Endocrine Abstracts

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 therapy, to target and disrupt hypersecreting aldosterone producing adenomas (APA), while also preserving adjacent normal adrenal cortex.

Methodology

Adrenocortical cell lines (H295R and HAC15) were treated with hyperthermia for 15 minutes using a water bath at temperatures between 37–65°C. Cell death and apoptosis were analysed immediately and at 24 hours and 48 hours post hyperthermia using Annexin V/Propidium Iodide (PI) (flow cytometry), and Calcein/PI (fluorescence microscopy). Steroidogenesis was analysed following hyperthermia (i) measuring cytosolic calcium flux in response to angiotensin II (ANGII) using Flou-4 staining (flow cytometry); (ii) analysing steroidogenic enzyme expression (RT-PCR/Western Blotting); and (iii) measuring cortisol and aldosterone in cell supernatants (chemiluminescent assays). Cells were also stimulated with forskolin for assays of steroidogenesis.

Results

Hyperthermia induced necrotic cell death without apoptosis. Percentage cell death was significantly higher at 50°C-65°C (95.41 ± 5.74% vs control 9.17 ± 3.01%, P < 0.0001). Cytosolic calcium changes in response to ANGII were lower above 55°C-65°C in H295R (–36.33 ± 7.06 Ca + Response vs control 10.67 ± 1.76 Ca + Response, P < 0.001) and above 45°C-65°C in HAC15 (–43.33 ± –12.36 Ca + Response vs control 4.43 ± 1.57 Ca + Response, P < 0.002). Intracellular stored calcium was also diminished with increasing hyperthermia in both cell lines. CYP11B1 (–31.6 ± 2.8 DDCt vs control, P < 0.0001), CYP11B2 (–11.3 ± 4.9 DDCt vs control, P < 0.0001), and HSD3B2 (–42 ± 9.8 DDCt vs control, P < 0.0001) enzyme gene expression was decreased at 45°C and above in HAC15 cells. CYP11B2 (–9.3 ± 2.5 DDCt vs control, P < 0.0001), and HSD3B2 (–15 ± 1.3 DDCt vs control, P < 0.0001) enzyme gene expression decreased significantly in H295R cells. Measurement of cortisol decreased with heat treatment ≥ 45°C (22.35 ± 4.49 nmol/l vs control 117.7 ± 25 nmol/l, P < 0.0001) while aldosterone output was unaffected at temperatures below 50°C.

Conclusion

Hyperthermia induces necrotic cell death in adrenocortical cell lines at temperatures > 50°C. Cortisol steroid secretion is more susceptible to sublethal hyperthermia when compared to aldosterone. Knowledge of the differential effects of hyperthermia at lethal and sublethal doses on steroidogenesis and steroid secretion is critical when designing thermal ablation systems for adrenal use and warrant consideration during treatment planning for thermal ablation of APA.