ENaC-mediated alveolar fluid clearance and lung fluid balance depend on the channel-activating protease 1

Abstract

Sodium transport via epithelial sodium channels (ENaC) expressed in alveolar epithelial cells (AEC) provides the driving force for removal of fluid from the alveolar space. The membrane-bound channel-activating protease 1 (CAP1/Prss8) activates ENaC in vitro in various expression systems. To study the role of CAP1/Prss8 in alveolar sodium transport and lung fluid balance in vivo, we generated mice lacking CAP1/Prss8 in the alveolar epithelium using conditional Cre-loxP-mediated recombination. Deficiency of CAP1/Prss8 in AEC induced in vitro a 40% decrease in ENaC-mediated sodium currents. Sodium-driven alveolar fluid clearance (AFC) was reduced in CAP1/Prss8-deficient mice, due to a 48% decrease in amiloride-sensitive clearance, and was less sensitive to beta(2)-agonist treatment. Intra-alveolar treatment with neutrophil elastase, a soluble serine protease activating ENaC at the cell surface, fully restored basal AFC and the stimulation by beta(2)-agonists. Finally, acute volume-overload increased alveolar lining fluid volume in CAP1/Prss8-deficient mice. This study reveals that CAP1 plays a crucial role in the regulation of ENaC-mediated alveolar sodium and water transport and in mouse lung fluid balance.

Figure 1. Generation of alveolar epithelium-specific CAP1/Prss8-deficient mice

1. Triple transgenic mice were generated that harbour the rtTA protein under the control of the SPC promoter in distal lung epithelial cells (SPC tg). Upon doxycycline (DOX) treatment, rtTA activates expression of the (tetO)₇-CMV-Cre recombinase transgene (Cre tg). Cre recombinase recognizes the loxP sites (black triangles) in the floxed CAP1/Prss8 gene locus, causing deletion of the exons 3–5 to generate a null allele (Prss8Δ). Coding (grey boxes) and noncoding exon sequences (white boxes) are indicated.

2. Expression of surfactant proteins A (sftpa) and C (sftpc), and of Cre recombinase (Cre) mRNA transcripts by semi-quantitative RT-PCR run at 25, 30 and 35 cycles in distal lung extracts from control (no tg: black bars; SPC tg^+/0: light grey bars; Cre tg^+/0: dark grey bars) and knockout (SPC tg^+/0; Cre tg^+/0, white bars) groups. Quantification of signals was performed after 30 cycles for sftpa and sftpc, and after 35 cycles for Cre. Results are expressed as the ratio of sftpa, sftpc or Cre mRNA/β-actin mRNA (n = 3 mice per group). ***: Significantly different from control groups (p < 0.001).

3. PCR analysis with primers A–C distinguish between lox (413 bp) and Δ (473 bp) alleles of CAP1/Prss8 gene locus (top). Note the shift of the Prss8lox allele into Prss8Δ allele in AEC harbouring the SPC and Cre transgenes (lane 1). Detection of SPC tg (middle) and Cre tg (bottom), and of myogenin (middle and bottom).

4. Quantification of CAP1/Prss8, CAP2 and CAP3 mRNA transcripts by qRT-PCR in AEC isolated from pooled control mice (no transgene or only SPC or Cre transgene: black bars) and knockout mice (SPC^tg+/0; Cre^tg+/0: white bars). Results are expressed as the ratio of CAP1/Prss8, CAP2/Tmprss4 or CAP3/Prss14 mRNA/β-actin mRNA (n = 4 mice per group).**: Significantly different from control group (p < 0.01).

Figure 2. Distal lung histology

1. Light photomicrographs of representative lung sections from control and knockout littermates stained with haematoxylin and eosin. The morphological aspect of blood vessels (V), bronchioles (BR), alveolar ducts (AD) and alveoli was normal in knockout mice. Scale bar, 100 µm.

2. Electron micrographs showing that the ultrastructure of alveolar septa and alveolar epithelium was normal in both groups. AS, alveolar space; AT1, alveolar type 1 cells; AT2, alveolar type 2 cells; LB, lamellar body; En, endothelial cell; CL, pulmonary capillary lumen; RBC, red blood cell. Scale bar, 2 µm.

Figure 3. Expression of ENaC subunits in distal lung epithelial cells

1. Quantification of mouse ENaC subunit mRNA transcript expression by semi-quantitative RT-PCR run at 30 cycles in distal lung homogenate extracts from control (no tg: black bars; SPC tg^+/0: light grey bars; Cre tg^+/0: dark grey bars) and knockout mice (SPC tg^+/0; Cre tg^+/0, white bars). Results are expressed as the ratio of α-, β- or γ-mENaC mRNA/β-actin mRNA (n = 3 mice per group).

2. Representative immunoblots showing the expression of ENaC subunit proteins and β-actin protein in distal lung homogenate extracts from control and knockout mice.

3. Quantification of α-, β- and γ-mENaC signals in pooled control (black bars) and knockout (KO, white bars) mouse lung homogenates was obtained using NIH image software. Results are expressed as the ratio of mENaC protein/β-actin protein (n = 4 mice per group). There was no significant difference between the two groups with respect to the mRNA transcript or protein expression levels.

Figure 4. Electrophysiological properties and expression of the tight junction protein occludin and ZO-1 in control and CAP1/Prss8-deficient mouse AEC

1. Transepithelial resistance (R_te) and (B) PD were measured under open-circuit conditions in control (black bars) and knockout (white bars) AEC monolayers grown on transwell filters for 5 days.

2. Equivalent short-circuit current (I_eq) was calculated from R_te and PD at baseline (total I_eq), and after apical treatment with amiloride (10 µM) (amiloride-insensitive I_eq) (9–12 filters per group from 3 independent cultures). Amiloride-sensitive I_eq is the difference between I_eq values in the absence and in the presence of amiloride. Transepithelial PD as well as total and amiloride-sensitive I_eq were significantly reduced in knockout AEC monolayers. *: Significantly different from control group (p < 0.05).

3. AEC from control (No tg) and knockout mice (SPC tg^+/0; Cre tg^+/0) were cultured for 5 days on transwell filters before immunofluorescent detection of occludin and ZO-1 was performed. Nuclei are counterstained with Sytox Orange. Occludin staining was localized at the periphery of the cells in both groups. Scale bar, 25 µm.

Figure 5. Alveolar fluid clearance under basal condition in control and CAP1/Prss8-deficient mice

1. Sodium-driven AFC was measured at baseline over a 15-min period in control (black, light grey and dark grey bars) and knockout (white bars) CAP1/Prss8^lox/lox littermates aged 2–5 months at 37°C using an in situ nonventilated model in which the airspace was instilled with an isoosmolar Ringer's lactate solution containing ¹²⁵I-albumin as a volume marker. Note that AFC was significantly lower in the knockout group than in the control groups (*: significantly different from control groups, p < 0.05).

2. AFC was measured in the absence (baseline) or presence of amiloride (final concentration: 1 mM) or hNE (final concentration: 33 µg/ml) in the alveolar instillate in pooled control littermates (black bars) and knockout (white bars) littermates.

3. Calculated values of amiloride-sensitive AFC (difference between AFC values in the absence and in the presence of amiloride) and hNE-mediated AFC (difference between AFC values in the absence and in the presence of hNE). Results are expressed as percentage fluid absorption at 15 min (5–13 mice per group for basal and hNE experiments, 4 mice per group for amiloride experiments. *, **, ***: Significant difference between groups as indicated (*, p < 0.05; **, p < 0.01; and ***, p < 0.001, respectively).

Figure 6. Alveolar fluid clearance under β₂-agonist-stimulated condition in control and CAP1/Prss8-deficient mice

1. Sodium-driven AFC was measured at baseline and in the presence of terbutaline (final concentration in alveolar instillate: 10⁻⁴ M) over a 15-min period in control (black, light grey and dark grey bars) and knockout (white bars) CAP1/Prss8^lox/lox littermates aged 2–5 months at 37°C as described in Methods. AFC values at baseline and in the presence of terbutaline were significantly lower in the knockout group than in control groups (*, **: significantly different from control groups, p < 0.05 and p < 0.01, respectively).

2. AFC was measured in the absence or presence of terbutaline, hNE (33 µg/ml), the elastase inhibitor EPI-hNE4 (50 µg/ml), or the serine protease inhibitor aprotinin (100 µg/mL) in the alveolar instillate in pooled control littermates (black bars) and knockout (white bars) littermates. Results are expressed as percentage fluid absorption at 15 min (n = 5–12 mice per group and per condition). *, **: Significantly different from respective control group (p < 0.05, p < 0.01, respectively); §, §§, §§§: significantly different from baseline value (no treatment) in the corresponding group (p < 0.05, p < 0.01 and p < 0.001, respectively).

Figure 7. Effect of acute volume-overload on lung fluid balance in control and CAP1/Prss8-deficient mice

The volume of the alveolar epithelial lining fluid was estimated from the change in ¹²⁵I-albumin concentration in the alveolar instillate at baseline and at the end of acute saline infusion (40% body weight over 2 h, volume-overload) in control (black bars) and knockout (white bars) littermates aged 2–6 months. Results are expressed in microliter (n = 5–7 mice per group and per condition). *: Significantly different from control group (p < 0.05). §, §§§: Significantly different from baseline value in the corresponding group (p < 0.05 and p < 0.001, respectively).