Unravelling salt transport in cystic fibrosis
=============================================

* P G Noone
* K W Southern

*   nasal potential difference
*   cystic fibrosis
*   ion transport
*   genotype/phenotype

Sodium hyperabsorption may be a key therapeutic target in CF

Cystic fibrosis (CF) lung disease is characterised by thick viscid airway secretions, the development of progressive airways obstruction and bronchiectasis, and colonisation with specific bacteria, notably *Pseudomonas aeruginosa*.1 Although the precise pathogenic pathways in CF are still debated (see below), airway epithelial ion transport has been known to be defective in CF for two decades. This can be assessed in the airway in vivo by measuring potential difference (PD)—that is, the voltage generated across an electrically tight epithelium by the active transport of charged sodium and chloride ions.2 In patients with CF the magnitude of sodium absorption across airway epithelia and the response to the sodium channel blocker amiloride are substantially increased compared with normal subjects, coupled with an inability to secrete chloride ions.3 In the 1990s the putative gene (the cystic fibrosis transmembrane conductance regulator (*CFTR*) gene) was cloned4 and the affected protein was identified as a gated chloride channel,5 supporting the hypothesis that CF is linked to abnormal transepithelial ion transport.

Despite this clear link between abnormal ion transport and CF, the pathogenesis of lung disease in CF is complex, and much effort has been expended trying to elucidate the pathways involved in the development of airways disease. One hypothesis suggests that lung disease in CF develops in large part because of the deranged ion transport, resulting in a reduction in airway surface liquid volume and compromised mucociliary clearance.6 These abnormal mechanisms set up a cycle of retained airway secretions, accumulation of mucus with infection and inflammation in the airways, ultimately leading to airway destruction, respiratory failure, and death from lung disease.

Recent in vitro studies on airway cell cultures grown to confluence with an air/liquid interface have yielded further insights into the impact of the CF ion transport defect on airway defence mechanisms.7 In the absence of CFTR, sodium absorption (through the epithelial sodium channel (ENaC)) is upregulated. Subsequent dehydration of the airway surface liquid results in abolition of normal ciliary function. Thus, sodium (and fluid) absorption appears to dominate the normal “steady state” in the airway; however, recent data have shown that, under certain circumstances, airway epithelium can shift its phenotype and chloride (and fluid) secretion becomes predominant.8 In CF airway cultures this ability to shift to a secretory phenotype is compromised.

### Which ion transport abnormality is most important in CF lung disease?

So which of these abnormalities—upregulation of basal fluid absorption or an inability to switch efficiently to a secretory phenotype—is most important for the development of CF lung disease? Both mechanisms could theoretically result in similar reductions in airway surface liquid volume with the resulting impact on mucociliary transport. This question is important, since potential therapeutic strategies currently target both loops of the cascade, for example, with sodium channel blockers and chloride secretagogues.9,10 It may be that both the sodium and chloride ion transport defects have a role (a double hit) in the pathogenesis of CF; however, murine models suggest that sodium hyperabsorption may be more important.

In the 1990s a number of *cftr* knockout mice were generated.11–13 These mice have a form of gastrointestinal disease but no overt lung disease. This has been explained by chloride secretion through alternative channels in the airway.14 Nasal PD in these “CF mice” is raised, as in humans with CF and consistent with sodium hyperabsorption; however, surprisingly, there is no increased PD in the lower airway.15

A major development has been the recent generation of a transgenic mouse with overexpression of the β subunit of the *ENaC* gene in the airways (driven by a lung specific promoter), but with normal *cftr* expression and function.16 These mice have an increased magnitude of PD throughout the airway, consistent with sodium hyperabsorption, and develop early respiratory distress with a significant number dying in the first month of life. Investigations reveal depletion of airway surface liquid and mucus accumulation with reduced airway and bacterial clearance. The striking similarity to human CF disease provides convincing evidence that sodium hyperabsorption may be a primary determinant of CF lung disease.

### Does the extent of ion transport abnormality in the airway determine the severity of CF lung disease?

Many groups have looked for a link between the degree of abnormality of airway ion transport (as determined by the nasal PD) and disease severity. Some have suggested relationships between the respiratory condition and sodium hyperabsorption (as determined by basal PD/response to amiloride),17 and others with chloride secretion (as determined by the change in PD under specific conditions—that is, following perfusion of a solution with chloride ions replaced by gluconate with a β agonist such as isoprenaline).18 These different results probably reflect small numbers and differing techniques. A European study reduced the confounding variables of environment and genotype by examining twins and siblings homozygous for ΔF508.19 A weak relationship between respiratory disease and chloride secretion was demonstrated (concordant sibling pairs with mild disease had a small but significantly increased level of chloride secretion compared with pairs with severe disease). Subsequent groups have not been able to identify a link between respiratory phenotype and chloride secretion.20,21

In this issue of *Thorax* Fajac and colleagues provide data which add to this debate.22 Using nasal PD, they measured ion transport in 79 adult patients with CF of varying severity and related PD outcomes to pancreatic status (pancreatic sufficient (n = 17) or pancreatic insufficient (n = 62)) and lung function (forced expiratory volume in 1 second (FEV1) >50% predicted (n = 49) or <50% predicted (n = 30)). All patients with CF had diagnostic sweat chloride levels except for four patients with mild genetic mutations (73/79 had two recognised *CFTR* mutations). At baseline, patients with CF either had a raised basal PD with increased sensitivity to amiloride (typical of CF) and/or a lack of response to perfusion with a low chloride solution with isoprenaline. They found a weak relationship between the severity of lung disease (as determined by FEV1) and increased sodium transport (as determined by basal PD and response of that PD to amiloride). With a univariate analysis, basal PD was slightly higher in subjects with severe lung disease (mean −54 mV) than in those with milder lung disease (mean −45 mV). The reduction with amiloride was also greater in the severe group although, in a multivariate analysis including chronic infection with *P aeruginosa*, the relationship between lung function and basal PD disappeared but remained for lung function and amiloride sensitivity (odds ratio 3.7). There was no relationship between FEV1 and chloride secretion (+2 mV in severe lung disease, +1 mV in mild lung disease).

### Where does this leave us in our understanding of the impact of ion transport on CF lung disease?

The bottom line is that, if a true relationship between ion transport (as measured by nasal PD) and the severity of respiratory disease exists, it is likely to be weak. These data are consistent with the notion of a “point of no return”—that is, the ion transport abnormality provides the setting for CF lung disease early in life but, once established, other factors such as non-*CFTR* gene modifiers, the response of the innate immune system, mucus secretory control mechanisms, or environmental factors are more important in determining disease severity.23

Is this concept important? Certainly; nasal PD has frequently been employed as a surrogate outcome measure for “proof of principle” trials of new treatments.24 If fundamental treatments are to work, these data suggest that early intervention is necessary as other factors may have a more profound influence on the eventual severity of lung disease. If ion transport is to be used as a surrogate outcome for fundamental treatments, then the data from murine studies suggest that correction of sodium hyperabsorption is the least required to halt the development of CF lung disease rather than correction of the chloride secretory defect alone.25

Although Fajac and colleagues did not find a strong relationship between ion transport and respiratory disease, they did find that pancreatic sufficient patients are significantly more likely to have evidence of chloride secretion in their nasal airway.22 If nasal PD reflects ion transport elsewhere, such as in the pancreatic ducts, then this is an important observation in terms of the pathogenesis of CF pancreatic disease.

From a practical point of view, this finding has a bearing on the interpretation of nasal PD measurement when used to determine a diagnosis in people with atypical clinical features and equivocal sweat test results. The combination of a raised magnitude of basal PD and a lack of cftr-mediated chloride response is strong supportive evidence of a CF diagnosis, with the latter having been advocated as the most reliable component of the nasal PD tracing for making a diagnosis of CF.26,27 However, groups have presented data demonstrating significant chloride secretion in some patients with “classic” CF.17,21 Therefore, in the presence of a high magnitude of basal PD (>40 mV), the finding of chloride secretion does not negate a diagnosis of CF; indeed, the data of Fajac *et al* suggest that, in patients who are pancreatic sufficient, this may regularly be the case.

From a treatment standpoint, it seems logical to focus efforts on modulating the sodium channel to restore volume to the airway surface liquid, improve mucociliary clearance, and prevent the first step towards CF lung disease early in life. A balance may be required between agents that stimulate chloride secretion and agents that block sodium absorption, in order to restore airway surface liquid volume and achieve adequate airway clearance mechanisms in CF. However, at present, the weight of evidence suggests that sodium hyperabsorption may be a key therapeutic target.

Sodium hyperabsorption may be a key therapeutic target in CF

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