Submersion and hypoxia inhibit alveolar epithelial Na+ transport through ERK/NF-κB signaling pathway

Abstract

Background: Hypoxia is associated with many respiratory diseases, partly due to the accumulation of edema fluid and mucus on the surface of alveolar epithelial cell (AEC), which forms oxygen delivery barriers and is responsible for the disruption of ion transport. Epithelial sodium channel (ENaC) on the apical side of AEC plays a crucial role to maintain the electrochemical gradient of Na⁺ and water reabsorption, thus becomes the key point for edema fluid removal under hypoxia. Here we sought to explore the effects of hypoxia on ENaC expression and the further mechanism related, which may provide a possible treatment strategy in edema related pulmonary diseases.

Methods: Excess volume of culture medium was added on the surface of AEC to simulate the hypoxic environment of alveoli in the state of pulmonary edema, supported by the evidence of increased hypoxia-inducible factor-1 expression. The protein/mRNA expressions of ENaC were detected, and extracellular signal-regulated kinase (ERK)/nuclear factor κB (NF-κB) inhibitor was applied to explore the detailed mechanism about the effects of hypoxia on epithelial ion transport in AEC. Meanwhile, mice were placed in chambers with normoxic or hypoxic (8%) condition for 24 h, respectively. The effects of hypoxia and NF-κB were assessed through alveolar fluid clearance and ENaC function by Ussing chamber assay.

Results: Hypoxia (submersion culture mode) induced the reduction of protein/mRNA expression of ENaC, whereas increased the activation of ERK/NF-κB signaling pathway in parallel experiments using human A549 and mouse alveolar type 2 cells, respectively. Moreover, the inhibition of ERK (PD98059, 10 µM) alleviated the phosphorylation of IκB and p65, implying NF-κB as a downstream pathway involved with ERK regulation. Intriguingly, the expression of α-ENaC could be reversed by either ERK or NF-κB inhibitor (QNZ, 100 nM) under hypoxia. The alleviation of pulmonary edema was evidenced by the administration of NF-κB inhibitor, and enhancement of ENaC function was supported by recording amiloride-sensitive short-circuit currents.

Conclusions: The expression of ENaC was downregulated under hypoxia induced by submersion culture, which may be mediated by ERK/NF-κB signaling pathway.

Keywords: Alveolar epithelial cell; Epithelial sodium channel; Extracellular signal-regulated kinase; Hypoxia; Nuclear factor κB.

Fig. 1

Hypoxia model was established in AEC. A Representative graphs of HIF-1α in different volumes (2 ml, 4 ml, 6 ml, and 8 ml) of culture medium at 24 h and HIF-1α in 8 ml at different time (12 h, 24 and 48 h) in A549 cells. B, C The statistical data obtained from Western Blot and quantified through gray analysis (HIF-1α/β-actin). ***P < 0.001, compared with 2 ml; ^&&P < 0.01, compared with 0 h. One-way ANOVA followed by Bonferroni’s test was used to analyze the difference of the means for significance. n = 5–6

Fig. 2

Hypoxia decreased the protein and mRNA of ENaC. A Representative graph of α/γ-ENaC in different volumes (2 ml, 4 ml, 6 ml, and 8 ml) of culture medium for 24 h in A549 cells. B, C The statistical data of α/γ-ENaC in different volumes (2 ml, 4 ml, 6 ml, and 8 ml) of culture medium for 24 h in A549 cells obtained from Western Blot and quantified through gray analysis (α/γ-ENaC/β-actin). D Representative graph of α/γ-ENaC in 8 ml culture medium at different times (12 h, 24 h, and 48 h) in A549 cells. E, F The statistical data obtained from Western Blot and quantified through gray analysis (α/γ-ENaC/β-actin). G–I α/β/γ-ENaC mRNA expression level of A549 cells after 12 h, 24 h, and 48 h in 8 ml culture medium (α/β/γ-ENaC/β-actin). ^&P < 0.05, compared with 2 ml group. *P < 0.05, **P < 0.01, ***P < 0.001, compared with 0 h group. One-way ANOVA followed by Bonferroni’s test was used to analyze the difference of the means for significance. n = 3–6

Fig. 3

Effect of hypoxia on ENaC and associated signal pathways on A549 and mouse AT2 cells. A, C Representative graph of HIF-1α, α-ENaC, ERK1/2, and NF-κB signaling pathways under hypoxia. B, D The statistical data obtained from Western Blot and quantified through gray analysis. *P < 0.05, **P < 0.01, ***P < 0.001, compared with Control group. Mann-Whitney U test with Bonferroni correction and One-way ANOVA followed by Bonferroni’s test were used to analyze the difference of the means for significance. n = 4–6

Fig. 4

Hypoxia-induced activation of NF-κB signaling pathway was downregulated by ERK inhibitor in A549 and AT2 cells. A–D Representative and statistical data of t-ERK1/2 and p-ERK1/2 after PD98059 (ERK inhibitor, 10 µM) treatment. E–H Representative and quantitative analyses of p-IκB/t-IκB. I–L Representative and quantitative analyses of p-p65/p65. *P < 0.05, **P < 0.01, ***P < 0.001, compared with Control group; ^&P < 0.05, ^&&P < 0.01, ^&&&P < 0.001, compared with Hypoxia group. One-way ANOVA followed by Bonferroni’s test was used to analyze the difference of the means for significance. n = 3–6

Fig. 5

Hypoxia-induced decrease of α-ENaC was mediated by the activation of the ERK1/2 signaling pathway. A, B Representative and statistical data of Western Blot of α-ENaC expression of A549 cells cultured under hypoxia (8 ml) for 24 h after PD98059 (10 µM) treatment. D, E Representative and statistical data of Western Blot of α-ENaC expression of AT2 cells cultured in hypoxic condition (8 ml) for 24 h after PD98059 (10 µM) treatment. C, F qRT-PCR results for α-ENaC mRNA in A549 and AT2 cells, and β-actin was used as internal reference. *P < 0.05, **P < 0.01, compared with Control group; ^&P < 0.05, compared with Hypoxia group. One-way ANOVA followed by Bonferroni’s test was used to analyze the difference of the means for significance. n = 3–6

Fig. 6

Hypoxia-induced decrease of α-ENaC was mediated by the activation of the ERK1/2 and NF-κB signaling pathways. A Immunofluorescence of α-ENaC (green) in AT2 cells cultured in hypoxic condition (8 ml) for 24 h after PD98059 (10 µM) treatment. The nucleus was stained with DAPI (blue). Scale bar = 20 μm. B, C Representative and statistical data of α-ENaC in AT2 cells cultured in hypoxic condition (8 ml) for 24 h after QNZ (NF-κB inhibitor, 100 nM) treatment. n = 4–6. D Representative Isc traces of Control, Hypoxia and Hypoxia + QNZ group in H441 cell monolayers, and amphotericin B (100 µM) was added to the basolateral compartment, followed by 100 µM amiloride applied to the apical side. E Statistic ASI, defined as the difference between the total current and the amiloride-resistant current, n = 4. ***P < 0.001, compared with Control group; ^&P < 0.05, ^&&&P < 0.001, compared with Hypoxia group. One-way ANOVA followed by Bonferroni’s test was used to analyze the difference of the means for significance

Fig. 7

Hypoxia-induced decrease of AFC and ENaC activity were associated NF-κB pathway. A NF-κB inhibitor (QNZ, 1 mg/kg) was administered intraperitoneally to mice 1 h before the exposure to hypoxic condition (8% O₂) for 24 h, and the effect of NF-κB inhibitor was assessed by AFC assay, n = 4–5. B The lung W/D ratio changed in QNZ-treated mice, n = 3–5. C Representative Isc traces of Control, Hypoxia and Hypoxia + QNZ group in mouse tracheal epithelium, and 1 mM amiloride was added to apical solution. D Statistic ASI, defined as the difference between the total current and the amiloride-resistant current, n = 3–4. *P < 0.05, **P < 0.01, ***P < 0.001, compared with Control group; ^&P < 0.05, ^&&P < 0.01, ^&&&P < 0.001, compared with Hypoxia group. One-way ANOVA followed by Bonferroni’s test was used to analyze the difference of the means for significance

Fig. 8

The schematic diagram depicts the potential mechanism about decreased expression of ENaC in AT2 cells during hypoxia. The accumulation of pulmonary edema fluid created a hypoxic environment, which stimulated the phosphorylation of ERK1/2, induced phosphorylation of IκB and NF-κB p65, and promoted nuclear translocation of p65. Proinflammatory cytokines may also affect ENaC activity and expression. Eventually, vectorial Na⁺ transport was suppressed, leading to edema formation. AT1/2 alveolar type 1/2, ERK1/2 extracellular signal-regulated kinase 1/2, ENaC epithelial sodium channel, HIF-1α hypoxia-inducible factor 1α, IκBα inhibitor κBα, NF-κB nuclear factor κB