Transgenically expressed human delta epithelial sodium channel facilitated fluid absorption in mouse fetal lung explants

Abstract

Epithelial sodium channels (ENaCs) are essential for sodium (Na⁺) transport and maintaining fluid balance, which is vital for the removal of fetal fluid at birth and the homeostasis of luminal fluid in the lungs. In mice, ENaC is composed of three subunits (α, β, and γ). However, in humans, a fourth δ-subunit is also expressed. This study investigated the physiological role of the δ-ENaC in fetal/neonatal lungs, an area that remains less explored despite its potential significance. We measured expansion in mouse E15 lung explants expressing human δ-ENaC (SCNN1D-Tg). We found that transgenic expression of δ-ENaC enhanced fluid absorption and significantly reduced the surface area increase compared with wild-type (WT) explants (142.30 ± 5.81% vs. 163.80 ± 5.95% expansion, P < 0.001). Amiloride treatments revealed that both α-ENaC and δ-ENaC contributed to fluid absorption. No statistical significance was observed in the amiloride-sensitive fraction of SCNN1D-Tg explants compared with WT preparations in the presence of 100 µM amiloride (P = 0.400). In contrast, a significant reduction in amiloride-sensitive fraction in SCNN1D-Tg explants was observed in the presence of 10 µM amiloride (P < 0.001). Furthermore, specific blocking of α-ENaC using α-13 inhibitory peptide resulted in a 2.12-fold growth increase in WT explants, compared with a 1.47-fold increase in SCNN1D-Tg explants (P < 0.001). In summary, this study provides evidence that δ-ENaC may contribute to fluid absorption in E15 and newborn lungs, highlighting its significance in alveolar fluid regulation in prenatal and postnatal lungs.NEW & NOTEWORTHY The findings of our study highlight the significance of δ-ENaC in lung fluid regulation. Transgenic expression of human δ-ENaC contributes to fluid absorption increase, supporting its potential as a pathway for alveolar fluid clearance in E15 and postnatal lungs.

Keywords: E15 lung explants; amiloride; epithelial sodium channels; lung explants; α-13 peptide.

Figure 1.. Area comparison of WT and SCNN1D-Tg E15lung explants ex vivo.

Distal lung explants from WT and SCNN1D-Tg embryo (E15) were cultured for three days and imaged every 24h. A. Experimental protocol. B. Representative DIC images of two sets of experiments. Lung explants were imaged 2h after seeding for the first time (day 0, X 60). Scale bar, 1 mm. C. Comparison of WT and Tg explant surface area on day 3. Mean ± S.E.M. Two tailed t-test . P<0.050, n=19 explants/7 WT embryos, n=15 explants/6 SCNN1D-Tg embryos. The surface area of lung explants was measured using ImageJ software version 1.54k. Explants showing less than 20 % growth on day 3 relative to day 0, or lacking clear alveolar structures, were excluded from the analysis.

Figure 2.. Amiloride inhibits δβγ-ENaC mediated fluid absorption in E15 lung explant in a dose-dependent manner.

Distal lung explants from WT and SCNN1D-Tg embryo (E15) were cultured for three days and imaged every 24h. A. Explants were exposed to amiloride 10 μM, or 100 μM. Representative DIC images of WT and SCNN1D-Tg (Tg) explants. Magnification 60 X, scale bar, 1 mm. B. Relative surface area on day 3 over day 0 measurement. Mean ± S.E.M. Two-Way ANOVA with the Tukey’s multiple comparisons test.. 2-sided. α, P< 0.0005 for WT+ 10 μM vs SCNN1D-Tg + 10 μM; β, P< 0.0001 for SCNN1D-Tg + 10 μM vs SCNN1D-Tg + 100 μM. C. Amiloride-sensitive fraction on day 3. n=16 explants/12 WT embryos and n=16 explants/11 SCNN1D-Tg embryos. Chi-square test with 2-sided p<0.050. The surface area of lung explants was measured using ImageJ software version 1.54k. Explants showing less than 20% growth on day 3 relative to day 0 or lacking clear alveolar structures were excluded from the analysis.

Figure 3.. Effects of α-type ENaC inhibitor (α−13 peptide 330 μM) on surface area of lung explants.

Distal lung explants from WT and SCNN1D-Tg embryo (E15) were cultured for three days and imaged every 24h. A. Representative images of lung explants from day 0 to day 3. Scale bar, 1 mm. Explants were incubated with α−13 peptide for 72 h. Controls were cultured in the absence of the peptide. B. Relative surface area over day 0 measurement. Mean ± S.E.M. Three -way ANOVA test with the Dunnetts’s multiple comparison test, , $, P<0.0001 for WT control vs Tg; & P<0.0001 for control vs. α−13, #, P<0.0001 for days vs WT & SCNN1D-Tg; P=0.0001 , days vs α−13; P<0.0001, WTx Tg vs. α−13; P=0.0002, Days x (WT vs Tg) x (A13 inhibition); P=0.007.C. α−13 peptide-inhibitable component of surface area. n=15 explants/12 WT embryos and n=15 explants/11 SCNN1D-Tg embryos. Chi-square test with 2-sided<0.05. P=0.225. The surface area of lung explants was measured using ImageJ software Version 1.54k. Explants showing less than 20% growth on day 3 relative to day 0, or lacking clear alveolar structures, were excluded from the analysis.

Figure 3.. Effects of α-type ENaC inhibitor (α−13 peptide 330 μM) on surface area of lung explants.

Distal lung explants from WT and SCNN1D-Tg embryo (E15) were cultured for three days and imaged every 24h. A. Representative images of lung explants from day 0 to day 3. Scale bar, 1 mm. Explants were incubated with α−13 peptide for 72 h. Controls were cultured in the absence of the peptide. B. Relative surface area over day 0 measurement. Mean ± S.E.M. Three -way ANOVA test with the Dunnetts’s multiple comparison test, , $, P<0.0001 for WT control vs Tg; & P<0.0001 for control vs. α−13, #, P<0.0001 for days vs WT & SCNN1D-Tg; P=0.0001 , days vs α−13; P<0.0001, WTx Tg vs. α−13; P=0.0002, Days x (WT vs Tg) x (A13 inhibition); P=0.007.C. α−13 peptide-inhibitable component of surface area. n=15 explants/12 WT embryos and n=15 explants/11 SCNN1D-Tg embryos. Chi-square test with 2-sided<0.05. P=0.225. The surface area of lung explants was measured using ImageJ software Version 1.54k. Explants showing less than 20% growth on day 3 relative to day 0, or lacking clear alveolar structures, were excluded from the analysis.