Gene transfer of MRCKα rescues lipopolysaccharide-induced acute lung injury by restoring alveolar capillary barrier function

Overexpression of MRCKα increases tight junction protein expression in healthy mouse lungs

We previously have reported that the induction of tight junction proteins and their membrane localization by the Na⁺, K⁺-ATPase β1 subunit in cultured alveolar epithelial cells is mediated through the kinase MRCKα and further that forced expression of MRCKα in these cells was sufficient to increase tight junction protein levels²³. To determine whether MRCKα overexpression also leads to increased tight junction protein levels in the healthy mouse lung, we delivered MRCKα-expressing plasmids to the lung by transthoracic electroporation and evaluated the relative expression of ZO-1 and occludin, two tight junction proteins 2 days later (Fig. 1). As we have shown previously in cells and mouse lungs, similar overexpression of the β1-Na⁺, K⁺-ATPase increased the levels of ZO-1 and occludin one to twofold (2.8 ± 0.04, p < 0.01, and 2.64 ± 0.64, p = 0.056, respectively), compared to naive, whereas gene transfer of a non-expressing empty plasmid (pcDNA3) had no statistically significant effect on the levels of either protein¹⁸. Overexpression of MRCKα increased ZO-1 and occludin expression relative to naïve similarly to that seen with β1 (3.1 ± 0.21, p < 0.001, and 2.69 ± 0.23, p < 0.05, respectively). To ask whether overexpression of both β1-Na⁺, K⁺-ATPase and MRCKα could lead to expression that was greater than either alone, both plasmids were delivered to mice (both plasmids delivered at the same levels as for individual delivery), but lead to similar overexpression as for either protein individually (3.32 ± 0.33, p < 0.001, and 3.16 ± 0.34, p < 0.05, respectively, compared to naïve). These results show that increased level of ZO-1 and occludin following overexpression of MRCKα alone was comparable to that caused by overexpression of β1-Na⁺, K⁺-ATPase alone or in combination with MRCKα, and are consistent with the engagement of β1-Na⁺, K⁺-ATPase/MRCKα axis observed in cultured alveolar epithelial cells to enhance tight junctions²³.

Overexpression MRCKα increases tight junction protein expression in healthy mouse lungs. Plasmids (100 µg each) expressing either no insert (pcDNA3), the β1 subunit of the Na⁺,K⁺-ATPase (β1), MRCKα, or β1 and MRCKα were delivered in 50 µl to the lungs of C75B6 mice (n = 3) by aspiration followed immediately by electroporation (8 pulses of 10 ms duration each and 200 V/cm). Two days later, lungs were perfused with PBS and lysates were prepared for analysis by Western Blot (A). Levels of expression were normalized to GAPDH as a loading control and the relative expression of ZO-1 (white bars) and Occludin (grey bars) are shown as mean ± SEM (B). All experiments were carried out three times and a representative experiment is shown. One-way ANOVA with post-hoc Tukey’s multiple comparisons was used for statistical analysis; a, p < 0.01 compared to naïve; b, p < 0.05 compared to naïve; c, p < 0.001 compared to naïve; d, p < 0.05 compared to pcDNA3; e, p < 0.01 compared to pcDNA3.

Overexpression of MRCKα alone or in combination with β1-Na⁺, K⁺-ATPase restores ZO-1 and occludin expression in previously injured mouse lungs

We have shown that electroporation-mediated gene transfer of the β1-Na⁺, K⁺-ATPase can treat pre-established LPS-induced lung injury in mice¹⁸. In LPS injured mouse lungs, tight junction expression levels are decreased, as is barrier function, but upon gene delivery of the β1-Na⁺, K⁺-ATPase to LPS-injured lungs, tight protein levels were partially restored¹⁸. Since the increased levels of tight junction proteins (ZO-1 and occludin) following gene delivery of the β1-Na⁺, K⁺-ATPase is mediated through MRCKα in cells²³ and in healthy lungs (Fig. 1), we next asked whether overexpression of MRCKα could similarly rescue the decreased level of ZO-1 and occludin in LPS injured mouse lungs. Lung injury was induced by intratracheal administration of LPS (5 mg/kg body weight) and 1 day later when edema, infiltrating inflammatory cells, and injury is present¹⁷, plasmids expressing the β1-Na⁺, K⁺-ATPase, MRCKα, or a mixture of the two were delivered by aspiration and electroporation (Fig. 2). Forty-eight hours later, the lungs were harvested for western blots (Fig. 3). As seen previously, levels of ZO-1 and occludin are both decreased in animals with LPS-induced lung injury at 3 days compared to naïve animals. Treatment of LPS-injured mice with either PBS alone (no electroporation) or with an empty plasmid (pcDNA3) had no beneficial effects on the levels of either protein, nor were they statistically more reduced compared to LPS alone. By contrast, electroporation-mediated delivery of MRCKα increased expression of both proteins by twofold compared to PBS or empty plasmid (p < 0.05 for ZO-1 or occludin compared to PBS or pcDNA3). Similarly, overexpression of β1-Na⁺, K⁺-ATPase gave a similar increase in the levels of both proteins compared to PBS or pcDNA3 (p < 0.05), as did the combined delivery and expression of both β1-Na⁺, K⁺-ATPase and MRCKα. These results indicate that MRCKα overexpression can increase levels of tight junction proteins in animals with existing lung injury. Further, since co-expression of both β1 and MRCKα did not lead to increases in ZO-1 or occludin expression beyond those seen with either gene alone, this would suggest that the increased expression seen of both proteins may be maximal in the context of the injured lung.

Experimental timeline for treatment of lung injury. Lung injury was established in mice by 5 mg/kg LPS administered by aspiration and 1 day later, plasmids (either β1, MRCKα, or a combination of the two; 100 µg in 50 µl PBS) were aspirated into the lungs and electroporated using electrodes placed on either side of the chest. Two days after gene delivery, lung injury was assessed.

Overexpression of MRCKα restores ZO-1 and occludin expression in previously injured mouse lungs. Lung injury was established in C57B6 mice (n = 6–8) by aspiration of LPS (5 mg/kg) and 1 day later plasmids (100 µg each) expressing either no insert (pcDNA3), the β1 subunit of the Na⁺,K⁺-ATPase (β1), MRCKα, or β1 and MRCKα were delivered in 50 µl to the lungs by aspiration followed immediately by electroporation (8 pulses of 10 ms duration each and 200 V/cm). Two days later (3 days after LPS administration), lungs were perfused with PBS and lysates were prepared for analysis by Western Blot (A). Levels of expression were normalized to GAPDH as a loading control and the relative expression of Occludin (B) and ZO-1 (C) are shown as mean ± SEM. All experiments were carried out three times and representative experiments are shown. One-way ANOVA with post-hoc Tukey’s multiple comparisons was used for statistical analysis; a, p < 0.05 compared to naïve; b, p < 0.05 compared to pcDNA3.

Electroporation-mediated gene transfer of MRCKα attenuates the increased lung permeability in LPS-injured mice

Our previously published data showed that overexpression of the β1-Na⁺, K⁺-ATPase attenuated the increased alveolar-capillary permeability seen in LPS-injured lungs, consistent with the ability of the β1 subunit to upregulate tight junction protein expression in mouse lungs with existing lung injury¹⁸. We also have shown in cultured AT1 cells that silencing of MRCKα by siRNA abrogates β1 increased alveolar epithelial barrier integrity (measured by TEER) and upregulation of tight junction proteins by β1²³. Further, gene transfer of MRCKα alone has been shown to be sufficient to increase the epithelial barrier integrity and tight junction complex formation in cultured ATI cells²³. These data suggest that MRCKα mediates the upregulation of tight junction proteins and barrier function either by its overexpression or by that of the β1 subunit, at least in vitro. While we also have shown that overexpression of β1 in cultured human pulmonary artery endothelial cells increases transcription and protein levels of ZO-1 and occludin, we have not evaluated whether gene transfer in vitro can protect endothelial cells from LPS-induced barrier disruption²³. Cultured mouse microvascular endothelial cells were transfected with plasmids expressing MRCKα and/or β1 and 48 h later challenged with LPS (1 µg/ml) for 5 h, at which point cells were fixed and stained for expression of VE-cadherin (Fig. 4). In naïve cells, VE-cadherin staining at the cell membrane is clear and is greatly decreased upon stimulation with LPS. However, when either MRCKα, β1, or a combination of the two genes was overexpressed in these cells, the LPS-induced decrease in VE-cadherin was largely attenuated, suggesting that both genes can regulate junctional complexes in endothelial cells as well as epithelial cells²³. Based on this, we evaluated whether MRCKα and/or β1 gene transfer to LPS pre-injured mouse lungs also resulted in endothelial delivery and restored the decreased junctional complexes in vivo. To this end, we quantified levels of VE-cadherin, a protein that has been shown to be a good indicator of endothelial cell barrier dysfunction, in mice that were injured with intratracheal LPS and 24 h later subjected to electroporation-mediated gene transfer. When lung homogenates were probed for VE-cadherin by Western blot, we found that gene transfer of either MRCKα, β1, or a mixture of MRCKα and β1, but not an empty plasmid (pcDNA3) reversed the LPS-induced reductions in VE-cadherin seen in mouse lungs (Fig. 5). This indicates that electroporation-mediated gene transfer can deliver genes to multiple cell layers in the lung and that MRCKα and β1 can rescue the decreased levels of endothelial junctional complexes.

Overexpression of MRCKα or the Na⁺,K⁺-ATPase β1 subunit in microvascular endothelial cells attenuates LPS-induced reduction of VE-cadherin expression. Human MVECs grown on coverslips were transfected with plasmids (2 µg/well) expressing either no insert (pcDNA3), MRCKα, the β1 subunit of the Na⁺,K⁺-ATPase (β1), or β1 and MRCKα. Forty-eight hours later, cells were treated with nothing (Naïve) or LPS (1 µg/ml) for 5 h prior to fixation and immunofluorescent staining for VE-cadherin. The experiment was carried out on triplicate coverslips in three different experiments and representative images from two different wells are shown.

Electroporation-mediated gene transfer of MRCKα to mice with existing lung injury attenuates LPS-induced reduction in VE-cadherin. Lung injury was established in C57B6 mice (n = 6–12) by aspiration of LPS (5 mg/kg) and 1 day later plasmids (100 µg each) expressing either no insert (pcDNA3), the β1 subunit of the Na⁺,K⁺-ATPase (β1), MRCKα, or β1 and MRCKα were delivered in 50 µl to the lungs by aspiration followed immediately by electroporation (8 pulses of 10 ms duration each and 200 V/cm). Two days later (3 days after LPS administration), lungs were perfused with PBS and lysates were used for Western blots of VE-cadherin expression (A). Levels of expression were normalized to GAPDH as a loading control and the relative expression of VE-cadherin (B) is shown as mean ± SEM. All experiments were carried out three times and representative experiments are shown. One-way ANOVA with post-hoc Tukey’s multiple comparisons was used for statistical analysis; a, p < 0.05 compared to LPS only; b, p < 0.01 compared to LPS only; c, p < 0.05 compared to pcDNA3; and d, p < 0.01 compared to pcDNA3.

To further determine whether overexpression of MRCKα could attenuate the increased pulmonary barrier leakage in pre-injured living animals, lung permeability was measured by the leakage of EBD labeled albumin from blood into airways³⁰. As in Fig. 2, mouse lungs were injured with LPS and 1 day later electroporated with MRCKα, β1, or control plasmids and 47 h later evaluated for extravascular EBD accumulation following tail-vein injection of EBD (Fig. 6). Compared to the naïve group (0.203 ± 0.015), LPS induced threefold more leakage of EBD into the lung (0.623 ± 0.039), indicating alveolar capillary barrier disruption (p < 0.0001). Gene transfer of the empty vector pcDNA3 into lungs injured 24 h prior with LPS resulted in no change in lung permeability to EBD (0.601 ± 0.039) compared to LPS only. However, gene transfer of MRCKα significantly reduced the LPS-induced lung leakage to 0.455 ± 0.035, compared with LPS only (p < 0.01) or with pcDNA3 (p < 0.05). Electroporation-mediated gene delivery of the β1-Na⁺, K⁺-ATPase to LPS-injured lungs showed similar activity regarding permeability as did MRCKα (0.465 ± 0.033, p < 0.05 compared to LPS or pcDNA3). Co-administration of plasmids expressing β1 with MRCKα showed no further reduction in permeability compared to either plasmid alone (0.439 ± 0.028, p < 0.01 compared to LPS or pcDNA3). Collectively, these results indicate that gene transfer of MRCKα alone improves the alveolar capillary barrier function, pointing to the potential of MRCKα activation/overexpression to treat acute lung injury.

Electroporation mediated gene transfer of MRCKα attenuates lung leakage in lungs of mice previously injured with LPS. Lung injury was established in C57B6 mice (n = 9–11) by aspiration of LPS (5 mg/kg) and 1 day later plasmids (100 µg each) expressing either no insert (pcDNA3), the β1 subunit of the Na⁺, K⁺-ATPase (β1), MRCKα, or β1 and MRCKα were delivered in 50 µl to the lungs by aspiration followed immediately by electroporation (8 pulses of 10 ms duration each and 200 V/cm). Forty-seven hours later, Evans Blue Dye (30 mg/kg) was administered by tail vein injection and one hour later, lungs were perfused with PBS and harvested for Evans Blue Dye extraction. Lung permeability was evaluated by quantifying the absorbance of extracted Evans Blue Dye and shown as mean ± SEM. All experiments were carried out three times and a representative experiment is shown. One-way ANOVA with post-hoc Tukey’s multiple comparisons was used for statistical analysis; a, p ≤ 0.0001 compared to naïve; b, p < 0.05 compared to LPS; c, p < 0.01 compared to LPS; d, p < 0.05 compared to pcDNA3; e, p < 0.01 compared to pcDNA3.

MRCKα gene transfer to mouse lungs with existing LPS-induced injury can reduce pulmonary edema

We next determined whether MRCKα could ameliorate the overall lung edema accumulation following lung injury. As above, lung injury was induced by LPS, 1 day later plasmids were transferred to the mice, and lungs were harvested for gravimetric analysis and wet to dry ratios 2 days after gene transfer (Fig. 7). Compared with the naïve group (4.298 ± 0.051), mice injured with LPS showed an increased wet to dry ratio of 4.853 ± 0.043 (p < 0.0001), indicating a significant accumulation of edema fluid in the lung. Consistent with our previously published data, gene transfer of the empty vector plasmid pcDNA3 mice showed no change in the wet to dry ratio (4.853 ± 0.071) compared with LPS alone, whereas gene transfer of the MRCKα plasmid significantly decreased the wet to dry ratio to 4.599 ± 0.044 (p < 0.05 and p < 0.01 compared to LPS alone and LPS + pcDNA3, respectively). Similar to mice receiving the MRCKα plasmid, lungs electroporated with β1 subunit plasmid also showed significantly reduced pulmonary edema 4.619 ± 0.022 (p < 0.05 compared to LPS or pcDNA3). While gene transfer of MRCKα in combination with β1 plasmid reduced the wet to dry ratio (4.679 ± 0.046), this only trended to significance (p < 0.066 and p < 0.068, compared to LPS alone or LPS + pcDNA3, respectively). Thus, MRCKα overexpression attenuates edema accumulation in the injured lungs.

Electroporation mediated gene transfer of MRCKα attenuates lung edema fluid accumulation in previously injured lungs. Lung injury was established in C57B6 mice (n = 5–8) by aspiration of LPS (5 mg/kg) and 1 day later plasmids (100 µg each) expressing either no insert (pcDNA3), the β1 subunit of the Na⁺,K⁺-ATPase (β1), MRCKα, or β1 and MRCKα were delivered in 50 µl to the lungs by aspiration followed immediately by electroporation (8 pulses of 10 ms duration each and 200 V/cm). Two days later (3 days after LPS administration), wet to dry ratios were determined as a measure of pulmonary edema fluid and shown as mean ± SEM. All experiments were carried out three times and a representative experiment is shown. One-way ANOVA with post-hoc Tukey’s multiple comparisons was used for statistical analysis; a, p < 0.0001 compared to naïve; b, p < 0.01 compared to naïve; c, p < 0.05 compared to LPS; d, p < 0.05 compared to pcDNA3; e, p < 0.01 compared to pcDNA3.

Gene transfer of MRCKα attenuates inflammation in LPS injured lungs

BAL fluid harvested from mouse lungs was used to analyze inflammation and injury by measuring cellularity and protein concentration as a measure of barrier dysfunction. As expected, instillation of LPS induced accumulation of infiltrating cells, most of which were PMNs, and increased the levels of serum protein in the BAL (Figs. 8 and 9). Compared with LPS injured mice which received empty vector pcDNA3, mice electroporated with plasmid encoding β1-Na⁺, K⁺-ATPase showed a significant reduction in the number of total cells and PMNs in BAL fluid (Fig. 8). Similarly, transfer of the β1 plasmid also reduced the concentration of total protein (p < 0.05) as well as that specifically of serum albumin in the BAL compared to LPS alone or LPS + pcDNA3 (p < 0.001 and p < 0.0001, respectively). More importantly, gene transfer of MRCKα alone or in combination with β1-Na⁺, K⁺-ATPase decreased the number of total cells in the BAL to 2.767 × 10⁶ ± 0.160 × 10⁶ and 2.500 × 10⁶ ± 0.305 × 10⁶, respectively, compared with mice with pcDNA3 delivery (4.220 × 10⁶ ± 0.336 × 10⁶; p < 0.05 and p < 0.01 for MRCKα or both plasmids) or LPS only (4.290 × 10⁶ ± 0.417 × 10⁶; p < 0.01 and p < 0.05 for MRCKα or both plasmids) (Fig. 8C). PMNs in the BAL were the predominant cell type and were markedly reduced to 2.365 × 10⁶ ± 0.139 × 10⁶ (MRCKα alone) or 1.983 × 10⁶ ± 0.253 × 10⁶ (MRCKα + β1- Na⁺, K⁺-ATPase), compared with 3.707 × 10⁶ ± 0.371 × 10⁶ of mice with control pcDNA3 (Fig. 5C). Total BAL protein and serum albumin were also significantly reduced in the lungs of mice that received either MRCKα alone or in combination with β1 plasmids (Fig. 9). However, gene transfer of both MRCKα and β1 plasmids failed to provide any significant benefit over either gene individually. Collectively, these results indicated that electroporation mediated gene transfer of MRCKα can treat injured lungs by reducing the numbers of infiltrating cells and decreasing extravasated serum protein in the BAL fluid.

Gene delivery of MRCKα to mice with existing lung injury attenuates inflammatory cell infiltration. Lung injury was established in C57B6 mice (n = 8–10) by aspiration of LPS (5 mg/kg) and 1 day later plasmids (100 µg each) expressing either no insert (pcDNA3), the β1 subunit of the Na⁺,K⁺-ATPase (β1), MRCKα, or β1 and MRCKα were delivered in 50 µl to the lungs by aspiration followed immediately by electroporation (8 pulses of 10 ms duration each and 200 V/cm). Naïve mice (n = 5) received no LPS or DNA. Two days later (3 days after LPS administration), lungs were lavaged with PBS and BAL fluid was collected and analyzed for cellularity by cytospin followed by Diff-quik staining (A). All experiments were carried out three times and a representative experiment is shown. Total cells were quantified in the BAL fluid and shown as mean ± SEM (B). One-way ANOVA with post-hoc Tukey’s multiple comparisons was used for statistical analysis; a, p < 0.0001 compared to naïve; b, p < 0.001 compared to naïve; c, p < 0.05 compared to LPS; d, p < 0.01 compared to LPS; e, p < 0.05 compared to pcDNA3; f, p < 0.01 compared to pcDNA3. The number of PMNs in the BAL fluid were also quantified and shown as mean ± SEM (C). One-way ANOVA with post-hoc Tukey’s multiple comparisons was used for statistical analysis; a, p < 0.0001 compared to naïve; b, p < 0.01 compared to naïve; c, p < 0.05 compared to LPS; d, p < 0.01 compared to LPS; e, p < 0.05 compared to pcDNA3;f, p < 0.01 compared to pcDNA3.

MRCKα gene transfer reduces BAL protein levels in previously injured mice. Lung injury was established in C57B6 mice (n = 8–9) by aspiration of LPS (5 mg/kg) and 1 day later plasmids (100 µg each) expressing either no insert (pcDNA3), the β1 subunit of the Na⁺,K⁺-ATPase (β1), MRCKα, or β1 and MRCKα were delivered in 50 µl to the lungs by aspiration followed immediately by electroporation (8 pulses of 10 ms duration each and 200 V/cm). Naïve mice (n = 5) received no LPS or DNA. Two days later (3 days after LPS administration), lungs were lavaged with PBS and BAL fluid was collected and analyzed for total protein content, shown as mean ± SEM (A). All experiments were carried out three times and a representative experiment is shown. One-way ANOVA with post-hoc Tukey’s multiple comparisons was used for statistical analysis; a, p < 0.0001 compared to naïve; b, p < 0.01 compared to naïve; c, p < 0.05 compared to LPS; d, p < 0.001 compared to LPS; e, p < 0.05 compared to pcDNA3; f, p < 0.001 compared to pcDNA3. The concentration of albumin in the BAL fluid was quantified by ELISA and shown as mean ± SEM (B). One-way ANOVA with post-hoc Tukey’s multiple comparisons was used for statistical analysis; a, p < 0.001 compared to naïve; b, p < 0.001 compared to LPS; c, p < 0.0001 compared to pcDNA3; d, p < 0.01 compared to LPS.

Levels of histologically evident lung injury in LPS injured lungs is reduced following gene transfer of MRCKα

To determine the overall injury in animals, we inflation fixed lungs and analyzed them histologically. Figure 8 shows random images taken of 5 different mice for each treatment cohort at low and high magnification. As expected, naïve mouse lungs show abundantly clear airspaces with undetectable infiltrating cells, thin alveolar walls, and no evidence of pulmonary edema or fibrin deposition in the alveoli, at either low or high magnification (Fig. 10). By contrast, when injured with LPS and no intervention, significant atelectasis, infiltrating cells (primarily PMNs), and thickened alveolar walls are clearly evident and relatively uniform throughout the lung, even at low magnification (Fig. 10A). Gene transfer of the empty plasmid pcDNA3 did not greatly affect any of these hallmarks of diffuse alveolar damage and ALI. However, animals receiving either MRCKα, β1, or a combination of the two, all showed greatly diminished injury in terms of reduced atelectasis, increased numbers of open and clear alveoli, and thin alveolar walls. While the lungs in all of these treated animals are not as pristine as those of naïve mice, they clearly show much less inflammation and damage than do the LPS-injured lungs of mice that received no therapeutic plasmids. Taken together with all the other measures of lung injury, these results demonstrate that degree of treatment of injured lungs following gene transfer of MRCKα is essentially comparable to that of lungs receiving the β1-Na⁺, K⁺-ATPase.

Electroporation-mediated gene transfer of MRCKα improves overall histology of mice with pre-existing LPS-induced lung injury. Lung injury was established in C57B6 mice (n = 6–8) by aspiration of LPS (5 mg/kg) and 1 day later plasmids (100 µg each) expressing either no insert (pcDNA3), the β1 subunit of the Na⁺,K⁺-ATPase (β1), MRCKα, or β1 and MRCKα were delivered in 50 µl to the lungs by aspiration followed immediately by electroporation (8 pulses of 10 ms duration each and 200 V/cm). Naïve mice (n = 7) received no LPS or DNA. Two days later (3 days after LPS administration), lungs were inflated to 20 cm H₂O with 10% buffered formalin and processed for paraffin-embedding, sectioning, and hematoxylin and eosin staining. Sections from 5 representative animals are shown at ×50 (A) and ×400 (B) magnification. All experiments were carried out three times and a representative experiment is shown. Scale bar is 400 µm (A) and 50 µm (B).

Unlike the β1-Na⁺, K⁺-ATPase, MRCKα does not affect alveolar fluid clearance rates

Historically, the leading therapeutic goal ARDS treatment has been to increase alveolar fluid clearance to promoting edema resolution. As the Na⁺, K⁺-ATPase is crucial for maintaining transepithelial osmotic pressure and continuously extrudes Na⁺, and hence, water, out of the alveoli and into the interstitium and capillaries, gene transfer of Na⁺, K⁺-ATPase subunits has been shown to significantly increase AFC in various experimental models^{14,15,16,18,31,32}. To further investigate whether overexpression of MRCKα could accelerate fluid clearance in the lung, thereby accounting for a potential mechanism for the treatment effects of MRCKα, AFC was measured in living mice 2 days after gene transfer in healthy mice (Fig. 11).

Electroporation mediated gene transfer of MRCKα has no effect on rates of alveolar fluid clearance. Plasmids (100 µg each) expressing either no insert (pcDNA3), the β1 subunit of the Na⁺,K⁺-ATPase (β1), MRCKα, or β1 and MRCKα were delivered in 50 µl to the lungs of C75B6 mice (n = 6–7) by aspiration followed immediately by electroporation (8 pulses of 10 ms duration each and 200 V/cm). Naïve mice received no DNA. Two days later, alveolar fluid clearance was measured in living mice and calculated based on the change in concentration of Evans Blue Dye-labeled albumin in an isosmolar (324 mOsm) instillate placed into the alveolar space and mechanically ventilated over a 30 min period. Procaterol (10⁻⁸ mol/L) was administered in the instillate and used as the positive control in a set of naïve mice. Rates of alveolar fluid clearance are shown as mean ± SEM. All experiments were carried out three times and a representative experiment is shown. One-way ANOVA with post-hoc Tukey’s multiple comparisons was used for statistical analysis; a, p < 0.01 compared to naïve; b, p < 0.001 compared to pcDNA3; c, p < 0.01 compared to MRCKα; d, p < 0.05 compared to MRCKα.

Gene transfer of the β1 subunit significantly enhanced AFC (34.65 ± 2.46) by 73.5% and 51% compared with naïve (20.08 ± 1.64, p < 0.01) and mice that received empty plasmid pcDNA3 (20.04 ± 1.21, p < 0.001), respectively. There was no significant difference in AFC rates between naïve and pcDNA3 mice. Procaterol (10⁻⁸ mol/L), a specific β₂-Adrenergic Receptor agonist, was used as a positive control and lead to a similar increase in AFC of 77.5% compared with naïve mice (p < 0.01). However, electroporation of MRCKα alone into mouse lungs failed to improve AFC (23.58 ± 1.16), compared with either naïve (p < 0.7859) or pcDNA3 (p < 0.9999) mice. By contrast, gene transfer of MRCKα in combination with β1 subunit plasmids increased AFC to 32.71 ± 1.70, comparable to that seen in mice treated with procaterol or those electroporated with the β1 plasmid alone. These results indicate that overexpression of MRCKα could not accelerate fluid clearance; the increased AFC in mice that received both MRCKα and β1 subunit plasmids was due to the enhanced ion transport activity caused by overexpression/activation of the Na⁺, K⁺-ATPase, independent of MRCKα.