Proceedings of The Physiological Society

Europhysiology 2018 (London, UK) (2018) Proc Physiol Soc 41, C020

Oral Communications

Complicated receptors in the collecting duct: regulation of ENaC-mediated Na+ transport by corticosteroids.

M. K. Mansley1, M. A. Bailey1

1. Centre for Cardiovascular Science, The University of Edinburgh, Edinburgh, United Kingdom.

ENaC-mediated Na+ reabsorption by principal cells (PC) in the renal collecting duct is stimulated by aldosterone and contributes importantly to Na+ and blood pressure homeostasis1. PCs express both mineralocorticoid (MR) and glucocorticoid receptors (GR) but the role of the GR is uncertain since 11βHSD2 is thought to metabolise active glucocorticoids within the cell. In vivo studies challenge this concept and in immortalised PC models, the GR agonist dexamethasone strongly stimulates ENaC. In this study we used the mouse-derived mCCDcl1 cells2 which respond to physiological concentrations of aldosterone3 to examine the relative roles of the MR and GR in mediating ENaC-mediated Na+ transport in response to the corticosteroids: aldosterone, dexamethasone and corticosterone. Transepithelial ion transport was assessed across polarised mCCDcl1 cells using epithelial-volt-ohm meter measurements of transepithelial voltage (Vt) and resistance (Rt) allowing calculation of equivalent short-circuit current (Ieq) by Ohm's law. Amiloride (10μM) was added at the end of experiments to assess ENaC-mediated Na+ transport and RNA was then isolated from cellular lysates to confirm expression of MR, GR and 11βHSD2 by qPCR. Data are shown as mean±S.D., statistical significance was determined by one-way ANOVA, with Tukey's post-hoc test. Basal Ieq in cells was -11.2±4.2µA/cm2 (n=164), ~95% of which was amiloride-sensitive: Ieq predominantly reflects ENaC-mediated Na+ transport. Application of aldosterone (3nM, 3h) stimulated Ieq by 2.8±1.7 fold (n=6) compared to 1.3±0.1 fold in cells treated with solvent vehicle (n=8). Pre-incubating cells with a non-steroidal MR antagonist PF-03882845 (1µM, 30min) abolished aldosterone-induced Ieq (n=5, p<0.001). Pre-incubation with mifepristone, a GR antagonist with no affinity for MR, inhibited aldosterone-induced Ieq by ~55% (n=6, p<0.001). Dexamethasone, a synthetic glucocorticoid, stimulated Ieq by 3.9±0.5 fold (n=8). This response was inhibited by ~70% by mifepristone (n=8) and ~20% by MR inhibition (n=5). Corticosterone (100nM, 3h) did not alter Ieq (n=8), consistent with the expression of 11βHSD2. When cells were pre-incubated with carbenoxolone (10µM, 30min), an inhibitor of 11βHSD2, corticosterone increased Ieq by 3.6±0.5 fold (n=8, p<0.001) and this was inhibited ~65% by MR blockade (n=5, p<0.001) and ~20% by GR antagonism (n=8, p<0.001). The key findings of this study are that the GR plays a permissive role in the regulation of ENaC by physiological concentrations of aldosterone. In contrast to dexamethasone, corticosterone predominantly engages MR rather than GR and necessitates inhibition of 11βHSD2 to stimulate ENaC. Thus, for endogenous corticosteroids, the interplay between MR and GR is complex and further work is required unpick the role of the GR in the regulation of ENaC in the distal nephron.

Where applicable, experiments conform with Society ethical requirements