the tools used for diagnosis or treatment are still those that originated in the 1970-1990s. Conversely, molecular discoveries have transformed our understanding of adrenal physiology and pathology. Many molecules and processes associated with constant adrenocortical renewal and interzonal metamorphosis also feature in aldosterone-producing adenomas and aldosterone-producing micronodules. The adrenal gland has one of the most significant rates of non-silent somatic mutations, with frequent selection of those driving autonomous aldosterone production, and distinct clinical presentations and outcomes for most genotypes. The disappearance of aldosterone synthesis and cells from most of the adult human zona glomerulosa is the likely driver of the mutational success that causes aldosterone-producing adenomas, but insights into the pathways that lead to constitutive aldosterone production and cell survival may open up opportunities for novel therapies.
Assuntos
Adenoma , Hiperaldosteronismo , Adulto , Humanos , Aldosterona/metabolismo , Hiperaldosteronismo/diagnóstico , Hiperaldosteronismo/genética , Hiperaldosteronismo/terapia , Saúde Pública , Medicina Molecular , Adenoma/complicações , Adenoma/metabolismoRESUMO
OBJECTIVE: Primary aldosteronism (PA) is one of the most frequent causes of secondary hypertension. Although clinical practice guidelines recommend a diagnostic process, details of the steps remain incompletely standardized. DESIGN: In the present SCOT-PA survey, we have investigated the diversity of approaches utilized for each diagnostic step in different expert centers through a survey using Google questionnaires. A total of 33 centers from 3 continents participated. RESULTS: We demonstrated a prominent diversity in the conditions of blood sampling, assay methods for aldosterone and renin, and the methods and diagnostic cutoff for screening and confirmatory tests. The most standard measures were modification of antihypertensive medication and sitting posture for blood sampling, measurement of plasma aldosterone concentration (PAC) and active renin concentration by chemiluminescence enzyme immunoassay, a combination of aldosterone-to-renin ratio with PAC as an index for screening, and saline infusion test in a seated position for confirmatory testing. The cutoff values for screening and confirmatory testing showed significant variation among centers. CONCLUSIONS: Diversity of the diagnostic steps may lead to an inconsistent diagnosis of PA among centers and limit comparison of evidence for PA between different centers. We expect the impact of this diversity to be most prominent in patients with mild PA. The survey raises 2 issues: the need for standardization of the diagnostic process and revisiting the concept of mild PA. Further standardization of the diagnostic process/criteria will improve the quality of evidence and management of patients with PA.
Assuntos
Hiperaldosteronismo , Hipertensão , Humanos , Aldosterona , Renina , Hipertensão/diagnóstico , Hipertensão/etiologia , Inquéritos e QuestionáriosRESUMO
Aldosterone-producing adenomas (APAs) are the commonest curable cause of hypertension. Most have gain-of-function somatic mutations of ion channels or transporters. Herein we report the discovery, replication and phenotype of mutations in the neuronal cell adhesion gene CADM1. Independent whole exome sequencing of 40 and 81 APAs found intramembranous p.Val380Asp or p.Gly379Asp variants in two patients whose hypertension and periodic primary aldosteronism were cured by adrenalectomy. Replication identified two more APAs with each variant (total, n = 6). The most upregulated gene (10- to 25-fold) in human adrenocortical H295R cells transduced with the mutations (compared to wildtype) was CYP11B2 (aldosterone synthase), and biological rhythms were the most differentially expressed process. CADM1 knockdown or mutation inhibited gap junction (GJ)-permeable dye transfer. GJ blockade by Gap27 increased CYP11B2 similarly to CADM1 mutation. Human adrenal zona glomerulosa (ZG) expression of GJA1 (the main GJ protein) was patchy, and annular GJs (sequelae of GJ communication) were less prominent in CYP11B2-positive micronodules than adjacent ZG. Somatic mutations of CADM1 cause reversible hypertension and reveal a role for GJ communication in suppressing physiological aldosterone production.
Assuntos
Neoplasias do Córtex Suprarrenal , Adenoma Adrenocortical , Hiperaldosteronismo , Hipertensão , Humanos , Aldosterona , Citocromo P-450 CYP11B2 , Junções Comunicantes , Mutação , Molécula 1 de Adesão CelularRESUMO
Most aldosterone-producing adenomas (APAs) have gain-of-function somatic mutations of ion channels or transporters. However, their frequency in aldosterone-producing cell clusters of normal adrenal gland suggests a requirement for codriver mutations in APAs. Here we identified gain-of-function mutations in both CTNNB1 and GNA11 by whole-exome sequencing of 3/41 APAs. Further sequencing of known CTNNB1-mutant APAs led to a total of 16 of 27 (59%) with a somatic p.Gln209His, p.Gln209Pro or p.Gln209Leu mutation of GNA11 or GNAQ. Solitary GNA11 mutations were found in hyperplastic zona glomerulosa adjacent to double-mutant APAs. Nine of ten patients in our UK/Irish cohort presented in puberty, pregnancy or menopause. Among multiple transcripts upregulated more than tenfold in double-mutant APAs was LHCGR, the receptor for luteinizing or pregnancy hormone (human chorionic gonadotropin). Transfections of adrenocortical cells demonstrated additive effects of GNA11 and CTNNB1 mutations on aldosterone secretion and expression of genes upregulated in double-mutant APAs. In adrenal cortex, GNA11/Q mutations appear clinically silent without a codriver mutation of CTNNB1.
Assuntos
Neoplasias do Córtex Suprarrenal/genética , Adenoma Adrenocortical/genética , Aldosterona/biossíntese , Subunidades alfa de Proteínas de Ligação ao GTP/genética , beta Catenina/genética , Adolescente , Neoplasias do Córtex Suprarrenal/patologia , Adenoma Adrenocortical/patologia , Adulto , Feminino , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/genética , Humanos , Hiperaldosteronismo/patologia , Masculino , Menopausa/metabolismo , Pessoa de Meia-Idade , Gravidez , Puberdade/metabolismoRESUMO
PURPOSE OF REVIEW: Aldosterone regulation in the adrenal plays an important role in blood pressure. The commonest curable cause of hypertension is primary aldosteronism. Recently, mutations in novel genes have been identified to cause primary aldosteronism. Elucidating the mechanism of action of these genetic abnormalities may help understand the cause of primary aldosteronism and the physiological regulation of aldosterone in the zona glomerulosa. RECENT FINDINGS: KCNJ5, ATP1A1, ATP2B3, CACNA1D, CTNNB1, and CACNA1H mutations are causal of primary aldosteronism. ARMC5 may cause bilateral lesions resulting in primary aldosteronism.LGR5, DACH1, and neuron-specific proteins are highly expressed in the zona glomerulosa and regulate aldosterone production. SUMMARY: Most mutations causing primary aldosteronism are in genes encoding cation channels or pumps, leading to increased calcium influx. Genotype-phenotype analyses identified two broad subtypes of aldosterone-producing adenomas (APAs), zona fasciculata-like and zona glomerulosa-like, and the likelihood of under-diagnosed zona glomerulosa-like APAs because of small size. Zona fasciculata-like APAs are only associated with KCNJ5 mutations, whereas zona glomerulosa-like APAs are associated with mutations in ATPase pumps, CACNA1D, and CTNNB1. The frequency of APAs, and the multiplicity of causal mutations, suggests a pre-existing drive for these mutations. We speculate that these mutations are selected for protecting against tonic inhibition of aldosterone in human zona glomerulosa, which express genes inhibiting aldosterone production.
Assuntos
Aldosterona/metabolismo , Hiperaldosteronismo/genética , Hiperaldosteronismo/metabolismo , HumanosRESUMO
Primary aldosteronism is present in ≈10% of hypertensives. We previously performed a microarray assay on aldosterone-producing adenomas and their paired zona glomerulosa and fasciculata. Confirmation of top genes validated the study design and functional experiments of zona glomerulosa selective genes established the role of the encoded proteins in aldosterone regulation. In this study, we further analyzed our microarray data using AmiGO 2 for gene ontology enrichment and Ingenuity Pathway Analysis to identify potential biological processes and canonical pathways involved in pathological and physiological aldosterone regulation. Genes differentially regulated in aldosterone-producing adenoma and zona glomerulosa were associated with steroid metabolic processes gene ontology terms. Terms related to the Wnt signaling pathway were enriched in zona glomerulosa only. Ingenuity Pathway Analysis showed "NRF2-mediated oxidative stress response pathway" and "LPS (lipopolysaccharide)/IL-1 (interleukin-1)-mediated inhibition of RXR (retinoid X receptor) function" were affected in both aldosterone-producing adenoma and zona glomerulosa with associated genes having up to 21- and 8-fold differences, respectively. Comparing KCNJ5-mutant aldosterone-producing adenoma, zona glomerulosa, and zona fasciculata samples with wild-type samples, 138, 56, and 59 genes were differentially expressed, respectively (fold-change >2; P<0.05). ACSS3, encoding the enzyme that synthesizes acetyl-CoA, was the top gene upregulated in KCNJ5-mutant aldosterone-producing adenoma compared with wild-type. NEFM, a gene highly upregulated in zona glomerulosa, was upregulated in KCNJ5 wild-type aldosterone-producing adenomas. NR4A2, the transcription factor for aldosterone synthase, was highly expressed in zona fasciculata adjacent to a KCNJ5-mutant aldosterone-producing adenoma. Further interrogation of these genes and pathways could potentially provide further insights into the pathology of primary aldosteronism.
Assuntos
Adenoma/genética , Aldosterona/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/genética , Hiperaldosteronismo/genética , Fatores de Transcrição/genética , Adenoma/fisiopatologia , Córtex Suprarrenal/patologia , Córtex Suprarrenal/fisiologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Hiperaldosteronismo/fisiopatologia , Feocromocitoma/genética , Feocromocitoma/fisiopatologia , Estudos de Amostragem , Regulação para Cima , Via de Sinalização Wnt , Zona Fasciculada/metabolismo , Zona Glomerulosa/metabolismoRESUMO
Aldosterone-producing adenomas (APAs) vary in phenotype and genotype. Zona glomerulosa (ZG)-like APAs frequently have mutations of an L-type calcium channel (LTCC) CaV1.3. Using a novel antagonist of CaV1.3, compound 8, we investigated the role of CaV1.3 on steroidogenesis in the human adrenocortical cell line, H295R, and in primary human adrenal cells. This investigational drug was compared with the common antihypertensive drug nifedipine, which has 4.5-fold selectivity for the vascular LTCC, CaV1.2, over CaV1.3. In H295R cells transfected with wild-type or mutant CaV1.3 channels, the latter produced more aldosterone than wild-type, which was ameliorated by 100 µM of compound 8. In primary adrenal and non-transfected H295R cells, compound 8 decreased aldosterone production similar to high concentration of nifedipine (100 µM). Selective CaV1.3 blockade may offer a novel way of treating primary hyperaldosteronism, which avoids the vascular side effects of CaV1.2-blockade, and provides targeted treatment for ZG-like APAs with mutations of CaV1.3.
Assuntos
Aldosterona/metabolismo , Canais de Cálcio Tipo L/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo L/genética , Linhagem Celular , Células Cultivadas , Genótipo , Humanos , Mutação , Nifedipino/farmacologia , Transporte ProteicoRESUMO
Common somatic mutations in CACNAID and ATP1A1 may define a subgroup of smaller, zona glomerulosa (ZG)-like aldosterone-producing adenomas. We have therefore sought signature ZG genes, which may provide insight into the frequency and pathogenesis of ZG-like aldosterone-producing adenomas. Twenty-one pairs of zona fasciculata and ZG and 14 paired aldosterone-producing adenomas from 14 patients with Conn's syndrome and 7 patients with pheochromocytoma were assayed by the Affymetrix Human Genome U133 Plus 2.0 Array. Validation by quantitative real-time polymerase chain reaction was performed on genes >10-fold upregulated in ZG (compared with zona fasciculata) and >10-fold upregulated in aldosterone-producing adenomas (compared with ZG). DACH1, a gene associated with tumor progression, was further analyzed. The role of DACH1 on steroidogenesis, transforming growth factor-ß, and Wnt signaling activity was assessed in the human adrenocortical cell line, H295R. Immunohistochemistry confirmed selective expression of DACH1 in human ZG. Silencing of DACH1 in H295R cells increased CYP11B2 mRNA levels and aldosterone production, whereas overexpression of DACH1 decreased aldosterone production. Overexpression of DACH1 in H295R cells activated the transforming growth factor-ß and canonical Wnt signaling pathways but inhibited the noncanonical Wnt signaling pathway. Stimulation of primary human adrenal cells with angiotensin II decreased DACH1 mRNA expression. Interestingly, there was little overlap between our top ZG genes and those in rodent ZG. In conclusion, (1) the transcriptome profile of human ZG differs from rodent ZG, (2) DACH1 inhibits aldosterone secretion in human adrenals, and (3) transforming growth factor-ß signaling pathway is activated in DACH1 overexpressed cells and may mediate inhibition of aldosterone secretion in human adrenals.
Assuntos
Carcinoma Adrenocortical/genética , Aldosterona/metabolismo , Proteínas do Olho/genética , Regulação Neoplásica da Expressão Gênica , RNA Neoplásico/genética , Fatores de Transcrição/genética , Fator de Crescimento Transformador beta/genética , Zona Glomerulosa/metabolismo , Carcinoma Adrenocortical/metabolismo , Carcinoma Adrenocortical/patologia , Progressão da Doença , Proteínas do Olho/biossíntese , Feminino , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais , Fatores de Transcrição/biossíntese , Fator de Crescimento Transformador beta/biossíntese , Células Tumorais Cultivadas , Zona Glomerulosa/patologiaRESUMO
CONTEXT: Aldosterone synthesis and cellularity in the human adrenal zona glomerulosa (ZG) is sparse and patchy, presumably due to salt excess. The frequency of somatic mutations causing aldosterone-producing adenomas (APAs) may be a consequence of protection from cell loss by constitutive aldosterone production. OBJECTIVE: The objective of the study was to delineate a process in human ZG, which may regulate both aldosterone production and cell turnover. DESIGN: This study included a comparison of 20 pairs of ZG and zona fasciculata transcriptomes from adrenals adjacent to an APA (n = 13) or a pheochromocytoma (n = 7). INTERVENTIONS: Interventions included an overexpression of the top ZG gene (LGR5) or stimulation by its ligand (R-spondin-3). MAIN OUTCOME MEASURES: A transcriptome profile of ZG and zona fasciculata and aldosterone production, cell kinetic measurements, and Wnt signaling activity of LGR5 transfected or R-spondin-3-stimulated cells were measured. RESULTS: LGR5 was the top gene up-regulated in ZG (25-fold). The gene for its cognate ligand R-spondin-3, RSPO3, was 5-fold up-regulated. In total, 18 genes associated with the Wnt pathway were greater than 2-fold up-regulated. ZG selectivity of LGR5, and its absence in most APAs, were confirmed by quantitative PCR and immunohistochemistry. Both R-spondin-3 stimulation and LGR5 transfection of human adrenal cells suppressed aldosterone production. There was reduced proliferation and increased apoptosis of transfected cells, and the noncanonical activator protein-1/Jun pathway was stimulated more than the canonical Wnt pathway (3-fold vs 1.3-fold). ZG of adrenal sections stained positive for apoptosis markers. CONCLUSION: LGR5 is the most selectively expressed gene in human ZG and reduces aldosterone production and cell number. Such conditions may favor cells whose somatic mutation reverses aldosterone inhibition and cell loss.
Assuntos
Glândulas Suprarrenais/metabolismo , Aldosterona/biossíntese , Receptores Acoplados a Proteínas G/fisiologia , Via de Sinalização Wnt/genética , Glândulas Suprarrenais/citologia , Contagem de Células , Regulação para Baixo/genética , Perfilação da Expressão Gênica , Humanos , Imuno-Histoquímica , Análise em Microsséries , Células Tumorais Cultivadas , Regulação para Cima/genética , Zona Fasciculada/metabolismo , Zona Glomerulosa/citologia , Zona Glomerulosa/metabolismoRESUMO
At least 5% of individuals with hypertension have adrenal aldosterone-producing adenomas (APAs). Gain-of-function mutations in KCNJ5 and apparent loss-of-function mutations in ATP1A1 and ATP2A3 were reported to occur in APAs. We find that KCNJ5 mutations are common in APAs resembling cortisol-secreting cells of the adrenal zona fasciculata but are absent in a subset of APAs resembling the aldosterone-secreting cells of the adrenal zona glomerulosa. We performed exome sequencing of ten zona glomerulosa-like APAs and identified nine with somatic mutations in either ATP1A1, encoding the Na(+)/K(+) ATPase α1 subunit, or CACNA1D, encoding Cav1.3. The ATP1A1 mutations all caused inward leak currents under physiological conditions, and the CACNA1D mutations induced a shift of voltage-dependent gating to more negative voltages, suppressed inactivation or increased currents. Many APAs with these mutations were <1 cm in diameter and had been overlooked on conventional adrenal imaging. Recognition of the distinct genotype and phenotype for this subset of APAs could facilitate diagnosis.
Assuntos
Doenças do Córtex Suprarrenal/genética , Canais de Cálcio Tipo L/genética , Hipertensão/genética , Mutação , ATPase Trocadora de Sódio-Potássio/genética , Doenças do Córtex Suprarrenal/complicações , Doenças do Córtex Suprarrenal/diagnóstico , Substituição de Aminoácidos , Canais de Cálcio Tipo L/química , Canais de Cálcio Tipo L/metabolismo , Análise por Conglomerados , Feminino , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/genética , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/metabolismo , Perfilação da Expressão Gênica , Humanos , Hipertensão/diagnóstico , Hipertensão/etiologia , Masculino , Conformação Proteica , ATPase Trocadora de Sódio-Potássio/química , ATPase Trocadora de Sódio-Potássio/metabolismoRESUMO
OBJECTIVE: Adrenal aldosterone-producing adenomas (APAs) are an increasingly recognized cause of primary aldosteronism, and somatic mutations within the KCNJ5 gene encoding an inwardly rectifying K(+) channel (also called GIRK4 or Kir3.4) have been identified by several groups including our own. We identified the previously noted G151R and L168R mutations in the region of a selectivity filter of the channel as well as a previously unreported 3-base deletion, delI157. Here, we report the functional properties of KCNJ5 channels carrying this novel delI157 mutation. METHODS: The delI157 mutation was introduced into wild-type KCNJ5 sequences to allow its expression in both H295R cells and Xenopus oocytes to study its expression and electrophysiology, respectively. RESULTS: In the adrenal cell line H295R, the delI157 mutant expresses and traffics normally to the cell surface. However, the current-voltage behavior of the mutant in oocytes is distinct from wild-type channels and mimics closely other selectivity filter mutations. In particular, its ability to support substantial current when extracellular K(+) is replaced by Na(+). We also report for the first time that the mutants have reduced sensitivity to the KCNJ5 inhibitor tertiapin-Q that binds to the external vestibule of the channel pore. CONCLUSION: This novel KCNJ5 mutation behaves like the three selectivity filter mutations previously reported in APAs depolarizing the cell and showing reduced cation selectivity. The reduced sensitivity to tertiapin-Q suggests that the abnormal Na(+) permeability of these selectivity mutations does indeed reflect structural changes around the mouth of the ion channel.
Assuntos
Adenoma/metabolismo , Aldosterona/biossíntese , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/genética , Mutação , Adenoma/genética , Linhagem Celular , Humanos , Técnicas de Patch-ClampRESUMO
Primary hyperaldosteronism, one cause of which is aldosterone-producing adenomas (APAs), may account for ≤5% to 10% of cases of essential hypertension. Germline mutations have been identified in 2 rare familial forms of primary hyperaldosteronism, but it has been reported recently that somatic mutations of the KCNJ5 gene, which encodes a potassium channel, are present in some sporadic nonsyndromic APAs. To address this further we screened 2 large collections of sporadic APAs from the United Kingdom and Australia (totalling 73) and found somatic mutations in the selectivity filter of KCNJ5 in 41% (95% CI: 31% to 53%) of the APAs (30 of 73). These included the previously noted nonsynonymous mutations, G151R and L158R, and an unreported 3-base deletion, delI157, in the region of the selectivity filter. APAs containing a somatic KCNJ5 mutation were significantly larger than those without (1.61 cm [95% CI: 1.39-1.83 cm] versus 1.04 cm [95% CI: 0.91-1.17 cm]; P<0.0001) but with substantial overlap in size between genotypes. The APAs carrying a mutation, but not those without, also consistently lacked a postural aldosterone response, suggesting a physiologically distinct subtype. Hence, somatic KCNJ5 mutations are not restricted to large APAs (>2 cm), and their frequency in our unselected series suggests they are common and could be important in the molecular pathogenesis of many sporadic cases of APA.
Assuntos
Adenoma/complicações , Neoplasias das Glândulas Suprarrenais/complicações , Aldosterona/biossíntese , DNA de Neoplasias/genética , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/genética , Mutação em Linhagem Germinativa , Hiperaldosteronismo/genética , Adenoma/sangue , Adenoma/genética , Neoplasias das Glândulas Suprarrenais/sangue , Neoplasias das Glândulas Suprarrenais/genética , Glândulas Suprarrenais/metabolismo , Glândulas Suprarrenais/patologia , Feminino , Predisposição Genética para Doença , Humanos , Hiperaldosteronismo/sangue , Hiperaldosteronismo/etiologia , Masculino , Pessoa de Meia-Idade , Fenótipo , Reação em Cadeia da Polimerase , PrognósticoRESUMO
CONTEXT: Identification of unilateral aldosterone-producing (Conn's) adenomas has traditionally required lateralization by the invasive and technically difficult procedure of adrenal vein sampling (AVS). (11)C-metomidate, a potent inhibitor of adrenal steroidogenic enzymes, is a positron emission tomography (PET) radiotracer that is selectively accumulated by Conn's adenomas. OBJECTIVE: The objective of the study was to compare the sensitivity and specificity of (11)C-metomidate PET-computed tomography (CT) against the current gold standard of AVS. DESIGN: The design of the study was within-patient comparison of diagnostic techniques. SETTING: The study was conducted at a single center-university teaching hospital. PATIENTS: Thirty-nine patients with primary hyperaldosteronism (PHA) and five with nonfunctioning adenomas (incidentalomas) participated in the study. INTERVENTION(S): The first six PHA patients were studied on three occasions to determine whether steroid pretreatment reduced (11)C-metomidate uptake by normal adrenal. Subsequent patients received dexamethasone for 3 d prior to injection of (11)C-metomidate 150-500 MBq. MAIN OUTCOME MEASURE(S): Maximum standardized uptake values (SUV(max)) over regions of interest determined from 35-45 min after injection were measured. RESULTS: Dexamethasone increased tumor to normal adrenal SUV(max) ratio by 25.6 ± 5.0% (P < 0.01). PET-CT visualized subcentimeter adenomas and distinguished hot from cold adenomas within a gland. In 25 patients with PHA and AVS lateralization to the side of an adenoma, SUV(max) over tumor (mean ± sem) of 21.7 ± 1.6 was greater than over normal adrenal, 13.8 ± 0.6 (P = 0.00003); this difference was absent in 10 patients without lateralization on AVS (P = 0.28) and in four of five incidentalomas. On receiver-operator characteristics analysis, an SUV(max) ratio of 1.25:1 provided a specificity of 87% [95% confidence interval (69, 104)] and sensitivity of 76% (59, 93); in tumors with SUV(max) greater than 17, the specificity rose to 100%. CONCLUSIONS: (11)C-metomidate PET-CT is a sensitive and specific noninvasive alternative to AVS in the management of PHA.
Assuntos
Neoplasias do Córtex Suprarrenal/diagnóstico por imagem , Adenoma Adrenocortical/diagnóstico por imagem , Aldosterona/metabolismo , Etomidato/análogos & derivados , Imagem Multimodal/métodos , Tomografia por Emissão de Pósitrons , Tomografia Computadorizada por Raios X , Neoplasias do Córtex Suprarrenal/metabolismo , Adenoma Adrenocortical/metabolismo , Adulto , Radioisótopos de Carbono , Humanos , Achados Incidentais , Pessoa de Meia-Idade , Curva ROC , Sensibilidade e EspecificidadeRESUMO
CONTEXT: Aldosterone-producing adenomas (APA) are heterogeneous. The recent finding of somatic KCNJ5 mutations suggests a genetic explanation. OBJECTIVES: The objectives of this study were the following: 1) to compare transcriptional profiles in APA and adjacent adrenal gland (AAG); 2) to test whether gene expression profile clusters with different cell histology; and 3) to measure the frequency of KCNJ5 mutations and determine the genotype-phenotype relationship. DESIGN/SETTING: The design of the study included laboratory analyses of 46 unselected APA. PATIENTS: The patients in this study had primary hyperaldosteronism with unilateral APA. INTERVENTIONS: The objectives of this study were the following: 1) Illumina beadchip analysis of RNA from eight paired APA-AAG; 2) a blinded review of cell histology for 46 APA; 3) laser capture microdissection of zona glomerulosa (ZG) and zona fasciculata (ZF) cells; and 4) sequencing of KCNJ5 in 46 APA. MAIN OUTCOME MEASURES: The main outcome measures of this study were the following: 1) a difference in gene expression profile and a correlation with histological markers of ZF; 2) a frequency of KCNJ5 mutations and phenotypic comparisons of wild type with mutant APA. RESULTS: The results of the study were the following: 1) a cluster analysis of microarray data separated APA from AAG. APA at opposite ends of the APA cluster had an approximately 800-fold difference in CYP17A1 mRNA expression, whereas histology showed 0% ZF-like cells in one vs. 100% in the other. A heat map ranking APA by CYP17A1 expression correctly predicted several genes (e.g. KCNK1, SLC24A3) to be enriched in laser capture microdissection samples of ZG; 2) known or novel mutations of KCNJ5 were found in 20 of 46 consecutive APA [43% (95% confidence interval [CI] (29, 58)%)]. The APA with KCNJ5 gene mutations were larger compared with tumors harboring the wild type, 1.63 [95% CI (1.37, 1.88)] vs. 1.14 [0.97, 1.30] cm (P = 0.0013), had predominantly ZF-like cells, and their CYP17A1 (log(2)-fold change) was higher than in wild type: -0.96 [95% CI (-0.07, -1.85)] vs. -2.54 [-1.61, -3.46], (P = 0.017). CONCLUSIONS: KCNJ5 mutations are common in APA, particularly those arising from ZF. The long-recognized heterogeneity among APA may have a genetic basis.
Assuntos
Adenoma/genética , Neoplasias das Glândulas Suprarrenais/genética , Aldosterona/biossíntese , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/genética , Hiperaldosteronismo/genética , Zona Fasciculada/metabolismo , Zona Glomerulosa/metabolismo , Adenoma/metabolismo , Adenoma/patologia , Neoplasias das Glândulas Suprarrenais/metabolismo , Neoplasias das Glândulas Suprarrenais/patologia , Adulto , Idoso , Feminino , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/metabolismo , Genótipo , Humanos , Hiperaldosteronismo/metabolismo , Hiperaldosteronismo/patologia , Masculino , Pessoa de Meia-Idade , Fenótipo , Zona Fasciculada/patologia , Zona Glomerulosa/patologiaRESUMO
The components of the classical renal aldosterone response pathway are expressed in human adrenocortical cells; however, studies in H295R cells have shown that pharmacological manipulation of this pathway has no effect on aldosterone production. We have characterised aldosterone and cortisol production by primary human adrenocortical cells and tested the hypothesis that a mineralocorticoid response pathway modulates aldosterone secretion. Aldosterone production by cells obtained from normal adrenal cortex was stimulated by angiotensin II, extracellular K(+) and a reduction in extracellular Na(+). Conn's adenoma cells, in comparison, produced higher aldosterone/cortisol ratios and were less responsive to angiotensin II and extracellular Na(+). Close coupling of aldosterone and cortisol secretion was observed in all adrenocortical cells. The mineralocorticoid receptor antagonists, eplerenone and potassium canrenoate, had no significant effect on aldosterone or cortisol production. In contrast, the glucocorticoid receptor antagonist, mifepristone, and the Na(+) uptake inhibitor, amiloride, had significant inhibitory effects on steroid production. Our current experiments do not support the hypothesis that an adrenal aldosterone-response pathway mediates the negative feedback of aldosterone on its own release, but do raise interest in the glucocorticoid receptor and downstream targets of the mineralocorticoid receptor as mediators of corticosteroid production.
Assuntos
Córtex Suprarrenal/metabolismo , Córtex Suprarrenal/patologia , Adenoma Adrenocortical/metabolismo , Adenoma Adrenocortical/patologia , Aldosterona/metabolismo , Tumores Neuroendócrinos/metabolismo , Tumores Neuroendócrinos/patologia , Adrenalectomia , Adenoma Adrenocortical/cirurgia , Aldosterona/biossíntese , Angiotensina II/farmacologia , Células Cultivadas , Espaço Extracelular/efeitos dos fármacos , Espaço Extracelular/metabolismo , Humanos , Hidrocortisona/biossíntese , Mineralocorticoides/metabolismo , Tumores Neuroendócrinos/cirurgia , Potássio/metabolismo , Sódio/metabolismoRESUMO
We have unexpectedly found expression of the epithelial Na(+) channel (ENaC) in human adrenocortical cells and tested the hypothesis that these cells contain the components of an aldosterone response pathway. Tissue was obtained from patients undergoing adrenalectomy and mRNA and protein expression of recognised components of an aldosterone-response pathway were determined by RT-PCR and Western blotting. The effects of mineralocorticoid receptor agonists and antagonists, amiloride analogues, and extracellular Na(+) on basal and stimulated aldosterone release from immortalised (H295R) cells were determined by radioimmunoassay. Expression of mRNA for alpha-, beta- and gamma-subunits of ENaC, the mineralocorticoid receptor, Nedd4L, Sgk1 and 11beta hydroxysteroid dehydrogenase type II was confirmed in human adrenal cortex. Using Western blotting alpha-, beta- and gamma-ENaC expression was demonstrated in adrenocortical cells. Measurements of 24 h aldosterone release from H295R cells showed stimulation by K(+) and angiotensin II, suppression by both Na(+) and high-concentration 5-(N-ethyl-N-isopropyl) amiloride (EIPA, blocker of Na(+)-H(+) exchange) and no change with benzamil (ENaC blocker). (22)Na-uptake into H295R cells was inhibited by EIPA, but not by benzamil. Our experiments suggest that the components of an aldosterone response pathway are present in human adrenal cortex. Studies in H295R cells, however, suggest that ENaC is not an important mediator of (22)Na-uptake or aldosterone production. Further studies are required to determine the importance of an adrenal aldosterone response pathway.