The volume-regulating hormone aldosterone stimulates Na⁺ reabsorption via the epithelial Na⁺ channel (ENaC) in the aldosterone-sensitive distal nephron (ASDN) [1], downstream of transcriptional processes via the mineralocorticoid (MR) and glucocorticoid receptor (GR). Aldosterone specificity is conferred to principal cells (PCs) of the ASDN by the enzyme 11βHSD2 which inactivates the more abundant cortisol. Both MR and GR have been localised to the ASDN [2], and recent evidence suggests that earlier portions of the ASDN contain “unprotected” ENaC [3]. However, the specific roles of these receptors in mediating steroid-induced ENaC activity remains unclear. The aim of this study was to assess the relative roles of MR and GR in mediating steroid-induced ENaC activity and the target genes involved.

mCCD_cl1 murine collecting duct (CD) cells [4] or primary CD cells (PCDs) [5] were seeded onto permeable filters for 9-11 days. Cells were treated with MR (PF-03882845, 100nM) or GR (mifepristone, 10μM) antagonists for 30min, or respective control, followed by ALDO (3nM), corticosterone (CORT, 10nM) or dexamethasone (DEX, 100nM) for 3h. For experiments involving CORT, 10µM carbenoxolone was included in the 30min treatment to inhibit 11βHSD2 activity. Changes in equivalent short-circuit current (ΔI_eq) were measured by epithelial volt-ohm-meter. RNA was extracted and expression of 3 ALDO-induced genes [5]: Sgk1, Zbtb16 and Rasd1 were measured by qRT-PCR. Results are mean±95% CI, statistical significance determined by two-way ANOVA and Tukey’s post-hoc test.

ALDO-induced ΔI_eq was -5.2±1.0 μA⋅cm^-2 in mCCD_cl1 cells (n=9-10, p<0.001) and -3.6±0.5 μA⋅cm^-2 in PCDs (n=6, p<0.001). MR antagonism near abolished this in both mCCD_cl1 and PCDs (p<0.001), whereas GR antagonism did not. When pre-treated with CBX, CORT-induced ΔI_eq was -6.4±1.0 μA⋅cm^-2 in mCCD_cl1 and -4.0±0.3 μA⋅cm^-2 in PCDs (p<0.001). These were partly inhibited by MR antagonism in mCCD_cl1 (p<0.05) and PCDs (p<0.001), however GR antagonism had no effect. DEX-induced ΔI_eq was -9.3±1.0 μA⋅cm^-2 in mCCD_cl1 cells and -4.7±0.5 μA⋅cm^‑2 in PCDs (p<0.001). GR, but not MR, antagonism inhibited this response in both mCCD_cl1 cells and PCDs (p<0.001).

Expression of Sgk1, Zbtb16 and Rasd1 was upregulated by ALDO, CBX+CORT and DEX (n=9-10, p<0.001 across all). MR antagonism reduced ALDO-induced expression of Sgk1 (p<0.001), Zbtb16 (p<0.001) and Rasd1 (p<0.05). Interestingly GR antagonism also reduced ALDO-induced expression of Sgk1 (p<0.001), Zbtb16 (p<0.001) and Rasd1 (p<0.05). MR, but not GR, antagonism reduced CORT-induced expression of Sgk1 (p<0.0001), Zbtb16 (p<0.05) and Rasd1 (p<0.05). Finally, GR, but not MR, antagonism near-abolished DEX-induced expression of Sgk1 (p<0.001), Zbtb16 (p<0.001), Rasd1 (p<0.001).

Whilst ALDO-induced ENaC activity was MR- but not GR-dependent, the expression of all target genes involved both MR and GR. These findings may reflect that mCCD_cl1 cells are a mixed population of principal and intercalated cells. Notably, CORT-induced ENaC activity and target gene expression was MR-dependent, with no effect of GR antagonism. In this setting, CORT may be considered a mineralocorticoid and the sole MR-dependence is at odds with the ALDO results. These findings reveal a complexity to the regulation of ENaC activity by corticosteroid hormones and the receptors involved. Further work is required to unpick these mechanisms.