Corticosteroid Regulation of ENaC‐Mediated Na+ Transport in a Cellular Model of the Cortical Collecting Duct
Authors: Mansley Morag K , Bailey Matthew A 
Published: June 2021

Na+ absorption in the distal nephron is under strict hormonal control, perhaps most notably by the mineralocorticoid aldosterone. The specificity of aldosterone action is conferred by the enzyme 11β‐hydroxysteroid dehydrogenase 2 (11βHSD2) which is thought to protect the mineralocorticoid receptor (MR) from illicit occupancy by glucocorticoids, which circulate freely at ~100 fold greater concentration. In vivo studies challenge this model e.g. mice haploinsufficient for 11βHSD2 exhibit salt‐sensitive hypertension associated with downregulated ENaC‐mediated Na+ absorption1. This downregulation can be reversed by glucocorticoid receptor (GR), but not MR, blockade, strongly indicating a role for the GR. The present study has therefore utilised a cellular model of the collecting duct to interrogate the molecular pathways enabling corticosteroids to regulate ENaC activity.ENaC‐mediated Na+ transport was quantified across monolayers of mCCDcl1 cells via electrometric measurements of amiloride‐sensitive current (Iami). SGK1 abundance and activity were monitored by Western blot analysis.Time course experiments revealed that both aldosterone (3nM) and the synthetic glucocorticoid dexamethasone (100nM) stimulated Iami within 1h. Whilst aldosterone‐induced Iami peaked at 3–6h before returning to baseline, dexamethasone‐induced Iami remained stimulated after 24h. Increasing concentrations of the endogenous glucocorticoid corticosterone (0.01–100nM) did not alter Iami, indicative of functional 11βHSD2. At concentrations ≥1μM however, Iami was stimulated indicating the capacity of the enzyme had been surpassed. Inhibition of 11βHSD2 with carbenoxolone (CBX) unmasked a concentration‐dependent stimulation of Iami by corticosterone appearing at 1nM. Changes in Iami correlated with increased abundance and activity of the serum and glucocorticoid‐induced kinase 1 (SGK1). Aldosterone also stimulated Iami, as well as SGK1 abundance and activity, in a concentration‐dependent manner but was unaffected by pre‐treatment with CBX confirming that aldosterone is not a substrate for 11βHSD2. To determine the steroid receptors involved in mediating the effects of corticosteroids, Iami was measured following pharmacological inhibition of the MR using the non‐steroidal compound PF‐03882845 or of the GR using mifepristone. Application of aldosterone (3nM), dexamethasone (100nM) or corticosterone (100nM, in the presence of CBX) for 3h, stimulated Iami by 2.8‐, 3.9‐ and 3.6‐fold, respectively. Both aldosterone‐ and corticosterone‐induced Iami were inhibited predominantly (>65%) by MR antagonism, but were also inhibited by GR antagonism, ~55% and ~20%, respectively. In contrast, dexamethasone‐induced Iami was inhibited predominantly by GR antagonism, with only a small component inhibited by MR blockade.Whilst stimulation of ENaC‐mediated Na+ transport by aldosterone and corticosterone, unlike dexamethasone, is predominantly mediated by MR, these data indicate that the GR may play a permissive role in mediating the Na+ retaining effects of endogenous corticosteroids in the distal nephron.Support or Funding InformationFunded by a Kidney Research UK Intermediate Fellowhship.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.