Departments of 1Pediatrics and 2Physiology, University of Maryland, Baltimore, Maryland 21201; and 3Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, 21949-900 Rio de Janeiro, Brazil
Submitted 4 April 2003 ; accepted in final form 30 October 2003
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ABSTRACT |
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lung development; chloride secretion
The cystic fibrosis transmembrane conductance regulator (CFTR) is an essential Cl- channel. Mutations of CFTR lead to recurrent lung infections and premature death in patients with cystic fibrosis. However, despite defects in CFTR expression and function, patients with cystic fibrosis are born with normal lung morphology (41), suggesting that another Cl- channel is responsible for Cl- and fluid secretion in the developing lung. Proposed alternative Cl- channels in the lung include the Ca2+-activated Cl- channel (11), the outwardly rectifying Cl- channel (16), or members of the voltage-gated CLC family of Cl- channels (CLC-2, -3, and -5) (8, 14, 21, 31).
CLC-2 is a member of the CLC family of voltage-gated Cl- channels that is widely expressed in epithelial cells, including those in the lung (42). Previous work in this laboratory revealed that protein expression of CLC-2 was most abundant in the canalicular and saccular phases of lung development and decreased significantly shortly after birth (31). In addition, immunocytochemical analysis revealed that CLC-2 protein is localized to the apical surface of the fetal airway epithelium (30, 31). The presence of CLC-2 protein in the epithelium of conducting airways during the canalicular and saccular phases of lung development coincides with the period of peak fluid secretion in the fetal lung (34).
The expression of CLC-2 during the fetal period supports the hypothesis that Cl- secretion by CLC-2 channels contributes to the secretion of fluid in the developing lung. In the present study, we tested this hypothesis in a fetal rat lung explant culture (24) by exposure to a CLC-2 antisense oligodeoxynucleotide (ODN). We found that fetal lungs in explant culture developed fluid-filled cysts over a 48-h period, as described by others (23, 26). In contrast, lungs treated with a CLC-2 antisense ODN for 48 h exhibited branching, but the size of the cysts was markedly reduced, suggesting fluid secretion was impaired. Transepithelial potential difference (PD) measurements across the membrane of the cysts were markedly reduced in the antisense-treated lungs, suggesting that inhibition of CLC-2 Cl- secretion was responsible for the reduction in fluid secretion. Analysis of CLC-2 protein and mRNA confirmed that CLC-2 channel formation was blocked by the antisense ODN after 48 h in culture. These results suggest that CLC-2 is an important factor in the regulation of fetal lung fluid secretion.
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MATERIALS AND METHODS |
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Timed-pregnant Sprague-Dawley rats (Zivic Miller) were killed on day 14 of gestation (22 days = term) by a 3-min exposure to 100% CO2. The fetuses were aseptically removed from uterine decidua and placed in sterile Hanks' balanced salt solution (HBSS) at 4°C. Fetal lung rudiments were microdissected and washed in HBSS at 4°C for 510 min. Three to four lungs from a single litter were submerged in 500 µl of Matrigel (Becton Dickinson) and incubated for 1 h in a humidified atmosphere at 37°C in 5% CO2 and 21% O2 before transfection.
Transfection of ODNs
ODNs targeted for transfection were determined previously by Kajita et al. (18) to inhibit CLC-2 expression and function in porcine choroid plexus. The antisense CLC-2 ODN corresponded to nucleotide sequence 2,4122,390 (accession number GI56705): 5'-TTC CAA TTC TGA CGT CGT CGA CT-3' and the random control ODN: 5'-AGA CGC TTG CGA CAG CAG ATC G-3'. The ODNs were synthesized by Oligos, Etc. (Wilsonville, OR) and modified by phosphorothiolation to improve stability against nuclease degradation in culture.
Transfection with either the antisense or the random control ODN into the lung explant was accomplished by a cation liposome transfection method. For each experiment the ODN was diluted in Opti-MEM (Life Technologies) and mixed with Lipofectamine 2000, at a final concentration of 45 µM ODN, and incubated at room temperature for 20 min. This oligo/Lipofectamine solution was carefully added to each well, and the cultures were allowed to incubate for 48 h at 37°C in 21% O2-5% CO2. For each litter, approximately six fetal lungs were used as mock-transfected controls, six were transfected with the antisense ODN, and six were transfected with the random control ODN. For mock-transfected control conditions, the transfection solutions were prepared as above, without any ODN added (only Lipofectamine in Opti-Mem was applied to the lung).
Lung Explant Morphology
Individual lung sections were photographed on a Nikon TS 100 inverted microscope with a x10 objective, using an Optronics camera and Twain 32 software. Images were imported into Photoshop 4.0.1. Morphological changes in lung cyst development were documented at 0, 24, and 48 h. These images were scanned into a densitometry program, MacBas, on a PowerMac G4. To measure the diameter of each air space, we identified the transect by cyst density and distance across the lumen measured in millimeters. The same cysts were measured for each condition over the 48-h period in culture.
CLC-2 mRNA Expression by Quantitative Polymerase Chain Reaction
Isolation of total RNA. To determine whether CLC-2 antisense ODNs inhibited CLC-2 mRNA expression, we performed semiquantitative reverse transcription polymerase chain reaction (RT-PCR) of explants at 48 h. Total RNA was extracted from pooled lung explants transfected on the same day with antisense CLC-2, random, or no ODN (mock-transfected controls, as described above) using TRIzol reagent (GIBCO-BRL, Grand Island, NY) according to the manufacturer's instructions. The isolated RNAs were treated with ribonuclease-free deoxyribonuclease DNase I (1 U/µl) for 1 h to eliminate contamination with genomic DNA.
RT-PCR. To prepare first-strand complementary deoxyribonucleic acid (cDNA), total RNA isolated from antisense, random, and mock-transfected lung explants above was first primed with oligodeoxythymidilic [oligo(dT)] primer and then reverse-transcribed with SuperScript (GIBCO-BRL) at 37°C for 60 min. Extraction with phenol-chloroform-isopropyl alcohol and precipitation with ethanol terminated the treatment.
PCR was used to amplify the synthesized cDNA with the following solution: 0.2 µmol primers, 0.2 µmol each deoxynucleotide triphosphate, 50 mmol KCl, 10 mmol Tris·Cl (pH 8.3), 1.5 mmol MgCl2, and 2.5 units AmpliTaq (Perkin Elmer). One pair of oligonucleotides was synthesized for CLC-2 amplification (A = 5'-TATGCCATCGCGTC TG-3' and B = 5'-GAAGTCGAGTCGGAACCG-3') corresponding, respectively, to nucleotides 345362 and 943960 of rat CLC-2 cDNA sequence (accession number GI8393137). PCR was performed with 35 cycles of denaturation (94°C, 1 min), annealing (55°C, 1 min), and extension (72°C, 1 min).
Semiquantitative RT-PCR was used to compare the expression of CLC-2 in 500 ng of total RNA isolated from mock-transfected control explants and cells treated with antisense or random ODNs. Oligo(dT) was used in the RT to obtain single-stranded DNA. PCR conditions were the same as described above. For the semiquantitative RT-PCR, GAPDH primers, predicted to amplify a 225-bp product, were added into the same RT-PCR reaction tubes, and their products were used as an internal control. The GAPDH primers were 5'-GTC TTC ACC ACC ATG GAG-3' and 5'-CGA TGC CAA AGT TGT CAT G-3', corresponding to nucleotides 301318 and 508526, respectively (accession number XM227654). PCR products were size fractionated with 10 g/l agarose gel electrophoresis. The expected CLC-2 and GAPDH bands from the same sample were densitometrically analyzed and normalized by dividing the CLC-2 values by the corresponding GAPDH values.
The semiquantitative method of RT-PCR was validated in preliminary experiments. First, the optimal PCR conditions that yielded a single band on agarose gel electrophoresis were determined for each gene in the same reaction tube. Second, to determine whether the method was semiquantitative, serial amounts (65.5, 125, 250, 500, 1,000, 2,000, and 4,000 ng) were used for RT-PCR amplification for both genes in the same reaction tube. Third, experiments were performed to determine the optimal number of PCR cycles that yielded PCR products in the linear phase of amplification. Finally, to ensure that the reactions were consistent, we performed PCR reactions at least twice. Only one of these reactions was included for final densitometric analysis, and the selection was arbitrary.
All reactions included a negative control RT(-) (cDNA made in the absence of reverse transcriptase). The identity of the amplification was confirmed by determination of the molecular size on agarose gel electrophoresis (1.6% agarose in buffer containing 40 mmol/l of Tris-acetate plus 1 mmol/l EDTA) and visualized by ethidium bromide staining (0.5 µg/ml) under ultraviolet light. The computer software Sigma Gel version 1.1 was used (Jandel Scientific) for densitometric analysis of the bands. Band density was calculated as densitometric mean values of CLC-2 compared with the mean of GAPDH.
CLC-2 Protein Expression by Western Analysis
To confirm that antisense ODN transfection inhibited not only CLC-2 mRNA but also protein expression, we performed Western analysis. Lung homogenates from explants transfected for 48 h were prepared in 250 mM sucrose-5 mM Tris-1 mM EDTA (STE) buffer with Complete Mini EDTA-free Protease Inhibitor Cocktail (Boehringer Mannheim). For each condition (antisense- vs. mock-transfected controls), approximately six fetal lungs were homogenized in 300 µl of STE buffer with a Polytron homogenizer (Brinkman Instruments). The homogenates were gently mixed for 1 h while maintained at 4°C and centrifuged at 10,000 g for 10 min, and the supernatant was removed for analysis.
Equivalent amounts of total protein (20 µg) from antisense and control homogenates were separated on an 8% SDS-PAGE gel. Proteins were transferred to nitrocellulose and immunoblotted with whole chicken serum CLC-2 COOH terminus antibody at a 1:3,000 dilution (31). Analysis of -actin, using monoclonal anti-
-actin mouse ascites fluid (Sigma Immunochemicals), on the same immunoblots, served as a measure of equivalent loading of samples. Band density was quantified on MacBas.
Immunolocalization of CLC-2 in Explants
Explants prepared, transfected, and incubated for 48 h as described above were washed three times in phosphate-buffered saline (PBS) and fixed in 4% paraformaldehyde in PBS, blocked in paraffin, and sectioned at 10 µm.
Sections on slides were deparaffinized, hydrated, and mordanted in hot citrate buffer 30 min, cooled to room temperature, and blocked in 5% donkey serum, 0.3% Triton X in PBS for 1 h. Sections were exposed to chicken anti-CLC-2 (31) at 1:100 in block solution for 1 h at room temperature and washed five times in PBS. Second antibody was tetramethylrhodamine isothiocyanate-conjugated rabbit anti-chicken IgG (Sigma) at 1:1,000 in PBS was added for 1 h at RT. After five washes in PBS, Hoechst 33342 counterstain at 1:30,000 was added for 10 min at room temperature, and the sections were washed again five times in PBS and mounted in Fluoromount G (Southern Biotechnical Associates).
Sections were viewed under a Zeiss LSM410 scanning confocal microscope (Facility for Confocal Microscopy, Department of Physiology, University of Maryland Baltimore) for emission at 568 nm (rhodamine) and 364 nm (Hoechst 33342) and saved on a Zeiss LSM-PC Image processing system.
Measurement of Lung Cyst Transepithelial PD
Explants to be used for microelectrode studies were prepared as above, with the added step of mounting the fetal lung in Matrigel on a glass coverslip before transfection. The lungs were examined after incubation for 48 h in the transfection media. The lung on the coverslip was mounted in an open chamber and perfused with a solution containing (in mM): 145 NaCl, 1 CaCl2, 1 MgCl2, 5 glucose, 60 sucrose, and 5 HEPES. The microelectrode studies were performed at 21°C. Glass microelectrodes (resistance 1050 M
) were filled with 3 M KCl, placed in a microelectrode holder containing a Ag-AgCl pellet, and advanced under direct observation using a dissection microscope (Olympus, Melville, NY) into a fetal lung cyst with a three-dimensional hydraulic micromanipulator (Newport). Transepithelial voltage (Vt) was measured through a high-input impedance electrometer (725B; Warner, Hamden, CT) with respect to a bath electrode ground (a stable flowing 3 M KCl junction connected to a Ag-AgCl half cell) and recorded on a strip chart recorder (model TA 240; Gould, Valleyview, OH). Impalements were considered acceptable if: 1) there was an abrupt negative deflection of the electrical PD when entering the cyst, 2) the PD reached a stable plateau that was maintained for at least 1 min, and 3) there was an abrupt return to baseline on withdrawal from the cyst. On each experimental day, at least three recordings were made for each condition, and all three conditions were tested.
Statistical Analysis
These studies were approved by the Institutional Animal Care and Use Committee at the University of Maryland, Baltimore. The data are given as means ± SE of n observations. Comparisons between mean values of treated and control explants were determined by one-way analysis of variance and the significance of the difference between means by t-tests. Newman-Keuls adjustment was used in multiple comparison of group means of outcomes for RNA data, and Tukey-Kramer adjustment was used in multiple comparison of group means for PD data. Statistical significance was determined for P < 0.05.
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RESULTS |
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Transfection with CLC-2 antisense ODN resulted in marked changes in lung morphology over 48 h (Fig. 1). Under control conditions the fetal lungs developed fluid-filled cysts by 24 h that further enlarged by 48 h similar to previous investigators (36, 40). In the presence of antisense CLC-2 ODNs, the fetal lungs continued to differentiate, but cyst lumen size was markedly reduced. In contrast, in the presence of the randomized control ODN, cyst size was comparable to the mock-transfected control.
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Quantitation of lung cyst size was accomplished by measurement of cyst lumen diameter in treated and untreated conditions at 0, 24, and 48 h. At 24 h, lung cysts increased 2.9, 1.8, and 2.3 times baseline in mock control, antisense-, and nonsense-transfected cysts (P < 0.01 antisense vs. nonsense and control). By 48 h, cyst diameter increased from baseline 4.9-fold in mock-transfected control cysts and 3.2-fold in random ODN-transfected controls compared with only 2.3 times in antisense CLC-2-treated cysts (P < 0.01 antisense vs. nonsense and control, Fig. 2, n = 46 cysts each condition from six separate experiments). Thus antisense treatment of CLC-2 interrupts lung cyst expansion, which presumably reflects a decrease in fluid production.
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Inhibition of CLC-2 mRNA Expression
To confirm that antisense ODNs inhibited CLC-2 mRNA expression, we used semiquantitative PCR. Amplification of CLC-2 cDNA from antisense and control explants yielded the expected 615-base pair product. Amplification of the internal control, GAPDH, yielded an expected 211-base pair product. Mean densitometric measurements for CLC-2 compared with GAPDH demonstrated that transfection with antisense CLC-2 ODN had the expected result and interrupted CLC-2 mRNA transcript expression (Fig. 3) compared with mock- and nonsense-transfected controls (P < 0.01 antisense vs. mock; P < 0.01 antisense vs. nonsense; P > 0.05 mock vs. nonsense, n = 4 separate experiments).
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Antisense CLC-2 ODNs Reduce Protein Expression
To investigate whether arrest of explant lung cyst development was also associated with interruption of CLC-2 protein expression, we performed Western analysis of lung homogenates collected 48 h after transfection. Total protein homogenates from explants cultured for 48 h in the presence or absence of CLC-2 antisense ODN were analyzed for both immunoreaction to a polyclonal CLC-2 antibody and -actin to normalize for variation in loading. We detected a reduction in a 94-kDa band using the CLC-2 antibody in antisense-treated explants compared with the 25-kDa band detected with the
-actin monoclonal antibody (Fig. 4). Quantification of CLC-2/
-actin band intensity for antisense- and control-treated explants demonstrated that this reduction is significant (P = 0.03 by t-test, n = 5 experiments).
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Antisense Inhibition of Apical CLC-2 Staining
CLC-2 expression was also examined in fetal lung explants by immunocytochemistry. Lung cysts that were treated with antisense CLC-2 oligos were smaller than controls. Immunostaining with CLC-2 polyclonal antibody demonstrated predominant localization in the apical compartment of the cyst epithelium (Fig. 5A). This immunolocalization was reduced in antisense-treated cysts (Fig. 5B). Localization of CLC-2 protein along the apical surface of the cysts supports the idea that this Cl- channel could play a role in the secretion of Cl- and fluid into the developing lung lumen.
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Inhibition of Vt
We also measured the impact of CLC-2 knockout on the transepithelial PD across the fluid-filled cysts to determine whether the decrease in lung cyst development was the result of inhibition of CLC-2 Cl- transport. A total of 38 recordings were made in mock-transfected control cysts, 32 in antisense ODN-treated cysts, and 30 in random ODN-treated control cysts. Figure 6 shows the mean results of these measurements. In mock-transfected explants, the PD recorded was -3.74 ± 0.3 mV (mean + SE). In lungs treated with the CLC-2 antisense ODN, the transepithelial PD was reduced to -1.49 ± 0.2 mV (mean + SE), which was significantly less than the mock-transfected control (Vt = -1.49 vs. -3.74 P < 0.0001). Finally, in lungs treated with the random ODN, the PD was -3.36 ± 0.3 mV (mean + SE), which was significantly greater than lungs treated with the antisense ODN (Vt = -1.49 vs. -3.36, P < 0.0001) but not significantly different from the untreated control (P > 0.05). Thus blocking the expression of CLC-2 resulted in a reduction of ion movement across the cyst membrane. Because treating the lungs with the random control ODN had no effect on transepithelial PD, the negative charge is likely carried by Cl- through CLC-2 Cl- channels. We conclude that Cl- movement through CLC-2 channels is an important contributor to fluid secretion in the developing lung.
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DISCUSSION |
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The airway epithelium is a complex system with multiple ion transport mechanisms necessary for lung development. Cl- transport across the basolateral surface of the airway epithelial cell is linked to Na+ and K+ via the Na+-K+-2Cl- cotransporter (NKCC1). The Na+-K+-ATPase on the basolateral surface of the airway epithelium exchanges Na+ for K+. The Cl- gradient generated by this energy-dependent process leads to passive secretion of Cl- across the apical surface of the cell. The best-studied Cl- channel, the CFTR, is expressed in the fetal lung (28) and is developmentally regulated (29). In the postnatal lung, CFTR is essential for host defense and, when defective, is responsible for chronic respiratory infections, lung destruction, and shortened lifespan. Although cAMP-mediated CFTR Cl- secretion is dysfunctional in utero (27, 28), its role in the developing lung is not crucial, because individuals with cystic fibrosis have normal lung morphogenesis (41). In addition, CFTR knockout mice have Cl- and fluid secretory pathways that can compensate for defective CFTR (10, 11). These non-CFTR Cl- secretory pathways (46, 22) may contribute to fetal lung development; however, the molecular identity of such fetal Cl- channels has not been elucidated.
In earlier studies, we have shown that acidic pH-activated Cl- channels in the fetal lung exist (8) that can be inhibited by the CLC-2 channel blocker cadmium chloride (38). In addition, CLC-2 is gestationally regulated and is predominantly expressed in fetal lung compared with the postnatal lung (30, 31). The expression of CLC-2 on the luminal surface of airway epithelia is consistent with a role in Cl- and fluid secretion in the developing lung. Keratinocyte growth factor induces CFTR-independent fluid accumulation in fetal lung explants (45) and is essential to CLC-2-like pH-sensitive Cl- secretion in fetal airway epithelia (8). We demonstrate here that CLC-2 Cl- secretion contributes to lung cyst expansion in a fetal lung explant model. The 14-day gestation fetal rat lung explant has a distal epithelial cell layer that has characteristics of type II cells (26). Using antisense ODN strategies similar to other investigators (18), we successfully inhibited CLC-2 mRNA and protein expression in the developing lung. Apical expression of CLC-2 in the epithelial cells of the explant cysts supports the potential role of CLC-2 in the developing lung. This CLC-2 knockout model led to a reduction in transepithelial PD and arrest of lung cyst development, suggesting that CLC-2 contributes to fetal lung fluid production.
On the basis of the findings of this study, we believe that CLC-2 may be an important fetal lung Cl- channel. Knockout of the CLC-2 channel in this explant model leads to inhibition of lung cyst development, which is linked to decreased Vt. CLC-2 knockout, however, may be insufficient to lead to complete lung dysgenesis, since the CLC-2 knockout mouse has no obvious respiratory distress at birth (9). Similarly, although the importance of NKCC1 for Cl- entry through the basolateral membrane of the fetal lung has been demonstrated, NKCC1 knockout mice are born with apparently normal lung function and have no detectable respiratory problems well into adulthood (17). There is likely redundancy to fetal ion and fluid transport as demonstrated in several knockout models, such that additional channels may compensate when CLC-2, CFTR, or NKCC1 is defective. Future studies using double knockout models may be able to address the contributions of these airway epithelial Cl- channels to lung development. In addition, knockout mice may inadequately model the consequences of ion transport abnormalities in the human (10, 13, 17, 33) and should not inhibit further pursuit of the pathogenesis of ion and fluid transport in human disease.
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ACKNOWLEDGMENTS |
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This work was funded by National Heart, Lung, and Blood Institute Grant K08-HL-03469 (C. J. Blaisdell).
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FOOTNOTES |
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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.
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REFERENCES |
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