Published ahead of print on August 21, 2008, doi:10.1165/rcmb.2007-0456OC
American Journal of Respiratory Cell and Molecular Biology. Vol. 40, pp. 211-216, 2009
© 2009 American Thoracic Society
DOI: 10.1165/rcmb.2007-0456OC
Epithelial Sodium Channel Inhibition in Primary Human Bronchial Epithelia by Transfected siRNA
Emanuela Caci1,*,
Raffaella Melani1,*,
Nicoletta Pedemonte1,*,
Guelnihal Yueksekdag2,
Roberto Ravazzolo1,
Joseph Rosenecker2,
Luis J. V. Galietta1 and
Olga Zegarra-Moran1
1 Laboratorio di Genetica Molecolare, Istituto Giannina Gaslini, Genova, Italy; and 2 Department of Pediatrics, Experimentelle Pneumologie und Therapieforschung, Klinikum der Universität München, München, Germany
Correspondence and requests for reprints should be addressed to Olga Zegarra-Moran, Laboratorio di Genetica Molecolare, Istituto Giannina Gaslini, L.go G. Gaslini, 5, Genova, I-16148, Italy. E-mail: ozegarra{at}unige.it
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Abstract
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Na+ absorption and Cl– secretion are in equilibrium to maintain an appropriate airway surface fluid volume and ensure appropriate mucociliary clearance. In cystic fibrosis, this equilibrium is disrupted by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene resulting in the absence of functional CFTR protein, which in turn results in deficient cAMP-dependent Cl– secretion and predominant Na+ absorption. It has been suggested that down-regulation of the epithelial sodium channel, ENaC, might help to restore airway hydration and reverse the airway phenotype in patients with cystic fibrosis. We used an siRNA approach to analyze the possibility of down-regulating ENaC function in bronchial epithelia and examine the resulting effects on fluid transport. siRNA sequences complementary to each of the three ENaC subunits have been used to establish whether single subunit down-regulation is enough to reduce Na+ absorption. Transfection was performed by exposure to siRNA for 24 hours at the time of cell seeding on permeable support. By using primary human bronchial epithelial cells we demonstrate that (1) siRNA sequences complementary to ENaC subunits are able to reduce ENaC transcripts and Na+ channel activity by 50 to 70%, (2) transepithelial fluid absorption decreases, and (3) these functional effects last at least 8 days. A decrease in ENaC mRNA results in a significant reduction of ENaC protein function and fluid absorption through the bronchial epithelium, indicating that an RNA interference approach may improve the airway hydration status in patients with cystic fibrosis.
Key Words: cystic fibrosis transmembrane conductance regulator • cystic fibrosis • membrane proteins • mRNA • function
The volume of the surface fluid covering the airways is maintained through a fine balance between ion and water secretion and absorption. This is obtained by exerting a tight control of the activity of ion channels and transporters localized on the apical and basolateral membranes of epithelial cells. In particular, Na+ absorption through the epithelial Na+ channel (ENaC), localized in the apical membrane, and the Na/K-ATPase, in the basolateral membrane, is in equilibrium with Cl– secretion through the cystic fibrosis transmembrane conductance regulator (CFTR) and other Cl– channels in the apical membrane, and the NKCC cotransporter in the basolateral membrane (1). An appropriate volume of periciliary fluid is essential not only to allow cilia beating, but also to maintain the hydration and therefore the proper viscoelastic characteristics of mucus. The correct performance of these two processes, absorption and secretion, ensures an effective mucociliary clearance.
In cystic fibrosis (CF), the equilibrium between absorption and secretion is disrupted by mutations in the CFTR Cl– channel. As a consequence, Cl– secretion is strongly reduced and Na+ absorption becomes predominant. Accordingly, airways of patients with CF are dehydrated, obstructed by thick mucus, inflamed, and frequently infected (2). This situation could last several years, but more often the lungs get colonized by opportunistic pathogens such as Pseudomonsa aeruginosa that are responsible for the destruction of the lung and the development of respiratory insufficiency and death (3).
The negative effects of the disequilibrium between Na+ absorption and Cl– secretion has been also demonstrated by the production of a transgenic mouse that hyperexpresses the β subunit of the ENaC (4). In this mouse, the increased Na+ and water absorption produces a CF-like lung disease, characterized by surface liquid depletion, increased mucus concentration and stasis, inflammation, and poor bacterial clearance.
It has been suggested that down-regulation of ENaC may help to restore airway hydration and mucus clearance, and to reverse, at least partially, the airway phenotype in patients with CF. As a consequence, double-blind clinical trials using the ENaC blocker amiloride were performed some years ago in patients with CF. Amiloride was given by topical administration in aerosol. However, while amiloride was reported to reversibly reduce the sodium reabsorption and increase the mucus clearance in subjects with CF (5, 6), the effects had short duration, probably because of a rapid amiloride removal from the epithelium surface (7). Short duration has also been reported by in vivo pharmacodynamic studies in sheep using the more potent ENaC blocker benzamil (8).
In recent years, RNA interference has been identified as a powerful molecular process used by cells to modulate gene expression by directing the RNA machinery to carry out mRNA degradation. The effect is mediated by small sequences of approximately 20 to 22 bases that complement a target mRNA (9). Here we have used a short interfering RNA (siRNA) approach to analyze the feasibility of down-regulating ENaC function in human bronchial epithelial cells. We have found that transfection of siRNA against each one of the three ENaC subunits produces a long-term down-regulation at the mRNA and functional protein levels. This effect results in a significant inhibition of fluid absorption.
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MATERIALS AND METHODS
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Cell Culture
The method to generate highly differentiated and polarized cultures of bronchial epithelia has been described elsewhere (10). To obtain polarized epithelia, human bronchial epithelial cells were plated at high density (5·105cells/cm2), in serum- and hormone-supplemented medium, on 0.4-µm porous permeable inserts (Snapwell; Corning Costar, Cambridge, MA). Cells were maintained at 37°C in a 5% CO2/95% air atmosphere. To obtain high activity of the ENaC channel, we kept the cells in liquid–liquid conditions (culture medium on both apical and basolateral sides). In fact, air–liquid interface conditions (no apical medium) strongly down-regulate ENaC activity (our unpublished results). Apical and basolateral media were replaced every 24 hours. Transepithelial resistance and potential difference were daily measured with an epithelial voltohmmeter (Millipore-ERS, Billerica, MA) using chopstick-like electrodes. If not otherwise stated, experiments were done 6 to 8 days after plating, when the transepithelial resistance and potential difference were approximately 1 k ·cm2 and –20 mV.
siRNA Sequences
We used commercially available siRNAs complementary to  , β, or  ENaC subunits either as pools of four siRNAs against the same subunit (Smart Pools; Dharmacon, Lafayette, CO) or as single siRNA. The sequences of the four oligos complementary to each subunit are indicated in Table E1 in the online supplement. As a control we used either pools or single nontargeting siRNAs.
Transfection
To down-regulate ENaC subunits, human bronchial epithelial cells were transfected with 100-nM siRNA duplexes using Lipofectamine 2000 (Invitrogen, Life Technologies, Carlsbad, CA) as transfection agent. Cells were detached from flasks, counted, and transfected in antibiotic-free Optimem medium (Gibco, Invitrogen, Life Technologies). Next, cells were seeded on Snapwell supports at a density of 5 · 105cells/cm2. The Lipofectamine 2000/siRNA complex remained on the apical side of the support for 24 hours. The medium was then replaced with a fresh one containing antibiotics. Apical and basolateral media were replaced daily. We first determined transfection efficiency using an siRNA marked with a fluorescent dye (Alexa fluor 488; Amersham, PerkinElmer, Waltham, MA). Transfection efficiency measured at 24 hours was higher than 80%, as estimated by eye from the fluorescence distribution on cells seeded on permeable supports. We did not use fluorescent reporters together with specific siRNAs, since the presence of the dye could alter the efficacy of oligos.
Quantitative Real-Time RT-PCR
The evaluation of ENaC subunits mRNA was assessed in human bronchial epithelial cells 7 days after seeding on permeable supports. Total RNA was extracted by lysing human bronchial epithelia directly on Snapwell supports (see online supplement). One microgram of spectrophotometer-quantified RNA was retro-transcribed with random primers and oligo(dT) using Advantage RT-for-PCR kit (BD-Clontech, Palo Alto, CA). Real-time quantitative PCR was performed using inventoried Assays-on-Demand from Applied Biosystems. PCR was done using the ABI Prism 7700 Sequence Detection System (Applied Biosystems, Foster City, CA). mRNA was quantified by using the comparative CT Method (Sequence Detection System Chemistry Guide; Applied Biosystems). Each sample was run in triplicate. Data were analyzed by using Sequence Detector Systems version 2.0 software (Applied Biosystems).
Transepithelial Na+ Currents
To analyze ENaC activity, Snapwell inserts were mounted into an Ussing chamber–like Vertical Diffusion Chamber. The apical and basolateral chambers contained identical solutions: 126 mM NaCl, 0.38 mM KH2PO4, 2.1 mM K2HPO4, 1 mM MgSO4, 1 mM CaCl2, 24 mM NaHCO3, and 10 mM glucose. Solutions were bubbled with air containing 5% CO2 and maintained at 37°C. Hemichambers were connected to a DVC-1000 voltage clamp (World Precision Instruments, Berlin, Germany) via Ag/AgCl electrodes and 1 M KCl agar bridges to measure short-circuit currents.
Transepithelial Fluid Transport Measurement
To quantify fluid absorption, bronchial epithelial cells were cultured as explained above. Seven days after seeding, the apical surface of epithelia was washed with a saline solution containing (in mM): 137 NaCl, 2.7 KCl, 8.1 Na2HPO4, 1.5 KH2PO4, 1 CaCl2, 0.5 MgCl2, with or without 10 µM amiloride. The apical medium was removed, then 500 µl of room temperature saline solution was added to the apical surface. Filters were rotated gently to remove the medium remaining at the walls of the cup, and then the fluid was recovered and eliminated. This process was repeated three times. After washing, the apical side of the epithelium was covered with 50 µl of the same solution and 150 µl of mineral oil to prevent evaporation (11). Cells were maintained at 37°C in a 5% CO2/95% air atmosphere. After 24 hours, the apical fluid was carefully removed, centrifuged to separate the mineral oil, and the volume of aqueous phase measured.
Statistics
Data are presented as representative traces or as means ± SEM. Significance was assessed using Student's t test for unpaired groups of data.
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RESULTS
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ENaC Expression
The expression of the three ENaC subunits was measured by quantitative real-time RT-PCR (for more details on the method, see the online supplement) 7 days after plating on Snapwell supports, a time at which epithelia were optimally polarized as indicated by high transepithelial resistance, potential difference (see MATERIALS AND METHODS), and ion transport properties (10, 12, 13). In addition, we measured ENaC expression also 48 and 72 hours after seeding. At 48 hours, β and ENaC mRNA were undetectable ( ENaC not done), while at 72 hours the expression of the two ENaC subunits was slightly higher than at 7 days after seeding (i.e., 0.045 instead of 0.023 A.U. for β ENaC and 0.005 instead of 0.003 A.U. for ). However, we decided to keep measuring the effects of siRNAs at Day 7 to allow comparison with functional experiments. Our results indicate that -ENaC was notably more abundant than the other two subunits (Figure 1, upper panel). Actually, -ENaC expression was 13-fold higher than that of β-ENaC and 110-fold higher than that of -ENaC (n = 5–6). To explore whether the relative expression of the three ENaC subunits could be a consequence of the culture conditions used (liquid–liquid; see MATERIALS AND METHODS), we measured the expression of ENaC in epithelia maintained in air–liquid interface (Figure 1, lower panel). In this condition, the expression of the three subunits was approximately 70% lower. However, the relative abundance was similar than in liquid–liquid interface, the expression of -ENaC being 16-fold higher than that of β-ENaC, and 276-fold higher than that of -ENaC (n = 2). These results indicate that the relative expression of ENaC subunits is a characteristic of the human bronchial epithelium and not a consequence of culture conditions.
Functional Down-Regulation of ENaC by siRNA
Aiming at identifying which siRNA sequences most efficiently reduce human ENaC activity and understanding if interfering with a single subunit is enough to abolish the protein function, we initially transfected cells with pools of four sequences directed against a specific ENaC subunit (for more details on the methods for making these measurements, see the online supplement). After 7 days, ENaC activity was evaluated looking at the amount of short-circuit current blocked by amiloride. After amiloride, epithelia were sequentially treated with forskolin and CFTRinh-172, to activate and block CFTR, respectively. Pools of siRNA molecules against each one of the ENaC subunits caused a significant reduction in the response to amiloride (Figures 2A and 2B) with respect to epithelia treated with the nontargeting siRNA pool. In fact, the measured short-circuit currents were 11.5 ± 1.5, 4.8 ± 0.5, 3.2 ± 0.5, and 6 ± 1.1 µA/cm2 for epithelia treated with nontargeting, , β, or siRNA, respectively. Amiloride-sensitive current was particularly reduced by the β-ENaC pool (70% reduction), though siRNA pools targeting the and subunits also caused a significant decrease in ENaC activity (53% and 42%, respectively). Very similar values were found on epithelia obtained from patients with CF (not shown). As expected, the activity of CFTR was not modified by siRNAs against ENaC subunits (see traces in Figure 2A). The current blocked by CFTRinh-172 was 10.8 ± 1.8 µA/cm2 (n = 11) in control conditions and 10.3 ± 2 (n = 9), 9.3 ± 2.5 (n = 8), and 6.1 ± 2.1 µA/cm2 (n = 6) in cells treated with , β, or siRNAs, respectively. For comparison, the current blocked by CFTRinh-172 in epithelia treated with siRNA for CFTR was 3.2 ± 0.5 µA/cm2 (n = 9).
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Figure 2. ENaC function. (A) Representative traces of human bronchial epithelial cell short-circuit currents (Iscs). ENaC and cystic fibrosis transmembrane conductance regulator (CFTR) function are estimated as the Isc blocked by amiloride, and the Isc activated by forskolin (fsk) and blocked by CFTRInh-172, respectively. (B) Mean results of 11 to 16 filters in each condition from eight different preparations. Bars are SEM. The Isc of epithelia receiving any of the three ENaC siRNAs were statistically lower than the control transfected with nontargeting sequences (P < 0.05).
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Specificity of ENaC Subunit Down-Regulation
We performed quantitative real-time RT-PCR to measure the extent of mRNA reduction for each ENaC subunit. Figure 3 depicts the relative expression of the three subunits from epithelia treated with specific siRNA sequences as compared with the expression of the same subunits from epithelia treated with nontargeting controls. In the presence of the specific siRNAs against and β ENaC subunits, the corresponding mRNAs were decreased to 30 to 35% of the value of the respective controls. The effect was less marked for ENaC siRNA, which was reduced to 50% of control. Interestingly, only the siRNA pool for β ENaC decreased exclusively the targeted subunit, causing no alteration in the expression of the other two. In contrast, siRNA pool against reduced also the expression of the subunit and vice versa. Neither nor siRNAs altered the expression of the β subunit. When we repeated these experiments in cultures maintained in air–liquid interface, the effects were very similar. Specific siRNAs reduced , β, and ENaC subunits to 35%, 61%, and 49%, respectively (n = 2, data not shown). We wondered whether the use of pools of four siRNAs was necessary to down-regulate efficiently each ENaC subunit, as suggested by the manufacturer, or if a similar result could be obtained using a single duplex. To answer this question we transfected cells independently with each of the four sequences that constituted the pools against and β ENaC (see Table E1). As shown in Figures 4A and 4B, all sequences but 2 were efficient in reducing ENaC current.
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Figure 3. Relative ENaC mRNA expression detected by real-time RT-PCR. Expression was normalized to that in epithelia transfected with nontargeting sequences and to β2-microglobulin (housekeeping gene). In each panel is shown the expression of the three ENaC subunits when a single subunit was targeted. The dashed lines show the expression level for each subunit in the absence of specific siRNA sequences. Asterisks indicate that the differences were statistically significant from controls (n = 4, P < 0.05).
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Figure 4. Amiloride blocked Isc in the presence of nontargeting siRNA sequences or of single sequences for or β ENaC, as indicated. Data were normalized for ENaC currents of epithelia treated with nontargeting sequences (indicated by dashed lines). The final concentration of siRNA in each condition was 100 nM. Asterisks indicate differences that resulted statistically significant from controls (P < 0.05).
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Expression of and ENaC Proteins
We analyzed the effect of siRNAs on ENaC proteins by Western blot. As shown in Figure 5, specific antibodies to human ENaC subunits showed the presence of (Figure 5A) and (Figure 5B) bands. We could not analyze the effect of siRNAs on the expression of β ENaC due to the lack of an efficient antibody for this subunit. Transfection of primary human bronchial epithelial cells with siRNA for subunit (Figure 5A) resulted in a reduction ( 50%) of ENaC as evidenced by a reduced density of the band compared with cells transfected with nontargeting (NT) siRNA sequences. In contrast, transfection of the cells with siRNA for the subunit did not result in a reduction of the ENaC subunit in comparison to control conditions (Figure 5B). Figure 5C shows the quantitative analysis of the protein bands in experiment of the type shown in Figures 5A and 5B.
ENaC-Dependent Fluid Transport
To evaluate the physiologic relevance of siRNA-mediated ENaC down-regulation, we measured fluid transport in bronchial epithelia treated with sequences complementary for the three ENaC subunits, in control conditions and in the presence of 10 µM amiloride. As shown in Figure 6, the presence of anti-ENaC siRNAs reduced fluid absorption from the apical side of the epithelium to 40 to 60% of the absorption measured in control epithelia. The extent of transport reduction was similar to that of control epithelia treated with amiloride. Addition of amiloride to siRNA-treated epithelia had no further effect on fluid transport.
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Figure 6. Transepithelial fluid transport through human bronchial epithelium. Fluid absorption was measured 7 to 8 days after cell treatment with a nontargeting sequence (control condition) or with sequences complementary to ENaC subunits. Incubation with 10µM amiloride reduced fluid absorption on control epithelia (nontargeting) but had no further effect on epithelia treated with siRNAs for ENaC subunits. Values are means of 5 to 13 experiments. Bars are SEM. In all conditions, independently of the presence of amiloride, fluid absorption was lower than in the presence of the nontargeting control (lower bar, *P < 0.05).
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DISCUSSION
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ENaC is an oligomeric membrane protein constituted by three homologous subunits, , β, and . A fourth subunit, delta, is expressed mainly in the brain and reproductive tissues in substitution of subunit. The first three subunits are widely expressed in the apical membrane of epithelial cells and together form the channel devoted to electrogenic Na+ absorption. The stoichiometry of the channel is believed to be that of a heterotrimer formed by one , one β, and one subunit (14). While the subunit alone is capable of producing low levels of a functional channel (15, 16), oligomerization seems to be necessary for a maximal amiloride-sensitive current to appear and for an efficient Na+ absorption. Actually, there is evidence that insufficient expression of one subunit limits the activity of ENaC, as demonstrated by mutations causing pseudohypoaldosteronism type 1 (17). However, the relative expression of ENaC subunits is not homogeneous and seems to be tissue specific. Moreover, the three subunits are often synthesized in differential manner, suggesting that one of them plays a role in modulating the functional expression of the ENaC channel. For example, the expression of the three subunits is equivalent in rat nasal gland ducts, while in rat trachea and subunits prevail over β (18). Regarding modulation, aldosterone stimulates the three ENaC subunits from rat colon, but only the subunit from rat kidney (19). Furthermore, dexamethasone increases β and protein subunits of alveolar A549 cells, while does not change (20). Also, we found that IL-4 causes a reduction of ENaC currents in the human bronchial epithelium by reducing mainly the mRNA of the and to a lesser extent of the β subunits without altering subunit expression (12).
It is conceivable that reduction of the less expressed subunit by RNA interference might have the more dramatic effect in reducing ENaC activity. Therefore, we determined the levels of expression of the three ENaC subunits in our epithelia. The relative expression found here, characterized by a preponderance of subunit followed by β and with low levels of , is similar to that described previously in human airways (23). Explanations for the unbalanced expression could be that excess in subunit, the only subunit able to form an active channel, might assure a certain level of ENaC function. In contrast, the low expression levels of β and could suggest that they are more prone to modulation by external factors, such as hormones, cytokines, and so on.
Given the high relative abundance of subunit (see Figure 1), we hypothesized that down-regulation of or β subunits would be the most effective in reducing ENaC current. However, this seemed not to be the case. In fact, the siRNA sequences for the three subunits were all able to down-regulate ENaC efficiently. The siRNA complementary to β ENaC caused the strongest reduction at the mRNA and current levels ( 70%; see Figures 2 and 3), though the difference with the effects of and siRNAs was not very large. The siRNA against to the less abundant subunit reduced the expression of messenger by approximately 50% and, accordingly, the ENaC current by 40%.
Unexpectedly, the siRNA for subunit also caused a reduction to half of ENaC currents. At the mRNA level, the mRNA reduction was approximately 65%. However, the 35% residual mRNA is still several fold higher in absolute terms than the mRNA of the other two subunits (see Figure 1). Therefore, oligomerization of the three subunits should have reached the same extent as in nontargeted epithelia, and amiloride-blocked currents should not have changed. It is possible that the current reduction is not due to a decrease in subunit expression, but mainly to the simultaneous reduction by 50% of mRNA observed in this condition (see Figures 2 and 3). Alternatively, the distribution of ENaC between intracellular and membrane pools might be tightly regulated resulting in a number of channels in the apical membrane that are a constant fraction of the total subunit. Another possibility is that part of the channels that we have measured were not typical heterotrimers but were formed prevalently by the subunit. In this regard, Jain and coworkers have found that treatment with antisense oligonucleotides to any of the three subunits of ENaC resulted in a significant decrease in the density of highly selective sodium channels in the apical membrane of rat alveolar type II cells (21), while only the antisense oligonucleotide targeting the -subunit resulted in a significant decrease in the density of nonselective cation channels (22). These results indicate that in these rat cells, beside being part of highly selective sodium channels, the -subunit of ENaC is a major component of nonselective cation channels.
It is not clear why the mRNA for subunit was reduced by siRNA complementary to and the subunit by siRNA complementary to . The sequences used were specific for each subunit and no unspecific effects were expected. Other transport systems in the epithelium do not seem to be affected by the siRNAs for ENaC. This is the case for CFTR and the NKCC cotransporter, as shown by a cAMP-dependent transepithelial current that does not change (response to forskolin and CFTRinh-172 in Figure 2A). Indirectly, it is also the case of the Na-K ATPase, because its inhibition and the consequent intracellular Na+ increase would have reduced the activity of the sodium-potassium-chloride cotransporter (24) and thus the response to forskolin. These data support the idea that the siRNAs used were specific and are in agreement with data from Li and Folkesson (25), who used an siRNA-generating plasmid DNA against ENaC in newborn rat lungs, finding that ENaC was reduced, while the β subunit and the Na-K ATPase mRNA did not change. In addition, in our experiments the sequences complementary to β caused no effect on the other two subunits. It is possible to speculate that and mRNAs are regulated in a coordinated fashion, so that reduction of one would decrease the expression of the other. In any case, from a functional point of view, targeting , β, or subunits produced similar effects, as siRNAs for the three ENaC subunits reduced the epithelium short-circuit current and fluid absorption.
The independent use of the siRNA sequences that constitute the subunits pools gave useful indications. While one sequence (2) failed to knock down the ENaC subunit, the other had similar or even better effect than the corresponding pool of siRNAs. However, an advantage of using pools of siRNAs is that they probably ensure that at least one of the sequences is functional. So, by using pools of siRNAs, we have obtained significant reduction of ENaC subunits mRNA and transepithelial short-circuit currents. The reduction of the subunit protein by at least 40% after treatment with the corresponding siRNA is also consistent with mRNA and Na+ current measurements. However, the effect of siRNA for ENaC was not so clear, since the blot could not demonstrate a reduction of the protein subunit. A possible explanation is that the antibody for ENaC has insufficient sensitivity to detect small changes in the expression of this low-expressing subunit. In spite of this result, we have found, with more sensitive and quantitative methods, that the siRNA for ENaC reduces mRNA, transepithelial current, and fluid absorption through the epithelium, suggesting that the protein subunit is also reduced.
In conclusion, our study demonstrates that siRNA sequences complementary to any of the ENaC subunits cause a long-term reduction of ENaC activity in human bronchial epithelia for at least 8 days after treatment. The long duration of this effect might depend on the fact that cells forming part of a differentiated epithelium are not dividing, and that therefore there is no dilution effect. The reduction of ENaC activity does have functional consequences on the airways, as demonstrated by the fluid measurements. Such experiments show that ENaC down-regulation causes a significant reduction in fluid absorption through the bronchial epithelium, suggesting that the periciliary fluid volume would increase and, as a consequence, there would be an improvement in mucociliary clearance. In agreement with this idea, Li and collaborators, using siRNA for ENaC on newborn rat lungs, have recently found that ENaC mRNA and protein were reduced in alveolar cells, while the lung water was increased (26). The impact of such increase in hydration levels on the airways of patients with cystic fibrosis still needs to be evaluated. Nevertheless, our results point out the potential beneficial action of reducing ENaC activity through an siRNA approach. Moreover, the long-lasting effects of interfering RNA targeting ENaC subunits on the human bronchial epithelium suggest that doses to treat patients with cystic fibrosis could be administered separated by several days or even weeks. However, before applying our results to patients, the nucleic acid transfer problem has to be addressed. In fact, we have transfected cells while seeding them on permeable supports because transfection efficiency on already-formed epithelia was extremely low, as measured by using a siRNA marked with a fluorescent dye (Alexa fluor 488; Amersham) or by measuring the functional down-regulation of CFTR channel by specific siRNA (data not shown). In addition, Griesenbach and coworkers (27) reported recently inefficient transfer of lipid-mediated siRNA to airway epithelial cells in vivo. Comparable problems have been experienced by researchers working on gene therapy and using different vectors to transfer healthy genes to the airways (28). One possibility to improve nucleic acid internalization is to infect cells with viral vectors carrying siRNAs. Alternatively, chemical modifications of siRNAs could improve the level of interfering RNAs penetration on resting cells. In addition, the lack of off-target effects has to be determined on a larger number of proteins.
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Footnotes
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* These authors contributed equally to this work. 
This project was supported by the European Commission grant LSHB-CT-2004–005213.
This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org
Originally Published in Press as DOI: 10.1165/rcmb.2007-0456OC on August 21, 2008
Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.
Received in original form December 19, 2007
Accepted in final form July 10, 2008
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Abstract
MATERIALS AND METHODS
