RESUMO
CONTEXT: The search for somatic mutations in adrenals resected from primary aldosteronism (PA) patients is being performed by Sanger sequencing, often implemented with immunohistochemistry (IHC)-guidance focused on aldosterone-producing (CYP11B2-positive) areas. OBJECTIVE: To investigate the impact of double IHC for CYP11B1 and CYP11B2 on Sanger and next generation sequencing (NGS). METHODS: We investigated 127 consecutive adrenal aldosterone producing adenoma from consenting surgically cured PA patients using double IHC for CYP11B1 and CYP11B2, Sanger sequencing and NGS. RESULTS: Double IHC for CYP11B2 and CYP11B1 revealed 3 distinct patterns: CYP11B2-positive adenoma (pattern 1), mixed CYP11B1/CYP11B2-positive adenoma (pattern 2), and adrenals with multiple small CYP11B2-positive nodules (pattern 3). Sanger sequencing allowed detection of KCNJ5 mutations in 44% of the adrenals; NGS revealed such mutations in 10% of those negative at Sanger and additional mutations in 61% of the cases. Importantly the rate of KCNJ5 mutations differed across patterns: 17.8% in pattern 1, 71.4% in pattern 2, and 10.7% in pattern 3 (χ2=22.492, p<0.001). CONCLUSIONS: NGS allowed detection of mutations in many adrenals that tested negative at Sanger sequencing. Moreover, the different distribution of KCNJ5 mutations across IHC patterns indicates that IHC-guided sequencing protocols selecting CYP11B2-positive areas could furnish results that might not be representative of the entire mutational status of the excised adrenal, which is important at a time when KCNJ5 mutations are suggested to drive management of APA patient.
RESUMO
BACKGROUND: Tubulointerstitial fibrosis, the final outcome of most kidney diseases, involves activation of epithelial mesenchymal transition (EMT). Endothelin-1 (ET-1) activates EMT in cancer cells, but it is not known whether it drives EMT in the kidney. We therefore tested the hypothesis that tubulointerstitial fibrosis involves EMT driven by ET-1. METHODS AND RESULTS: Transgenic TG[mRen2]27 (TGRen2) rats developing fulminant angiotensin II-dependent hypertension with prominent cardiovascular and renal damage were submitted to drug treatments targeted to ET-1 and/or angiotensin II receptor or left untreated (controls). Expressional changes of E-cadherin and α-smooth muscle actin (αSMA) were examined as markers of renal EMT. In human kidney HK-2 proximal tubular cells expressing the ETB receptor subtype, the effects of ET-1 with or without ET-1 antagonists were also investigated. The occurrence of renal fibrosis was associated with EMT in control TGRen2 rats, as evidenced by decreased E-cadherin and increased αSMA expression. Irbesartan and the mixed ET-1 receptor antagonist bosentan prevented these changes in a blood pressure-independent fashion (P < 0.001 for both versus controls). In HK-2 cells ET-1 blunted E-cadherin expression, increased αSMA expression (both P < 0.01), collagen synthesis, and metalloproteinase activity (P < 0.005, all versus untreated cells). All changes were prevented by the selective ETB receptor antagonist BQ-788. Evidence for involvement of the Rho-kinase signaling pathway and dephosphorylation of Yes-associated protein in EMT was also found. CONCLUSIONS: In angiotensin II-dependent hypertension, ET-1 acting via ETB receptors and the Rho-kinase and Yes-associated protein induces EMT and thereby renal fibrosis.
Assuntos
Actinas/efeitos dos fármacos , Antagonistas de Receptores de Angiotensina/farmacologia , Caderinas/efeitos dos fármacos , Endotelina-1/farmacologia , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Hipertensão/metabolismo , Nefropatias/metabolismo , Actinas/metabolismo , Animais , Animais Geneticamente Modificados , Compostos de Bifenilo/farmacologia , Bosentana , Caderinas/metabolismo , Antagonistas do Receptor de Endotelina B/farmacologia , Endotelina-1/antagonistas & inibidores , Fibrose , Humanos , Hipertensão/complicações , Irbesartana , Rim/patologia , Nefropatias/etiologia , Nefropatias/patologia , Túbulos Renais Proximais/citologia , Túbulos Renais Proximais/efeitos dos fármacos , Oligopeptídeos/farmacologia , Piperidinas/farmacologia , Ratos , Receptor de Endotelina B/metabolismo , Transdução de Sinais , Sulfonamidas/farmacologia , Tetrazóis/farmacologia , Quinases Associadas a rho/metabolismoRESUMO
BACKGROUND: A high aldosterone-renin ratio (ARR) is commonly used to identify primary aldosteronism, but the ARR is high when renin is low, even if plasma aldosterone concentration values are normal, suggesting the existence of 'normoaldosteronemic' primary aldosteronism. However, most such cases did not undergo adrenalectomy; moreover, because of the lack of antibody for the human CYP11B2 (aldosterone synthase), conclusive demonstration of a normoaldosteronemic aldosterone-producing adenoma was not possible thus far. METHOD: In 2003, a lady presented with severe hypertension a right adrenal nodule, low renin, high ARR, but normal plasma aldosterone concentration. As adrenal vein sampling showed lateralized aldosterone secretion, she underwent left adrenalectomy, which consistently normalized blood pressure (BP) and renin during 11-year follow-up. RESULT AND CONCLUSION: The development of a novel monoclonal antibody for the human CYP11B2 in 2014 allowed immunochemically identification of a CYP11B2-positive adenoma in the resected adrenal. Moreover, this case unequivocally demonstrates for the first time the existence of normoaldosteronemic aldosterone-producing adenoma, which suggests that many cases of 'low renin-essential hypertension' might instead have a surgically curable form of primary aldosteronism.
Assuntos
Adenoma/sangue , Adenoma/química , Neoplasias das Glândulas Suprarrenais/sangue , Neoplasias das Glândulas Suprarrenais/química , Aldosterona/sangue , Citocromo P-450 CYP11B2/análise , Renina/sangue , Adenoma/cirurgia , Neoplasias das Glândulas Suprarrenais/cirurgia , Adrenalectomia , Adulto , Pressão Sanguínea , Feminino , Humanos , Hiperaldosteronismo/sangue , Hiperaldosteronismo/cirurgia , Hipertensão/etiologia , ImunoquímicaRESUMO
Fertile women have lower blood pressure and cardiovascular risk than age-matched men, which suggests that estrogens exert cardiovascular protective effects. However, whether 17 ß-estradiol (E2) blunts aldosterone secretion, and thereby affects the gender dimorphism of blood pressure, is unknown. We therefore sought for the estrogen receptor (ER) subtypes in human adrenocortical tissues ex vivo by performing gene and protein expression studies. We also investigated the effect of E2 on aldosterone synthesis and the involved receptors through in vitro functional experiments in the adrenocortical cells HAC15. We found that in the human adrenal cortex and aldosterone-producing adenoma cells, the most expressed ERs were the ERß and the G protein-coupled receptor-1 (GPER-1), respectively. After selective ERß blockade, E2 (10 nmol/L) markedly increased both the expression of aldosterone synthase and the production of aldosterone (+5- to 7-fold vs baseline, P < .001). Under the same condition, the GPER-1 receptor agonist 1-[4-(6-bromo-benzo (1, 3)dioxol-5-yl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c] quinolin-8-yl]-ethanone (G-1) (10 nmol/L) mimicked this effect, which was abrogated by cotreatment with either the GPER-1 receptor antagonist (3aS*,4R*,9bR*)-4-(6-Bro-mo-1,3-benzodioxol-5-yl)-3a,4,5,9b-3H-cyclopenta[c]quinoline (G-15), or a selective protein kinase A inhibitor 8-Bromo-2-monobutyryladenosine-3,5-cyclic mono-phosphorothioate, Rp-isomer. Silencing of the ERß significantly raised aldosterone synthase expression and aldosterone production. Conversely, silencing of the GPER-1 lowered aldosterone synthase gene and protein expression. Moreover, it blunted the stimulatory effect of E2 on aldosterone synthase that was seen during ERß blockade. These results support the conclusion that in humans, E2 inhibits aldosterone synthesis by acting via ERß. Pharmacologic disinhibition of ERß unmasks a potent secretagogue effect of E2 that involves GPER-1 and protein kinase A signaling.
Assuntos
Aldosterona/biossíntese , Estradiol/farmacologia , Receptor beta de Estrogênio/metabolismo , Receptores de Estrogênio/fisiologia , Receptores Acoplados a Proteínas G/fisiologia , Córtex Suprarrenal/metabolismo , Receptor beta de Estrogênio/agonistas , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Lactente , Ligantes , Masculino , Redes e Vias Metabólicas/efeitos dos fármacos , Redes e Vias Metabólicas/genética , Interferência de RNA , Receptores de Estrogênio/antagonistas & inibidores , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Células Tumorais CultivadasRESUMO
CONTEXT: Understanding the function of the KCNJ5 potassium channel through characterization of naturally occurring novel mutations is key for dissecting the mechanism(s) of autonomous aldosterone secretion in primary aldosteronism. OBJECTIVE: We sought for such novel KCNJ5 channel mutations in a large database of patients with aldosterone-producing adenomas (APAs). METHODS: We discovered a novel somatic c.446insAAC insertion, resulting in the mutant protein KCNJ5-insT149, in a patient with severe drug-resistant hypertension among 195 consecutive patients with a conclusive diagnosis of APA, 24.6% of whom showed somatic KCNJ5 mutations. By site-directed mutagenesis, we created the mutated cDNA that was transfected, along with KCNJ3 cDNA, in mammalian cells. We also localized CYP11B2 in the excised adrenal gland with immunohistochemistry and immunofluorescence using an antibody specific to human CYP11B2. Whole-cell patch clamp recordings, CYP11B2 mRNA, aldosterone measurement, and molecular modeling were performed to characterize the novel KCNJ5-insT149 mutation. RESULTS: Compared with wild-type and mock-transfected adrenocortical cells, HAC15 cells expressing the mutant KCNJ5 showed increased CYP11B2 expression and aldosterone secretion. Mammalian cells expressing the mutated KCNJ5-insT149 channel exhibited a strong Na(+) inward current and, in parallel, a substantial rise in intracellular Ca(2+), caused by activation of voltage-gated Ca(2+) channels and reduced Ca(2+) elimination by Na(+)/Ca(2+) exchangers, as well as an increased production of aldosterone. CONCLUSIONS: This novel mutation shows pathological Na(+) permeability, membrane depolarization, raised cytosolic Ca(2+), and increased aldosterone synthesis. Hence, a novel KCNJ5 channelopathy located after the pore α-helix preceding the selectivity filter causes constitutive secretion of aldosterone with ensuing resistant hypertension in a patient with a small APA.