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Am J Physiol Lung Cell Mol Physiol 287: L608-L615, 2004. First published May 28, 2004; doi:10.1152/ajplung.00031.2004
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Expression of epithelial sodium channel {alpha}-subunit mRNAs with alternative 5'-untranslated regions in the developing human lung

Katharine Banasikowska, Martin Post,1,2,3 Ernest Cutz,3 Hugh O'Brodovich,1,2 and Gail Otulakowski1

Canadian Institutes for Health Research Group in Lung Development, Research Institute of the Hospital for Sick Children and Departments of 1Pediatrics, 2Physiology, and 3Laboratory Medicine and Pathobiology of the University of Toronto, Ontario, Canada M5G 1X8

Submitted 4 February 2004 ; accepted in final form 22 May 2004


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 
In preparation for birth, lung epithelia must switch from net fluid secretion, required for lung development, to net absorption, which prepares the lungs for postnatal gas exchange. The apical membrane amiloride-sensitive epithelial Na channel (ENaC) is the rate-limiting step for Na+ and fluid absorption. Expression of {alpha}-ENaC mRNA has been detected in human lung as early as the embryonic stage of development. However, humans express multiple transcripts for {alpha}-ENaC, containing differing 5'-untranslated regions (UTR) with unknown effects on protein translation, and different ontogenies for individual transcripts could provide a novel mechanism for developmental regulation of ENaC function. To assess the relative expression of the two most abundant {alpha}-ENaC transcripts ({alpha}-ENaC1 and {alpha}-ENaC2) during lung development, we performed nonradioactive in situ hybridization using probes specific to the alternative 5'-UTRs. Both transcripts were expressed throughout intrauterine lung development (8 to 40 wk gestation), and expression was localized to the surface epithelial cells of the conductive and respiratory airways in both ciliated cells and nonciliated Clara cells. {alpha}-ENaC mRNA expression was also identified in the serous cells of the submucosal glands surrounding the proximal airways. In the mature prenatal lung, subsets of alveolar type II (ATII) cells expressed one or both of the {alpha}-ENaC transcripts. Our observations demonstrate that a developmentally regulated switch between {alpha}-ENaC 5'-UTR variants is not the trigger by which the developing human lung becomes a fluid-absorbing organ at birth, that individual ATII cells express neither, one, or both of the {alpha}-ENaC transcripts, and that the overall expression is linked to epithelial cell differentiation and lung maturation.

fluid absorption; lung maturation; postnatal gas exchange

THE FETAL LUNG ACTIVELY secretes fluid that is important for lung growth and development in utero. At the time of birth, this pulmonary fluid must be absorbed to allow a successful postnatal transition to an air-breathing existence. Failure of lung air space fluid clearance can lead to neonatal respiratory distress syndrome (nRDS) (4). Early studies in fetal lambs determined that lung fluid absorption depends on amiloride-sensitive Na+ transport across the epithelia lining the distal airways (21). The rate-limiting step for transepithelial Na+ absorption is the apically located amiloride-sensitive epithelial Na channel (ENaC), which consists of three homologous subunits {alpha}-, {beta}-, and {gamma}-ENaC (6, 7). Functional expression studies of ENaC subunits in Xenopus laevis oocytes show that coexpression of all three subunits is necessary for maximal channel activity, although small currents arise when {alpha}-ENaC is expressed alone or paired with either {beta}- or {gamma}-ENaC (7). Gene-targeting experiments in mice have shown that {alpha}-ENaC (but not {beta}- or {gamma}-ENaC) knockout mice die within 2 days of birth, are unable to clear their fetal lung fluid, and exhibit severe nRDS, suggesting that the {alpha}-subunit is most critical for neonatal lung fluid clearance in this model (3, 14, 18). Studies in humans have shown that the amiloride-sensitive decrease in potential difference between the nasal epithelium and the subcutaneous space, a surrogate measure for Na+ transport and ENaC activity, is significantly reduced in premature human infants with nRDS (1) and transient tachypnea of the newborn (11).

The ability of the developing lung to switch from fluid secretion to fluid absorption in response to circulating catecholamines is dependent on the degree of lung maturation (21), and the response can be primed in the immature lung by the combined administration of thyroid and glucocorticoid hormones (2). Consistent with these observations, Northern analyses indicate there are only very low levels of {alpha}-ENaC mRNA during early stages of lung development in rats (20) and mice (9), which can be upregulated by steroid treatment (29). Studies in developing mouse lung, using in situ hybridization for {alpha}-ENaC mRNA, did not detect this transcript before fetal day 16 (28). In contrast, in the developing human lung {alpha}-ENaC mRNA has been detected from early lung bud onward (26). Early expression of {beta}- and {gamma}-ENaC proteins during human airway development, beginning at 17 wk gestation, has been reported (10). The observation that there is early expression of ENaC mRNAs and protein when the lung is a secretory, and not an absorptive, organ suggests that in the developing human lung posttranscriptional regulation may be important in controlling ENaC function.

Human lung epithelium expresses four transcripts for {alpha}-ENaC mRNA, which differ in their 5'-untranslated regions (UTR); one of these transcripts also contains an upstream in-frame AUG, creating a variant protein with an extended NH2 terminus ({alpha}-ENaC2) that nevertheless exhibits biophysical characteristics very similar to the originally described subunit ({alpha}-ENaC1) (30). However, 5'-UTRs can influence the efficiency of translation of the associated mRNA (31). {alpha}-ENaC mRNA transcripts with different 5'-UTRs are also found in the rat, where it has been recently shown that development influences the lung utilization of different 5'-UTRs for {alpha}-ENaC (22). The relative expression of individual {alpha}-ENaC mRNAs during human lung development and their influence on {alpha}-ENaC protein expression are unknown and may represent a mechanism for developmental control of ENaC function. The primary goal of this study was to establish the spatial and temporal expression of the two most abundant {alpha}-ENaC transcripts, {alpha}-ENaC1 and {alpha}-ENaC2, in the developing human lung. We analyzed archival human lung tissue from the pseudoglandular to the alveolar stages of lung development using in situ hybridization with cRNA probes specific to the alternate 5'-UTRs. To identify the epithelial cell types expressing {alpha}-ENaC, immunohistochemical detection of Clara cell 10-kDa protein (CC10) and surfactant protein B precursor (pro-SPB) was carried out, as well as periodic acid-Schiff (PAS) staining for mucus-producing cells. Using these techniques, we show that both {alpha}-ENaC1 and {alpha}-ENaC2 mRNAs are expressed at the same times throughout human lung development and in the same cell types in the airway, consistent with coexpression during lung development. However, individual alveolar type II (ATII) cells appear to express either one or both of the transcripts.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Human fetal tissue material. Normal lung tissue was obtained from pregnancies terminated by prostaglandin induction or dilation and evacuation, from spontaneous abortions, or from stillbirths. Pseudoglandular samples (n = 2, 8–12 wk gestation) and canalicular samples (n = 3, 18–19 wk gestation) were obtained from pregnancies terminated for therapeutic reasons performed by dilation and evacuation. Two of the normal saccular stage tissue samples (n = 3, 25–32 wk gestation) were obtained at autopsy (less than 24 h after death) from stillborn infants, and the third sample was obtained from a deceased prematurely born infant. The alveolar samples (n = 4, 38–40 wk gestation) were obtained from stillbirth due to perinatal asphyxia. All material from human subjects was used with the approval of the Human Subjects Review Committee of The Hospital for Sick Children, Toronto, Canada.

Anatomic terminology. The airways referred to as proximal or large (≥1 mm in fully developed lungs) included all the bronchi (1°, 2°, 3°) characterized by surrounding cartilaginous rings, smooth muscle cell layer, and submucosal glands. These airways were lined by pseudostratified columnar epithelium, which in turn was composed of ciliated, goblet, neuroendocrine, and basal cells. The smaller conducting airways (≤1 mm in fully developed lungs) included the bronchioles, terminal bronchioles, and respiratory bronchioles. Bronchioles were identified by the absence of cartilage, submucosal glands, and goblet cells but had surrounding smooth muscle cells. They were lined by columnar, low columnar, and cuboidal epithelium, which was composed of ciliated, nonciliated, or Clara cells, and neuroendocrine cells.

The peripheral lung area or alveolar region consisted of alveolar ducts, alveolar sacs, and alveoli and was lined by cuboidal type II and squamous type I cells.

Sample preparation. The tissues were all fixed in 10% (vol/vol) formalin and embedded in paraffin. Five-micrometer serial sections were cut and used for in situ hybridization and immunohistochemistry.

{alpha}-ENaC1 and {alpha}-ENaC2 cRNA riboprobe synthesis. cDNAs corresponding to nucleotides 1,762–2,055 ({alpha}-ENaC1 specific) and 2,180–2,484 ({alpha}-ENaC2 specific) of the human {alpha}-ENaC gene (Genbank U81961) were generated by RT-PCR and subcloned into pGEM 3Zf (Promega, Madison, WI). The probe regions chosen are located in the 5'-UTRs and contain no significant stretches of nucleotide sequence homology either to each other or to other human ENaC mRNAs. All plasmids containing the cDNA probe inserts were linearized using the restriction endonuclease HindIII for in vitro transcription of the sense strand and EcoRI for the antisense strand. Single-strand sense and antisense digoxigenin (DIG)-labeled cRNA probes were in vitro transcribed, using 1 µg of linearized template and 40 U of the appropriate polymerase (T7 RNA polymerase for the sense strand, SP6 RNA polymerase for the antisense strand) with DIG-labeled uridine triphosphate using the DIG RNA Labeling Kit according to the manufacturer's directions (Roche Diagnostics, Laval, PQ, Canada).

In situ hybridization. Tissue sections were deparaffinized in a xylene series and then rehydrated through a decreasing ethanol series diluted in dimethyl pyrocarbonate-treated water. Sections were heated in a pressure cooker filled with 1 liter of 0.1 M Tris·HCl, pH 8.0, in a 900-W microwave at maximum power for 14 min. The slides were cooled in the closed pressure cooker for 15 min and for an additional 30 min with the lid off (26). The slides were prehybridized for 2 h at 37°C in 50% (vol/vol) deionized formamide and 1x SSC (0.15 M NaCl, 0.015 M trisodium citrate, pH 7.0) before hybridization with 250 ng/ml of the sense or antisense {alpha}-ENaC1 or {alpha}-ENaC2 cRNA DIG-labeled probes in a humid chamber at 55°C for 18 h. Positive controls consisting of normal human kidney sections were included in each set of hybridized slides. Hybridized slides were washed in increasingly stringent preheated washes of 2x SSC, 1x SSC, 0.5x SSC, and 0.1x SSC for 15 min at 65°C, repeated twice at each concentration. Detection was carried out following incubation with an alkaline phosphatase-conjugated anti-DIG antibody using nitroblue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl phosphate according to the manufacturer's instructions (Roche Diagnostics). Slides were counterstained with methyl green, dehydrated, and mounted.

Immunohistochemistry. Tissue sections were deparaffinized in xylene, rehydrated in descending concentrations of ethanol (100 to 50% ethanol), and washed in 1x PBS. Antigen retrieval was performed in 10 mM sodium citrate buffer (pH 6.0), in which the slides were boiled for 5 min at medium high setting in the microwave, allowed to cool to room temperature with the jar lid on, then heated again and cooled with the lid off for another 20 min. After a further wash in 1x PBS, endogenous peroxidase activity was blocked with 1% (vol/vol) hydrogen peroxide in absolute methanol for 30 min at room temperature followed by 1x PBS washes. Nonspecific binding sites were blocked with 4% (vol/vol) normal donkey serum and 1% (wt/vol) BSA in 1x PBS for 1 to 2 h at room temperature in a humidified chamber. For CC10 detection, slides were incubated overnight at 4°C with a 1:100 dilution of affinity-purified goat anti-human CC10 polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA) in blocking solution. After being washed in 1x PBS, biotinylated secondary donkey anti-goat IgG (1:500 in blocking solution) was incubated on the slides for 2 h at room temperature. After further washes, sections were incubated with avidin biotin peroxidase complex (ABC) solution following the manufacturer's instructions (Vector Laboratories, Burlingame, CA). The peroxidase enzyme reaction was developed in diaminobenzidine tetrahydrochloride/0.01% hydrogen peroxide for 10 min at room temperature. The slides were counterstained with hematoxylin and mounted. Immunohistochemical detection of surfactant protein-B (SP-B) was performed on the NEXES autoimmunostainer (Ventana Medical Systems, Tucson, AZ) using a monoclonal antibody against pro-SP-B at a dilution of 1:20 (Research Diagnostics, Flanders, NJ). All tissues were treated with heat-induced epitope retrieval and blocked for both endogenous peroxidase and biotin. Immunodetection was carried out using the ABC system employing the Ventana DAB (3–3'-diaminobenzidine) detection system. Sections were counterstained with hematoxylin for nuclear detail.

PAS stain. Slides previously processed for in situ hybridization detection of {alpha}-ENaC were oxidized in 0.5% (vol/vol) periodic acid for 5 min, washed in distilled water, and stained with Schiff's reagent for 15 min. Stained slides were rinsed quickly under running water and then for 5 min in lukewarm tap water for color development. Hematoxylin was used for nuclear staining.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Characterization of {alpha}-ENaC1 and {alpha}-ENaC2 cRNA probes. Northern analysis of adult human kidney and lung RNAs demonstrated that DIG-labeled antisense cRNAs specific to {alpha}-ENaC1 and {alpha}-ENaC2 each hybridized to a single band of 3.7 kb (Supplementary Figure S1A)1, appropriate for the larger of the two {alpha}-ENaC transcripts typically reported in human tissue (17, 32); no hybridization signal was detected in RNA from liver or when sense cRNA probes were used. It has been previously shown that although probes homologous to the protein coding region detect both transcripts, probes specific to {alpha}-ENaC1 and {alpha}-ENaC2 5'-UTRs detect only the larger transcript (30). To confirm specificity of the probes and the in situ hybridization protocol, archival human kidney tissue was analyzed by in situ hybridization with {alpha}-ENaC1 and {alpha}-ENaC2 riboprobes (Supplementary Figure S1B). {alpha}-ENaC1 and {alpha}ENaC2 were detected using the antisense riboprobes in the epithelial cells of the distal nephron of the kidney; no significant reaction was seen with sense riboprobes. (Supplementary Figures S1 and S2 are available online at the AJP—Lung web site at http://ajplung.physiology.org/cgi/content/full/00031.2004/DC1.1)



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  		Fig. 1. Expression of epithelial Na channel transcripts ({alpha}-ENaC1 and {alpha}-ENaC2) in pseudoglandular and canalicular stage human lung by nonradioactive in situ hybridization. Serial sections of pseudoglandular lung at 8 wk gestation hybridized with probes specific for {alpha}-ENaC1 (A) and {alpha}-ENaC2 (B) show predominantly apical and some basolateral labeling of cells lining the developing airway (a). Apparent subcellular localization is presumably due to the relatively large percentage of the cell occupied by the nuclei. C and D: a similar pattern was seen in 10- to 12-wk gestation lung. During canalicular stage (19 wk gestation), expression of {alpha}-ENaC1 (E) and {alpha}-ENaC2 (F) was seen in all cells lining distal airways (d) but had become patchy in proximal airways (p). Magnification x250.

 
Competitive in situ hybridization was performed in newborn human lung to demonstrate that {alpha}-ENaC1 and {alpha}-ENaC2 bound to different target sequences (Supplementary Figure S2). The signal from DIG-labeled antisense cRNA specific to {alpha}-ENaC1 was not competed out by excess unlabeled {alpha}-ENaC2 antisense cRNA and vice versa.



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  		Fig. 2. Expression of {alpha}-ENaC1 and {alpha}-ENaC2 in proximal airway during alveolar stage of development (38 wk gestation). In situ hybridization detection of {alpha}-ENaC1 (A) and {alpha}-ENaC2 (B) in serial sections at low magnification (x100) revealed similar expression patterns for the 2 mRNAs in cells lining a large airway and in submucosal (sm) gland acini. At higher magnification (C and D), well-differentiated ciliated cells are visible lining the large airway. The pattern of expression of {alpha}-ENaC1 (C) was similar to {alpha}-ENaC2 (D) in serial sections. Not all ciliated cells expressed {alpha}-ENaC1 or {alpha}-ENaC2 (arrows). Magnification x400.

 
{alpha}-ENaC1 and {alpha}-ENaC2 mRNA expression in the developing human lung. To provide the most precise comparison of the spatial and temporal expression of {alpha}-ENaC1 and {alpha}-ENaC2 mRNAs, serial sections were used to minimize cellular and structural differences among the lung tissues examined. In addition, immunohistochemistry was employed to identify specific differentiated cell types within the lung sections. CC10 has been widely used to identify Clara cells in human bronchioles (8, 13, 19, 24, 25, 33), whereas pro-SP-B is a cellular marker for type II pneumocytes and Clara cells in the human lung (15, 27, 33).

Pseudoglandular stage. In tissue sections from 8- to 12-wk gestation (pseudoglandular stage) human fetal lung (Fig. 1, A–D), developing airways were evident, but a pseudostratified appearance was not yet present in the lining epithelium. Using in situ hybridization, both {alpha}-ENaC1 and {alpha}-ENaC2 mRNAs were evident in the undifferentiated developing airways at 8 wk gestation (Fig. 1, A and B) and at 10–12 wk gestation (Fig. 1, C and D). The signal was strong and uniform within the airway epithelia, with a trend for localization in the basal or apical aspect of the cell probably due to the minimal amount of cytoplasm in those cells.

Canalicular stage. The canalicular stage of development is associated with the appearance of the gas-exchanging region and the differentiation of epithelial cells into mature phenotypes. The cell differentiation process is not uniform, but it begins in the trachea and moves outward toward the distal lung. In tissue sections from 19-wk-gestation human fetal lung, the proximal airway epithelium [indicated by (p) in Fig. 1, E and F] displayed a strong but patchy signal for both {alpha}-ENaC1 and {alpha}-ENaC2, characteristic of these mRNAs being present in a select population of cells. In contrast, the developing bronchioles [distal airway (d) in Fig. 1, E and F] showed a more uniform pattern of {alpha}-ENaC1 and {alpha}-ENaC2 signal, comparable to what was observed in the developing airways of pseudoglandular human lung.

Saccular stage. During the saccular stage, the terminal airways widen to form saccules and the mature phenotypes of the epithelial cells appear. In lung samples from the saccular stage, proximal airway epithelium was well differentiated, and the cuboidal epithelium lining the distal lung unit had begun to flatten. In situ hybridization signals for {alpha}-ENaC1 and {alpha}-ENaC2 were present in a select population of ciliated cells of the proximal airway and in the serous cells of the submucosal glands (data not shown). Examination of the distal lung unit indicated that {alpha}-ENaC1 and {alpha}-ENaC2 mRNAs were present in a subset of epithelial cells within the terminal saccules of the peripheral lung; serial sections showed that similar regions of the terminal airway stained for both {alpha}-ENaC mRNAs and for CC10 (data not shown).

Alveolar stage. Tissue sections from alveolar stage (38–40 wk gestation) developing human lung possessed a typical well-differentiated, tall columnar ciliated epithelium in the proximal airways with numerous submucosal glands (Fig. 2). The signal for both {alpha}-ENaC1 and {alpha}-ENaC2 was very strong in the proximal airways and in the submucosal glands (Fig. 2, A and B). Higher magnification analysis of corresponding regions in serial sections showed a very similar staining pattern for {alpha}-ENaC1 (Fig. 2C) and {alpha}-ENaC2 (Fig. 2D) within a select population of ciliated cells of the proximal airway. However, not all ciliated cells were positive, and {alpha}-ENaC1 and {alpha}-ENaC2 did not always coexpress in the same ciliated cells, giving rise to the patchy staining pattern. The submucosal glands surrounding the proximal airway appeared to coexpress {alpha}-ENaC1 and {alpha}-ENaC2 only in serous cells. To confirm that the {alpha}-ENaC signal was present in the serous cells of the submucosal glands but absent from the mucus-producing cells of the airway and submucosal glands, PAS staining was performed on slides previously used for {alpha}-ENaC2 in situ hybridization. As shown in Fig. 3, cells of the submucosal gland that clearly failed to stain with the {alpha}-ENaC2 cRNA probe (Fig. 3B) were strongly positive for mucus (Fig. 3D). In addition, mucus-containing cells of the proximal airway (Fig. 3C) failed to hybridize to the {alpha}-ENaC2 cRNA (Fig. 3A).



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  		Fig. 3. Lack of {alpha}-ENaC mRNA expression in mucus-producing cells of proximal airways at 38 wk gestation. Magnification x250. A: expression of {alpha}-ENaC2 by in situ hybridization in cells lining the proximal airway (p). C: cells without {alpha}-ENaC2 mRNA signal (arrows) correspond to mucus-producing cells (similar results for {alpha}-ENaC1 not shown). B: submucosal glands of proximal airways showed similar areas without {alpha}-ENaC2 signal (arrow). Signal for {alpha}-ENaC was confined to serous cells of the duct epithelium (arrowheads). The presence of mucus was confirmed by periodic acid-Schiff staining (C and D, purple color).

 
Distal airways were lined by cuboidal epithelium containing multiple cells that stained positively for CC10 and pro-SP-B (Fig. 4). Alveolar structures could be seen in distal lung parenchyma with developing secondary septa and pro-SP-B-positive ATII cells (Figs. 4 and 5). Immunohistochemistry directed against CD68 confirmed that alveolar macrophages were not present in these sections (data not shown). Sections of lung from the alveolar stage showed both CC10 and pro-SP-B-immunoreactive cells, indicating the presence of Clara cells in the now well-differentiated terminal airways (Fig. 4, A and B). Serial sections of this region exhibited {alpha}-ENaC1 and {alpha}-ENaC2 staining within the terminal airways and the surrounding acinar region (Fig. 4, C and D) in a pattern that was very similar to {alpha}-ENaC expression in saccular lung. Under higher magnification, {alpha}-ENaC1 and {alpha}-ENaC2 were expressed in low columnar cells located in similar regions of the same airway (Fig. 4, C and D, inset). In serial sections of the same airway, CC10-immunoreactive cells were low columnar in phenotype and their cytoplasm protruded into the airway lumen (Fig. 4A, inset). Pro-SP-B-immunoreactive cells were also low columnar (Fig. 4B, inset); however, CC10 and pro-SP-B immunoreactivity did not always seem to appear in all of the same cells. It was possible to locate cells, such as the one denoted as (a) in Fig. 4, which showed coexpression of {alpha}-ENaC1, {alpha}-ENaC2, CC10, and pro-SP-B. Several cells could be identified coexpressing either {alpha}-ENaC1 or {alpha}-ENaC2 with CC10 or pro-SP-B [cells labeled b and c, respectively]. Both {alpha}-ENaC1 and {alpha}-ENaC2 were also expressed in a subpopulation of ciliated cells within the terminal airway (arrows, Fig. 4, C and D). This heterogeneity of expression in the ciliated cells was similar to that of the proximal airways.



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  		Fig. 4. Coexpression of {alpha}-ENaC1 and {alpha}-ENaC2 mRNA with 10-kDa Clara cell protein (CC10) and surfactant protein B (SP-B) in distal human lung at 38 wk gestation. Immunohistochemistry detected CC10-immunoreactive cells (A) in the terminal airways (TA) and cells reacting with SP-B precursor (pro-SP-B) antibody (B) in both TA and adjacent parenchyma of the developing alveoli (al). Serial sections subjected to in situ hybridization demonstrated both {alpha}-ENaC1 (C) and {alpha}-ENaC2 (D) mRNAs were expressed in very similar regions of the superficial epithelium lining the TA and in individual cells of the parenchyma. A-D: magnification x100. A-D, insets: magnification x400. Rare cells positive for all 4 signals could be identified (for example, cell labeled a), as well as cells expressing {alpha}-ENaC1 with both immunohistochemical markers (cells labeled b), or {alpha}-ENaC1 with only pro-SP-B (labeled d). Similarly, {alpha}-ENaC2-positive cells could be detected coexpressing both markers (labeled c). {alpha}-ENaC1 and {alpha}-ENaC2 were also expressed in ciliated cells (arrows) of the terminal bronchiole. *Cell at edge of invagination used as landmark to align serial sections.

 


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  		Fig. 5. Expression of {alpha}-ENaC1 and {alpha}-ENaC2 mRNA in alveolar region of 38-wk-gestation human lung. A: immunohistochemistry for pro-SP-B labeled numerous alveolar type II (ATII) cells in this region. In situ hybridization with probes specific for {alpha}-ENaC1 (B) and {alpha}-ENaC2 (C) in adjacent serial sections demonstrated {alpha}-ENaC mRNAs in only a subpopulation of the ATII cells (magnification x250). For most cells expressing {alpha}-ENaC1, a corresponding signal for pro-SP-B could be found (for example, cells labeled b, d, f, and g). The same is true for cells expressing {alpha}-ENaC2 (cells labeled a and g). However, it was also possible to locate {alpha}-ENaC-expressing cells such as (c) and (e) in regions where no cells stained positively for pro-SP-B. D-G: identification of individual cells was carried out using images obtained at magnification x1,000. Individual cells (arrows, cells f and a) expressing SP-B by immunonhistochemistry (D and E) can be aligned with cells expressing {alpha}-ENaC1 (F) or {alpha}-ENaC2 (G).

 
The alveolar region of the lung showed positive pro-SP-B staining in cuboidal cells in scattered locations consistent with identification as ATII cells (Fig. 5A). Cells expressing {alpha}-ENaC1 (Fig. 5B) or {alpha}-ENaC2 (Fig. 5C) on serial sections of the same region were much less numerous than the pro-SP-B-stained cells. {alpha}-ENaC1 and {alpha}-ENaC2 mRNAs were evident in the cytoplasm of a small population of cuboidal cells lining the alveoli and these cells appeared to correspond to pro-SP-B-immunoreactive cells on an adjacent section as demonstrated in Fig. 5, D–G, see for example cell (a) positive for {alpha}-ENaC2 and pro-SP-B, and cell (f) positive for {alpha}-ENaC1 and pro-SP-B. Although cells such as (c) in Fig. 5 could be found which expressed both {alpha}-ENaC mRNAs with no pro-SP-B-immunoreactive cells in the corresponding area of the adjacent section, the section thickness (5 µm) is such that multiple adjacent sections may not contain identical cell populations. When the {alpha}-ENaC1 and {alpha}-ENaC2 signals to the overall distribution of ATII cells in serial sections are compared (Fig. 5), it appears that the majority of ATII cells that expressed {alpha}-ENaC mRNA expressed either {alpha}-ENaC1 or {alpha}-ENaC2, but not both.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The aim of this study was to determine the temporal and spatial expression of the 5'-UTR variant {alpha}-ENaC mRNAs, {alpha}-ENaC1 and {alpha}-ENaC2, in the developing human lung. We used archival tissue samples and nonradioactive in situ hybridization to determine the mRNA expression pattern of both transcripts in serial sections. Our results show that both {alpha}-ENaC1 and {alpha}-ENaC2 were expressed in a very similar pattern consistent with coexpression throughout development, that this pattern of expression was linked to epithelial cell differentiation and lung maturation, and that individual ATII cells express {alpha}-ENaC1 or {alpha}-ENaC2 or both of the transcripts. Although we have not investigated the possible influence of the different 5'-UTR regions on translation of {alpha}-ENaC mRNA into protein in this study, it seems clear that a developmentally regulated switch between {alpha}-ENaC 5'-UTR variants is not the trigger by which the developing human lung becomes a fluid-absorbing, rather than fluid-secreting, organ at the time of birth.

Although uniform expression of both {alpha}-ENaC1 and {alpha}-ENaC2 mRNA was seen throughout the epithelia of 8- to 12-wk pseudoglandular stage lungs examined in this study, we observed patchy expression of both {alpha}-ENaC mRNAs in proximal lung at 19 wk gestation, suggesting a loss of {alpha}-ENaC mRNA expression in a subset of lung epithelial cells possibly related to cell differentiation or maturation. The uniform {alpha}-ENaC mRNA signal observed in the distal airways of 19-wk gestation lung is consistent with such a hypothesis because distal airways of human lungs only begin to differentiate at 19–20 wk of fetal development. At the saccular stage of development, the patchy expression pattern of {alpha}-ENaC1 and {alpha}-ENaC2 extended from proximal to distal airways; expression in the saccules was very limited. CC10, {alpha}-ENaC1, and {alpha}-ENaC2 staining could be detected in corresponding regions of distal airways in serial sections, and some of the {alpha}-ENaC1- and {alpha}-ENaC2-staining cells presented a phenotype typical of Clara cells, i.e., columnar cells of the distal airways with apical cytoplasm protruding into the airway lumen.

In the alveolar stage lung, proximal airway expression of {alpha}-ENaC1 and {alpha}-ENaC2 mRNAs was very similar, with both DIG-labeled cRNA probes hybridizing to serous cells of the submucosal glands and to the majority of ciliated cells. In distal airways, {alpha}-ENaC1 and {alpha}-ENaC2 mRNAs were abundant and could be detected in both Clara cells and a subset of ciliated cells. In the alveolar region, {alpha}-ENaC1 and {alpha}-ENaC2 mRNA signal was found within a population of cuboidal cells consistent with ATII cell expression; however, adjacent sections subjected to pro-SP-B immunohistochemistry to specifically identify ATII cells indicated that the pro-SP-B-containing cells were much more numerous than either {alpha}-ENaC1- or {alpha}-ENaC2-positive cells. Thus it appears that {alpha}-ENaC expression lags behind pro-SP-B expression in differentiating ATII cells, even at term gestation. The alveolar and distal airway epithelia are believed to be the primary sites of lung fluid clearance; however, the relative contribution of alveolar vs. distal airway cells to lung fluid clearance is unknown (16). Our results in full-term human lung indicating a high abundance of {alpha}-ENaC-expressing cells in small airways, while relatively few pro-SP-B-expressing cells stained positive for {alpha}-ENaC, would lend support to the role of distal airways over ATII cells in newborn infants.

Previous in situ hybridization studies from our laboratory (26) investigated {alpha}-ENaC mRNA expression in human fetal lung using a probe from the common 3'-UTR. Our results using two additional probes directed against the 5'-UTRs of {alpha}-ENaC1 and {alpha}-ENaC2 are consistent with the previous publication. We extended the previous study using PAS staining to confirm the absence of {alpha}-ENaC mRNA in mucus-containing cells of the proximal airway and immunohistochemical detection of CC10 and pro-SP-B to confirm expression of {alpha}-ENaC mRNA in Clara cells and ATII cells in the distal lung. Our results indicate that {alpha}-ENaC mRNA is expressed in the undifferentiated epithelial cells lining the early developing lung's airways but is lost from certain cell types during development.

As determined by in situ hybridization, expression patterns for {alpha}-ENaC in lungs of full-term human infants are very similar to observations in adult lungs (5, 23). Gaillard et al. (10) previously reported immunohistochemical localization of {beta}- and {gamma}-ENaC protein in developing human airways in a pattern very comparable to our observation of {alpha}-ENaC mRNA. Together, these studies suggest the developing human lung may possess the capacity to form functional heterotetrameric {alpha}2{beta}{gamma}-ENaC channels from at least the early canalicular stage of development. There are many potential points of control that could be responsible for the maintenance of net fluid secretion during lung development. The amount of {alpha}-ENaC mRNA may be insufficient to support net fluid absorption, as suggested by recent PCR data indicating that preterm infants (gestational age ~27 wk) with RDS express lower levels of all three ENaC subunits in airway epithelium than healthy full-term infants (12). In addition, in situ hybridization does not provide evidence that the protein is present or functional. The capacity of the {alpha}-ENaC mRNA to be translated in the fetal lung is still unknown and may be regulated by its long, complex 5'-UTR. However, our results here clearly showed that a developmentally regulated switch between {alpha}-ENaC 5'-UTR variants is not the trigger by which the developing human lung becomes a fluid-absorbing, rather than fluid-secreting, organ at the time of birth. At birth, increases in ENaC mRNA or protein synthesis, increased delivery to the apical membrane, or changes in Cl– channel expression could all help tip the balance to net Na+ absorption. A complete understanding of the regulation of ENaC expression and function in the developing human lung will foster the development of novel therapies for nRDS.


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This work was supported by a Canadian Institutes for Health Research Group grant in Lung Development and Operating grant to H. O'Brodovich and by an Ontario Thoracic Society Grant-in-Aid to G. Otulakowski.


   FOOTNOTES
 

Address for reprint requests and other correspondence: G. Otulakowski, Programme in Lung Biology Research, Hospital for Sick Children Research Institute, 555 University Ave., Toronto, Ontario, Canada M5G 1X8 (E-mail: gail.otulakowski{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.

1 The Supplementary Material for this article (Figures S1 and S2) is available online at http://ajplung.physiology.org/cgi/content/full/00031.2004/DC1. Back


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