The role of CFTR in transepithelial liquid transport in pig alveolar epithelia

James F. Collawn, Sadis Matalon

in a recent issue of AJP-Lung, Li et al. (22) report that the cystic fibrosis transmembrane conductance regulator (CFTR) is required for cAMP-stimulated liquid absorption in the alveolar epithelia. Using the cystic fibrosis (CF) pig model, they examined the ion and water transport properties of isolated type II alveolar epithelial cells (AEC). Although there have been studies on the role of CFTR in fluid transport in intact rabbit (29), rat (17, 35), mouse lung (9), and cultured human type II AEC (10), Li et al. (22) examined this process in the newly developed pig model for CF, which not only allows for a species comparison but also offers the exciting potential for following ion and fluid transport in an animal model for CF that appears to mimic many of the pathological features of lung disease found in CF patients (1, 27, 28, 30, 36); for an excellent review of the porcine lung as a model for CF see Ref. 32.

Fluid transport and regulation in the lung are critical for a number of reasons including during the transition from fetus to newborn, for lung surface liquid homeostasis, and for recovery from pulmonary edema. Active salt transport typically drives osmotic water transport, and this process promotes fluid clearance in the distal alveolar epithelia (reviewed in Ref. 26). Support for this view comes from studies that illustrate that blocking sodium transport inhibits alveolar fluid clearance (6, 25, 34). The epithelial sodium channel (ENaC) has been shown to be essential for this process since elimination of the alpha subunit of ENaC inhibits perinatal removal of alveolar fluid in the mouse lung (12). The role of chloride transport in this process, however, is less clear. Jiang et al. (15) reported that an apical CFTR-like chloride channel in type II AEC was important for driving sodium uptake (15). At the time, this report stimulated discussion about whether the sodium transport was due to hyperpolarization of the membrane due to Cl influx or whether cAMP directly stimulated both Cl and Na+ channels (21); more recent data support the latter hypothesis (20).

Both ENaC and CFTR are expressed in alveolar epithelia (8, 31), and therefore Matthay and colleagues (9) first tested a direct role for CFTR in fluid transport using DeltaF508 mice and using intact mouse lung found that cAMP-dependent fluid absorption in the distal airways involves CFTR function. This analysis, however, did not identify the cell types involved, which could also include type I AEC (2, 16). In subsequent studies, they found that CFTR was expressed in human type II AEC and that amiloride inhibited both basal and cAMP-stimulated fluid transport in type II AEC monolayers, whereas CFTRinh-172 was able to inhibit only stimulated fluid transport (10). This demonstrated that basal fluid transport (apical to basolateral) in type II AEC required ENaC, but not CFTR, whereas cAMP-stimulated fluid transport required both (10).

In CF, loss of CFTR leads to a lung disease characterized by dehydrated mucus, chronic bacterial infections, and airway obstruction and eventually ends with respiratory failure (7). Finding a suitable animal model for CF has proven to be difficult since CFTR knockout mice do not develop lung disease (11), although, interestingly, mice overexpressing the beta subunit of ENaC do (23). Beta-ENaC-overexpressing mice do show many of the pathological features of CF lung disease (23, 24); however, this in turn has generated a controversy over the role of ENaC in CF (3–5, 13, 14, 18, 19, 33, 37, 38). The CF pig model utilized by Li et al. (22) in this issue is important since many of the predominant features of CF lung disease are demonstrated in both the CFTR knockout animals (CFTR−/−) and DeltaF508 CFTR animals (30). Following these animals from birth through the development of CF lung disease and comparing the results to the lung pathologies found in CF patients is an important aspect that should not be underestimated since this model can serve to resolve a number of controversies that still exist in the CF field as well as providing an in vivo model for potential therapeutic interventions.

Li et al. (22) used this model to examine the ion and liquid transport properties in isolated type II AEC and in ex vivo studies of nonperfused lungs. Their analyses included wild-type (CFTR+/+), heterozygous (CFTR+/−), and null (CFTR−/−) pigs. The type II AEC were isolated from the lungs of newborn piglets within 12 h after birth and the ex vivo newborn pig lungs were isolated 8–15 h after birth (22), strikingly illustrating one of the many advantages of a large animal model.

In the Ussing chamber analysis of type II AEC from CFTR+/+, CFTR+/−, and CFTR−/− animals, Li et al. (22) demonstrated several important points. First, the amiloride-inhibited short-circuit currents were not different between the three different type II AEC, suggesting that the ENaC activities were not affected by the loss of CFTR. Second, inhibition of CFTR currents with GlyH101 was not different between the CFTR+/+ and CFTR+/−, suggesting that CFTR was not rate limiting for chloride transport (22). And third, addition of DIDS had no effect on the short-circuit currents, suggesting the absence of calcium-activated chloride channels (22). The addition of the CFTR+/− type II AEC was interesting since an intermediate phenotype might have been the expected result. In contrast to the data above from the pig lung, CFTR does seem to regulate ENaC activity in type II AEC in mouse lung slices, suggesting perhaps species differences in this regulatory process (20).

In analyzing liquid transport under open-circuit conditions, Li et al. (22) found that under basal conditions the liquid absorption rate was the same between CFTR+/+ and CFTR−/− type II AEC, indicating that CFTR played no role under basal conditions, and this is consistent with the results of Matthay and colleagues using human type II AEC (10). And as was seen with the human type II AEC, CFTR was required for the cAMP-mediated increase in fluid transport (22). The cAMP activation did increase fluid transport in the CFTR−/− type II AEC, but at an approximately four- to fivefold lower level than the CFTR+/+ type II AEC (22). And again, there was no difference in the cAMP-stimulated fluid transport between the CFTR+/+ and CFTR+/− type II AEC. The ex vivo analysis of CFTR−/− newborn pig lungs supported the in vitro findings that CFTR is required for cAMP-stimulated liquid absorption, but not basal fluid transport. To test for the role of CFTR in liquid secretion, Li et al. (22) examined type II AEC grown at an air-liquid interface and found the subphase liquid height was not affected by the presence or absence of CFTR in the type II AEC under basal conditions, whereas cAMP-activation did significantly increase the height in the CFTR+/+ but not the CFTR−/− type II AEC, highlighting that CFTR is a key regulator for both liquid absorption and secretion.

The results are significant because they are consistent with the earlier results of Matthay and colleagues using human type II AEC (10) and illustrate the usefulness of the pig model. Furthermore, this model provides an opportunity to analyze the role CFTR function at later time points during pig development and during the initial stages of CF lung pathology. It is important to remember that the lung pathology in CFTR−/− pigs mimics many of the clinical features of the human disease, including the bacterial infections (32, 36). For the future, closely following how well the model compares to the human disease and understanding how pig and human lungs differ in disease pathology and physiology will be an important consideration for using the pig model for basic ion and liquid transport properties and, perhaps just as importantly, how valid the model is for testing different CF therapeutic interventions.

GRANTS

This work was supported by grants from the National Institutes of Health (DK060065 to J. F. Collawn) and (5U01ES015676 and HL031197 to S. Matalon).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

Author contributions: J.F.C. and S.M. drafted manuscript.

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