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ENaC and Nedd4-2 in the conversion from lung fluid secretion to fluid absorption at birth in the rat as assayed by RNA interference analysisDepartment of Physiology and Pharmacology, Northeastern Ohio Universities College of Medicine, Rootstown, Ohio
Submitted 16 April 2007 ; accepted in final form 7 August 2007
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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ENaC and Nedd4-2. siRNA-generating plasmid DNA (pDNA) was administered via trans-thoracic intrapulmonary (ttip) injection 24 h before ENaC and Nedd4-2 expression, extravascular lung water, and mortality were measured. ENaC mRNA and protein were specifically reduced by 
65% after pSi-4 injection. Nedd4-2 mRNA and protein were reduced by 60% after pSi-N1 injection. Interestingly, ENaC and 
ENaC mRNA and protein expression were increased after Nedd4-2 silencing. Extravascular lung water was significantly increased after ENaC silencing and reduced after Nedd4-2 silencing. ENaC silencing resulted in a fourfold increase in newborn mortality, whereas silencing Nedd4-2 did not affect mortality. We also isolated distal lung epithelial (DLE) cells after in vivo ENaC or Nedd4-2 silencing and measured ENaC or Nedd4-2 expression in freshly isolated DLE cells. In these DLE cells, there were attenuated ENaC or Nedd4-2 mRNA and protein, thus demonstrating that ENaC and Nedd4-2 silencing occurred in alveolar epithelial cells after ttip injection. We also looked for pDNA by PCR to determine pDNA presence in the lungs and found strong evidence for pDNA presence in both lungs. Thus we provide evidence that ENaC and Nedd4-2 are involved in the transition from lung fluid secretion to fluid absorption near term and at birth. distal lung epithelium; ion transport; lung development; newborn; ubiquitination
ENaC membrane expression is regulated by steroid hormones; in the lung, glucocorticoids increase transepithelial Na+ absorption primarily by increasing ENaC presence at the cell surface (18, 36). Experimental data suggest a role for neural precursor expressed, developmentally downregulated protein 4-2 (Nedd4-2) in this regulation. Studies focused on Nedd4-2 interactions with ENaC have shown that Nedd4-2 is phosphorylated by the serum and glucocorticoid-regulated kinase (SGK; see Refs. 13 and 47); SGK is induced by cortisol and a key mediator of ENaC regulation downstream from steroid hormones (14, 23). Nedd4-2 phosphorylation inhibits Nedd4-2 and decreases its ENaC binding and thus reduces Nedd4-2-mediated ENaC inhibition (13, 47). In summary, this suggests that steroid hormones may regulate transepithelial Na+ transport via negative Nedd4-2 regulation. Thus reduced cytoplasmic Nedd4-2 levels may prove beneficial in stimulating fetal lung fluid absorption at term and at preterm delivery.
ENaC function has been assessed in ENaC-deficient (ENaC–/–; see Refs. 4, 24, and 37) mice, but these results may be confounded by various in vivo compensatory mechanisms. In the present study, our first aim was to adapt and use our recently developed RNA interference (RNAi) technique (34) to silence ENaC in newborn rats by trans-thoracic intrapulmonary (ttip) injection and to explore functional in vivo responses to ENaC silencing. Our second aim was to study what silencing of Nedd4-2 would do to ENaC expression and function in newborn rats. Thus, by determining protein and mRNA expression, as well as functional endpoints such as extravascular lung water, and mortality after ENaC and/or Nedd4-2 silencing, we examined the roles of ENaC and its interactions with Nedd4-2 at the conversion of the lung from fluid secretion to fluid absorption.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Timed-pregnant Sprague-Dawley rats (wt 200–250 g, n = 27; Charles River, Wilmington, MA) were used in the study. The rats were housed separately in their cages in a temperature- and humidity-controlled environment (20 ± 2°C and 55 ± 10% relative humidity). The rats were kept at a 12:12-h day-night rhythm and had free access to standard rat chow (Purina, Copley Feed, Copley, OH) and tap water. All studies were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) at the Northeastern Ohio Universities College of Medicine (Rootstown, OH).
Plasmid Construction
Small-interfering RNA (siRNA) generating plasmid DNA (pDNA) was constructed using a commercial plasmid (pSilencer 3.0-H1; Ambion, Austin, TX) with standard techniques (43). Recombinants were sequenced (CEQ 2000XL; Beckman, Palo Alto, CA) to verify correct oligonucleotide frames and sequences. pDNA was amplified in Escherichia coli DH5 and purified using the Wizard PureFection pDNA Purification System (Promega, Madison, WI). This pDNA isolation kit has a specific resin-binding procedure to remove endotoxin from the pDNA. After isolation and purification, pDNA concentration and purity (ratio 1.7–1.8) was measured at 260/280 nm, and samples were stored at -80°C.
Rat ENaC mRNA (GenBank accession no.: NM_031548) secondary folding structure was predicted based on the principle of minimizing free energy, using the RNA structure v. 3.71 software (34). In our earlier study, we generated four pDNA constructs named pSi-1 to pSi-4 for ENaC silencing. Pilot studies demonstrated that pSi-4 was the most effective pDNA construct (34) and was thus selected for these studies. The sequence corresponds to rat ENaC cDNA nucleotide positions 1617–1635. The target was specific for rat ENaC and did not match other GenBank sequences. For construction of siRNA-generating pDNA, two complementary oligonucleotides (forward and reverse) containing a sense strand, followed by a short spacer (5'-TTCAAGAGA-3'), an antisense strand, and a RNA polymerase III termination signal (5'-TTTTTTGGAAA-3') were synthesized, annealed, and ligated into pSilencer 3.0-H1. Synthesized oligonucleotides with BamHI and HindIII overhangs were as follows: pSi-4, forward: 5'-GATCCGTTACACTATTAACAACAAATTCAAGAGATTTGTTGTTAATAGTGTAATTTTTTGGAAA-3'; pSi-4, reverse: 5-AGCTTTTCCAAAAAATTACACTATTAACAACAAATCTCTTGAATTTGTTGTTAATAGTGTAACG-3'. As a negative control, we used a nonsilencing sequence, 5'-GATCCGTTACACTTTTTTGGAAA-3' (scramble, which does not correspond to any known transcript) with BamHI and HindIII overhangs, inserted in pSilencer 3.0-H1, named pSi-0.
The same strategy was used to calculate rat Nedd4-2 mRNA (GenBank accession no.: XM_001064485) secondary folding structure. For candidate siRNA-generating pDNA construction, two complementary oligonucleotides (forward and reverse) containing a sense strand, followed by a short spacer and an antisense strand were synthesized, annealed, and ligated into the pSilencer 3.0-H1 plasmid. The sequences correspond to rat Nedd4-2 cDNA nucleotide positions 166–186 (pSi-N1) and 1384–1404 (pSi-N2). The targets were specific for rat Nedd4-2 and did not match other GenBank sequences. The sequences for the candidate Nedd4-2 oligonucleotides with BamHI and HindIII overhangs were as follows: pSi-N1, forward: 5'-GATCCGACAATTAAAAAGACGCTGTTCAAGAGACAGCGTCTTTTTAATTGTCTTTTTTGGAAA-3'; pSi-N1, reverse: 5'-AGCTTTTCCAAAAAAGACAATTAAAAAGACGCTGTCTCTTGAACAGCGTCTTTTTAATTGTCG-3'; pSi-N2, forward: 5'-GATCCACCACAACACAAAGTCACATTCAAGAGATGTGACTTTGTGTTGTGGTTTTTTTGGAAA-3'; pSi-N2, reverse: 5'-AGCTTTTCCAAAAAAACCACAACACAAAGTCACATCTC- TTGAATGTGACTTTGTGTTGTGGTG-3'. As the negative control, we used the same nonsilencing sequence as used for the pSi-4 silencing studies of ENaC (pSi-0).
Solutions
After measuring pDNA concentration, the pDNA solution was either concentrated by a vacuum centrifuge (SVC100H; Savant Instrument, Farmingdale, NY) or diluted with sterile deionized water to the required concentration. pDNA pretreatment solution osmolality was measured by a Vapor Pressure Osmometer 5500 (Wescor, Logan, UT) and, if needed, adjusted with sterile NaCl or deionized water to 100 mosmol/kgH2O. The pDNA pretreatment solution was freshly prepared by mixing pDNA with Lipofectamine 2000 (Invitrogen, Carlsbad, CA) under optimized transfection conditions (32): pDNA (µg)-Lipofectamine 2000 (µl) ratio 1:1, generating a pretreatment solution with the concentration 4 µg/g body wt in a final volume of 40 µl/g body wt for each newborn rat.
pDNA Delivery
Timed-pregnant rats were observed for signs of labor and delivery. Newborn rats were removed from the dams within 1 h after birth. Freshly prepared pDNA/Lipofectamine solution was then delivered trans-thoracically via the right pleural cavity to both lungs using a 30-gauge needle. The general success rate for this procedure measured as rats surviving >1 h after the ttip pDNA delivery was 85 ± 6% (15 of 100 newborn rats died within 1 h after the ttip injection). Newborn rats were placed on a 37°C temperature-controlled pad after pDNA injection. The newborn rats were then allowed to recover in cages with their respective dams where they remained for the 24-h study.
Specific Protocols
All newborn rats were divided into the following six groups. All groups contained rat pups randomly assigned for the various treatments from at least 3 litters each. Lungs and kidneys were collected from the rats that survived the 24-h study period. Rats that died within the 24-h study period were excluded from the mRNA and protein expression studies and extravascular lung water measurements.
Pilot studies.
The pDNA sequence for ENaC silencing (pSi-4) was selected based on the results in the prior studies (34). For selecting the best siRNA-generating pDNA sequence for Nedd4-2, we generated two candidate pDNAs, pSi-N1 and pSi-N2 (see Plasmid Construction) and injected six newborn rats with each construct. After all analyses (RT-PCR of Nedd4-2 mRNA expression) were carried out on these rats (Fig. 1), it was determined that pSi-N1 had the best overall silencing efficiency (60%) and was used for the remaining studies. pSi-N2, however, had some silencing effect (30%) on Nedd4-2. Both constructs demonstrated the increased ENaC expression observed after Nedd4-2 silencing (Fig. 1).
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Control siRNA pretreatment. Newborn rats were injected with irrelevant pDNA (pSi-0, n = 86) 24–48 h before the extravascular lung water, mortality, RT-PCR, and Western blot studies.
ENaC siRNA pretreatment.
Newborn rats were injected with ENaC siRNA-generating pDNA (pSi-4, n = 62) 24–48 h before the extravascular lung water, mortality, RT-PCR, and Western blot studies.
Nedd4-2 siRNA pretreatment. Newborn rats were injected with Nedd4-2 siRNA-generating pDNA (pSi-N1, n = 18) 24 h before the extravascular lung water, mortality, RT-PCR, and Western blot studies.
ENaC ± Nedd4-2 siRNA pretreatment.
Newborn rats were injected with ENaC siRNA-generating pDNA and Nedd4-2 siRNA-generating pDNA (n = 6) simultaneously 24 h before RT-PCR and Western blot studies.
Distal Lung Epithelial Cell Isolation and RNAi Localization
Distal lung epithelial (DLE) cells were isolated from newborn rats (27) injected with pSi-0 (N = 6), pSi-4 (N = 6), and pSi-N1 (N = 6) for 24 h. Lungs and hearts were excised en bloc (heart removed and discarded) immediately after decapitation. Blood was collected. Lungs from each litter were pooled, rinsed two times in ice-cold Hanks' balanced salt solution (HBSS; without Mg2+ and Ca2+), and minced to 1 mm3. Lung tissue was digested in HBSS containing 0.125% trypsin (Mediatech, Herndon, VA) and 25 µg/ml DNase I (MP Biochemicals, Aurora, OH) 20 min at 37°C. After 20 min, collagenase (USB, Cleveland, OH) and additional DNase I were added to final concentrations of 0.1% and 50 µg/ml, respectively, and digestion was continued for 20 min. Enzymes were neutralized by adding 2 ml FBS (Atlanta Biologicals, Lawrenceville, GA) at +4°C. Cell suspensions were transferred to new tubes by tituration to break up cell clumps. Dispersed cell solutions were filtered through 100-µm cell strainers (Becton Dickinson Labware, Franklin Lakes, NJ) and then through 70-µm cell strainers. DLE cells were collected by centrifugation (420 g; 6 min) and resuspended in 15 ml DMEM-F-12 (50:50; Cellgro, Herndon, VA). DLE cells were purified by differential adherence steps. Cells were plated 2 x 30 min to remove fibroblasts. Cell yield was determined by a Beckman Coulter Z1 Coulter particle counter. For RNAi localization, the cells were collected immediately after isolation by centrifugation 5 min at 1,000 g and snap-frozen in liquid N2 for downstream PCR analyses.
PCR
A PCR was used to detect pDNA in lung tissue after in vivo pSi-0 transfection. Template DNA was isolated from lung tissue using a DNA isolation kit (Promega). A pair of primers was synthesized targeting a pSi-0-specific sequence: pSi-0+, 5'-CACTCGGATCCGTTACACTT-3' and pSi-0–, 5'-TAGTCCTGTCGGGTTTCG-3'. PCR was done using a PCR Master Mix kit (Promega). Reaction volume was 25 µl, with 50 ng of template DNA and 0.1 µM of each primer were added under optimized conditions: 95°C for 30 s, 55°C for 30 s, and 72°C for 1.5 min for 30 cycles and a final extension for 5 min at 72°C. PCR amplification would yield a 127-bp pSi-0-specific fragment. PCR products were resolved in 1.5% agarose gel containing 1 µg/ml ethidium bromide. Gels were scanned by a Typhoon 8610 scanner (Molecular Dynamics).
RT-PCR
Total RNA was extracted from lung and kidney tissue using a Versagene RNA isolation kit from Gentra (Minneapolis, MN). RNA yield and purity was determined spectrophotometrically at 260/280 nm, and RNA integrity was verified by agarose gel electrophoresis. A competitive RT-PCR was carried out using the One-Step RT-PCR kit (EMD Biosciences, San Diego, CA). Total volume of reaction was 25 µl, containing 50 ng of total RNA, 1x PCR buffer, 0.2 mM of each dNTP, 2.5 mM MgSO4, 0.1 µM of each primer, and 1.5 units recombinant Thermus thermophilus DNA polymerase. RT-PCR conditions were optimized as follows: 60°C for 30 min for reverse transcription followed by 40 cycles at 94°C for 45 s, 60°C for 2 min, and final extension for 7 min at 60°C. We tested in preliminary experiments 30 and 40 amplification cycles for pSi-4 and pSi-N1. We elected to use 40 cycles after a careful initial analysis of outcome vs. number of cycles and because we found that this amplification generated repeatable results for both pSi-4 and pSi-N1 and the control genes. Five primer pairs (+, sense; –, antisense) were derived from GenBank sequences and synthesized for competitive RT-PCR: ENaC (NM_031548
[GenBank]
), ENa+: 5'-CATGATGTACTGGCAGTTCGC-3' (731–751), ENa–: 5'-TCCCTTGGGCTTAGGGTAGAAG-3' (1751–1772); ENaC (NM_012648
[GenBank]
), ENb+: 5'-CCGTGACTGAGTGGTACATCC-3' (690–710), ENb–: 5'-CTCGTGGAGCATCAGCCTAG-3' (996–1015); Nedd4-2 (XM_001064485), Ned+: 5'-TGCTAAGGGACGCACATACT-3' (1107–1126), Ned–: 5'-TGTAGTTGTCCGTGGCAGAG-3' (1974–1993); 1-Na-K-ATPase (NM_012504
[GenBank]
), NaK+: 5'-TGGCTGTTTCTCCTATTATCA-3' (429–449), NaK–: 5'-TTCTTCACCAGGCAGTTCTT-3' (1060–1079); glyceraldehydes-3-phosphate dehydrogenase (GAPDH; NM_017008
[GenBank]
), GAPD+: 5'-ACCACAGTCCATGCCATCAC-3' (1369–1388), GAPD–: 5'-TCCACCACCCTGTTGCTGTA-3' (1801–1820). Amplification of this competitive RT-PCR yields a 1,042-bp ENaC fragment, a 326-bp ENaC fragment, a 887-bp Nedd4-2 fragment, a 651-bp 1-Na-K-ATPase fragment, and a 452-bp GAPDH fragment (internal control). RT-PCR products were resolved in 1.5% agarose gels stained with 1 µg/ml ethidium bromide. Gels were scanned by a Typhoon 8610 Scanner. Densitometric analysis was done using TotalLab software (Nonlinear Dynamics, Newcastle upon Tyne, UK).
Western Blot
Lung tissue from newborn rats was homogenized in T-Per Reagent (Pierce, Rockford, IL) containing protease inhibitors, aprotinin (30 µg/ml; Sigma, St. Louis, MO), and leupeptin (1 µg/ml; Sigma), with a homogenizer (Tissue Tearor) on ice. The homogenate was centrifuged at 13,000 g for 5 min at +4°C. Supernatant (membrane and cytosol) was collected, separated into aliquots in multiple vials, and snap-frozen in liquid nitrogen. One vial was used for determining total protein concentration of the sample to ensure equal loading of the electrophoresis gel. Aliquots were stored at -80°C until analyzed.
PAGE and transfer to nitrocellulose membranes (Pierce) were carried out using standard protocols. After the PAGE and transfer, the nitrocellulose membranes were blocked [SuperBlock Dry Blend blocking buffer in Tris-buffered saline (TBS); Pierce] for 1 h at room temperature. After blocking, membranes were incubated with primary antibodies on an orbital shaker overnight at +4°C.
ENaC
Primary ENaC and ENaC antibodies were purchased from Alpha Diagnostics International (San Antonio, TX), directed against NH2-termini of ENaC and ENaC, and used at a 1:1,000 dilution. The antibodies recognize membrane proteins of appropriate sizes (85–95 kDa) in rats. After incubation, membranes were washed 5 x 10 min with wash buffer (pH = 7.5; TBS with 0.1% Tween 20). Membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:1,000 dilution; goat anti-rabbit IgG) for 1 h at room temperature. After incubation, membranes were washed again. Substrate solution (SuperSignal West Femto; Pierce) was added and incubated for 5 min. The luminescence signal was detected using a Kodak image analyzer and densitometrically analyzed using TotalLab software.
Nedd4-2
Anti-Nedd4 antibody was obtained from Cell Signaling Technology (Danvers, MA), directed against a synthetic peptide obtained from human Nedd4, and used at a 1:1,000 dilution. The antibody detects endogenous levels of Nedd4 and also recognizes Nedd4-2 of rat origin. After incubation, the membrane was washed as above and incubated with HRP-conjugated secondary antibodies (1:1,000) for 1 h at room temperature. Next, substrate solution (SuperSignal West Femto) was added to the blot and incubated for 5 min. Luminescence was detected and densitometrically analyzed as above.
Na-K-ATPase
Anti-Na-K-ATPase antibodies were obtained from Upstate Cell Signaling Solutions (Waltham, MA), directed against residues 338–518 of the 1-subunit of the Na-K-ATPase, and used at a 1:1,000 dilution. These antibodies specifically recognize a membrane protein of appropriate size (95 kDa for the 1-subunit) in rats. A rat heart microsomal protein preparation was always run on the gel as a positive control. After incubation, the membrane was washed as above and incubated with HRP-conjugated secondary antibodies (1:1,000) for 1 h at room temperature. Next, substrate solution (SuperSignal West Femto) was added to the blot and incubated for 5 min. Luminescence was detected and densitometrically analyzed as above.
GAPDH
We used a rabbit monoclonal anti-GAPDH antibody (dilution 1:1,000) from Cell Signaling Technology that detects endogenous levels of GAPDH of rat origin as loading and transfer control. After incubation, membranes were washed and incubated with HRP-conjugated secondary antibodies (1:1,000) as above. Next, substrate solution (SuperSignal West Femto) was added to the blot, and luminescence was detected and densitometrically analyzed as above.
Extravascular Lung Water
To measure extravascular lung water in newborn rat lungs, we modified the original method described previously (45). Extravascular lung water was measured in untreated (n = 20), pSi-0-injected (n = 10), pSi-4-injected (n = 15), and pSi-N1-injected (n = 8) newborn rats from three to four litters each. The lungs were excised rapidly (hearts were removed) and placed in preweighed sample tubes and reweighed. Water (250 µl) was added, and lungs were weighed again and homogenized using the Tissue Tearor. If extravascular lung water determinations were not done on the day of lung harvest, collected lungs were weighed, water (250 µl) was added, and lungs were reweighed and stored frozen at -20°C until analysis. Parts of lung homogenates were centrifuged for 5 min at 14,000 g. Blood was collected from a small number of newborn rats after decapitation to obtain a hemoglobin (Hb) value for newborn rat blood. Hb content was measured on supernatants obtained after centrifugation of the lung homogenates, and blood volumes of newborn rat lungs were calculated from homogenate supernatant Hb concentration relative to blood Hb concentration. Newborn rat blood wet-dry weight was determined. Lung wet-to-dry weights were corrected for blood volume. Drying of lung homogenates, lung homogenate supernatants, and newborn rat blood was carried out using a moisture analyzer (Sartorius, Edgewood, NY) that continuously recorded water loss as samples dried. Each sample was dried at 80–120°C until dry weights reached stability. Typically, this procedure required 15 min/sample. Nonspecific water loss of wet samples and nonspecific rehumidification of dried samples, as may occur when small samples are measured by traditional extravascular lung water techniques, were prevented in this analysis. We verified the technique by comparing it with traditional techniques (45) in adult rat lungs. We found an excellent correlation in extravascular lung water between both techniques (data not shown).
Statistics
All data are presented as means ± SD. Data were analyzed with one-way ANOVA with Tukey's test as post hoc or Student's t-test as appropriate. A G-test of independence followed by a Chi square analysis for individual group comparisons was used for the mortality data analysis (48). Differences were considered significant when P < 0.05.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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ENaC, ENaC, 1-Na-K-ATPase, and Nedd4-2 Expression After ENaC and Nedd4-2 Silencing
Because we knew from earlier studies (17, 19) that ENaC remains elevated in the newborn rat lung for >24 h after birth, we elected to carry out our studies in newborn rats (within 1 h from actual delivery). Thus we investigated if pSi-4 (siRNA-generating pDNA for ENaC) would silence ENaC and affect Nedd4-2 and ENaC expression in newborn rats. After ttip pSi-4 injection (24 h), as shown in Fig. 2 and Fig. 3, ENaC mRNA and protein in the total lung extracts were both decreased by 65%; pSi-4 ttip injection did not affect Nedd4-2 expression or ENaC expression in the total lung extract.
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ENaC and ENaC expression in the total lung extract. The ttip injection of pSi-N1 (siRNA-generating pDNA for Nedd4-2) increased ENaC (120%) and ENaC (80%) mRNA expression (Fig. 2) and reduced Nedd4-2 mRNA expression by 60% (Fig. 2). Similarly, ttip pSi-N1 injection decreased Nedd4-2 protein expression by 50% (Fig. 3). Moreover, ENaC and ENaC protein expression were both increased by 65% and 30%, respectively, after ttip pSi-N1 injection (Fig. 3). GAPDH mRNA and protein expression, which were used as loading and transfer controls, remained unchanged between all experimental conditions (Figs. 2 and 3).
We also carried out confirmatory studies in total lung extracts where ENaC and Nedd4-2 were silenced simultaneously and mRNA expression was measured by RT-PCR to determine if silencing ENaC interfered with Nedd4-2 silencing or vice versa. In these confirmatory studies, the combined pSi-4 and pSi-N1 ttip injection resulted in ENaC and Nedd4-2 gene silencing, reduced by 65% and 55%, respectively, not different from that when pSi-4 or pSi-N1 were given alone to the newborn rats.
We also examined whether the altered ENaC expression affected 1-Na-K-ATPase mRNA expression in the total lung extracts and found no changes in 1-Na-K-ATPase mRNA (Fig. 4A) or protein (Fig. 4B) expression between pSi-0-, pSi-4-, and pSi-N1-injected rats.
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ENaC, ENaC, and Nedd4-2 mRNA expression. There were no differences in mRNA expression of either ENaC subunit or Nedd4-2 in the kidney following silencing of ENaC and/or Nedd4-2 (Fig. 5).
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Extravascular lung water is a good measure of excess lung water (i.e., pulmonary edema or presence of fetal lung fluid in the airspaces; see Ref. 36), and thus we measured extravascular lung water as the principal functional physiological endpoint from ENaC and Nedd4-2 silencing in vivo. pSi-0-injected newborn rats displayed the same extravascular lung water as normal, untreated newborn rats (Fig. 6). Extravascular lung water in ENaC-silenced newborn rat lungs was significantly increased after ttip siRNA-generating pDNA injection (Fig. 6). In contrast, extravascular lung water in Nedd4-2-silenced newborn rat lungs was significantly decreased below the extravascular lung water content in the pSi-0-injected or untreated control newborn rat lungs (Fig. 6). For comparison, extravascular lung water in adult (n = 6) dry rat lungs was 3.91 ± 0.06 g water/g dry lung.
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ENaC and Nedd4-2 Silencing
Because ENaC knockout mice demonstrated a newborn mortality of 100% in mice within the first 40 h of newborn life (24), we also studied if ttip pSi-0, pSi-4, or pSi-N1 injection affected the mortality of newborn rats. Newborn rats that died within 1 h after ttip pDNA injection were excluded as injection-related abnormalities. When tabulated, newborn rats that died after >1 h demonstrated the following mortality: untreated control: 2 of 67 rats died; pSi-0: 4 of 108 rats died; pSi-4: 14 of 83 rats died (P < 0.05 compared with the other groups); and pSi-N1: 2 of 40 rats died. The data demonstrate mortality rates of <4% in untreated control rats and pSi-0-injected newborn rats, 17% in pSi-4-injected newborn rats, and 5% in pSi-N1-injected newborn rats.
Localization of RNAi Silencing
To investigate in vivo transfection ability of pDNA during these conditions, we detected lung pSi-0 pDNA existence by PCR. As shown in Fig. 7A, there was a single clear band representing pSi-0 in pSi-0-transfected lung tissue, and that band was of equal intensity in both right and left lung.
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ENaC. DLE cells were isolated from pSi-0-, pSi-4-, and pSi-N1-pretreated newborn rats. As can be seen in Fig. 7B, ENaC mRNA, as determined by RT-PCR, was absent in freshly isolated DLE cells after in vivo ttip pSi-4 injection but visible after in vivo ttip pSi-0 injection. Nedd4-2 mRNA was clearly decreased in freshly isolated DLE cells after in vivo ttip pSi-N1 injection but present after in vivo ttip pSi-0 injection (Fig. 7B). |
DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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ENaC siRNA-generating pDNA (pSi-4) increased extravascular lung water and mortality of newborn rats simultaneously with specifically decreasing ENaC mRNA and protein in vivo in newborn rat lungs. Second, Nedd4-2 silencing (pSi-N1) increased lung ENaC expression and decreased extravascular lung water in newborn rat lungs, thus suggesting that Nedd4-2 is involved in regulation of ENaC function and expression at the cell surface near term.
The majority of infants make the transition from intrauterine life to postnatal life without complications, but only hours before birth, lungs are filled with an essentially protein-free isosmolar solution that has been actively secreted by the lung epithelium. The normal rate of lung fluid absorption in newborn rats has been determined earlier (19). In that study, lung fluid absorption was not apparent before birth, reached a high rate immediately after birth, and decreased to the rate seen in adult rats by 40 h of newborn life. There was no amiloride-sensitivity before birth, thus suggesting that a rapid increase in ENaC expression and function took place at birth. The molecular mechanism responsible for perinatal lung fluid absorption has also been proposed to be ENaC dependent, since mice deficient in ENaC expression die within 40 h of birth from failure to clear the lungs from fetal lung fluid (24). mRNA for ENaC is found earliest at gestation day 19, whereas both - and 
-subunits are not expressed until at or after birth (50). This expression pattern agrees well with the observed amiloride sensitivity and function of fetal rat lungs at birth in the earlier study (19), especially since the ENaC channel requires all three subunits to become fully functional. Recent studies have demonstrated that failure in lung fluid absorption at birth may be associated with ENaC deficiency (3, 17, 39). In some cases, such as congenital diaphragmatic hernia (CDH), ENaC deficiency may have serious impact on the lung fluid absorption rate at birth and drastically affect the ability to oxygenate newborns and newborn survival (17). In our current study, silencing ENaC with pSi-4 was associated with elevated extravascular lung water and an increased mortality, thus strengthening the assumption of ENaC being important for the transition from fluid-filled fetal lungs to air-filled newborn lungs at birth. An earlier study in rats has reported that fetal lung fluid absorption was mediated by -adrenergic receptor (-AR) stimulation (19). However, the elevated lung fluid absorption in 22-day-gestation rat fetuses was only minimally amiloride sensitive and increased in amiloride sensitivity during the first 40 h of postnatal life (19). ENaC maintained a relatively high expression from birth to postnatal day 1 and subsequently reached adult levels at the fifth postnatal day, thus supporting the results of a rapid increase of an amiloride-sensitive lung fluid absorption immediately after birth in the rat (19).
In our earlier study (34), ENaC gene silencing in adult rat lungs was achieved by intrapulmonary siRNA generating pDNA pretreatment, where pDNA was delivered conjugated with liposomal complexes via intratracheal instillation. This method was originally developed by Folkesson and colleagues (21) and took advantage of the anatomic characteristics of the lung, which consists of a series of highly branched hollow tubes ending blindly in functional structures, i.e., alveoli. To deliver pDNA in the original study (34), we used a modification of the discoveries by Sawa and colleagues (44) where they demonstrated that intraluminal water instillation in the lung airspaces increased transfection efficiency. However, this instillation technique is not suitable for newborn rats because of their small size. It has been demonstrated that DNA can be delivered directly to skeletal muscle by intramuscular injection of "naked" pDNA, but gene transfer was, however, restricted to muscle cells adjacent to the route of injection (53). A more recent study by Bhargava and colleagues (7) demonstrated that site-specific transient gene knockdown can be achieved by local double-strand RNA hypothalamic injection. In our current study, we modified the siRNA delivery methods by developing a repeatable ttip injection technique using a pDNA (µg)-to-liposome (µl) ratio of 1:1 with a pDNA concentration of 4 µg/g body wt in a final volume of 40 µl/g body wt and with a low osmolality of 100 mosmol/kgH2O. Our results showed a reproducible specific siRNA-mediated ENaC silencing, similar in silencing efficiency of ENaC to what was observed in the earlier study in the adult rat lung (34), and Nedd4-2 silencing, 60–65% for ENaC and Nedd4-2 mRNA and protein in newborn rat lungs by this method. Moreover, our results confirmed that the siRNA-generating pDNA reached both lungs after the ttip injection procedure.
The fact that ttip injection of pSi-4 did not affect ENaC or 1-Na-K-ATPase expression indicated that the gene silencing of ENaC was specific in both ENaC subunit silencing and protein specificity of the siRNA-generating pDNA. The results from the combined pSi-4 and pSi-N1 administration further support that these effects were specific to the individual siRNA-generating pDNAs used. Thus, because pSi-4 silenced ENaC in the presence of pSi-N1 and pSi-N1 silenced Nedd4-2 in the presence of pSi-4, this strengthens our hypothesis that Nedd4-2 affects ENaC expression in the membrane by potentially increasing ENaC membrane presence.
As a functional endpoint, we evaluated changes in extravascular lung water and mortality following ttip pSi-4 and pSi-N1 injections. Extravascular lung water was increased significantly after ttip pSi-4 injection, clearly suggesting that there had been an impairment of lung fluid absorption following ENaC silencing. In contrast, in pSi-N1-injected rats, extravascular lung water was decreased significantly, even below the level in the untreated newborn control rats. Because Nedd4-2 is believed to be important in the ubiquitination process of proteins and ultimately leads to the degradation of ubiquitinated proteins, this strongly implies that silencing Nedd4-2 attenuated ENaC targeting for degradation and prolonged the membrane survival time of ENaC, enabling the lung to more effectively remove the fetal lung fluid than what was possible in the control rats. Moreover, ENaC silencing by ttip pSi-4 injection resulted in a fourfold increase in mortality, demonstrating the importance of ENaC for this process in vivo. These results, thus, strongly indicate that ENaC is involved in the transition from lung fluid secretion to lung fluid absorption at birth. The mortality rate, however, was not the same as in ENaC gene knockout mice studies (24), possibly because the siRNA-generating pDNA was administered after birth, in contrast to a gestational knockout. It may also be associated with the fact that ttip pSi-4 injection only silenced ENaC in the lung and thus we avoided unspecific systemic side effects from ENaC knockdown in other organs, i.e., the kidney and the gastrointestinal tract. A third possibility is that siRNA-mediated ENaC knockdown was incomplete and left 35% residual ENaC expression in the lung. When a rescue model with CMV promoter-driven rat ENaC expression in the ENaC–/– mouse lung was used, a very low ENaC was also encountered, and this was apparently sufficient to rescue 50% of the mouse pups at birth (25).
The increased plasma corticosteroid concentration observed normally late during gestation and near term leads to lung maturation and induction of fetal lung fluid absorption via an ENaC-dependent mechanism (2, 51, 54). An important downstream regulator induced by glucocorticosteroids is SGK (11, 38), and SGK is posttranslationally activated by phosphorylation (11, 38, 42). It has been shown that SGK integrates a variety of signals that may modulate ENaC function and expression at the cell surface (1, 52), among them phosphorylating and inactivating Nedd4-2 (47). Glucocorticosteroids also affect ENaC expression at both the mRNA and protein level (12, 20), possibly via SGK-induced Nedd4-2 inhibition (47). Thus a potential reduction in Nedd4-2 expression and function, as observed in our studies after silencing Nedd4-2, may explain the increase in ENaC and ENaC mRNA.
Factors that have been implied to regulate lung fluid absorption and ENaC expression in the newborn include ubiquitination, -AR agonists, glucocorticoid hormones, thyroid hormones, and oxygen concentrations (2, 5, 16, 19, 31). The key Na+ transport proteins involved are the basolateral Na-K-ATPases, which provide the driving force, and ENaC, since it provides the apical Na+ entry pathway in the epithelial cells and has been recognized as the rate-limiting step in transepithelial Na+ absorption (22). Regulation of ENaC expression at the epithelial cell surface has been suggested to involve ENaC binding to Nedd4-2, an E3 ubiquitin-protein ligase that targets ENaC for degradation (31). In Liddle's syndrome, mutations in ENaC preventing ENaC-Nedd4-2 interactions increase Na+ transport across the apical cell membrane by reducing ENaC degradation (31). Endogenous factors, such as SGK (47) and the AMP-regulated kinase (6), also affect ENaC function via functional Nedd4-2 regulation. SGK phosphorylates Nedd4-2 in vivo, and this phosphorylation inactivates Nedd4-2, thus leading to a prolonged ENaC membrane expression (46). Thus we postulated that interfering with, i.e., reducing, Nedd4-2 via siRNA in the developing rat lung would enhance fetal lung fluid absorption at birth. After silencing Nedd4-2, we found that both ENaC and ENaC expression levels were increased significantly and extravascular lung water was decreased significantly. Evidence that suggested that Nedd4-2-mediated ubiquitination and tagging for ENaC degradation was involved in regulating membrane expression of ENaC and fetal lung fluid removal from the newborn rat lung. Moreover, our data suggest that a balanced Nedd4-2 interference could be one pathway to potentially stimulate reabsorption of fetal lung fluid in the preterm born.
In conclusion, our data demonstrate by using selective siRNA inhibition of ENaC and Nedd4-2 expression that ENaC and Nedd4-2 both may be involved in the transition from lung fluid secretion to lung fluid absorption at birth. Our data also suggest that ENaC may be the principal agent stimulating lung fluid absorption at birth. Use of agents that result in a balanced upregulation of ENaC in the newborn lung, i.e., such as interfering with Nedd4-2 expression and function, may prove beneficial in stimulating the reabsorption of fetal lung fluid at birth and after preterm delivery.
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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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 T. Li, S. Koshy, and H. G. Folkesson IL-1{beta}-induced cortisol stimulates lung fluid absorption in fetal guinea pigs via SGK-mediated Nedd4-2 inhibition Am J Physiol Lung Cell Mol Physiol, March 1, 2009; 296(3): L527 - L533. [Abstract] [Full Text] [PDF]
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 E. Caci, R. Melani, N. Pedemonte, G. Yueksekdag, R. Ravazzolo, J. Rosenecker, L. J. V. Galietta, and O. Zegarra-Moran Epithelial Sodium Channel Inhibition in Primary Human Bronchial Epithelia by Transfected siRNA Am. J. Respir. Cell Mol. Biol., February 1, 2009; 40(2): 211 - 216. [Abstract] [Full Text] [PDF]
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). GAPDH was amplified as an internal control. GAPDH, glyceraldehydes-3-phosphate dehydrogenase; OD, optical density; bp, base pairs; M, DNA marker; NS, no sample.
).