Epithelial Sodium Channel Silencing as a Strategy to ... - ATS Journals

Epithelial Sodium Channel Silencing as a Strategy to Correct the Airway Surface Fluid Deficit in Cystic Fibrosis Ambra Gianotti1, Raffaella Melani1, Emanuela Caci1, Elvira Sondo1, Roberto Ravazzolo1,2, Luis J. V. Galietta1, and Olga Zegarra-Moran1 1 Laboratorio di Genetica Molecolare, Istituto Giannina Gaslini, Genoa, Italy; and 2Dipartimento di Neuroscienze, Riabilitazione, Oftalmologia, Genetica e Scienze Materno-Infantili, University of Genoa, Genoa, Italy

In the respiratory system, Na1 absorption and Cl2 secretion are balanced to maintain an appropriate airway surface fluid (ASF) volume and ensure efficient mucociliary clearance. In cystic fibrosis (CF), this equilibrium is disrupted by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, resulting in the absence of functional CFTR-dependent Cl2 secretion. The consequences of defective Cl2 transport are worsened by the persistence of Na1 absorption, which contributes to airway surface dehydration. We asked whether normal ASF can be restored to an equal extent by recovering Cl2 secretion from mutated CFTR or by reducing Na1 absorption. This is highly relevant in the selection of the best strategy for the treatment of patients with CF. We analyzed the ASF thickness of primary cultured bronchial CF and non-CF epithelia after silencing the epithelial Na1 channel (ENaC) with specific short, interfering RNAs (siRNAs) and after the pharmacological stimulation of CFTR. Our results indicate that (1) single siRNAs complementary to ENaC subunits are sufficient to reduce ENaC transcripts, Na1 channel activity, and fluid transport, but only silencing both the a and b ENaC subunits at the same time leads to an increase of ASF (from nearly 7 mm to more than 9 mm); (2) the ASF thickness obtained in this way is about half that measured after maximal CFTR stimulation in non-CF epithelia (10–14 mm); and (3) the pharmacological rescue of mutant CFTR increases the ASF to the same extent as ENaC silencing. Our results indicate that CFTR rescue and ENaC silencing both produce a significant and long-lasting increase of airway hydration in vitro. Keywords: CFTR; ENaC; siRNA; airway surface fluid; cystic fibrosis

Cystic fibrosis (CF) is an autosomal recessive genetic disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. The mutations reduce or abolish the function of CFTR protein, an epithelial anionic channel, leading to ion transport defects in the pancreas, liver, intestine, vas deferens, sweat ducts, and airways. In particular, lung disease results in a reduction of airway surface hydration, and thus inefficient mucociliary clearance. This condition facilitates lung infection and colonization of the airways by opportunistic pathogens, leading to persistent inflammation, progressive airway obstruction, and lung function decline (1). Therefore, one major therapeutic challenge involves the recovery of airway surface hydration.

(Received in original form October 9, 2012 and in final form April 16, 2013) This project was supported by Italian Cystic Fibrosis Foundation grant FFC 7/2009, with contributions from Work in Progress Communication SRL and Brook Brothers Retail Brand Alliance Europe SRL, and by Telethon Foundation grant GGP10026. R.M. and A.G. have been Italian Cystic Fibrosis Foundation contract holders. Correspondence and requests for reprints should be addressed to Olga ZegarraMoran, M.D., Laboratorio di Genetica Molecolare, Istituto Giannina Gaslini, L.go G. Gaslini 5, Genoa I-16148, Italy. E-mail: [email protected] This article has an online supplement, which is accessible from this issue’s table of contents at www.atsjournals.org Am J Respir Cell Mol Biol Vol 49, Iss. 3, pp 445–452, Sep 2013 Copyright ª 2013 by the American Thoracic Society Originally Published in Press as DOI: 10.1165/rcmb.2012-0408OC on April 19, 2013 Internet address: www.atsjournals.org

CLINICAL RELEVANCE The pharmacological rescue of the mutated cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is a very promising approach in the treatment of cystic fibrosis (CF). However, many types of CF mutations (e.g., frame-shift mutations) cannot be directly treated with CFTR pharmacological modulators. We demonstrate that the inhibition of the epithelial Na1 channel (ENaC) by RNA interference is highly effective and comparable, in terms of airway surface hydration, to CFTR rescue. These results support the role of ENaC as an effective alternative target for CF.

The airway epithelium is basically absorptive, and this process is regulated by the activities of Na/K-ATPase in the basolateral membrane, and the epithelial Na1 channel (ENaC) in the apical membrane. However, Cl2 secretion and hence water are necessary to counterbalance situations of excessive absorption or epithelial dehydration. Thus, Na1 absorption and Cl2 secretion are carefully regulated to maintain an appropriate airway surface fluid (ASF) volume and ensure efficient mucociliary clearance. In CF, the balance of the system is disrupted by mutations in the CFTR gene, resulting in the absence of functional CFTRdependent Cl2 secretion (2, 3). The negative effects of the disequilibrium between Na1 absorption and Cl2 secretion have also been demonstrated by the production of a transgenic mouse that hyperexpresses the b subunit of the ENaC (4). In this mouse, the increased Na1 and water absorption produce a CF-like lung disease, characterized by surface liquid depletion, increased mucus concentration, inflammation, and poor bacterial clearance. CF mutations affect the CFTR through a variety of molecular mechanisms that produce different functional defects, namely, (1) an absence of functional protein synthesis, (2) defective protein processing, (3) altered channel gating, (4) reduced anion permeability, and (5) reduced amounts of protein. A single mutation, the deletion of phenylalanine in position 508 (F508del), is present in at least one chromosome in 50–90% of patients with CF. F508del displays a severe processing defect (Class II) with an almost total absence of the protein in the plasma membrane. However, when the processing defect is corrected in vitro by incubation at a low temperature, the F508del CFTR also exhibits an altered gating defect (Class III). In the past several years, many small molecules called potentiators, which are able to recover the activity of Class III CF mutations, have been identified (5–11). A Phase 3 clinical trial with the potentiator Ivacaftor (VX-770) showed that after 48 weeks, patients who carry the G551D mutation (Class III) demonstrated lung function improvement and reduced pulmonary exacerbations (12). However, the number of patients carrying Class III mutations who could derive benefit from this treatment is relatively low. Up to now, the compounds identified as correctors of the F508del processing defect have shown only partial activity in bronchial

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AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL 49 2013 TABLE 1. TARGET SiRNA SEQUENCES AGAINST a AND b EPITHELIAL Na1 CHANNEL SUBUNITS AND PROSTASIN Name SA1 SA2 SA3 DA1 DA3 SB1 SB2 SB3 DB2 DB3 SP1 SP2 SP3 DP1 DP2

Gene

Sense Sequence

Antisense Sequence

Source

SCNN1A SCNN1A SCNN1A SCNN1A SCNN1A SCNN1B SCNN1B SCNN1B SCNN1B SCNN1B PRSS8 PRSS8 PRSS8 PRSS8 PRSS8

59GCUCUUUGACCUGUACAAA 59GAGAAAUGGAGUGGCCAAA 59CUUUACCCUUCAAAGUACA 59UUGUAGUUCAGCUCCUUGA 59UCAAGGAGCUGAACUACAA 59CCAAUAACAUCGUCUGGCU 59CAGUGUACCUUCCGGAACU 59GAGACAACCACAAUGGCUU 59UUGUACUGGGUGUCAUUGC 59UAGUUGCAGGGCUCAGCUC 59CCAUCACCUUCUCCCGCUA 59CCAUGUGUGUGGUGGCUCU 59CACUUUGUCCAAGAGGACA 59AUGUUGUACAGGCAGUUAC 59UCCUUGUGGUGCUCGCUGG

59UUUGUACAGGUCAAAGAGC 59UUUGGCCACUCCAUUUCUC 59UGUACUUUGAAGGGUAAAG 59UCAAGGAGCUGAACUACAA 59GCAGUGAUGUUCCUGUUGA 59AGCCAGACGAUGUUAUUGG 59AGUUCCGGAAGGUACACUG 59AAGCCAUUGUGGUUGUCUC 59GCAAUGACACCCAGUACAA 59GAGCUGAGCCCUGCAACUA 59UAGCGGGAGAAGGUGAUGG 59AGAGCCACCACACACAUGG 59UGUCCUCUUGGACAAAGUG 59GUAACUGCCUGUACAACAU 59UCCUUGUGGUGCUCGCUGG

Sigma-Aldrich Sigma-Aldrich Sigma-Aldrich Dharmacon Dharmacon Sigma-Aldrich Sigma-Aldrich Sigma-Aldrich Dharmacon Dharmacon Sigma-Aldrich Sigma-Aldrich Sigma-Aldrich Dharmacon Dharmacon

Definition of abbreviations: PRSS, prostasin; SCNN, epithelial Na1 channel subunit; siRNA, short, interfering RNA.

primary epithelial cells (11, 13), and a recent clinical study showed that corrector VX-809 did not improve the respiratory function of patients with F508del (14). Until very recently, a well accepted model for CF lung disease postulated that, in addition to defective Cl2 secretion, a second major abnormality consists of increased water and Na1 absorption because of the hyperactivity of ENaC (15). However, recent studies on newborn CFTR2/2 pigs and on human airway epithelia indicated that sodium absorption in the airways is not increased (16, 17). Irrespective of whether an up-regulation of Na1 absorption occurs in CF lungs, it is reasonable to postulate that ENaC down-regulation by pharmacological or genetic maneuvers may counteract the dehydration of CF airways. First, decreasing Na1 absorption might in itself expand the ASF. Second, reducing ENaC activity will hyperpolarize the apical membrane and increase the driving force for Cl2 secretion, either through the mutated CFTR (residual function) or through alternative Cl2 channels. Previous attempts to treat CF airway disease by aerosol applications of amiloride led to a reduction of sodium reabsorption and an increase in mucus clearance (18, 19). However, the effects were of short duration, probably because of the rapid removal of amiloride from the epithelial surface (20). Short duration has also been reported by in vivo pharmacodynamic studies in sheep, using the more potent ENaC blocker benzamil (21). Apparently, a more rapid absorption of benzamil offset its greater potency. Another potent ENaC blocker, P552-02, was found to improve airway hydration for longer times in vitro because of its slower absorption rate (22), and a Phase IIa clinical trial seems to indicate that the drug is safe. Another way to reduce ENaC activity has been achieved using inhibitors of Kunitz-type serine proteases. BAY 39-9437 was found to inhibit ENaC in monolayers of both wild-type and CF human bronchial epithelia (23). The protease inhibitor camostat produced a potent and prolonged attenuation of ENaC function in vitro and increased mucociliary clearance in sheep for at least 5 hours (24). More recently, we used pools of short, interfering RNA (siRNA) targeting ENaC subunits in vitro. This strategy leads to specific Na1 transport inhibition that can be maintained for several days (25). Here we sought to determine whether it is possible to recover the surface hydration of CF and non-CF primary human bronchial epithelia (HBE) by silencing ENaC using specific siRNAs. We found that the siRNA-based knockdown of single ENaC subunits is enough to produce a long-term down-regulation at the mRNA and functional levels. However, to observe a significant increase in ASF thickness, a more marked knockdown of two subunits is required. We compared the level of hydration obtained in this way with that measured after rescuing F508del with a corrector

and potentiators. Our results indicate that both the increase of CFTR activity and ENaC silencing produce a significant and long-lasting increase of airway hydration in vitro.

MATERIALS AND METHODS Cell Culture and siRNA Transfection Highly differentiated bronchial epithelia with high transepithelial electrical resistance were generated on a porous membrane (Snapwell; Corning Costar, Milan, Italy), as described previously (26) and in the online supplement. Epithelial monolayers of H441 cells were also studied. The transfection of siRNAs against ENaC subunits and prostasin (PRSS8; Table 1) was performed using Lipofectamine 2000 (Life Technologies, Milan, Italy). Evaluation of target silencing was performed using quantitative real-time RT-PCR and Western blotting (for details, see the online supplement).

Functional Assays Transepithelial ion transport in primary human bronchial epithelial cells (HBECs) and H441 cells was performed using short-circuit current

Figure 1. Expression levels of the epithelial Na1 channel (ENaC) and serine protease transcripts. (A) Relative expression (normalized by the expression of b2-microglobulin) of ENaC subunits from cultured primary human bronchial epithelia (HBE). Note that the ENaC d subunit (SCNN1D) is almost completely absent from these cells. (B) Expression of different serine proteases in primary human bronchial epithelium. The expression levels of prostasin-8 (PRSS8), PRSS15, PRSS22, and PRSS23 were comparable. Bars represent the means 6 SEMs of three experiments. A.U., arbitrary units.

Gianotti, Melani, Caci, et al.: Efficacy of ENaC Silencing in Cystic Fibrosis

measurements. Analysis of fluid absorption was performed by adding a fixed fluid volume (150 ml) on the apical surface of epithelia, and measuring the remaining fluid after 24 hours. ASF thickness was measured using a confocal microscope by staining with Texas Red dye conjugated with dextran (27). All technical details are reported in the online supplement.

Chemicals Texas Red–10 kD–dextran and 29,79-bis-(2-carboxyethyl)-5-(and-6)carboxyfluorescein (BCECF)/acetoxymethyl ester were obtained from Invitrogen (Carlsbad, CA). CFTRinh172 was obtained from Cystic Fibrosis Foundation Therapeutics (Bethesda, MD), and VX-770 and VX-809 were obtained from Selleck (Milan, Italy). Unless otherwise indicated, the remaining compounds were obtained from Sigma-Aldrich (Milan, Italy).

Statistical Analysis Data are presented as representative traces or as means 6 SEMs. Significance was assessed using the Student t test for unpaired groups of data.

RESULTS ENaC Subunit Expression and Regulation

The functional ENaC channel is composed of three homologous subunits, termed a (SCNN1A), b (SCNN1B), and g (SCNN1G).

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In some tissues, a-ENaC may be replaced by a d subunit (SCNN1D). Each of the ENaC subunits would be a suitable target for RNA interference, and a combination strategy is likely to be even more effective. As shown in Figure 1A, in HBECs, a-ENaC is markedly more expressed than b-ENaC, and the g subunit concentrations are very low. The d subunit seems to be absent from primary bronchial cells. ENaC expression and activity are regulated in multiple ways, including hormonal factors, extracellular Na1, pH, and proteolysis (28–30). Proteolysis is particularly interesting because a number of studies have demonstrated that the proteolytic cleavage causing ENaC activation may aggravate symptoms of CF during lung infections associated with the local production of proteases. To evaluate the effects of ENaC activation by proteolysis, we measured the amiloride-dependent short-circuit currents (Isc) in control samples and after 24-hour incubation with aprotinin, a nonspecific Kunitz-type serine protease inhibitor. This treatment strongly reduced ENaC activity by 65% (not shown). We have evaluated the expression levels of serine proteases, and found that at least four serine proteases are expressed at similar concentrations in HBEC cells, namely, prostasin-8 (PRSS8), PRSS15, PRSS22, and PRSS23 (Figure 1B). Airway prostasin seems to be a major activator of ENaC and thus another good target for therapeutic inhibition to reduce the fluid absorption in cystic fibrosis airways (31, 32).

Figure 2. Functional evaluation of ENaC knockdown by short, interfering RNA (siRNA). ENaC activity was measured as the amplitude of short-circuit current (Isc) blocked by amiloride. (A) Dose–response relationships of two siRNA sequences against the ENaC b subunit from different commercial sources, Sigma-Aldrich (SB1; Milan, Italy) and Dharmacon (DB3; Milan, Italy) on H441 cells or on primary human bronchial epithelial cells (HBECs). (B) Representative experiment on primary HBECs shows the reduction of amiloride-dependent Isc after transfection with siRNA for b ENaC (DB1) but not for prostasin (DP2). In fact, amiloride reduces Isc by 7.5 mA (from 14.4 to 6.9 mA), 2.1 mA (from 8.9 to 6.8 mA), and 6.6 mA (from 10.7 to 4.1 mA) in epithelia transfected with nontargeting (NT; control), b ENaC, and prostasin-8 (PRSS8) siRNAs, respectively. The cystic fibrosis transmembrane conductance regulator (CFTR) was activated and subsequently inhibited by 5 mM forskolin (FSK) and 5 mM CFTRinh-172, respectively. (C) Semiquantitative RT-PCR shows that a single sequence of siRNA at a 10-nM concentration reduced specific mRNAs to approximately 40% of nontargeting control samples. Bars represent the means of 2–4 epithelia 6 SEMs. (D) Western blotting shows protein expression levels of a ENaC after treatment with siRNA against the a subunit (left), and two gels show PRSS8 protein after treatment with siRNA against PRSS8 (right). NT siRNA was used as control. Actin was used to normalize the specific bands. Numbers at the right side of the gels indicate the molecular weight. (E) Normalized ENaC-dependent Isc in CF and non-CF epithelia transfected with the indicated siRNA sequences at 10, 20, or 25 nM. We observed that after the use of siRNA against the a or b subunit at a 10-nM concentration, the Isc was more markedly reduced than in A. This probably indicates that the DB3 oligonucleotide, which was used in A, was less efficient than the other siRNAs. All Dharmacon siRNAs have been used to obtain the results in E, including DB3. Bars represent the means of 6–12 monolayers from 4–5 different preparations. (F) Isc was blocked by amiloride in control non-CF and CF cells (n ¼ 18–19; **P , 0.005).

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Silencing ENaC with Low siRNA Concentrations

High doses of siRNA may result in the modest, unspecific knockdown of several genes (33). To reduce this possibility and to find the lowest effective siRNA concentration to silence ENaC, dose– response relationships were ascertained by measuring amiloridedependent Isc. The siRNA sequences used throughout this study are shown in Table 1. As shown in Figure 2A, at concentrations close to 20 nM, siRNAs against b-ENaC, particularly from Dharmacon, were significantly effective at reducing the ENaCdependent current. Therefore, subsequent experiments were performed on HBECs, using oligonucleotides from this source and at concentrations of 10, 20, or 25 nM. Figure 2B illustrates a representative experiment on epithelia generated from primary HBECs in which 20 nM siRNA for b-ENaC specifically reduced the Isc blocked by amiloride, compared with the epithelium treated with an irrelevant nontargeting (NT) sequence. The anti-ENaC siRNA did not affect CFTR activity, as demonstrated by the lack of effect on the amplitude of the current activated by forskolin and inhibited by CFTRinh-172. In contrast, the same concentration of siRNAs for prostasin was ineffective. The lack of effect by siRNAs for prostasin was not attributable to a lack of hybridization of the oligonucleotides to the target protein, because the mRNA concentrations of prostasin were reduced to the same extent as a or b mRNA in the presence of their respective siRNAs (Figure 2C), and prostasin protein concentrations were reduced by 31% 6 7% (n ¼ 3), not too different from the reduction observed for a ENaC when cells were treated with siRNA for the a subunit (38% 6 9%; Figure 2D). An application of trypsin before amiloride produced a similar Isc increase (z 2 mA/cm2; not shown) in both NT-silenced and PRSS8-silenced epithelia, indicating that the fraction of noncleaved ENaC is the same after each treatment. We have previously shown that silencing each one of the ENaC subunits exerts similar functional effects, and that siRNAs for a or g ENaC reduce both a and g subunit mRNAs, whereas siRNA for the b subunit results in the highly specific knockdown of b ENaC mRNA (25). Therefore, here we used siRNAs for a and for b subunits only. Figure 2E shows the functional effects of siRNA sequences at different concentrations, either isolated or in combination, on CF and non-CF epithelia. These data clearly show that the reduction of ENaC activity depended on the concentration of siRNA. CF epithelia showed higher basal Isc, significantly higher amiloride-dependent Isc with respect to non-CF epithelia (Figure 2F), and a lack of CFTR activity. Nevertheless, silencing a or b ENaC subunits led to similar effects in CF and non-CF cells. Of note, the combined silencing of a and b ENaC with 10 nM siRNAs resulted in a more marked inhibition than using a single siRNA at 20 or 25 nM (Figure 2E). Fluid Absorption of HBECs

ENaC inhibition should be accompanied by a decrease in the water absorption rate. The fluid absorption rate of non-CF epithelia was 1.94 6 0. 15 ml/hour/cm2 (n ¼ 3). As shown in Figure 3A, incubation with amiloride to decrease ENaC activity significantly reduced the net fluid absorption to 1.35 6 0.08 ml/hour/ cm2 (n ¼ 3, P , 0.05). This effect was similar to that obtained stimulating fluid secretion through wild-type CFTR by using 59N-ethylcarboxamidoadenosine (NECA), an adenosine receptor agonist that raises intracellular cyclic adenosine monophosphate (cAMP) (34). Indeed, under this condition, net absorption was 1.39 6 0.11 ml/hour/cm2, and net absorption reverted to control values in the presence of the receptor antagonist theophylline (1.97 6 0. 21 ml/hour/cm2, n ¼ 3). The net fluid absorption rate of control CF epithelia (treated with NT siRNA) was 1.74 6 0.06 ml/hour/cm2 (n ¼ 10), not statistically different from non-CF cells. In contrast, CF epithelia

Figure 3. Fluid absorption in cultured bronchial epithelia. (A) Net fluid absorption rate across non-CF epithelia under control conditions (untreated) or in the presence of 100 mM amiloride, 1 mM NECA, or NECA plus 20 mM theophylline. (B) Net fluid absorption measured in CF epithelia treated either with control siRNA (NT) or with siRNAs targeting a and b ENaC subunits (DA3 and DB1 siRNAs) at 10-nM concentrations. Bars represent the means of 4–10 monolayers from different preparations. *P , 0.05.

treated with 10 nM siRNA for a ENaC plus 10 nM for b ENaC demonstrated a reduced net absorption rate of 1.19 6 0.09 ml/hour/cm2 (n ¼ 10, P , 0.005; Figure 3B). These results indicate that siRNAs for ENaC constitute an efficient way to reduce significantly the net absorption of fluid to approximately 60%, a value similar to that obtained after blocking ENaC with amiloride or by the stimulation of CFTR with NECA in non-CF epithelia. Evaluation of ASF Height in ENaC-Silenced Epithelia

We asked whether changes in fluid absorption/secretion really translate into a change in ASF. Therefore, the thickness of ASF after ENaC silencing was measured directly on a confocal microscope (Figure 4). To better distinguish cells from fluid, in some experiments the cells were stained with a vital green dye (BCECF; Figure 4A), whereas in others, cells were transfected with an enhanced green fluorescent protein (Figure 4B). The height of the ASF was obtained as the mean of the entire imaged area (Figure 4C and the online supplement). We were concerned that the addition of dye could stress the cells triggering ATP release and/or intracellular Ca21 increase, with the consequent activation of transport systems (35). To avoid unwanted responses that could arise from manipulation (i.e., the addition of 2 mg/ml Texas Red to the ASF), the epithelium was permitted to equilibrate for 1 hour before initiating measurements in the confocal microscope. We evaluated the effects of siRNAs as the mean ASF height at 75 6 3 minutes after dye application. The ASF height of control non-CF and CF epithelia was similar, at 6.8 6 0.5 mm and 7.4 6 0.5 mm, respectively (n ¼ 6–9 epithelia; Figures 4D and 4F). Importantly, although siRNAs against single ENaC subunits were able to reduce the activity of ENaC when measured as transepithelial currents, the same concentrations (10–20 nM) did not increase the height of the ASF. The combination of siRNAs for a ENaC and for PRSS8 was also ineffective (Figure 4D). In contrast, the combination of siRNAs targeting a and b ENaC met our expectations, resulting in significant ASF expansion (Figures 4D–4F). The fluid layer increased significantly to 9.8 6 0.6 mm and 9.6 6 0.8 mm in non-CF and CF epithelia, respectively (n ¼ 6–9; P , 0.05). The different values in ASF thickness between


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Figure 4. Confocal microscopy analysis of airway surface fluid (ASF) in siRNA-treated cells. Representative xy images were obtained from epithelia with cells stained with BCECF (A) or transfected with an enhanced green fluorescent protein (B) at the indicated magnifications. At the bottom are shown xz reconstructions illustrating green cells covered by the red ASF stained with Texas Red–10 kD–dextran. (C) Three-dimensional reconstruction of ASF from A, and at right, the plot of fluorescence intensity across the space and the respective derivative curves with Gaussian fits. The vertical lines in the inset indicate the points of faster fluorescence increase and decrease that were taken as the inferior and superior limits, respectively, of the ASF layer. (D) Means 6 SEMs of the ASF thickness of non-CF epithelia in the presence of indicated siRNA sequences (n ¼ 4–15 per condition). (E) Representative xz fluorescence section of the ASF of an epithelium where ENaC was silenced with siRNAs for the a and b subunits, in comparison with a control epithelium treated with an irrelevant NT siRNA. Scale bar, 20 mm. (F) Means 6 SEMs of the ASF thickness of CF epithelia in the presence of indicated siRNA sequences (n ¼ 9 per condition). (G) Time course of the ASF thickness of control (open symbols) and ENaC-silenced (solid symbols) CF (black squares) and non-CF (gray triangles and circles) epithelia (n ¼ 6–9). DA1, DA2, DA3, DB1, and DB2 were used without distinction for these measurements. Each point represents a single measurement. Lines represent exponential fits to data, continuous lines represent silenced epithelia, and dashed lines represent control epithelia. *P , 0.05.

control and ENaC-silenced epithelia were maintained for at least 150 minutes (Figure 4G). Evaluation of ASF Height after Maximal CFTR Stimulation

Maximal activation of CFTR is expected to expand the ASF (34). In fact, we found that the acute incubation of non-CF epithelia with cAMP-elevating agents, either NECA (1 mM) or a cocktail consisting of isoproterenol (25 nM), forskolin (5 mM), and the CFTR potentiator genistein (20 mM) (IFG), increased the height of the ASF from approximately 7 mm to 10–14 mm, as illustrated in Figure 5A. As expected, expansion was precluded when NECA was applied together with the adenosine receptor antagonist theophylline (20 mM), or when the IFG cocktail was combined with CFTRinh-172 (5 mM). As already mentioned, correctors of the F508del CFTR trafficking defect and potentiators of gating mutations are already undergoing advanced clinical trials. We have incubated CF epithelia for 24 hours with the corrector VX-809 (3 mM), and found that although it significantly increased the CFTR-dependent Isc by fourfold and caused a significant ENaC-dependent Isc reduction of 20% (Figures 5C and 5D), it exerted no effect on ASF height (Figure 5B). However, the acute stimulation of VX-809–corrected

epithelia with IFG or with a cocktail in which genistein was replaced by VX-770 (1 mM) resulted in a significant ASF expansion to approximately 11 mm. This effect, importantly, was blocked by CFTRinh-172.

DISCUSSION For many years, the efficacy of maneuvers targeting ENaC as a strategy to improve CF airway surface hydration has been difficult to determine. The most widely used ENaC blocker, amiloride, and its derivates are rapidly removed from the ASF, precluding the direct measurement of their effects on ASF height (18–21). RNA interference is a powerful molecular process used by cells to modulate gene expression by directing the RNA machinery to perform mRNA degradation. This effect, mediated by small sequences of approximately 20–22 bases that complement a target mRNA, maintain reduced concentrations of the transcript for several days if cells are not dividing (36). We have taken advantage of this tool to reduce the activity of ENaC in primary HBECs, and to determine whether the reduction of ENaC transcripts may help expand the ASF layer in CF. This technique permitted us to transfect cells while seeding them on permeable supports, and to wait 1 week to allow the epithelium

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Figure 5. ASF thickness from non-CF and CF epithelia. (A) Means 6 SEMs of the ASF thickness from non-CF epithelia under control conditions and after CFTR stimulation by NECA (1 mM), isoproterenol (25 nM), or a forskolin (5 mM) plus genistein (20 mM) (IFG) cocktail in the presence or absence of theophylline (20 mM) or CFTRinh-172 (5 mM). (B) ASF measured in CF epithelia under control conditions, on epithelia incubated for 24 hours with medium containing the corrector Lumacaftor (VX-809) (3 mM) without further stimulus, or after the maximal activation of CFTR with IFG or with genistein replaced by VX-770 (1 mM) in the presence or absence of CFTRinh-172. Bars represent the means 6 SEMs of 6–18 epithelia. *P , 0.05. (C and D) The values of ENaC-dependent (C) and CFTR-dependent (D) Isc from non-CF, CF, and CF epithelia treated with VX-809. Bars represent the means 6 SEMs of 11–13 epithelia. *P , 0.05. **P , 0.005.

to form and polarize before measuring the consequences of silencing. It is well-known that siRNAs may cause off-target effects, namely, the broad silencing of several genes as a consequence of the hybridization of seed sequences in the sense or the antisense siRNA filament to mRNAs (33). To reduce these unspecific effects as much as possible, we looked at the lowest effective concentrations, finding that single siRNA sequences against a or b ENaC subunits at a 10–20 nM concentration produced a significant reduction of the transcripts and of ENaC-mediated shortcircuit currents, without altering the integrity of the epithelium,

the activity of CFTR (Figure 2C), or the activity of the Ca21 -activated Cl2 channel, measured as the Isc response to an apical application of uridine 59-triphosphate (not shown). At 20 or 25 nM, a single siRNA against a or b ENaC subunits was able to reduce the ENaC Isc to approximately 40%. However, combinations of 10 nM siRNA for the a and b subunits further reduced the activity of ENaC to less than 30%. To our surprise, siRNAs for prostasin did not modify the activity of ENaC, and an application of trypsin led to a similar Isc increase in both NT-silenced and PRSS8-silenced epithelia, indicating that the fraction of noncleaved ENaC is the same after each treatment. However, prostasin mRNA and protein were reduced to a similar extent as the a or b subunits in the presence of their respective siRNAs (Figures 2C and 2D). A possible explanation states that aprotinin-sensitive serine proteases able to activate ENaC proteolytically may be redundant in the airways in such a way that when the prostasin concentration is reduced, other proteases may cleave ENaC. The catalytic activity of the nonsilenced prostasin may also be enough to cleave ENaC. Although we found clear functional differences between nonCF and CF epithelia (i.e., a lack of CFTR activity and a higher amiloride-dependent Isc in CF), no difference in ASF height was found between CF and non-CF epithelia. This probably depends on the fact that the difference in the activity of ENaC is not high enough in 1-week old epithelia. The difference in ENaC activity has to be more marked, as seen between NT control samples and epithelia treated with combinations of siRNAs against a and b ENaC (Figure 2E), to translate into a different ASF height. Interestingly, we found that a combination of siRNAs for a and b subunits reduced net fluid absorption in CF epithelia to a similar extent as that obtained by stimulating CFTR-mediated Cl2 secretion in non-CF epithelia (Figure 3). These results suggest that a marked reduction of ENaC activity in CF airways might lead to an increase in ASF hydration levels that could compensate for the lack of functional CFTR. This assumption seems to be confirmed by our direct measurements of ASF thickness (Figure 4). Silencing the a or b subunits alone exerted no effect on the ASF. However, silencing a and b ENaC at the same time led to a long-lasting increase in this layer of 2.5–3 mm. To offer a probable explanation for the finding that siRNAs against single a or b ENaC subunits, even at 20 nM, reduced ENaC Isc but not the ASF, we think that the residual ENaC activity (40%, measured as Isc) was enough to maintain an absorption rate similar to that of the control epithelium. In contrast, when we combined siRNAs against a and b ENaC (at 10 nM), the residual ENaC (less than 30%; Figure 2E) was probably insufficient to maintain the same absorption rate as the control samples. This indicates that fluid absorption has to be markedly reduced to produce a change on the ASF. Whether the ASF expansion obtained by ENaC knockdown is enough to improve mucociliary clearance in CF is difficult to establish. However, a comparison of ASF height after ENaC silencing with that after maximal CFTR activity in a wild-type epithelium (6-mm increase when using NECA; Figure 5A) suggests that the effect may be valuable. The potent stimulation of ion secretion and the increase in ASF thickness by NECA (greater than the stimulation achieved with isoproterenol, forskolin and a potentiator) are hardly explained by an intracellular cAMP increase alone. A recent report indicates that mucosal adenosine stimulates basolateral Ca21-activated K1 channels in Calu-3 cells through phospholipase C/Ca21 signaling, increasing in this way the driving force for transepithelial anion secretion (37). A similar simultaneous action of NECA on apical CFTR and basolateral K1 channels may explain our results. We note that the ASF expansion caused by ENaC silencing is comparable to that obtained by treating F508del cells with the


corrector and a potentiator (3–4 mm expansion). The results of ongoing clinical trials with VX-809 and the potentiator VX-770 (now on the market with the name Ivacaftor; Vertex Pharmaceuticals Inc., Cambridge, MA) on patients with F508del will show whether the hydration level achieved in this way improves the lung function of patients with CF. In conclusion, this study mainly sought to establish whether it is possible to recover the surface hydration of CF airways by reducing ENaC activity, and if so, to compare the effect obtained with that achieved by stimulating CFTR. Our results demonstrate that a combination of siRNAs for a and b ENaC subunits at low concentrations can significantly reduce ENaC activity and expand the ASF, and that the height of the ASF obtained in this way in CF cells is similar to that obtained with a combination of a F508del corrector and a potentiator, and about half the ASF of non-CF cells when CFTR is maximally activated. This finding is particularly relevant because many types of CF mutations (e.g., frameshift mutations) cause a CFTR loss of function that cannot yet be treated directly with small molecules. Therefore, strategies aiming at alternative targets may be highly valuable. The development of efficient nanocarriers to deliver siRNA to the lung, a pursuit that currently involves different laboratories and pharmaceutical companies (38), is the next step needed to test and develop RNA interference as a therapeutic tool for CF. Author disclosures are available with the text of this article at www.atsjournals.org.

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