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Am J Physiol Lung Cell Mol Physiol 293: L537-L545, 2007. First published May 18, 2007; doi:10.1152/ajplung.00373.2006
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EDITORIAL FOCUS

The role of {alpha}-, beta-, and {gamma}-ENaC subunits in distal lung epithelial fluid absorption induced by pulmonary edema fluid

Nael Elias,1 Bijan Rafii,1 Muhammad Rahman,1 Gail Otulakowski,1,4 Ernest Cutz,2,3,6 and Hugh O'Brodovich1,4,5

1Program in Lung Biology, 2Division of Pathology, and 3Department of Pediatric Laboratory Medicine, Hospital for Sick Children Research Institute, Toronto; and Departments of 4Pediatrics, 5Physiology, and 6Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada

Submitted 21 September 2006 ; accepted in final form 14 May 2007


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Edema fluid (EF) increases epithelial Na+ transport by rat fetal distal lung epithelia (FDLE) and induces net lung fluid absorption in fetal mouse lung explants [Rafii B, Gillie DJ, Sulowski C, Hannam V, Cheung T, Otulakowski G, Barker PM, O'Brodovich H. J Physiol (Lond) 544: 537–548, 2002]. We now show that EF increases fluid absorption across monolayers of rat FDLE in a dose-dependent manner. To study the role of subunits of the epithelial Na+ channel (ENaC) in the phenomena, we cultured explants from the distal lungs of 16-day gestational age wild-type (WT) or {alpha}-, beta-, or {gamma}-ENaC knockout or heterozygote (HT) mice. WT explants cultured in media continuously expanded over time as a result of net fluid secretion. In contrast, when explants were exposed to EF for 24 h, net fluid absorption occurred. EF-exposed explants had normal histology, but marked changes were seen after Triton X-100 or staurosporine exposure. Transmission electron microscopy showed EF promoted lamellar body formation and abundant surfactant in the explants' lumens. EF-induced changes in explant size were similar in {alpha}-ENaC knockout, WT, and HT littermate fetal lung explants (P > 0.05). In contrast, EF's effect was attenuated in beta- and {gamma}-ENaC knockouts (P < 0.05) vs. WT and HT littermate fetal lung explants. EF exposure slightly decreased or had no effect on mRNA levels for {alpha}-ENaC in various mouse genotypes but decreased expression of beta- and {gamma}-ENaC subunit mRNAs (P < 0.01) across all genotype groups. We conclude that beta- and {gamma}-, but not {alpha}-, ENaC subunits are essential for EF to exert its maximal effect on net fluid absorption by distal lung epithelia.

epithelium; Na+ transport; lung development; amiloride-sensitive epithelial Na+ channel

PULMONARY EDEMA can mainly occur as a result of one or both of the following mechanisms: increased transvascular pressure in the pulmonary microvasculature, as occurs in congestive heart failure, or increased permeability of the alveolar-capillary membrane to solutes, as occurs in adult respiratory distress syndrome. The ability of the lungs to clear this air space fluid has important clinical implications. For example, others have demonstrated that the active absorption of air space fluid in adults with pulmonary edema correlates with improved survival (20).

Air space fluid clearance is actively driven by epithelial Na+ transport, which crosses the apical membrane and is then extruded across the basolateral membrane by Na+-K+-ATPase (18, 19). The rate-limiting step in active Na+ transport by epithelia is the number and activity of the apical membrane Na+ channels. One of these Na+ channels is the amiloride-sensitive epithelial Na+ channel (ENaC) that is composed of {alpha}-, beta-, and {gamma}-subunits (6). However, it has been shown that normal mammalian lung epithelia have both an amiloride-sensitive and an amiloride-insensitive component to their active Na+ transport both in vivo (24) and in vitro (8, 25). Presently, the molecular basis of this amiloride-insensitive Na+ transport is poorly understood. Regardless, if one could determine mechanisms underlying the regulation of this amiloride-sensitive and -insensitive Na+ transport, one could in the future identify novel approaches for the therapy of patients with pulmonary edema.

It has been previously shown by our group that edema fluid (EF), prepared from rats with acute cardiogenic pulmonary edema, increases amiloride-insensitive Na+ transport across rat fetal distal lung epithelia (FDLE) in a time- and dose-dependent manner and increases net fluid absorption in lung explants prepared from wild-type (WT) and {alpha}-ENaC-deficient fetal mice (30). The following study was conducted to determine the dependency of EF-induced lung fluid net absorption on each of the {alpha}-, beta-, or {gamma}-ENaC subunits. We found, and in contrast to what might be predicted from previous studies carried out in Xenopus laevis oocytes (6), that beta- and {gamma}-, but not {alpha}-ENaC, subunits are required for EF to have its full effect in mammalian lung epithelia.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Lung explant preparation. WT C57B6 or mice that were heterozygote (HT) for a deficiency in one of the {alpha}- (12), beta- (21), or {gamma}- (3) ENaC subunits (provided by Dr. E. Hümmler, Lausanne, Switzerland) were maintained in the Hospital for Sick Children's central animal facilities. HT mice were interbred to generate WT, HT, or homozygous-deficient (knockout or KO) littermates in one of the {alpha}-, beta-, or {gamma}-ENaC subunits. In some experiments, WT mice only were bred and used. The pregnant mice were killed at 16 days gestational age by cervical dislocation. An abdominal incision was performed, and all fetuses were removed, weighed, and placed in F-12 media on ice. Each fetus was further dissected to harvest the left lung, which was placed in F-12 media on ice. Under the microscope, the peripheral-most ribbon of left lung was dissected and cut into small pieces (lung explants). Lung explants (4–5/membrane) were placed on 24-mm Corning Transwell-Clear permeable supports (Fisher Scientific, Nepean, ON) containing 1:1 F-12-DMEM solution supplemented with bovine serum albumin (1 µg/ml–1) and incubated at 37°C in a 95% air-5% CO2 environment. In ENaC KO mice experiments, explants from each fetus were distributed onto two Transwells for subsequent exposure to EF and media, and the liver of each fetus was harvested for genotyping and allocation to either WT, HT, or KO group.

All experiments involving animals were reviewed and approved by the Hospital for Sick Children's Animal Care Committee.

Materials. All tissue culture media and reagents were from Invitrogen (Burlington, ON). Unless specified otherwise, all other reagents and proteins were obtained from Sigma Chemical (Oakville, ON).

Study protocol. Before genotyping and after 24 h of being cultured in media, lung explants were assigned to exposure to EF-50%. Other reagents were as described below or assigned to DMEM/F-12 (control group). In some experiments, we utilized different percentages of EF to establish the dose dependency of EF's effect on fluid movement across WT fetal murine distal lung explants. Digital photographs of explants were taken immediately after they were placed in culture (time = 0 h). Twenty-four hours later, additional photographs of the explants were taken, and explants were placed into fresh media (DMEM/F-12-control) or into DMEM/F-12 containing EF or other reagents. After an additional 24-h "test period," photographs of lung explants were taken, and RNA was isolated as described below. A cross-sectional area of lung explant was measured by pixel count using Adobe Photoshop. Percent of size change was calculated per each explant. Explants growing less than 20% in their cross-sectional area during the first 24 h were excluded. The main outcome was percent change in explant cross-sectional area over the test period. Previous work has demonstrated that this increase in cross-sectional area of explant reflects net lung fluid secretion (2, 11).

In additional experiments, the timeframe of which was similar to that described above, lung explants were exposed to control media or media containing EF, Triton X-100 (0.1%), or staurosporine (1 µM) and prepared for light and transmission electron microscopic examination. For histology studies, explants were fixed with 10% formalin, and ultrathin sections were stained with hematoxylin and eosin for light microscopy. For transmission electron microscopy (TEM) studies, explants were fixed in a solution containing 4% formaldehyde and 1% glutaraldehyde in phosphate buffer and then postfixed in 1% osmium tetroxide. The specimens were then dehydrated in a graded series of acetone and subsequently infiltrated and embedded in Epon-Araldite epoxy resin. The processing steps from postfixation to polymerization of resin blocks were carried out in a microwave oven, Pelco BioWave 34770 (Pelco International, Clovis, CA), using similar procedures, with slight modification, as recommended by the manufacturer. Ultrathin sections were cut with a diamond knife on the Reichert Ultracut E (Leica Microsystems, Richmond Hill, ON). Sections were stained with uranyl acetate and lead citrate before being examined in the JEM-1011 (JEOL USA, Peabody, MA). Digital electron micrographs were acquired directly with a 1,024 x 1,024-pixel charge-coupled device camera (AMT, Danvers, MA) attached to the TEM.

DNA extraction and genotyping. DNA was extracted from tails in adult mice by standard phenol extraction following proteinase K digestion and from liver tissue in fetuses by using DNeasy tissue kit (Qiagen, Mississauga, ON). Fetuses were genotyped by PCR using oligonucleotides specific to WT and KO genomic sequences. Mouse genotype was determined after photographs of the test period were taken.

Real-time PCR. At the end of the 24-h test period, RNA was extracted from lung explants using an RNeasy minikit (Qiagen) and quantified using Ribo green (Quant-iT Ribogreen RNA assay kit, Invitrogen) followed by fluorescence reading (F-2500, Fluorescence spectrophotometer, Hitachi). Reverse transcription, followed by real-time PCR (ABI Prism 7700 Sequence Detection System; Applied Biosystems, Foster City, CA), was done on each sample using comparative CT method with cytokeratin 18 as an endogenous reference control for relative quantitation of {alpha}-, beta-, and {gamma}-ENaC mRNA. Cytokeratin 18 was chosen because it is only expressed in epithelial cells. Taqman Gene Expression Assay kits (Applied Biosystems) were used for all targets according to the manufacturer's recommended protocols (assay ID numbers: {alpha}ENaC Mm00803386_m1; betaENaC Mm00441213_m1; {gamma}ENaC Mm00441228_m1; cytokeratin 18 Mm01601702_g1). Dilution series of adult mouse lung RNA were used to validate equal efficiencies of target and reference amplifications.

EF preparation. EF was prepared as previously described by our lab (30). In brief, male Sprague-Dawley rats (350 g) were anesthetized and ventilated via tracheostomy. A thoracotomy was performed, and the aorta was clamped for 30 s after a 15 ml x kg–1 Ringer solution was infused. The resultant EF was collected through a tracheal catheter within a few minutes, and the rat was killed with an overdose of anesthetic. EF was immediately centrifuged at 10,000 g for 30 min at 4°C and stored at –85°C. Before each experiment, EF was thawed and filtered through a 0.45-µm Millex-HA (Millipore, Nepean, ON, Canada). For heat-treated EF experiments, EF was heated at 80°C for 20 min and centrifuged at 10,000 g for 10 min at 4°C before use.

Fetal lung DNA content. We observed that KO explants grown in media were larger than WT explants (see RESULTS), and therefore we wondered whether or not there was enhanced fluid secretion with increased distention of the lungs in utero. If this were the case, one would expect lung hyperplasia. Therefore, we performed additional experiments where the entire lungs of individual fetuses of 16-day gestational age were isolated for the measurement of their total DNA content (36) to determine if the lungs of KO fetuses, relative to WT fetuses, had a greater total cell content.

FDLE primary culture. FDLE from 20-day gestation Wistar rat fetuses (term = 22 days) were isolated as previously described (26, 30). FDLE were seeded at 1 x 106 cells/cm2 onto 0.4-µm pore size Snapwell for Ussing chamber studies and on 6.5-mm Transwell permeable cell culture inserts (Corning Costar, Cambridge, MA) for fluid transport measurements. All cells were grown as submersion cultures in DMEM (4.5 g/l glucose with 2 mM L-glutamine and 110 mg/l sodium pyruvate) supplemented with 10% FBS (Cansera, Rexdale, ON), 100 U/ml penicillin G sodium, and 100 µg/ml streptomycin sulfate. The culture media was replaced 24 h after seeding to remove unattached cells, and then 24 h later, the media was replaced by EF, and bioelectric assessments were performed 18–24 h later.

Ussing chamber bioelectric measurements. Epithelial cells were studied in Ussing chambers at 37°C maintained under open-circuit conditions and then switched to short-circuit current (Isc) with transepithelial potential difference, Isc, and transepithelial resistances (R) determined intermittently with voltage/current clamps (30, 30). Addition of amiloride (final concentration = 10–4 M) to the apical side of the monolayers yielded amiloride-sensitive and -insensitive Isc.

Fluid transport across FDLE. Fluid transport was measured using methods similar to those previously published for adult type II alveolar epithelium (9). Two days postseeding, FDLE cultured on 6.5-mm Transwell permeable inserts were treated with media, 10%, 25%, or 50% EF diluted in DMEM/10% FBS (total 130 µl added above cells, 600 µl below), in the presence and absence of 100 µM amiloride added both apically and basolaterally. Since the basolateral well of the Transwells has a volume that is significantly greater than the apical volume of fluid, we added amiloride to both sides of the monolayer to ensure that the concentration of amiloride that was present on the apical side of the Transwell remained constant. Fluid transport was calculated via a direct quantitation of fluid added at time 0 and recovered after 48 h. To minimize/avoid potential pipetting error, fluid was quantitated by weight at both time points, and volume transported was calculated from the difference between time 0 and time 48 h, assuming a density of 1 g/ml. Corrections were also made for fluid loss due to evaporation using blank wells treated with the same volume of media per surface area.

Data analysis. Student's t-test or analysis of variance followed by the Tukey-Kramer multiple comparisons test were used to examine significant differences between the experimental groups (P < 0.05 considered significant). Data are expressed as means ± SE.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
WT fetal lung explants. Sixteen-day gestational age fetal lung explants prepared from WT mice continuously expand while being cultured in DMEM/F-12 (Figs. 1 and 2). This expansion reflects net fluid secretion (2).


Figure 1
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  		Fig. 1. Distal lung explants from 16-day gestational age fetal mice continuously increased in size while being cultured in media (DMEM/F-12) over time (0–63 h). Time 0 is when explants were placed into the culture media. N = 21 for all time points, except for 63-h time point, where n = 16, n is the number of lung explants. Each treatment group contained explants derived from multiple fetuses in 3 separate experiments.

 

Figure 2
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  		Fig. 2. Change in the size of lung explants cultured in DMEM/F-12 media or edema fluid (EF). A: all lung explants increased in size during their initial 24 h of culture in DMEM/F-12 media (media 0–24 h). During the subsequent 24-h test period, explants maintained in media (media 24–48 h) continued to increase their size, whereas explants exposed to EF (EF 24–48 h) markedly decreased in size. Magnification, x60. B: EF's ability to induce a decrease in explant size was dose dependent. Percent change refers to the change in size over the test period as defined in A. Media: n = 49, EF-10%: n = 20, EF-25%: n = 19, EF-50%: n = 15, EF-100%: n = 13 (P < 0.05 for all groups compared with control group).

 
In contrast, when lung explants were cultured in media containing 50% EF during the 24-h test period, they decreased their size, reflecting increased net lung fluid absorption (Fig. 2A). This EF-induced net fluid absorption was dose dependent (Fig. 2B).

To support our contention that EF was inducing net fluid absorption and was not just a reflection of toxicity or the induction of apoptosis, we exposed some WT explants to 0.1% Triton X-100, a toxic reagent, or to 1 µM staurosporine, an apoptosis-inducing reagent. Light microscopic examination of the hematoxylin and eosin-stained sections of fetal lung explants maintained in control culture media showed well-preserved fetal lung parenchyma composed of epithelium-lined tubes separated by mesenchymal cells (Fig. 3A). Similarly well-preserved histology was seen in lung explants cultured with EF (Fig. 3B). Explants maintained in the control culture medium showed much larger lumen of the tubes (reflecting net fluid secretion) compared with EF explants where the lumina were smaller and there seemed to be more tubules for the same surface area. This latter finding likely reflects net fluid absorption accounting for reduced size of explants. In contrast, explants exposed to Triton X-100 showed generalized cell necrosis of both epithelial and mesenchymal components consistent with cellular toxicity (Fig. 3C). The explants incubated with staurosporine showed a marked increase in apoptosis affecting both epithelial and mesenchymal cells (Fig. 3D). Explants exposed to either Triton X-100 or staurosporine did not change in size (Fig. 4).


Figure 3
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  		Fig. 3. Representative hematoxylin and eosin-stained histological sections of explants exposed to media (A), EF-50% (B), 0.1% Triton X-100 (TX-100) (C), and staurosporine (STAU) (D). Explants exposed to media or EF show well-preserved fetal lung parenchyma with intact epithelial and mesenchymal components. Explants exposed to TX-100 show massive necrosis of all cell types, whereas explants exposed to staurosporine exhibit extensive apoptosis affecting both epithelial and mesenchymal cells. Scale bar, 20 µm.

 

Figure 4
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  		Fig. 4. During exposure to either Triton X-100 (n = 30) or staurosporine (n = 29), the explants did not change in size, which was in marked contrast to explants exposed to either media alone (n = 34) or EF (n = 25). P < 0.001 for both Triton X-100 and staurosporine vs. EF group.

 
In two separate experimental preparations, we examined explants using TEM. TEM revealed that there was no evidence of toxicity, but surprisingly, TEM demonstrated that EF-exposed explants had an increased number of lamellar bodies within type II alveolar epithelia and very abundant surfactant in air spaces (Fig. 5). These ultrastructural observations indicate that EF has a significant maturational effect on the 16-day gestational age murine fetal lung.


Figure 5
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  		Fig. 5. Transmission electron microscopy of wild-type murine lung explants cultured with media (A) or in EF-50% (B). Lamellar bodies (LB) and air space surfactant (S) are abundant in EF-exposed explants. These observations were present in each explant studied within 2 different experimental preparations.

 
To investigate whether EF's effect on lung explants was a nonspecific effect arising from the exposure to proteins, we grew some explants in media containing 50% heat-treated EF or 99% pure rat serum albumin (RSA). The final RSA concentration was matched to the EF-50% total protein concentration of 26.6 mg/ml. Explants exposed to either RSA or heat-treated EF demonstrated net fluid secretion comparable to the control group (Fig. 6, P > 0.05), which was in marked contrast to net fluid absorption observed when explants were exposed to EF-50% (P < 0.01).


Figure 6
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  		Fig. 6. Percent size change (during the test period as defined in Fig. 2A) of lung explants exposed to media (n = 49), EF-50% (n = 15), heated EF-50% (HEF; n = 16), and rat serum albumin (RSA; n = 15). Neither heated EF nor RSA induced net fluid absorption. See text for details. *P < 0.001, P > 0.05 = not significant (ns) compared with media.

 
Dose-response effect of EF on ion transport by FDLE. Increasing concentrations of EF progressively increased amiloride-insensitive Isc, an effect that reached significance at concentrations of 25% or above. In contrast, amiloride-sensitive current (ASC) was significantly increased at moderate (10–25%) concentrations of EF, but remained at basal levels when cells were exposed to 100% EF (Fig. 7).


Figure 7
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  		Fig. 7. Amiloride-sensitive current (ASC) across fetal distal lung epithelia (FDLE) is EF-dose dependent. Shown are dose-response curves for the effects of EF on FDLE (n = 8–12, r = 1,792 ± 90 {Omega}xcm2). Amiloride-insensitive current (AIC) data were previously published (30); ASC and total short-circuit current (Isc) data are from same experiment, previously unpublished. Cells were cultured in submersion on permeable membranes in DMEM + 10% FBS and were exposed to varying concentrations of EF for 20 h. N refers to the number of different monolayers obtained from a minimum of 3 independent cell preparations. *P < 0.001 comparing EF vs. media; §P > 0.05 comparing 100% EF vs. media; P < 0.001 for 100% EF vs. 50% EF.

 
Net fluid absorption across monolayers of distal lung epithelia. Net fluid transport was assessed across FDLE monolayers exposed to increasing concentrations of EF in the presence or absence of amiloride (Fig. 8). Net fluid absorption from the apical to basolateral side was increased in cells exposed to ≥10% EF compared with cells cultured in media alone. Although there was a trend towards increased amiloride-sensitive fluid transport in FDLE exposed to 10% or 25% EF, this increase did not reach statistical significance and was not observed in cells exposed to 50% EF. In contrast, FDLE exposed to 50% EF exhibited significantly increased amiloride-insensitive fluid transport compared with control monolayers.


Figure 8
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  		Fig. 8. Net fluid transport (µl·cm–2·h–1) by FDLE from their apical (AP) to basolateral (basal) side while being cultured in media (Con) or increasing concentrations of EF in the absence or presence of 0.1 mM amiloride. Exposure of FDLE to EF increased their total net apical to basolateral fluid transport at all EF concentrations tested. *P < 0.01 EF vs. Con. Amiloride-insensitive (Amil-Insensitive) net fluid transport was significantly increased in 50% EF. #P < 0.001 50% EF vs. Con. N = 24 monolayers from 4 independent cell preparations.

 
Explants from {alpha}-, beta-, or {gamma}-ENaC-deficient fetal murine lungs. The genotypes for all fetuses of all litters were consistent with Mendelian inheritance [{alpha}-ENaC line (WT-30%, KO-20%, HT-50%), beta-ENaC line (WT-25%, KO-29%, HT-46%), and {gamma}-ENaC line (WT-33%, KO-22%, HT-45%)].

We found that when explants from {alpha}-, beta-, or {gamma}-ENaC-deficient lungs were cultured in media, they had significantly increased net fluid secretion relative to WT lungs (Fig. 9). We then conducted additional experiments and found that the DNA content of intact WT, HT, and KO whole fetal lungs were similar (Table 1), suggesting that while in utero, there was no prolonged distention of the fetal lungs by excess fluid. There are at least two potential explanations. First is that in utero, the distal lung fluid secretion was not increased. However, we believe a more likely explanation is that the extra fluid could relatively easily flow out of the developing lungs in utero compared with the closed-off lung explants, and thus did not induce the increase in transpulmonary pressure seen in models involving in utero tracheal occlusion and which is required to induce fetal lung hyperplasia.


Figure 9
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  		Fig. 9. Effect of EF on distal lung explants from ENaC knockout mice. Explants from each fetus were distributed onto 2 Transwells for subsequent exposure to EF and media to ensure representation of all genotypes in both treatment groups. A: percent size change (during the test period as defined in Fig. 2A) of lung explants from {alpha}-ENaC knockout ({alpha}-KO; n = 11), wild-type (WT; n = 15), and heterozygous (HT; n = 15) mice when these explants were exposed to media or EF-50%. The increase in size of explants from {alpha}-ENaC KO mice was significantly greater than those from WT or HT mice when exposed to media alone. In contrast, but in agreement with our previous report (30), the effect of EF was similar in each of the {alpha}-ENaC KO, WT, and HT. ***P < 0.001, ns. B: percent size change of lung explants from beta-ENaC knockout (beta-KO; n = 13), wild-type (WT; n = 10), and heterozygous (HT; n = 25) mice when these explants were exposed to media or EF-50%. The increase in size of explants from beta-ENaC KO mice was significantly greater than that from WT or HT mice when exposed to media alone. Explants from beta-ENaC KO mice, relative to WT or HT mice, showed a statistically significant abrogation of the EF's ability to induce a decrease in explant size. *P < 0.05, **P < 0.01, ***P < 0.001. C: percent size change of lung explants from {gamma}-ENaC knockout ({gamma}-KO; n = 14), wild-type (WT; n = 16), and heterozygous (HT; n = 27) mice when these explants were exposed to media or EF-50%. The increase in size of explants from {gamma}-ENaC KO mice was significantly greater than that from WT or HT mice when exposed to media alone. Explants from {gamma}-ENaC KO mice, relative to WT mice, showed a statistically significant abrogation of the EF's ability to induce a decrease in explant size, but not significantly different from HT mice. *P < 0.05, ***P < 0.001.

 

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  		Table 1. Total DNA content of 16-day gestational age murine fetal lungs

 
Explants that were obtained from {alpha}-ENaC KO, HT, or WT fetal murine lungs all switched to net fluid absorption when cultured in EF-containing media (Fig. 9A). In contrast, lung explants prepared from either beta- (Fig. 9B) or {gamma}-ENaC (Fig. 9C) KO fetal mice had significantly less net fluid absorption when cultured in EF-50% relative to their WT fetal littermates.

The average total amount of RNA extracted from lung explants (4–5 explants/insert) was 370 ng (range: 21–1,324 ng). The steady-state level of {alpha}-ENaC mRNA was either unaffected or slightly decreased in EF-exposed explants from mice of different genotypes (Fig. 10A). The steady-state levels of beta- and {gamma}-ENaC mRNA were significantly decreased in EF-exposed lung explants prepared from WT, HT, or KO fetal mice (Fig. 10, B and C).


Figure 10
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  		Fig. 10. ENaC subunit mRNA expression in lung explants from KO, WT, and HT littermate mice exposed to media or EF-50%. {alpha}-ENaC subunit expression was not affected or slightly decreased in WT, HT, and KO explants exposed to EF. In contrast, regardless if the explants were derived from WT, HT, or KO mice, the beta- and {gamma}-ENaC subunit expression decreased when exposed to EF. *P < 0.05, **P < 0.01, ***P < 0.0001, ns P > 0.05. A: {alpha}-, beta-, and {gamma}-ENaC mRNA expression in {alpha}-KO, {alpha}-WT, and {alpha}-HT littermates. B: {alpha}-, beta-, and {gamma}-ENaC mRNA expression in beta-KO, beta-WT, and beta-HT littermates. C: {alpha}-, beta-, and {gamma}-ENaC mRNA expression in {gamma}-KO, {gamma}-WT, and {gamma}-HT littermates.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Our previous discovery (30) that EF induced Na+ transport in primary cultures of rat FDLE and net fluid absorption in distal lung explants from fetal mice provided us with the opportunity to assess the relative contributions of the {alpha}-, beta-, and {gamma}-ENaC subunits in the fetal lung's net air space fluid movement in response to EF exposure. Our present study, which utilized fetal mice that were genetically deficient in one of the three ENaC subunits, demonstrated that under control conditions distal lung explants deficient in any of the three subunits secreted fluid at a greater rate than their WT or HT littermates. In contrast, we found that in the fetal murine lung, the beta- and {gamma}-, but not {alpha}-, ENaC subunits are necessary for EF to maximally induce lung epithelial net fluid absorption.

Distal lung fluid absorption, arising from the active transport of Na+ by alveolar epithelia, is clinically important in late gestation and at birth when lung fluid must be cleared (22, 23, 27) and in patients with pulmonary edema. The clearance of edema fluid from air spaces has been correlated with improved survival in adult patients (33, 35). Alveolar fluid absorption is driven by active Na+ transport through epithelial cation permeant ion channels, with Cl– and water following (reviewed in Refs. 18, 19, 22).

In the present study, we used an organotypic murine lung explant model to assess net lung fluid transport across the epithelia lining the air spaces (2, 11). We have shown that 16-day gestation distal lung explants prepared from WT mice continue to expand in size over time, reflecting net fluid secretion, but when exposed to EF, they markedly decrease in size reflecting their conversion to net fluid absorption. We have further tested the validity of this model by exposing lung explants to both toxic and apoptotic agents and comparing their size and histological and ultrastructural appearance to lung explants exposed to EF or media. We found that EF's effect on lung explants was neither toxic nor did it induce apoptosis. Further support for a "non-toxic" effect is derived by the dose dependency of the EF effect. We also found that the EF effect does not merely reflect nonspecific protein osmotic or other effects as lung explants did not convert to net fluid absorption in response to heat-treated EF or albumin. However, to our surprise, we found that EF-cultured lung explants, compared with media control, were more mature as indicated by their increased lamellar bodies and surfactant. Potential explanations include a role for fetal growth factors or hormones present in EF or a response to the mechanical contraction of lung explants induced by the onset of net fluid absorption. Further investigation is required to fully document the maturation phenomena and to determine the mechanism(s) that are involved.

To obtain further support for our contention that EF induced net fluid absorption, we studied the effect of EF on fluid transport across monolayers of rat distal lung epithelia using techniques reported by others (9). We have shown that EF induced a significant increase in net total fluid absorption in a dose-dependent manner, in agreement with our observations in distal lung explants. Although the lower concentrations tested showed a trend towards increasing amiloride-sensitive fluid transport, this effect was not seen in FDLE treated with 50% EF. Qualitatively, this trend is in agreement with our observation that at high (100%) or low (10%) concentrations of EF, the ASC is unchanged in FDLE, but ASC increases with moderate (25–50%) concentrations of EF. In contrast, the amiloride-insensitive component of net fluid transport showed a gradual increase from 10% to 50% EF, very similar to the effect of increasing concentrations of EF on amiloride-insensitive Isc.

Na+ channel activity represents the rate-limiting step in lung epithelial Na+ transport (19). An amiloride-sensitive ENaC participating in Na+ movement across the apical cell membrane has been cloned and well characterized (6, 34). ENaC is composed of three subunits, entitled {alpha}-, beta-, and {gamma}-ENaC, with suggested stoichiometry of two {alpha}-, one beta-, and one {gamma}-subunit (10). The relative mRNA expression of these three subunits may vary between species and tissues (colon, kidney, and lung) (31), even within different regions of the lung (29).

The relative importance of each subunit to ENaC's function may depend on the organ and the (patho)physiological state. For example, it has been shown in X. laevis oocytes that the {alpha}-subunit is the critical subunit for inducing amiloride-sensitive Na+ currents (6), and knockout experiments have shown that neonatal mice that are deficient for the {alpha}-ENaC subunit die shortly after birth due to their inability to clear fluids from the lungs (12), whereas beta- or {gamma}-ENaC knockout neonatal mice can clear fluid from the lungs at birth, although at a slower rate than that in WT mice, with death resulting from defective renal electrolyte reabsorption (3, 21). In contrast, our results using these three knockout lines indicated that beta- and {gamma}-ENaC subunits appeared to be more important than {alpha}-ENaC for a maximal fluid absorptive response following EF exposure. Possibly, the model we are using, of EF-induced fluid absorption in immature lung explants, acts via different mechanisms than the one at birth, which occurs in a mature lung in response to many triggers including steroid hormones, catecholamines, and changes in oxygen atmosphere. Interestingly, transgenic mouse experiments have shown that overexpression of the beta-, but neither {alpha}- nor {gamma}-, ENaC subunit results in a cystic fibrosis-like phenotype with excessive Na+ absorption by respiratory epithelia (17). These latter observations in mice provide some support for our present findings using distal lung explants from knockout mice. Further support for our findings can be found in the study by Richard et al. (32) where they showed that an increase in Isc was associated with an increase in beta- and {gamma}-ENaC mRNA levels.

The relative importance of each subunit may also vary between mammalian species. Consistent with findings in {alpha}-ENaC KO mice, Li and Folkesson (16) have shown the importance of {alpha}-ENaC subunit in alveolar fluid clearance in the adult rat lung. However, pseudohypoaldosteronism patients, the "human {alpha}-ENaC subunit knockout," do not have respiratory distress syndrome at birth (7) and have a lung phenotype characterized during childhood by increased fluid within their small airways (15).

We found that the steady-state levels of mRNA coding for beta- and {gamma}-ENaC subunits significantly decreased in EF-exposed lung explants prepared from WT, HT, or KO fetal mice. The decrease in beta- and {gamma}-ENaC mRNA expression following EF treatment was particularly surprising since these were the two subunits necessary for a maximal induction of the absorptive response by EF in KO explants. However, this inverse relationship is not without precedent since Otulakowski et al. (28) have previously reported an inverse correlation between {gamma}-ENaC mRNA levels and amiloride-sensitive potential difference in the human inferior nasal turbinate. One potential explanation is that despite the decrease in mRNA level of these subunits, there was no decrease in ENaC protein levels within the cell or at the membrane as a result of the different mechanisms of posttranscriptional regulation. How the EF induces its effect, presumably on the cell's apical membrane Na+ channel activity, is unknown, but one can speculate on potential mechanisms ranging among altered gene transcription, translational efficiency, or trafficking to and from the apical membrane. Also, it could be a direct effect on the DLE or a paracrine effect from adjacent nonepithelial cells contained within the explant.

Lung explants prepared from {alpha}-, beta-, or {gamma}-ENaC KO fetal mice and cultured in media had significantly more net fluid secretion relative to explants from WT littermates. Since increased fluid volume in utero is associated with lung hyperplasia (1), we measured the DNA content of WT, HT, and {alpha}-, beta-, or {gamma}-ENaC KO mice as a surrogate for total lung cell number. Since there was no difference between lung weight and DNA content of KO and WT fetal mice, we speculate that either this phenomena was not occurring in utero or that the excess fluid is drained out of the fetal lungs without distending the lungs. In the original report of {alpha}-ENaC KO (12), fetal lung development was said to be similar in both KO and WT mice; however, no quantitative data were provided in that report. Regardless, we did observe an increase in net fluid secretion in the ENaC KO fetal lung explants, and this would occur by a change in balance between fluid secretion and absorption (4, 5). There are at least two possible explanations. One is that the KO results in the loss of a low-level Na+ transport that is normally present in WT mice leading to less fluid absorption that favors net fluid secretion. Another potential explanation is that the presence of a functional ENaC decreases Cl– secretion, and when it is absent, as is the case in ENaC KO fetal mice, there is increased fluid secretion. This could occur either via direct inhibition of Cl– channels (e.g., CFTR) by ENaC or indirectly, i.e., if there is no ENaC activity and hence no entry of Na+ into the cell, then the apical membrane potential would become hyperpolarized, thereby increasing the driving force for Cl– to exit through the apical membrane. Finally, another potential but speculative explanation is that there are some cells secreting fluid and others absorbing via ENaC in the normal fetal lung, and if ENaC is not there, perhaps it affects the relative number or activity of Cl–-secreting cells.

In conclusion, we have shown that beta- and {gamma}-, but not {alpha}-, ENaC subunits play an important role in EF-induced lung net fluid absorption and that ENaC subunit KO fetal lung explants have increased net fluid secretion.


   GRANTS
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 ABSTRACT
 METHODS
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 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by the Heart and Stroke Foundation of Ontario (H. O'Brodovich).


   ACKNOWLEDGMENTS
 
We thank E. Hümmler of Lausanne, Switzerland, for providing male and female heterozygote mice that were genetically deficient in one of the {alpha}-, beta-, and {gamma}-ENaC subunits. We also thank Wenming Duan and Rose Belcastro for assistance in performing the genotyping and total lung DNA measurements.

Present address of N. Elias: Division of Pediatric Pulmonology, Meyer Children's Hospital, Rambam Medical Center, Haifa 31096, Israel.


   FOOTNOTES
 

Address for reprint requests and other correspondence: H. O'Brodovich, Univ. of Toronto, Hospital for Sick Children, 555 Univ. Ave., Toronto, Ontario, Canada M5G 1X8 (e-mail: hugh.obrodovich{at}sickkids.ca)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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H. O'Brodovich, P. Yang, S. Gandhi, and G. Otulakowski
Amiloride-insensitive Na+ and fluid absorption in the mammalian distal lung
Am J Physiol Lung Cell Mol Physiol, March 1, 2008; 294(3): L401 - L408.
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