Teresa Maria Seccia

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.

The Adrenal Vein Sampling International Study (AVIS) for Identifying the Major Subtypes of Primary Aldosteronism

CONTEXT In patients who seek surgical cure of primary aldosteronism (PA), The Endocrine Society Guidelines recommend the use of adrenal vein sampling (AVS), which is invasive, technically challenging, difficult to interpret, and commonly held to be risky. OBJECTIVE The aim of this study was to determine the complication rate of AVS and the ways in which it is performed and interpreted at major referral centers. DESIGN AND SETTINGS The Adrenal Vein Sampling International Study is an observational, retrospective, multicenter study conducted at major referral centers for endocrine hypertension worldwide. PARTICIPANTS Eligible centers were identified from those that had published on PA and/or AVS in the last decade. MAIN OUTCOME MEASURE The protocols, interpretation, and costs of AVS were measured, as well as the rate of adrenal vein rupture and the rate of use of AVS. RESULTS Twenty of 24 eligible centers from Asia, Australia, North America, and Europe participated and provided information on 2604 AVS studies over a 6-yr period. The percentage of PA patients systematically submitted to AVS was 77% (median; 19-100%, range). Thirteen of the 20 centers used sequential catheterization, and seven used bilaterally simultaneous catheterization; cosyntropin stimulation was used in 11 centers. The overall rate of adrenal vein rupture was 0.61%. It correlated directly with the number of AVS performed at a particular center (P = 0.002) and inversely with the number of AVS performed by each radiologist (P = 0.007). CONCLUSIONS Despite carrying a minimal risk of adrenal vein rupture and at variance with the guidelines, AVS is not used systematically at major referral centers worldwide. These findings represent an argument for defining guidelines for this clinically important but technically demanding procedure.

Vascular Remodeling and Duration of Hypertension Predict Outcome of Adrenalectomy in Primary Aldosteronism Patients

Remodeling of the resistance arteries is a hallmark of arterial hypertension and predicts cardiovascular events, but it was unknown whether it could also predict the blood pressure response to adrenalectomy of patients with an aldosterone-producing adenoma. Therefore, we investigated the outcome of adrenalectomy as a function of vascular remodeling in the context of the preoperative features of aldosterone-producing adenoma patients. At 2 referral centers for hypertension, we prospectively measured the media:lumen ratio of small arteries from fat tissue of 50 consecutive aldosterone-producing adenoma patients treated with adrenalectomy. The blood pressure response to adrenalectomy was assessed by considering the blood pressure values and the number and dosages of antihypertensive medications. Adrenalectomy significantly (P<0.001) lowered plasma aldosterone (from 27.3±4.9 ng/dL to 8.3±11.2 ng/dL), the aldosterone:renin ratio (from 117±35 to 11±2), and blood pressure (from 163±22/98±2 mm Hg to 133±2/84±1 mm Hg), even despite a reduction (from 141±14 to 100±15; P=0.02) of the score of antihypertensive treatment. It provided cure of hypertension in 30% of the aldosterone-producing adenoma patients, normotension with less antihypertensive therapy in 52%, and improved blood pressure control in the rest. The media:lumen ratio and the known duration of hypertension significantly predicted the blood pressure response to adrenalectomy at univariate and multivariate analyses. Because a long duration of hypertension and/or the presence of vascular remodeling imply lower chances of blood pressure normalization at long-term follow-up postadrenalectomy, these findings emphasize the importance of an early diagnosis of aldosterone-producing adenoma.

Adrenocorticotropic Hormone Stimulation During Adrenal Vein Sampling for Identifying Surgically Curable Subtypes of Primary Aldosteronism. Comparison of 3 Different Protocols

Adrenocorticotropic hormone administration was proposed to overcome the biases associated with pulsatile aldosterone secretion during adrenal venous sampling, but the usefulness of different protocols of stimulation was never systematically assessed. We, therefore, compared the effects of a high dose (HD; 250 &mgr;g IV as a bolus), a very low dose (VLD; 250 pg IV), and an intermediate dose (ID; 50 &mgr;g/h) of adrenocorticotropic hormone on the selectivity index (SI) and the lateralization index in primary aldosteronism patients, using the diagnosis of aldosterone-producing adenoma, based on pathology and follow-up data, as a reference. The HD (n=47) significantly increased plasma cortisol concentration in infrarenal inferior vena cava (+79%) blood and the SI on both sides (SIRIGHT +113% and SILEFT +131%), as compared with baseline values. The ID (n=14) also markedly increased both plasma cortisol concentration inferior vena cava (+93%) and the SI (SIRIGHT +690% and SILEFT +410%); the very low dose (n=6) had no effect on either the plasma cortisol concentration or SI. In the patients with unilateral aldosterone-producing adenoma, the increase of selectivity with the HD and ID was counterbalanced by a confounding effect on the correct identification of the aldosterone-producing adenoma side, which was attributed to the wrong side in 3.0% and 12.5% with HD and ID, respectively. In conclusion, the HD and the ID, but not the very low dose, adrenocorticotropic hormone stimulation protocol facilitated the ascertainment of selectivity of adrenal vein catheterization. However, this favorable effect was overridden by a confounding effect on the identification of lateralized aldosterone excess to the aldosterone-producing adenoma side. Hence, we do not recommend adrenocorticotropic hormone stimulation.

Cardiac fibrosis occurs early and involves endothelin and AT-1 receptors in hypertension due to endogenous angiotensin II.

OBJECTIVES We investigated if endothelin (ET)-1 and the renin-angiotensin-aldosterone system play a role in cardiac fibrosis. BACKGROUND Angiotensin II (Ang II) can induce cardiac fibrosis, but the underlying mechanisms are incompletely understood. METHODS Four-week-old transgenic (mRen2)27 rat (TGRen2) received for four weeks a placebo, the mixed ET(A)/ET(B) endothelin receptor antagonist bosentan, the angiotensin II type I receptor (AT-1) antagonist irbesartan, the ET(A) endothelin receptor antagonist BMS-182874, and a combined treatment with irbesartan plus BMS-182874. We measured collagen density on Sirius red-stained serial sections of the left ventricle (LV) with a photomicroscope equipped with specific software and assessed the gene expression of procollagen alpha1(I), atrial natriuretic peptide (ANP), transforming growth factor-beta 1 (TGFbeta1), endothelin converting enzyme, and ET(B) receptor. RESULTS In the placebo group, hypertension was associated with LV hypertrophy and cardiac fibrosis (LV weight: 4.0 +/- 0.3 mg/g body weight; collagen density: 2.21 +/- 0.16%), which were all prevented with irbesartan (2.3 +/- 0.1, 1.30 +/- 0.13, p < 0.001), but not with BMS-182874 (4.0 +/- 0.2, 2.41 +/- 0.22). Bosentan also prevented fibrosis (1.39 +/- 0.18) but not hypertension and LV hypertrophy (3.38 +/- 0.27). Combined irbesartan and BMS-182874 treatment prevented LV hypertrophy (2.9 +/- 0.1) but not fibrosis (2.52 +/- 0.16). Collagen density correlated (r = 0.414, p < 0.05) with plasma aldosterone levels. In TGRen2 with LV hypertrophy, the gene expression of ANP and ET(B) but not that of TGFbeta1 and procollagen alpha1(I) was increased. CONCLUSIONS In Ang II-dependent hypertension, cardiac fibrosis was associated with LV hypertrophy and was hindered by both mixed ET(A)/ET(B) blockade and AT-1 blockade. Only the latter treatment prevented both hypertension and LV hypertrophy. Thus, there is a dissociation between the mechanisms of cardiac fibrosis and hypertension, which do and do not entail ET-1, respectively.

Reciprocal regulation of endothelin-1 and nitric oxide: relevance in the physiology and pathology of the cardiovascular system.

The endothelium plays a crucial role in the regulation of cardiovascular structure and function by releasing several mediators in response to biochemical and physical stimuli. These mediators are grouped into two classes: (1) endothelium-derived constricting factors (EDCFs) and (2) endothelium-derived relaxing factors (EDRFs), the roles of which are considered to be detrimental and beneficial, respectively. Endothelin-1 (ET-1) and nitric oxide (NO) are the prototypes of EDCFs and EDRFs, respectively, and their effects on the cardiovascular system have been studied in depth. Numerous conditions characterized by an impaired availability of NO have been found to be associated with enhanced synthesis of ET-1, and vice versa, thereby suggesting that these two factors have a reciprocal regulation. Experimental studies have provided evidence that ET-1 may exert a bidirectional effect by either enhancing NO production via ETB receptors located in endothelial cells or blunting it via ETA receptors prevalently located in the vascular smooth muscle cells. Conversely, NO was found to inhibit ET-1 synthesis in different cell types. In vitro and in vivo studies have started to unravel the molecular mechanisms involved in this complex interaction. It has been clarified that several factors affect in opposite directions the transcription of preproET-1 and NO-synthase genes, nuclear factor-KB and peroxisome proliferator-activated receptors playing a key role in these regulatory mechanisms. ET-1 and NO interplay seems to have a great relevance in the physiological regulation of vascular tone and blood pressure, as well as in vascular remodeling. Moreover, an imbalance between ET-1 and NO systems may underly the mechanisms involved in the pathogenesis of systemic and pulmonary hypertension and atherosclerosis.

Heterogeneity of Aldosterone-Producing Adenomas Revealed by a Whole Transcriptome Analysis

Aldosterone-producing adenomas (APAs) are a common cause of arterial hypertension, but the underlying molecular mechanisms are unknown, although a transcriptional modulation of aldosterone synthase (CYP11B2) has been suggested. Aldosterone synthesis involves 2 main rate-limiting steps: cholesterol transport into mitochondria and CYP11B2 gene transcription. Evidence supports a role of Ca2+/calmodulin-dependent protein kinases (CAMKs) in the regulation of angiotensin II- and potassium-stimulated aldosterone production. CAMK-I mediates CYP11B2 transcription via cAMP response element binding protein and activating transcription factor 1 transcription factors and nuclear receptor Nur-related factor 1. CAMK-II affects cholesterol transport into mitochondria by acting on steroidogenic acute regulatory protein and/or cytoskeleton proteins. We analyzed the whole transcriptome of APAs as compared with a pool of normal human adrenocortical tissues. Based on steroidogenic enzyme gene expression profiles, we identified 2 APA subgroups: 1 featuring overexpression of CYP11B2, CAMK-I, 11-&bgr;-hydroxylase, 3-&bgr;-hydroxysteroid dehydrogenase, and 21-hydroxylase and the underexpression of CAMK-IIB and the other one with an opposite profile. The low CYP11B2 group exhibited a longer known duration of hypertension and a lower rate of long-term cure. Thus, aldosterone overproduction in APAs involves complex alterations of aldosterone synthesis regulation rather than simply increased aldosterone synthase gene expression. Whether the molecular signature of APA carries prognostic information is worth further investigation.

Impact of Accessory Hepatic Veins on Adrenal Vein Sampling for Identification of Surgically Curable Primary Aldosteronism

Adrenal vein sampling is the gold standard for identification of surgically curable primary aldosteronism, but its accuracy might be hindered by blood dilution from accessory vein blood. We prospectively investigated the presence of accessory veins draining into adrenal veins and their effect on the selectivity index (SI) in 74 consecutive patients undergoing adrenal vein sampling. On the right side, the venous anatomic pattern could be conclusively determined in 91.8% of the cases: we detected hepatic accessory veins in 12.1%, no accessory veins in 42.4%, and renal capsular veins in 45.5%. On the left side there was a phrenico-adrenal trunk in 89.4% and renal capsular accessory veins in 10.6% of the cases. On both sides, renal capsular and phrenic accessory veins did not affect the SI. At variance, on the right side, hepatic accessory veins were associated with SI values ≈3-fold lower than that found when such accessory veins were absent (median: 3.10 [range: 0.80 to 84.2] versus median: 1.10 [range: 0.70 to 2.20]; P=0.01). However, superselective adrenal catheterization resulted into higher SI values (median: 23.88; range: 4.80 to 84.20) in these cases. Thus, hepatic accessory veins sharing egress into the inferior vena cava with the right adrenal vein occurred in ≈12% of the patients and imply a low SI, likely because of adrenal blood dilution by hepatic blood carrying a low cortisol concentration. In the presence of this anatomic variation, superselective catheterization of the right adrenal vein should be undertaken to determine the lateralization of aldosterone secretion.

Within-Patient Reproducibility of the Aldosterone:Renin Ratio in Primary Aldosteronism

The plasma aldosterone concentration:renin ratio (ARR) is widely used for the screening of primary aldosteronism, but its reproducibility is unknown. We, therefore, investigated the within-patient reproducibility of the ARR in a prospective multicenter study of consecutive hypertensive patients referred to specialized centers for hypertension in Italy. After the patients were carefully prepared from the pharmacological standpoint, the ARR was determined at baseline in 1136 patients and repeated after, on average, 4 weeks in the patients who had initially an ARR ≥40 and in 1 of every 4 of those with an ARR <40. The reproducibility of the ARR was assessed with Passing and Bablok and Deming regression, coefficient of reproducibility, and Bland-Altman and Mountain plots. Within-patient ARR comparison was available in 268 patients, of whom 49 had an aldosterone-producing adenoma, on the basis of the “4-corner criteria.” The ARR showed a highly significant within-patient correlation (r=0.69; P<0.0001) and reproducibility. Bland-Altman plot showed no proportional, magnitude-related, or absolute systematic error between the ARR; moreover, only 7% of the values, for example, slightly more than what could be expected by chance, fell out of the 95% CI for the between-test difference. The accuracy of each ARR for pinpointing aldosterone-producing adenoma patients was ≈80%. Thus, although it was performed under different conditions in a multicenter study, the ARR showed a good within-patient reproducibility. Hence, contrary to previously claimed poor reproducibility of the ARR, these data support its use for the screening of primary aldosteronism.

Clinical Use of Laboratory Tests for the Identification of Secondary Forms of Arterial Hypertension

The prevalence of secondary hypertension can be underestimated if appropriate tests are not performed. The importance of selecting patients with a high pre-test probability of secondary forms of hypertension is first discussed. The laboratory tests currently used for seeking a cause of hypertension are critically reviewed, with emphasis on their operative features and limitations. Strategies to identify primary aldosteronism, the most frequent form of secondary hypertension, and to determine its unilateral or bilateral causes are described. Treatment entails adrenalectomy in unilateral forms, and mineralocorticoid receptor blockade in bilateral forms. Renovascular hypertension is also a common, curable form of hypertension, that should be identified as early as possible to avoid the onset of cardiovascular target organ damage. The tests for its confirmation or exclusion are discussed. The various tests available for the diagnosis of pheochromocytoma, which is much rarer than the above but extremely important to identify, are also described, with emphasis on recent developments in genetic testing. Finally, the tests for diagnosing some rarer monogenic forms and other renal and endocrine causes of arterial hypertension are explored.