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Updated: May 28, 2025
A Novel Method: Super-selective Adrenal Venous Sampling
Published on: September 15, 2017
Modulation of Calcium Signaling on Demand to Decipher the Molecular Mechanisms of Primary Aldosteronism
Bakhta Fedlaoui1, Teresa Cosentino1, Zeina R Al Sayed1
1Université Paris Cité, INSERM, PARCC (Paris Cardiovascular Research Center), France (B.F., T.C., Z.R.A.S., I.G.-D., N.F., M.F., J.-S.H., S.T.-A., F.L.F.-R., M.-C.Z., S. Boulkroun).
Background:
Primary aldosteronism is the most common form of secondary hypertension. The most frequent genetic cause of aldosterone-producing adenomas is somatic mutations in the potassium channel KCNJ5. They affect the ion selectivity of the channel, with sodium influx leading to cell membrane depolarization and activation of calcium signaling, the major trigger for aldosterone biosynthesis.
Methods:
To investigate how KCNJ5 mutations lead to the development of aldosterone-producing adenomas, we established an adrenocortical cell model in which sodium entry into the cells can be modulated on demand using chemogenetic tools [H295R-S2 α7-5HT3-R (α7-5HT3 receptor) cells]. We investigated their functional and molecular characteristics with regard to aldosterone biosynthesis and cell proliferation.
Results:
A clonal cell line with stable expression of the chimeric α7-5HT3-R in H295R-S2 (human adrenocortical carcinoma cell line, Strain 2) cells was obtained. Increased sodium entry through α7-5HT3-R upon stimulation with uPSEM-817 (uPharmacologically Selective Effector Molecule-817) led to cell membrane depolarization, opening of voltage-gated Ca2+ channels, and increased intracellular Ca2+ concentrations, resulting in the stimulation of CYP11B2 expression and increased aldosterone biosynthesis. Increased intracellular sodium influx did not increase proliferation but rather induced apoptosis. RNA sequencing and steroidome analyses revealed unique profiles associated with Na+ entry, with only partial overlap with Ang II (angiotensin II) or potassium-induced changes.
Conclusions:
H295R-S2 α7-5HT3-R cells are a new model reproducing the major features of cells harboring KCNJ5 mutations. Increased expression of CYP11B2 and stimulation of the mineralocorticoid biosynthesis pathway are associated with a decrease of cell proliferation and an increase of apoptosis, indicating that additional events may be required for the development of aldosterone-producing adenomas.
Insights
Primary aldosteronism, caused by KCNJ5 mutations, involves increased sodium influx, leading to aldosterone overproduction. This study developed a cell model showing sodium influx stimulates aldosterone but induces apoptosis, not proliferation, suggesting other factors in adenoma development.
Area of Science:
- Endocrinology
- Molecular Biology
- Cell Biology
Background:
- Primary aldosteronism is the most common secondary hypertension, frequently caused by KCNJ5 mutations in aldosterone-producing adenomas.
- KCNJ5 mutations alter ion selectivity, promoting sodium influx, cell depolarization, and aldosterone biosynthesis.
Purpose of the Study:
- To investigate the functional and molecular consequences of KCNJ5 mutations in a novel adrenocortical cell model.
- To understand how altered sodium entry impacts aldosterone production and cell behavior.
Main Methods:
- Development of a chemogenetically inducible H295R-S2 α7-5HT3-R cell line to modulate sodium entry.
- Utilized uPSEM-817 to stimulate sodium influx and assessed aldosterone biosynthesis, cell proliferation, and gene expression via RNA sequencing and steroidome analysis.
Main Results:
- Stimulated sodium influx mimicked KCNJ5 mutation effects, increasing CYP11B2 expression and aldosterone biosynthesis.
- Increased sodium entry led to cell membrane depolarization, elevated intracellular calcium, and apoptosis, but not proliferation.
- RNA sequencing and steroidome analyses revealed unique molecular profiles distinct from angiotensin II or potassium-induced changes.
Conclusions:
- The H295R-S2 α7-5HT3-R cell model effectively replicates key features of KCNJ5-mutated cells.
- While sodium influx drives aldosterone production, it also induces apoptosis, suggesting additional factors are necessary for adenoma development.
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