Selective inhibition of aldosterone production modulates human adrenocortical (HAC15) cell fate

Background Aldosterone, produced in the zona glomerulosa (ZG) by aldosterone synthase (CYP11B2), regulates sodium balance and blood pressure. In adult adrenals, CYP11B2-expressing ZG cells are sparse, likely reflecting chronic suppression of the renin–angiotensin system under high-salt conditions. Whether reduced aldosterone synthesis directly influences ZG cell behavior remains unclear. This study examined the impact of CYP11B2 inhibition on human adrenal cell fate and function. Methods CYP11B2 was inhibited in HAC15 cells using siRNA (siCYP11B2) or baxdrostat, a selective CYP11B2 inhibitor. Forty-eight hours post-treatment, steroid hormone levels, RNA sequencing profiles, and functional outputs (EZMTT metabolic assay, xCELLigence real-time cell analysis, and Annexin V/SYTOX flow cytometry for apoptosis/necrosis) were evaluated. Results siCYP11B2 reduced aldosterone production by 70% ± 2.9% ( n = 18, p < 0.001) and suppressed mRNA expression of CYP11B2 (88% ± 10.8%), HNRNPA1 (33% ± 17.6%), and UHMK1 (42% ± 13.7%) ( n = 9, p < 0.05). Transcriptomic analysis revealed differential expression signatures enriched in autophagy- and mitophagy-related pathways. Treatment with U0126 (MEK1/2 inhibitor) dose-dependently reduced angiotensin II-stimulated aldosterone and cortisol production ( n = 12, p < 0.001), and SB203580 (p38 MAPK inhibitor) increased the aldosterone-to-cortisol ratio (4.5 ± 0.5-fold, n = 12, p < 0.001), at concentrations previously reported to inhibit (25–50 µM U0126) or induce autophagy (1 µM SB203580), respectively. EZMTT assays demonstrated increased metabolic activity in siCYP11B2-treated cells at 96 h (Δ0.141 ± 0.02 absorbance units, p < 0.001), which was not observed following baxdrostat treatment. Baxdrostat treatment altered overall cell-death responses (FC = 1.42 ± 0.05, n = 144 and p = 1.3 × 10 −10 ); however, the relative distribution of apoptotic and necrotic populations varied across independent experiments. Conclusion Reduced aldosterone synthesis is associated with transcriptional signatures involving autophagy- and mitophagy-related pathways. Pharmacological treatment using U0126 and SB203580 altered steroidogenic output independently of CYP11B2 transcript expression, providing hypothesis-generating evidence linking cellular homeostasis and mitochondrial quality control processes to adrenal steroidogenesis. Furthermore, pharmacological CYP11B2 inhibition using baxdrostat produced variable alterations in cell-death profile, highlighting important considerations for long-term therapeutic targeting.