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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.

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 ET B 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 ET B 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 ET B receptors and the Rho‐kinase and Yes‐associated protein induces EMT and thereby renal fibrosis.

Background: Primary Aldosteronism (PA), the most common form of endocrine hypertension, is usually identified by a high aldosterone-renin ratio (ARR) alongside elevated plasma aldosterone concentration (PAC) values. Normo-aldosteronemic PA is also being diagnosed when only the high ARR was driven by low renin because PAC was normal. However, whether this entity truly exists remains contentious since most such cases did not undergo surgical confirmation and even when adrenalectomy was performed no demonstration of an aldosterone-producing adenoma (APA) could be obtained at immunohistochemistry. Case Description: In 2003 a young lady presented with severe hypertension, low plasma renin activity (PRA), but consistently normal PAC values. She undertook a magnetic resonance that revealed a small adenoma on the right side, but adrenal vein sampling (AVS) showed left lateralization of aldosterone secretion. Therefore, she underwent left laparoscopic adrenal surgery, which determined complete normalization of blood pressure and PRA long term follow up (12 years). In 2014 the development of novel monoclonal antibodies for the human aldosterone synthase CYP11B2 and 11β-hydroxylase CYP11B1, allowed us to immunochemically characterize the resected adrenal gland. Double immunostaining for CYP11B2 and CYP11B1 showed a small CYP11B2-positive adenoma, thus unequivocally proving the presence of an APA. Conclusions: This case provides compelling evidence for the existence of normo-aldosteronemic APA and suggests that many cases that we dismiss as “low renin-essential hypertension” might instead have an undetected surgically curable APA.

We tested the hypothesis that tubulo-interstitial fibrosis (TIF), the final outcome of most kidney diseases, involves activation of epithelial mesenchymal transition (EMT) through angiotensin II- and endothelin-1 (ET-1)-mechanisms. In a transgenic model of fulminant angiotensin II-dependent hypertension with early prominent renal damage, the TG(mRen2)27 rat (TGRen2), we found that the development of renal damage implied a decrease of the epithelial marker E-cadherin and an increase of the mesenchymal markers alpha SMA and vimentin, which indicated the occurrence of EMT. As treatment with the angiotensin II type 1 receptor antagonist irbesartan, or with the mixed ET-1 receptor antagonist bosentan, prevented these changes an involvement of both angiotensin II and ET-1 in EMT is suggested. To confirm this contention we exposed HK-2 human kidney tubular cells to ET-1. This showed that ET-1 blunted the expression of E-cadherin, increased that of alpha SMA and vimentin, enhanced the synthesis of collagen, and also the activity of metalloproteinases (MMP). These changes implicated activation of Rho-kinase signaling pathway and de-phosphorylation of Yes-associated protein (YAP). Hence, ex vivo and in vitro experiments demonstrated that EMT is a fundamental process in the TIF that accompanies the development of angiotensin II-dependent hypertension. Moreover, they suggested that EMT involves ET-1 acting via ETA and ETB receptors through activation of Rho-kinase and de-activation of YAP pathways.

Primary aldosteronism (PA) entails a group of disorders characterized by excess aldosterone production, relatively independent from the renin–angiotensin system.1 Because PA is the most common form of secondary hypertension and patients with PA have higher cardiovascular morbidity and mortality than age- and sex-matched patients with essential hypertension and the same degree of blood pressure elevation,2–5 early recognition of PA is of utmost importance to cure the high blood pressure and prevent deleterious cardiovascular effects. The 2 major PA subgroups are the unilateral forms, mainly aldosterone-producing adenoma (APA), and bilateral forms, mostly adrenal zona glomerulosa (ZG) hyperplasia, which require surgical or medical treatment, respectively. Adrenal vein sampling (AVS) is the best available method to distinguish between unilateral and bilateral aldosterone hypersecretion.6–8 In medical centers where AVS is available and strict cutoff values for the lateralization index are used,9 almost two thirds of PA cases are attributed to an APA, whereas one third remain classified as bilateral idiopathic forms. The opposite is seen when AVS is unavailable.10 Hence, the availability of AVS greatly affects the recognition of unilateral forms of PA that can be successfully treated with adrenalectomy. After surgery, demonstration of an adenoma at pathology is needed to reach a conclusive diagnosis of the only PA subtype that can be unequivocally diagnosed, eg, APA. After the introduction of the 4 corner criteria,10 an APA can be diagnosed when (1) there is evidence of PA at the screening tests, with an inappropriately high aldosterone/renin ratio and confirmation as needed; (2) aldosterone secretion is lateralized at AVS; (3) an adenoma is detected at pathology; and crucially important, (4) PA is corrected by adrenalectomy. However, at pathology, the identification of an APA may be challenging …

Background: Primary Aldosteronism (PA) is commonly due to an Aldosterone-Producing Adenoma (APA), but until now the lack of a specific antibody for human aldosterone synthase (hCYP11B2) has prevented the identification of APA in the excised adrenal gland. Objectives: To investigate the usefulness of a novel monoclonal antibody for hCYP11B2 for the immuno-phenotypic characterization of a large series of consecutive adrenal glands from APA patients diagnosed by the “four corners criteria” fully characterized for KCNJ5 mutations. Methods: To generate the antibodies, spleen cells from mice immunized with peptides against hCYP11B2 were fused to mouse myeloma cells. CYP11B2 and CYP11B1 were detected with immunohistochemistry in the adrenals from 49 consecutive APA patients. Based on results of a pilot study the following classification was developed: Pattern1: adenoma with uniform CYP11B2 staining + few areas of CYP11B1 staining; Pattern2: adenoma with diffuse CYP11B2 staining + few areas of CYP11B1 staining + some cells co-expressing CYP11B2/CYP11B1; Pattern3: adenoma expressing CYP11B1 + clusters of cells CYP11B2; Pattern4: no adenoma + multiple clusters of sub-capsular CYP11B2 cells; Pattern5: multi-nodular adrenal cortex + clusters of sub-capsular CYP11B2 cells. Results: KCNJ5 mutations were found in 38% of the cases (19/49). With CYP11B2/CYP11B1 immunostaining the following results were found: pattern 1 16.3% (8/49); pattern2 32.7% (15/49); pattern 3 16.3% (7/49); pattern 4 22.4% (10/49); pattern 5 12.2% (5/49). Conclusions: The highly variable steroidogenic enzymes (CYP11B2, CYP11B1) expression in a series of consecutive APA show the existence of at least 5 different patterns, thus challenging the classical notion that PA entails only APA or bilateral hyperplasia.

Context: Understanding the function of the Kir3.4 (KCNJ5 gene) potassium channel through characterization of occurring novel mutations is key for dissecting the mechanism(s) of autonomous aldosterone secretion in primary aldosteronism. Objective: To identify novel KCNJ5 channel mutations and functionally characterize them in a large database of patients with aldosterone-producing adenomas (APA). Methods: We sequenced the APA and germinal DNA of 195 consecutive patients, diagnosed with the four corners criteria of the PAPY study. Among the 24.6% (48/195) APA patients who showed somatic KCNJ5 mutations we discovered a novel c.446insAAC insertion resulting in the mutant protein KCNJ5-insT149 in a patient with severe drug-resistant hypertension. The mutated cDNA generated by site-directed mutagenesis was transfected along with KCNJ3 cDNA in mammalian cells. 17α-hydroxylase, CYP11B1, and CYP11B2 were immunochemically localized in the excised adrenal gland. Whole-cell patch clamp recordings, CYP11B2 mRNA, aldosterone and intracellular Ca2+ measurement (Fura-2), and molecular modeling were performed to characterize the KCNJ5-insT149 mutation. Results: The patient’s high blood pressure was long-term cured; his LVMI fell from 168 to 106 g/m2 after 2 years follow-up. Compared to wild type and mock-transfected HAC15 adrenocortical cells, those expressing the mutant KCNJ5 showed increased CYP11B2 expression (expression fold change: 2.9±0.3, p Conclusions: We identified a novel mutation of the Kir3.4 channelopathy located after the pore α-helix preceding the selectivity filter, which causes pathological Na+ permeability, membrane depolarization, raised cytosolic Ca2+, and constitutive hypersecretion of aldosterone resulting in ensuing pseudo resistant hypertension.

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.We sought for such novel KCNJ5 channel mutations in a large database of patients with aldosterone-producing adenomas (APAs).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.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.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.

Background. Fertile women have lower blood pressure and are held to be at lower risk for cardiovascular events than age-matched man. However, whether this gender dimorphism involves a modulation of aldosterone synthesis by 17 beta-estradiol (E2) is unknown. Aims. i) To investigate estrogen receptor subtypes gene expression in the normal human adrenal cortex (NAC), in aldosterone producing adenoma (APA) and in a human adrenocortical carcinoma cell line (HAC15); ii) To assess the effect of E2 on aldosterone synthase (CYP11B2) gene expression and to identify the receptor subtypes involved in this effect. Methods. We measured the expression of alpha (ERa), beta (ERb) and of G protein-coupled receptor (GPER-1)-1 in NAC and in APA tissue, and in HAC15 cells by real time RT-PCR. After demonstration that HAC15 cells express ERa, ERb, and GPER-1 we stimulated cells with 10-7M E2 alone, or after ERb selective blockade with 10-5M tetrahydrochrysenediol (THC), ERa selective blockade with 10-5M MPP dihydrochloride (MPP), non selective ERa and ERb blockade with 10-5M ICI 182.780, or after selective GPER-1 receptor blockade with 10-5M G-15. The cells were also exposed to the GPER-1 agonist G-1, alone or in the presence of MPP, THC, or Fulvestrant, and/or G-15. Changes of expression of CYP11B2 mRNA, measured with RT RT-PCR, was the experimental endpoint. Results. The quantitative expression of estrogen receptor subtypes was ERb > GPER-1 >> ERα in NAC, GPER-1 > ERb> ERa in APA, and ERb>ERa=GPER-1 in HAC15 cells. E2 alone or on top of selective ERa antagonism did not alter CYP11B2 expression. By contrast, E2 significantly increased CYP11B2 expression (+500 to + 700% from baseline, p Conclusion. E2 potently stimulates aldosterone synthase expression via GPER-1 subtype receptor activation when the ERb is blocked. The ERb-mediated tonic inhibition of aldosterone synthase could contribute to explaining both the lower BP and CV risk of fertile women and the increase of BP after when this tonic ERb-mediated inhibition wanes during menopause or estrogen-modulation treatment.

Objective:Primary Aldosteronism (PA) is commonly associated with the presence of an Aldosterone-Producing Adenoma (APA), but due to the lack of a specific antibody for human aldosterone synthase (hCYP11B2) the identification of aldosterone-producing nodules in the excised adrenal gland has not been