C Recarti

Isolation of human adrenocortical aldosterone-producing cells by a novel immunomagnetic beads method.

We detected intense CD56 immunostaining in the zona glomerulosa (ZG) and medulla of the normal human adrenal gland and therefore identified CD56, the neural cell adhesion molecule, as a membrane antigen specific for the ZG, aldosterone-producing adenoma (APA), and chromaffin cells. The APA and pheochromocytoma cells, which are histogenetically derived from the ZG and medulla, respectively, also showed intense CD56 immunostaining. Based on these findings we developed a strategy for isolating cells from the ZG and APA using CD56 immunobinding to magnetic beads. Morphology, gene expression studies, and aldosterone measurement confirmed that CD56 positive (+) cells were ZG and APA cells. Analysis of CD56+ cells under light and phase contrast microscopy evidenced that these cells formed clumps, as the ZG cells usually do; with electron microscopy they showed multiple features typical of a steroidogenic phenotype. Expression levels of the CD56 and the aldosterone synthase (CYP11B2) gene were markedly higher in CD56+ cells than CD56- cells (+1600 and +2100% increase, respectively). Moreover, aldosterone secretion was higher (+1380%) from CD56+ cells than from CD56- cells. Hence, this novel methodology allows isolation of a pure population of ZG and APA cells exhibiting multiple characteristics of the aldosterone-producing cells.

Expression and functional role of the prorenin receptor in the human adrenocortical zona glomerulosa and in primary aldosteronism.

Objectives: Prorenin can be detected in plasma of hypertensive patients. If detected in patients with primary aldosteronism could implicate prorenin in the development of primary aldosteronism. To address this issue, we measured the plasma prorenin levels in primary aldosteronism patients, the expression of the prorenin receptor (PRR) in the normal human adrenocortical zona glomerulosa and aldosterone-producing adenoma (APA), and we investigated the functional effects of PRR activation in human adrenocortical cells. Method: Plasma renin activity, aldosterone, and active and total trypsin-activated renin were measured in primary aldosteronism patients, essential hypertensive patients, and healthy individuals, and then prorenin levels were calculated. Localization and functional role of PRR were investigated in human and rat tissues, and aldosterone-producing cells. Results: Primary aldosteronism patients had detectable plasma levels of prorenin. Using digital-droplet real-time PCR, we found a high PRR-to-porphobilinogen deaminase ratio in both the normal adrenal cortex and APAs. Marked expression of the PRR gene and protein was also found in HAC15 cells. Immunoblotting, confocal, and immunogold electron microscopy demonstrated PRR at the cell membrane and intracellularly. Renin and prorenin significantly triggered both CYP11B2 expression (aldosterone synthase) and ERK1/2 phosphorylation, but only CYP11B2 transcription was prevented by aliskiren. Conclusion: The presence of detectable plasma prorenin in primary aldosteronism patients, and the high expression of PRR in the normal human adrenal cortex, APA tissue, CD56+ aldosterone-producing cells, along with activation of CYP11B2 synthesis and ERK1/2 phosphorylation, suggest that the circulating and locally produced prorenin may contribute to the development or maintenance of human primary aldosteronism.

HIGH EXPRESSION OF THE PRO-RENIN RECEPTOR IN ALDOSTERONE PRODUCING ADENOMA CAUSING HUMAN PRIMARY ALDOSTERONISM: 4D.02

Objective: Primary Aldosteronism (PA) is the most prevalent form of endocrine hypertension but its underlying mechanisms are unknown. The detection of prorenin, despite the suppression of renin, in plasma of PA patients suggests that prorenin, by acting via the Pro-Renin Receptor (PRR), could play a pathophysiologic role in PA by causing adrenocortical cell growth and hyperaldosteronism. Hence, we hypothesized that the PRR is expressed in the human zona glomerulosa (ZG) and in aldosterone producing adenoma (APA). Design and Method: To test this hypothesis we investigated the presence of the PRR by using beforehand a whole transcriptome analysis approach (in 24 APA). We then used Real time RT-PCR to quantify more precisely the PRR transcript in APA (n = 12), in two adrenocortical carcinoma cell lines (H295 and HAC15) and in immunomagnetic bead separated CD56+ human adrenocortical ZG cells (Caroccia, Endocrinology 2010). To confirm the expression of the PRR at the protein level immunohistochemistry and immunoblotting were also performed. Results and Conclusions: Microarray analysis evidenced the expression of PRR in all APAs. Quantitative gene expression studies demonstrated a level of expression of the PRR gene in APA which was on average much higher than that of the established adrenocortical house-keeping gene PBGD (PRR Ct = 22,69 ± 1,11; PBGD Ct = 27,49 ± 1,69 p < 0.0001). The expression of the PRR in the human adrenal cortical tissue was confirmed in CD56+ ZG cells, and in H295 and HAC15 cells. Immunohistochemistry confirmed the expression of the PRR at the protein level in all these cells. It also allowed to localize it more precisely to the adrenocortical ZG. These results are consistent with the hypothesis that circulating prorenin can activate the PRR in PA patients. Therefore, experiments are ongoing to investigate the functional relevance of these findings with the ultimate goal of demonstrating the role of the PRR in human PA.

ALDOSTERONE-PRODUCING ADENOMA CAUSING HUMAN PRIMARY ALDOSTERONISM ARE CHARACTERISED BY UNDER- EXPRESSION OF THE TWIK-RELATED ACID-SENSITIVE K+ CHANNEL 2 (TASK-2) GENE: PP.21.321

Background: Primary aldosteronism is a common cause of arterial hypertension, but its underlying molecular mechanisms are unknown. However, it is well known that K+ is a key regulator of aldosterone secretion, which interacts with physiological secretagogues of aldosterone as angiotensin II (Ang II) and endothelin-1. Genetic manipulation of a widely distributed channels class, which generate background or “leak” potassium (K+) currents, the Twik-related Acid-Sensitive K channel 1 (TASK-1) and 3 (TASK-3), generated a phenotype that mimics human primary aldosteronism. Objective: To measure the expression of the TASK family in a series of aldosterone producing adenoma (APA). Methods: Whole transcriptome of 24 APA was compared to that of a normal adrenocortical tissue pool (n = 10) by oligomicroarray technique. Data were validated by quantitative real time RT PCR. Immunohistochemistry was used to investigate TASKs protein expression by adrenocortical tissues. Results and Conclusions: The most expressed TASK channel gene in both the normal ZG and in APAs was TASK-1, followed by TASK-2 while the expression of TASK-3 was scant. With a whole transcriptome analysis followed by confirmation with quantitative real time RT-PCR we found a consistent and marked under expression of TASK-2 channel in all studied APA (n = 24), while TASK-1 and TASK-3 were heterogeneously expressed, as compared to the normal adrenal cortex. Immunohistochemistry was used to confirm the expression of the 3 TASK K+ channels in the adrenal cortex at the protein level and quantitative immnunoblotting will be used to confirm the differences detected at the transcript level. The underexpression of TASK-2 channels in APA suggests blunted leak” K+ currents, with ensuing lowering of the membrane potential and thereby increased likelihood of opening of the voltage-gated T-type calcium channels. The latter is known to render the adrenocortical zona glomerulosa cells more sensitive to aldosterone secretagogues. Hence, our ex vivo findings, while pointing to TASK-2 channels under-expression as an important mechanism of primary aldosteronism, mandates further studies to ascertain the molecular mechanisms by which this underexpression develops.