Mineralocorticoid effects in the late gestation ovine fetal lung

Abstract

This study was designed to determine the effects of corticosteroids at MR in the late-gestation fetal lung. Since both the mineralocorticoid receptor (MR) and the glucocorticoid receptor (GR) are expressed at relatively high levels in the fetal lung, endogenous corticosteroids may act at MR as well as GR in the preterm fetal lung. The GR agonist, betamethasone, the MR agonist, aldosterone, or both were infused intravenously for 48 h in ovine fetuses of approximately 130 days gestation. Effects on airway pressures during stepwise inflation of the in situ lung, expression of ENaC alpha (SCNN1A), ENaC beta (SCNN1B), and Na,K ATPase (ATP1A1), and elastin and collagen content were determined after the infusions. We found that aldosterone significantly reduced the airway pressure measured during the initial step in inflation of the lung, although aldosterone had no overall effect on lung compliance, nor did aldosterone induce expression of ENaCα, ENaCβ or Na,K ATPaseα1. Betamethasone significantly increased expression of the epithelial sodium channel (ENaC) subunit mRNAs, and collagen and elastin content in the lungs, although this dose of betamethasone also had no effect on lung compliance. There was no synergy between effects of the MR and GR agonists. Transcriptomic analysis suggested that although aldosterone did not alter genes in pathways related to epithelial sodium transport, aldosterone did alter genes in pathways involved in cell proliferation in the lungs. The results are consistent with corticosteroid-induced fluid reabsorption at birth through GR rather than MR, but suggest that MR facilitates lung maturation, and may contribute to inflation with the first breaths via mechanisms distinct from known aldosterone effects in other epithelia.

Keywords: Fetus; glucocorticoid; lung; mineralocorticoid.

Figure 1.

(A–D) Volume–Pressure relationships in ovine fetal lung after treatment with Aldo (red circles and bars, n = 5), 0.25Beta (green squares and bars, n = 4), 0.75Beta (blue triangles and bars, n = 4), and Aldo/0.25Beta (pink squares and bars, n = 4), Aldo/0.75Beta (purple triangles and bars, n = 4), or control twins (black circles, n = 9). Panels A and C, peak pressure changes during first and second sets of inflations, respectively; Panels B and D, steady‐state relaxation pressures during first and second sets of inflations, respectively. *indicates pressure in aldosterone‐treated lungs different than control group at same volume, P < 0.05 Panels E–F, calculated compliance during inflation using peak pressure and injected volumes during the first (E) and second (F) sets of inflations. a, different than 10 mL; b, different from 20 mL; c, different from 30 mL during same series of inflations. *indicates different from control group at same volume. All data are expressed as mean ± SEM.

Figure 2.

Top panels: Relative fold changes in mRNA of ENaC‐α, ENaC‐β, and Na,K ATPase‐α1. Expression of mRNA is expressed as mean fold change in each group (indicated below each bar) relative to expression in the control group: black bars, betamethasone (Beta); green bars, 0.25Beta; blue bars, 0.75Beta; red bars, aldosterone (Aldo), pink bars, Aldo/0.25Beta; purple bars, Aldo/0.75Beta. Lower panels: Changes in ENaC‐α and ENaC‐β protein expression. Protein was quantified as optical density of bands with equal loading of membrane (M) and whole cell (WC) protein (70 μg); estimated molecular weights for each band that was quantified are indicated. Differences between groups for each measure are indicated: *different from control; ^#different from aldosterone alone; ^†differences between 0.25Beta and 0.75Beta treatment with the same aldosterone treatment; ^‡difference between with aldosterone and without aldosterone treatment at same dose of betamethasone; P < 0.05. n's are as in Fig. 1.

Figure 3.

Representative images of lung depicting MR (green) and ENaCα (red) localization in lungs from (A) control fetuses, or fetuses after infusion of: (B) 0.25Beta, (C) 0.75Beta, (D) Aldo, (E) Aldo/0.25Beta, and (F) Aldo/0.75Beta. Large airway staining is shown in the large image in each panel; the smaller inserts are images of alveoli. All images are shown at 400×; the scale for all figures is shown by the white bar in panel E. Examples of MR staining are shown with solid arrowheads and ENaC‐α is shown with open arrowheads with long tails.

Figure 4.

Representative images of staining for elastin (black against yellow tissue staining; upper panels within each set) and collagen (red, in lower panels in each set) in lungs from (A) control fetuses, or fetuses after infusion of: (B) 0.25 mg Beta, (C) 0.75 mg Beta, (D) Aldo, (E) Aldo/0.25Beta, and (F) Aldo/0.25Beta.

Figure 5.

Fold changes in gene expression in four sets of twin pregnancies with one aldosterone‐treated fetus (red bars) relative to the mean values in the control twins (black). *difference between groups, P < 0.05.

Figure 6.

Fold changes in expression of AKR1C3, NCAPH, CXCR4, GMZB, SC5, and CATHL1B in the six groups of fetuses. Expression of mRNA is expressed as mean fold change in each group (indicated below each bar) relative to expession in the control group: black bars, betamethasone (Beta); green bars, 0.25Beta; blue bars, 0.75Beta; red bars, aldosterone (Aldo), pink bars, Aldo/0.25Beta; purple bars, Aldo/0.75Beta. *different from control; ^#different from aldosterone alone; ^†differences between 0.25Beta and 0.75Beta treatment with the same aldosterone treatment.