Maria Verena Cicala

Prevalence, Clinical, and Molecular Correlates of KCNJ5 Mutations in Primary Aldosteronism

Primary aldosteronism is the most common form of secondary hypertension. Mutations in the KCNJ5 gene have been described recently in aldosterone-producing adenomas (APAs). The aim of this study was to investigate the prevalence of KCNJ5 mutations in unselected patients with primary aldosteronism and their clinical, biological and molecular correlates. KCNJ5 sequencing was performed on somatic (APA, n=380) and peripheral (APA, n=344; bilateral adrenal hyperplasia, n=174) DNA of patients with primary aldosteronism, collected through the European Network for the Study of Adrenal Tumors. Transcriptome analysis was performed in 102 tumors. Somatic KCNJ5 mutations (p.Gly151Arg or p.Leu168Arg) were found in 34% (129 of 380) of APA. They were significantly more prevalent in females (49%) than males (19%; P<10−3) and in younger patients (42.1±1.0 versus 47.6±0.7 years; P<10−3) and were associated with higher preoperative aldosterone levels (455±26 versus 376±17 ng/L; P=0.012) but not with therapeutic outcome after surgery. Germline KCNJ5 mutations were found neither in patients with APA nor those with bilateral adrenal hyperplasia. Somatic KCNJ5 mutations were specific for APA, because they were not identified in 25 peritumoral adrenal tissues or 16 cortisol-producing adenomas. Hierarchical clustering of transcriptome profiles showed that APAs with p.Gly151Arg or p.Leu168Arg mutations were indistinguishable from tumors without KCNJ5 mutations. In conclusion, although a large proportion of sporadic APAs harbors somatic KCNJ5 mutations, germline mutations are not similarly causative for bilateral adrenal hyperplasia. KCNJ5 mutation carriers are more likely to be females; younger age and higher aldosterone levels at diagnosis suggest that KCNJ5 mutations may be associated with a more florid phenotype of primary aldosteronism.

Effect of KCNJ5 Mutations on Gene Expression in Aldosterone-Producing Adenomas and Adrenocortical Cells

CONTEXT Primary aldosteronism is a heterogeneous disease that includes both sporadic and familial forms. A point mutation in the KCNJ5 gene is responsible for familial hyperaldosteronism type III. Somatic mutations in KCNJ5 also occur in sporadic aldosterone producing adenomas (APA). OBJECTIVE The objective of the study was to define the effect of the KCNJ5 mutations on gene expression and aldosterone production using APA tissue and human adrenocortical cells. METHODS A microarray analysis was used to compare the transcriptome profiles of female-derived APA samples with and without KCNJ5 mutations and HAC15 adrenal cells overexpressing either mutated or wild-type KCNJ5. Real-time PCR validated a set of differentially expressed genes. Immunohistochemical staining localized the KCNJ5 expression in normal adrenals and APA. RESULTS We report a 38% (18 of 47) prevalence of KCNJ5 mutations in APA. KCNJ5 immunostaining was highest in the zona glomerulosa of NA and heterogeneous in APA tissue, and KCNJ5 mRNA was 4-fold higher in APA compared with normal adrenals (P < 0.05). APA with and without KCNJ5 mutations displayed slightly different gene expression patterns, notably the aldosterone synthase gene (CYP11B2) was more highly expressed in APA with KCNJ5 mutations. Overexpression of KCNJ5 mutations in HAC15 increased aldosterone production and altered expression of 36 genes by greater than 2.5-fold (P < 0.05). Real-time PCR confirmed increases in CYP11B2 and its transcriptional regulator, NR4A2. CONCLUSIONS KCNJ5 mutations are prevalent in APA, and our data suggest that these mutations increase expression of CYP11B2 and NR4A2, thus increasing aldosterone production.

Hypertension in Cushing’s Syndrome: From Pathogenesis to Treatment

Hypertension is one of the most distinguishing features of endogenous Cushing’s syndrome (CS), as it is present in about 80% of adult patients whereas in children its prevalence is about 47%. Hypertension in CS is significantly correlated with the duration of hypercortisolism and results from the interplay between several pathophysiological mechanisms regulating plasma volume, peripheral vascular resistance and cardiac output, all of which are increased in this state. Glucocorticoids cause hypertension through several mechanisms: their intrinsic mineralocorticoid activity; through activation of the renin-angiotensin system; by enhancement of vasoactive substances, and by causing suppression of the vasodilatory systems. In addition, glucocorticoids may exert some hypertensive effects on cardiovascular regulation through the CNS via both glucocorticoid and mineralocorticoid receptors. Hypertension in CS usually resolves with surgical removal of the tumor, but some patients require pharmacological antihypertensive treatment both pre- and postoperatively. Thiazides and furosemide should be avoided, while adrenergic blockade and calcium channel antagonists are usually ineffective. Mineralocorticoid receptor antagonists, Ang II blockers and ACE inhibitors are good anti-hypertensive options; PPAR-γ agonists may help in many aspects of the insulin resistance syndrome. The relatively selective glucocorticoid receptor antagonist Mifepristone (RU 486) could reduce blood pressure in patients with CS. Neuromodulatory agents such as the serotonin inhibitors cyproheptadine and ritanserin, valproid acid, dopamine agonists, somatostatin analogs may occasionally be effective, as well as drugs acting directly at the adrenal levels, such as Ketoconazole and aminoglutetimide or even opDDD. Treating hypertension in CS remains a difficult task and a big challenge, in order to decrease the morbidity and mortality associated with the disease.

Combination of sorafenib and everolimus impacts therapeutically on adrenocortical tumor models

Treatment options are insufficient in patients with adrenocortical carcinoma (ACC). Based on the efficacy of sorafenib, a tyrosine kinase inhibitor, and everolimus, an inhibitor of the mammalian target of rapamycin in tumors of different histotype, we aimed at testing these drugs in adrenocortical cancer models. The expression of vascular endothelial growth factor and its receptors (VEGFR1-2) was studied in 18 ACCs, 33 aldosterone-producing adenomas, 12 cortisol-producing adenomas, and six normal adrenal cortex by real-time PCR and immunohistochemistry and by immunoblotting in SW13 and H295R cancer cell lines. The effects of sorafenib and everolimus, alone or in combination, were tested on primary adrenocortical cultures and SW13 and H295R cells by evaluating cell viability and apoptosis in vitro and tumor growth inhibition of tumor cell line xenografts in immunodeficient mice in vivo. VEGF and VEGFR1-2 were detected in all samples and appeared over-expressed in two-thirds of ACC specimens. Dose-dependent inhibition of cell viability was observed particularly in SW13 cells after 24 h treatment with either drug; drug combination produced markedly synergistic growth inhibition. Increasing apoptosis was observed in tumor cells treated with the drugs, particularly with sorafenib. Finally, a significant mass reduction and increased survival were observed in SW13 xenograft model undergoing treatment with the drugs in combination. Our data suggest that an autocrine VEGF loop may exist within ACC. Furthermore, a combination of molecularly targeted agents may have both antiangiogenic and direct antitumor effects and thus could represent a new therapeutic tool for the treatment of ACC.

Somatic mutations in the KCNJ5 gene raise the lateralization index: implications for the diagnosis of primary aldosteronism by adrenal vein sampling.

CONTEXT Somatic mutations in the selectivity filter of KCNJ5 K(+) channel were found to be associated with higher plasma aldosterone concentrations in the patients with an aldosterone-producing adenoma (APA). OBJECTIVE We investigated whether plasma aldosterone levels and the lateralization index are higher from the side with the APA with the mutation, as compared with those without the mutation. DESIGN From 170 consecutive APA patients with comprehensive clinical and KCNJ5 data and a conclusive diagnosis, we recruited 91 patients with adrenal vein sampling and follow-up data. We measured CYP11B1 and CYP11B2 mRNA in APA tissue and plasma aldosterone (PAC) and plasma cortisol concentrations (PCC) in adrenal vein blood. To determine whether KCNJ5 mutations affected aldosterone output from the APA, we calculated the lateralization index (defined as the ratio of PAC to PCC at the APA side over the PAC to PCC ratio at the contralateral side). We also calculated two indexes of the aldosterone production from the APA side and the contralateral suppression index. RESULTS The mRNA content of CYP11B2, but not of CYP11B1, and, accordingly, the lateralization index was higher (29.9 ± 7.4 vs. 10.3 ± 3.6, P < 0.02) in the APA with the mutation than in the APA without the mutation. CONCLUSIONS APA patients with the somatic KCNJ5 mutations showed a higher production of aldosterone than those without such mutations, which translates in a higher lateralization index. Thus, they are more likely to be identified at adrenal vein sampling and therefore to receive adrenalectomy.

KCNJ5 gene somatic mutations affect cardiac remodelling but do not preclude cure of high blood pressure and regression of left ventricular hypertrophy in primary aldosteronism.

Objective: Aldosterone exerts detrimental cardiovascular effects, and patients with an aldosterone-producing adenoma (APA) carrying somatic mutations in the KCNJ5 K+ channel (mutAPA) have higher plasma aldosterone concentration than wild-type APA (wtAPA) patients. We therefore investigated whether mutAPA patients develop a more prominent cardiovascular damage than wtAPA patients. Methods and findings: From 257 consecutive primary aldosteronism patients, we identified 176 who had both a diagnosis of APA by the ’four corners’ criteria and high-quality echocardiographic data. Of them, 129 with KCNJ5 sequencing information and long-term follow-up data were compared for echocardiographic changes according to presence (mutAPA, 26%) or absence (wtAPA, 74%) of the KCNJ5 mutations. At baseline, the mutAPA were similar to the wtAPA for blood pressure (BP) and need for antihypertensive medications. However, they had higher left ventricular mass index (59 ± 19 vs. 51 ± 13 g/h2.7; P < 0.05) and plasma aldosterone concentration [49 (32–68) vs. 36 (25–52) ng/dl); P = 0.048] than the wtAPA patients. In spite of their more prominent cardiac involvement, the mutAPA patients exhibited a fall of BP and plasma aldosterone similar to wtAPA, and a regression of left ventricular mass index. Conclusions: Compared to the wild-type APA patients those with KCNJ5 mutations showed more prominent cardiovascular damage. Notwithstanding this, their chances of being cured from the hyperaldosteronism and the high BP, and of regression of left ventricular hypertrophy after adrenalectomy, were not compromised by the presence of these mutations.

Response to Effectiveness of Adrenalectomy and Aldosterone Antagonists for Long-Term Treatment of Primary Aldosteronism

Our data demonstrated the efficacy of mineralocorticoid receptor (MR) blockers for the treatment of primary aldosteronism (PA).1 However, by no means did we interpret our findings to show the superiority of adrenalectomy over MR blocker therapy. The higher rate of cure of hypertension seen with surgery was intrinsic with the definition of cure: 42% of our adrenalectomized versus none of our medically treated patients could withdraw pharmacological treatment at long term. Hence, when guided by results of adrenal vein sampling, adrenalectomy provides long-term cure of hypertension and allows tapering, or withdrawal, of the antihypertensive medications. Comparing adrenalectomy and MR blockade would require randomization of PA patients to either treatment. Neither the study of Reincke et …

Aldosterone-Producing Adrenocortical Carcinoma

In 1955 Conn described the syndrome of primary hyperaldosteronism (PHA) [1], which has come to be known as “Conn’s Syndrome” [2]. PHA is a group of disorders in which aldosterone production is inappropriately high, relatively autonomous from the renin-angiotensin system, and nonsuppressible by sodium loading. Such inappropriate production of aldosterone causes cardiovascular damage, suppression of plasma renin, hypertension, sodium retention, and potassium excretion that if prolonged and severe may lead to hypokalemia. PHA is commonly caused by adenoma (35%), bilateral adrenal hyperplasia (60%), or (rarely) by aldosterone producing adrenocortical carcinoma (APAC) (<1%) (Table 27.1) [3]. The first APAC was reported in 1955 by Foye and Feichtmeir [4] shortly after Conn’s original report. Aldosterone hypersecretion in adrenocortical carcinoma (ACC) is rare with only 58 patients being reported in a recent review [5]. The reported numbers of APAC amongst ACCs vary significantly. In one large series of ACCs, only 2.5% had developed hyperaldosteronism [6]. In a single center analysis on ACCs, which were subjected to operative management at the Mayo Clnic, the portion of APACs was 11%. Conversely, it has been estimated that hyperaldosteronism is due to APAC in only 1% of patients with PHA [7].

Primary Aldosteronism: Diagnosis and Treatment

PA is a common form of hypertension that is potentially curable. The ARR ratio is a rapid method for screening selective hypertensive patients for PA, and it is available in most medical centers. The value of the ARR depends on an appreciation of factors (such as diet, posture, time of day, presence of hypokalemia, medications, age, and renal function) which can affect the result, on the care with which these factors are either controlled or their effects taken into account, and on access to reliable and reproducible assays for renin and aldosterone. Even then, physiological day to day variability reduces the value of a single estimation, and repeat testing is recommended. The optimal cut-off however, is highly dependent on the type and quality of the aldosterone and renin assays and should be defined in every Center. Autonomous aldosterone secretion should in most cases be established by confirmatory testing Four confirmatory test to definitively confirm or exclude the diagnosis of PA are in common use: oral sodium loading, saline infusion, fludrocortisone suppression, and captopril challenge. However, there is currently insufficient direct evidence to recommend one over the others. It is than mandatory that the subtypes are identified. Although adrenal CT has several limitations all patients with primary aldosteronism should undergo adrenal computed tomography (CT) as the initial study in subtype testing and to exclude large masses that may represent adrenocortical carcinoma. The use of AVS is almost due in cases who are candidate for surgery since it will improve patient care and outcome, but, due to its practical difficulties must be justified on a case-by-case basis, and be undertaken in centers of excellence to achieve optimal sensitivity.

LONG-TERM CHANGES OF LEFT VENTRICULAR DIASTOLIC FUNCTION AFTER ADRENALECTOMY OR MEDICAL TREATMENT FOR PRIMARY ALDOSTERONISM: PP.18.177

Objectives: To evaluate the long-term effects on left ventricular (LV) diastolic function indexes in surgically or medically-treated patients with documented primary aldosteronism (PA). Design and Methods: After a baseline Doppler echocardiography assessment (DEA) of the LV geometry and global LV diastolic function, 136 PA patients were assigned to either adrenalectomy or medical therapy based on lateralization of aldosterone excess at adrenal vein sampling and entered a prospective follow-up study with serial DEA. Pulsed Doppler recordings at the level of the mitral valve tip were obtained from the apical 4-chamber view to measure early and late-wave diastolic filling velocities, their ratio (E/A ratio) and the ratio of their integrals (Ei/Ai), the E wave deceleration time (DT), the LV isovolumic relaxation time and the atrial contribution to LV filling. Aldosterone producing adenoma (APA) was diagnosed by the “4 corners criteria”. Results and Conclusions: Of the PA patients (age 50.7 ± 12.3 yrs, 46%F), 65% had APA and 35% had idiopathic hyperaldosteronism (IHA) and showed a high (51%) prevalence of inappropriate LV mass. At baseline adrenalectomized (n = 89) and medically-treated patients (n = 47) exhibited similar cardiac diameters and a similar E/A ratio (1.02 ± 0.04 vs. 1.01 ± 0.05). Blood pressure (BP) fell with either treatment to similar values (136 ± 15/85 ± 8 mmHg) after 3.05 yrs of follow-up and this was associated with LV remodeling around a significantly smaller diameter. At follow-up the DT increased significantly (from 218 ± 6 to 245 ± 9 msec, p = 0.003) in the adrenalectomized but not in the medically-treated patients. No significant changes in all the other indexes of LV diastolic filling were found. Therefore, in PA patients without overt evidences of LV diastolic dysfunction surgical or medical treatment reduced BP and caused inward LV remodeling without affecting significantly the diastolic function as assessed by the transmitral Doppler flow indexes, with the exception of the DT that increased in the adrenalectomized patients.