Hyperthermia as a Steroidogenic Inhibitor of Adrenodortical Cells, an Adrenal Sparing Treatment for Primary Aldosteronism?

Abstract

Introduction: Primary Aldosteronism (PA) is the commonest secondary cause of hypertension. Mainstay therapy, adrenalectomy resects both hypersecreting and adjacent normal tissue. It is therefore only suitable for patients with unilateral disease (40% cases), whom are surgical candidates. Thermal therapy presents a plausible minimally invasive therapeutic, to target and disrupt hypersecreting adrenal nodules in primary aldosteronism, while also preserving adjacent normal adrenal cortex. Methodology: Adrenocortical cell lines (H295R and HAC15) were treated with hyperthermia using a water bath at temperatures between 37-650C for 15 minutes. Cell death and apoptosis were analysed immediately, 24 hours, and 48 hours post hyperthermia using Annexin V / Propidium Iodide (PI) (flow cytometry), and Calcein / PI imaging techniques. Steroidogenic potential was also analysed post hyperthermia by (i) measuring cytosolic calcium flux in response to angiotensin II (ANGII) using Flou-4 staining (flow cytometry); (ii) measurement of steroidogenic enzyme expression (RT-PCR); (iii) by measurement of cortisol and aldosterone in cell supernatants (chemiluminescent assays). Cells were also stimulated with forskolin for steroid enzyme and output assays. Results: Hyperthermia induced mainly necrotic cell death not apoptotic. Percentage cell death was significantly increased from 500C-65 0C (95.41+/- 5.74% versus control 9.17+/- 3.01%, p<0.0001). Cytosolic calcium changes in response to ANGII were lowered from 550C-650C in H295R (-36.33+/-7.06 Ca+ Response versus control 10.67+/- 1.76 Ca+ Response, p<0.001) and from 450C-650C in HAC15 (-43.33+/-12.36 Ca+ Response versus control 4.43+/-1.57 Ca+ Response, p<0.002), intracellular stored calcium was also diminished with increased hyperthermia exposures in both cell lines. CYP11B1 (-31.6+/-2.8 ααCt versus control, p<0.0001), CYP11B2 (-11.3+/-4.9 ααCt versus control, p<0.0001), and HSD3B2 (-42+/-9.8 ααCt versus control, p<0.0001) enzyme gene expression was decreased following 450C for 15 minutes in HAC15 cells while only CYP11B2 (-9.3+/-2.5 ααCt versus control, p<0.0001), and HSD3B2 (-15+/-1.3 ααCt versus control, p<0.0001) enzyme gene expression was decreased in H295R cells. Steroid output was also shown to be impaired at 450C for 15 minutes (22.35+/- 4.49 nmol/L versus control 117.7+/- 25 nmol/L, p<0.0001). Conclusion: Hyperthermic therapy delivered at sublethal temperatures and exposure times inhibits steroidogenesis. Inhibiting steroidogenesis by applying targeted thermal therapy to functional adrenocortical nodules represents a novel adrenal sparing approach to definitive management of primary aldosteronism.