- Email: [email protected]
Sodium Channels and Cystic Fibrosis
CHEST Translating Basic Research Into Clinical Practice Sodium Channels and Cystic Fibrosis* Scott H. Donaldson, MD; and Richard C. Boucher, MD
The epithelial sodium channel (ENaC) represents the rate-limiting step of sodium absorption across airway epithelia and thereby constitutes the major pathway for volume absorption from the airway surface liquid compartment. ENaC dysregulation leads to dehydration of airway surfaces in patients with cystic fibrosis, which in turn disrupts the primary innate lung defense mechanism, mucus clearance. The development of treatment strategies that address this defect is a logical and promising means of preventing or delaying the onset of this lethal lung disease. (CHEST 2007; 132:1631–1636) Key words: cystic fibrosis; epithelial sodium channel Abbreviations: ASL ⫽ airway surface liquid; ATP ⫽ adenosine triphosphate; CACC ⫽ calcium-activated chloride channel; cAMP ⫽ cyclic adenosine monophosphate; CF ⫽ cystic fibrosis; CFTR ⫽ cystic fibrosis transmembrane conductance regulator; ENaC ⫽ epithelial sodium channel; MCC ⫽ mucociliary clearance; PCL ⫽ periciliary liquid
fibrosis (CF) is an autosomal recessive C ystic disorder that results from mutations in the CF transmembrane conductance regulator (CFTR) gene. Although CFTR is known to function as an apical epithelial chloride channel, dysregulated sodium transport is an additional, well-described phenomenon that is proposed to play a major role in the pathophysiology of CF lung disease. As a result, a significant effort has been made to better understand the role that the epithelial sodium channel (ENaC) plays in both lung health and CF lung disease. A number of in vitro studies,1–3 as well as in vivo observations in an animal model of sodium hyperabsorption, have established the link between ion transport activities, airway surface liquid (ASL) volume, and mucus clearance. ENaC, therefore, appears to play a critical role in normal airway defense *From the Cystic Fibrosis Research and Treatment Center, University of North Carolina at Chapel Hill, Chapel Hill, NC. Dr. Boucher holds equity and has served as a consultant for Inspire Pharmaceuticals, Parion Sciences, and Respirics. Dr. Donaldson has received research grants from Parion Sciences. Manuscript received January 31, 2007; revision accepted May 17, 2007. Reproduction of this article is prohibited without written permission from the American College of Chest Physicians (www.chestjournal. org/misc/reprints.shtml). Correspondence to: Scott H. Donaldson, MD, Assistant Professor of Medicine, 6007B Thurston Bowles Building, CB# 7248, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599; e-mail: [email protected] DOI: 10.1378/chest.07-0288 www.chestjournal.org
and the pathogenesis of CF lung disease. The development of specific, clinically effective inhibitors of ENaC provides hope that correction of this aspect of CF disease pathogenesis will prevent the cascade of impaired mucus clearance, chronic airways infection, and bronchiectasis that causes death in the majority of patients.
CF Ion Transport Defects Prior to the cloning of the CFTR gene in 1989, a number of investigators4 –7 recognized that CF epithelia possessed abnormal ion transport properties. Because the concentrations of Na⫹ and Cl⫺ were noted to be elevated in sweat from affected individuals, studies7 on isolated sweat glands were performed and revealed an abnormally low Cl⫺ permeability and reduced rates of Cl⫺ and Na⫹ reabsorption in CF glands. In the respiratory tract, in vivo measurements of ion transport revealed evidence for markedly elevated sodium channel activity and reduced chloride permeability.8 In fact, in vivo nasal potential difference measurements, which reveal heightened amiloride responses (owing to ENaC hyperactivity) and reduced responses to conditions that favor CFTR-mediated chloride transport, have become a useful tool for the diagnosis of CF when classic criteria are not met. A plethora of in vitro studies5,6 confirmed these findings in airway CHEST / 132 / 5 / NOVEMBER, 2007
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epithelia but left open the controversy over the primary CF defect (ie, reduced Cl⫺ permeability or increased Na⫹ absorption). After the CFTR gene was cloned in 1989, it was quickly established that this gene did indeed encode a cyclic adenosine monophosphate (cAMP)-regulated chloride channel. Interestingly, however, in vitro reconstitution of CFTR function in airway epithelia via adenovirusmediated expression of a normal CFTR gene resulted not only in restored chloride channel activity but also alleviated sodium hyperabsorption.9 Furthermore, CFTR expression was found to regulate the open probability of native epithelial sodium channels and to invert their response to phosphorylation by protein kinase A.10,11 These data, therefore, suggest that CFTR acted both as a chloride channel and as a regulator of other apical ion transport processes, including sodium transport. Ongoing research further supports the notion that both ion transport defects are important contributors to the development of CF lung disease.12
Structure and Regulation of the ENaC Cloning of the ␣, , and ␥ subunits of the ENaC via a functional complementation assay in Xenopus oocytes13 has allowed us to learn an enormous amount in the last decade about the structure and regulation of the ENaC. The channel is expressed in a variety of epithelial lined organs and has recognized functional importance in the kidney, colon, sweat duct, and lung. ENaC also plays important roles in sensory organs, including those mediating the perception of salt taste14 and mechanosensation.15 The channel is now known to be a heteromultimer that is likely composed of two ␣, one , and one ␥ subunits.16 Other combinations of these subunits (eg, ␣22, or ␣2␥2) or the inclusion of the more recently identified ␦ subunit are also possible and may underlie functionally important differences in channel activity/regulation in different lung regions.17 The regulation of the ENaC has been intensively studied and a theme of organ-level specificity for these regulatory pathways has emerged. Whereas ENaC activity in organs involved in total body volume homeostasis (ie, kidney, colon) is regulated in part by systemic levels of mineralocorticoids and their downstream effectors, ENaC regulation in airways is largely refractory to these “global” signals.18 Rather, ENaC in the airways appears to be regulated by local signals that reflect the status of the ASL compartment that bathes airway surfaces (Fig 1). One local signal that normal airway epithelia respond to by altering ENaC activity is the concentration of purine nucleotides and nucleo1632
Figure 1. Local regulators of ion transport in airway epithelia. ENaC activity is inhibited by extracellular ATP, through P2Y2 receptor-mediated signaling, and by CFTR activity in normal airways. Released ATP is also metabolized by ectonucleotidases to form adenosine (ADO), which activates CFTR by signaling through the A2B receptor. ENaC is activated by extracellular serine proteases, which may be membrane bound or released into the ASL. These channel activating proteases are, in turn, inhibited by endogenous Kunitz-type protease inhibitors.
sides (eg, adenosine triphosphate [ATP] and adenosine) in the ASL compartment. Extracellular ATP binds to P2Y2 and possibly P2X receptors,19 raises intracellular calcium levels, and induces transepithelial chloride secretion. An increase in local ATP concentration also inhibits ENaC activity.20,21 Importantly, both ATP release and extracellular metabolic pathways are present and responsive to various stimuli that may be encountered at the airway surface.22,23 Adenosine, a metabolic product of ATP released into the ASL, binds to A2B adenosine receptors, stimulates cAMP production, and activates CFTR in a protein kinase A-dependent fashion. In the presence of CFTR, adenosine also inhibits ENaC.10 However, in the absence of CFTR, cAMP stimulation actually increases ENaC activity and may worsen ASL volume depletion.5,11 Unfortunately, however, it remains unclear how CFTR regulates ENaC activity, and a number of mechanisms that range from altered cellular trafficking of ENaC to direct protein/ protein interactions remain under investigation.24 –27 A third ASL signal used by normal airways epithelia is the local concentration/activity of specific “channelactivating proteases.” These extracellular serine proteases activate ENaC by converting a “silent” channel at the apical membrane into a channel that is actively gating between open and closed states.28 Interestingly, a variety of proteases that are expressed under normal conditions (eg, prostasin) or various disease states (eg, neutrophil elastase) may be involved in this regulatory pathway.29 –31 Clearly, the level of endogenous antiproteases (eg, hepatocyte growth factor activator inhibitors 1 and 2) will also be an important factor that is involved in setting ENaC activity levels as well.32 Of note, there Translating Basic Research Into Clinical Practice
is growing evidence that the balance between channelactivating protease activities and their inhibitors may in fact be altered in CF, favoring heightened ENaC activity.30,33 Relationship Between Na⫹ Hyperabsorption and CF Lung Disease The volume of liquid on airway surfaces is vanishingly small (roughly 10 mL) relative to the large surface area (approximately 2 m2) it covers, yet its precise regulation has proven to be vitally important. The ASL compartment, comprised of a thin (7 to 30 m) liquid layer surrounding cilial shafts and an overlying mucus layer, fulfills important functions that are critical to the maintenance of mucociliary and cough clearance.34 First, the periciliary liquid (PCL) layer provides a low-viscosity environment in which cilia can beat effectively and, therefore, propel the overlying mucus layer toward the mouth. The PCL may also prevent the mucus layer from having direct contact with the epithelial surface, thereby preventing the development of mucus adhesion and formation of mucus plaques/plugs.1,3 Finally, the mucus layer itself traps inhaled pathogens/particles, allowing their removal via mucociliary clearance without the need to trigger a potentially injurious inflammatory response. The hydration status of the mucus layer is a critical determinant of the viscoelastic properties of the mucus gel required for efficient clearance. Because ENaC and CFTR control both PCL and mucus hydration, these channels are primary determinants of mucociliary clearance and careful regulation of their activities is necessary.35 Confocal imaging of airway epithelia that are cultured under conditions that mirror the growth/ differentiation/function of in vivo airway epithelia have been central to our ability to link changes in ENaC and CFTR activity to alterations in ASL volume and mucus clearance. In normal airway epithelia, the activity of ENaC and CFTR are reciprocally regulated such that the demand for increased or decreased ASL volume is met through coordinated changes in ENaC and CFTR activities.23 The well-documented inhibitory effect of CFTR over ENaC is likely central to the ability to up-regulate and down-regulate ENaC activity in response to local ASL conditions. Calcium-activated chloride channels (CACCs) are also available to rapidly respond to local stimuli (ie, extracellular ATP) with increased chloride and water secretion.23 As a result, normal airways epithelia are able to maintain a relatively constant volume of ASL and thereby maintain mucociliary clearance (MCC) under a variety of physiologic conditions (Fig 2, top left, A, and top right, B). In the setting of CF lung disease, the absence of CFTR-mediated chloride secretion and, hence, the www.chestjournal.org
ability to add volume to the ASL layer in response to stimuli that signal through intracellular cAMP levels, is lost. Fortunately, CACCs partially buffer against the loss of CFTR chloride channel activity. Equally important, the inhibitory effect that CFTR exerts on ENaC is lost, and sodium/water absorption proceeds at an unregulated rate, predisposing the airway to further dehydration. The reserve capacity to secrete chloride/water afforded by ATP signaling is vulnerable to insults (eg, viral infection) that reduce the capacity to balance sodium absorption and CACCmediated chloride secretion.23 During these insults, ion transport abnormalities manifest as PCL volume depletion and mucus layer dehydration.1 Both of these phenomena contribute to reductions in mucociliary and cough clearance, as cilial movement is restricted in PCL that is more shallow than the height of the cilia themselves, and because the mucus layer is less transportable when dehydrated and ultimately adheres to the airway surface (Fig 2, bottom left, C, and bottom right, D). Mucus stasis results, and adherent mucus is the nidus for the onset of first intermittent, and then chronic bacterial airway infection. In some models of CF pathogenesis, inherent glandular defects or inflammatory responses are believed to be key determinants of lung pathology. In any event, secondary steps of CF lung disease pathogenesis then result from the ensuing robust inflammatory response that, while failing to clear the underlying infection, destroys airway walls and lung parenchyma, ultimately leading to bronchiectasis and respiratory failure in the majority of patients. Powerful additional proof that excessive ENaC activity will not only deplete ASL volume but also reduce mucus clearance and cause a CF-like lung disease comes from a mouse model.3 In this mouse, ENaC subunits were overexpressed specifically in airway surface epithelia using a cell-specific promoter. The expected increase in amiloride-sensitive sodium currents was observed, and indeed this change translated into a more shallow ASL layer (revealed by electron microscopy), slowed mucociliary clearance (revealed by an in vivo microdialysis technique), and caused the development of mucus plugging in airways, spontaneous airway inflammation, and early death of these animals.3 Therapeutic Importance of ENaC in CF With the realization that altered ion transport and ASL volume depletion constitute initiating events of CF lung disease pathogenesis comes the opportunity to devise treatment strategies aimed at the cause of this disease, rather than downstream events. Toward this end, multiple strategies are being devised to improve airway surface hydration and thereby deCHEST / 132 / 5 / NOVEMBER, 2007
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Figure 2. The relationship between ion transport, ASL volume, and mucus transport in normal and CF epithelia. Top left, A: In normal epithelia, sodium absorption (through ENaC) and chloride secretion (via CFTR and CACC) constitute the major apical membrane ion transport pathways. Top right, B: When confronted with an excess of ASL volume, normal airway epithelia actively absorb this liquid until an approximately 7 m height is reach, approximating that of an outstretched cilium. This ASL height is then maintained through the coordinated activities of ENaC and CFTR, thus preserving an environment that is conducive for MCC. Bottom left, C: CF epithelia demonstrate accelerated sodium absorption through ENaC and lack CFTR-mediated chloride transport. A reduced ability to add volume to the ASL layer is maintained through the activation of CACCs. Bottom right, D: When challenged with an excess volume of ASL experimentally, CF epithelia rapidly absorb this liquid but are unable to appropriately adjust the rate of sodium transport to match the existing ASL conditions. As a result, the ASL volume becomes depleted and is, therefore, unable to normally support MCC.
fend against collapse of mucociliary clearance. Perhaps the most successful approach to date is the use of inhaled hypertonic saline solution to osmotically draw water into the ASL compartment. This approach has been shown to increase rates of mucociliary clearance in a sustained fashion, improve lung function, reduce pulmonary exacerbations, and improve quality of life.36,37 Other major approaches being developed include the use of agents that increase chloride secretion via non-CFTR pathways,38 restorers of CFTR function in the setting of mutant CFTR protein (pharmacologically39 or via gene transfer40), and inhibitors of ENaC. We will focus on the latter. The classical inhibitor of ENaC that has been used extensively in vitro and in human studies is amiloride. Despite some evidence41 that amiloride mod1634
estly improves mucociliary clearance, clinical trials42,43 of inhaled amiloride did not yield robust improvements in lung function. Further, when used prior to hypertonic saline solution in the attempt to prolong its duration of action, amiloride pretreatment actually prevented the accrual of benefits experienced by subjects treated with hypertonic saline solution alone.36 As a result, this agent has largely been abandoned as a CF therapeutic, and potential antagonistic interactions between ENaC inhibitors and osmotic airway hydrators are being carefully scrutinized. Closer examination of the pharmacokinetic profile of inhaled amiloride may explain its failure as a therapeutic for CF. This compound is rapidly absorbed from airway surfaces, leading to a very short duration of action.44 Therefore, longeracting and more potent amiloride analogs are being Translating Basic Research Into Clinical Practice
developed specifically for the treatment of CF lung disease. These agents (approximately 100 times more potent than amiloride) are designed to not only block ENaC more effectively and selectively, but also dissociate from the channel more slowly, increasing their duration of action.45 These agents are also designed to minimize the renal route of elimination, thus circumventing the potential problem of hyperkalemia that is typical of potassium-sparing diuretics. One such “third-generation” amiloride analog (PS55202; Parion Sciences; Durham, NC) has entered into phase 1 and 2 clinical trials in normal and CF subjects. Although conceptually exciting, the efficacy of these ENaC blockers with improved pharmacodynamic profiles remains to be determined. Conclusions Intense study of the pathogenesis of CF lung disease has elucidated the role that ENaC plays in this process. This knowledge is important because it provides a therapeutic target in CF with the potential for intervening early in the disease cascade. In addition, our understanding of CF lung disease pathogenesis, including ASL depletion as a consequence of dysregulated ENaC and CFTR pathways, may have unexpected relevance to other lung diseases, including chronic bronchitis exacerbations and other syndromes associated with reductions in mucociliary clearance, thereby creating the possibility for novel treatment strategies. Ultimately, it is hoped that therapies targeted at ENaC will substantially alter the trajectory of lung disease progression in CF. References 1 Matsui H, Grubb B, Tarran R, et al. Evidence for periciliary liquid layer depletion, not abnormal ion composition, in the pathogenesis of cystic fibrosis airways disease. Cell 1998; 95:1005–1015 2 Tarran R. Regulation of ariway surface liquid volume and mucus transport by active ion transport. Proc Am Thorac Soc 2004; 1:42– 46 3 Mall M, Grubb BR, Harkema JR, et al. Increased airway epithelial Na⫹ absorption produces cystic fibrosis-like lung disease in mice. Nat Med 2004; 10:487– 493 4 Boucher RC, Cheng EH, Paradiso AM, et al. Chloride secretory response of cystic fibrosis human airway epithelia: preservation of calcium but not protein kinase C- and A-dependent mechanism. J Clin Invest 1989; 84:1424 –1431 5 Boucher RC, Stutts MJ, Knowles MR, et al. Na⫹ transport in cystic fibrosis respiratory epithelia: abnormal basal rate and response to adenylate cyclase activation. J Clin Invest 1986; 78:1245–1252 6 Knowles MR, Stutts MJ, Spock A, et al. Abnormal ion permeation through cystic fibrosis respiratory epithelium. Science 1983; 221:1067–1070 7 Quinton PM. Chloride impermeability in cystic fibrosis. Nature 1983; 301:421– 422 www.chestjournal.org
8 Knowles M, Gatzy J, Boucher R. Increased bioelectric potential difference across respiratory epithelia in cystic fibrosis. N Engl J Med 1981; 305:1489 –1495 9 Johnson LG, Boyles SE, Wilson J, et al. Normalization of raised sodium absorption and raised calcium-mediated chloride secretion by adenovirus-mediated expression of cystic fibrosis transmembrane conductance regulator in primary human cystic fibrosis airway epithelial cells. J Clin Invest 1995; 95:1377–1382 10 Stutts MJ, Canessa CM, Olsen JC, et al. CFTR as a cAMPdependent regulator of sodium channels. Science 1995; 269: 847– 850 11 Stutts MJ, Rossier BC, Boucher RC. Cystic fibrosis transmembrane conductance regulator inverts protein kinase Amediated regulation of epithelial sodium channel single channel kinetics. J Biol Chem 1997; 272:14037–14040 12 Boucher RC. New concepts of the pathogenesis of cystic fibrosis lung disease. Eur Respir J 2004; 23:146 –158 13 Canessa CM, Horisberger JD, Rossier BC. Epithelial sodium channel related to proteins involved in neurodegeneration. Nature 1993; 361:467– 470 14 Feldman GM, Mogyorosi A, Heck GL, et al. Salt-evoked lingual surface potential in humans. J Neurophysiol 2003; 90:2060 –2064 15 Corey DP, Garcia-Anoveros J. Mechanosensation and the DEG/ENaC ion channels. Science 1996; 273:323–324 16 Firsov D, Gautschi I, Merillat AM, et al. The heterotetrameric architecture of the epithelial sodium channel (ENaC). EMBO J 1998; 17:344 –352 17 Ji HL, Su XF, Kedar S, et al. ␦-Subunit confers novel biophysical features to ␣  ␥-human epithelial sodium channel (ENaC) via a physical interaction. J Biol Chem 2006; 281:8233– 8241 18 Knowles MR, Gatzy JT, Boucher RC. Aldosterone metabolism and transepithelial potential difference in normal and cystic fibrosis subjects. Pediatr Res 1985; 19:676 – 679 19 Liang L, Zsembery A, Schwiebert EM. RNA interference targeted to multiple P2X receptor subtypes attenuates zincinduced calcium entry. Am J Physiol Cell Physiol 2005; 289:C388 –C396 20 Devor DC, Pilewski JM. UTP inhibits Na⫹ absorption in wild-type and DeltaF508 CFTR-expressing human bronchial epithelia. Am J Physiol 1999; 276:C827–C837 21 Mall M, Wissner A, Gonska T, et al. Inhibition of amiloridesensitive epithelial Na(⫹) absorption by extracellular nucleotides in human normal and cystic fibrosis airways. Am J Respir Cell Mol Biol 2000; 23:755–761 22 Donaldson SH, Lazarowski ER, Picher M, et al. Basal nucleotide levels, release, and metabolism in normal and cystic fibrosis airways. Mol Med 2000; 6:969 –982 23 Tarran R, Button B, Picher M, et al. Normal and cystic fibrosis airway surface liquid homeostasis: the effects of phasic shear stress and viral infections. J Biol Chem 2005; 280:35751–35759 24 Suaud L, Yan W, Carattino MD, et al. Regulatory interactions of N1303K-CFTR and ENaC in Xenopus oocytes: evidence that chloride transport is not necessary for inhibition of ENaC. Am J Physiol Cell Physiol 2007; 292:C1553–C1561 25 Schreiber R, Boucherot A, Murle B, et al. Control of epithelial ion transport by Cl- and PDZ proteins. J Membr Biol 2004; 199:85–98 26 Hallows KR, Fitch AC, Richardson CA, et al. Up-regulation of AMP-activated kinase by dysfunctional cystic fibrosis transmembrane conductance regulator in cystic fibrosis airway epithelial cells mitigates excessive inflammation. J Biol Chem 2006; 281:4231– 4241 27 Ji HL, Chalfant ML, Jovov B, et al. The cytosolic termini of CHEST / 132 / 5 / NOVEMBER, 2007
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the - and ␥-ENaC subunits are involved in the functional interactions between cystic fibrosis transmembrane conductance regulator and epithelial sodium channel. J Biol Chem 2000; 275:27947–27956 Caldwell RA, Boucher RC, Stutts MJ. Serine protease activation of near-silent epithelial Na⫹ channels. Am J Physiol Cell Physiol 2004; 286:C190 –C194 Tong Z, Illek B, Bhagwandin VJ, et al. Prostasin, a membraneanchored serine peptidase, regulates sodium currents in JME/ CF15 cells, a cystic fibrosis airway epithelial cell line. Am J Physiol Lung Cell Mol Physiol 2004; 287:L928 –L935 Tarran R, Trout L, Donaldson SH, et al. Soluble mediators, not cilia, determine airway surface liquid volume in normal and cystic fibrosis superficial airway epithelia. J Gen Physiol 2006; 127:591– 604 Donaldson SH, Hirsh A, Li DC, et al. Regulation of the epithelial sodium channel by serine proteases in human airways. J Biol Chem 2002; 277:8338 – 8345 Bridges RJ, Newton BB, Pilewski JM, et al. Na⫹ transport in normal and CF human bronchial epithelial cells is inhibited by BAY 39 –9437. Am J Physiol Lung Cell Mol Physiol 2001; 281:L16 –L23 Myerburg MM, Butterworth MB, McKenna EE, et al. Airway surface liquid volume regulates ENaC by altering the serine protease-protease inhibitor balance: a mechanism for sodium hyperabsorption in cystic fibrosis. J Biol Chem 2006; 281: 27942–27949 Knowles MR, Boucher RC. Mucus clearance as a primary innate defense mechanism for mammalian airways. J Clin Invest 2002; 109:571–577 Tarran R, Grubb BR, Gatzy JT, et al. The relative roles of passive surface forces and active ion transport in the modulation of airway surface liquid volume and composition. J Gen Physiol 2001; 118:223–236 Donaldson SH, Bennett WD, Zeman KL, et al. Mucus
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clearance and lung function in cystic fibrosis with hypertonic saline. N Engl J Med 2006; 354:241–250 Elkins MR, Robinson M, Rose BR, et al. A controlled trial of long-term inhaled hypertonic saline in patients with cystic fibrosis. N Engl J Med 2006; 354:229 –240 Deterding R, Retsch-Bogart G, Milgram L, et al. Safety and tolerability of denufosol tetrasodium inhalation solution, a novel P2Y2 receptor agonist: results of a phase 1/phase 2 multicenter study in mild to moderate cystic fibrosis. Pediatr Pulmonol 2005; 39:339 –348 Van GF, Straley KS, Cao D, et al. Rescue of ␦F508-CFTR trafficking and gating in human cystic fibrosis airway primary cultures by small molecules. Am J Physiol Lung Cell Mol Physiol 2006; 290:L1117–L1130 Ziady AG, Davis PB. Current prospects for gene therapy of cystic fibrosis. Curr Opin Pharmacol 2006; 6:515–521 Kohler D, App E, Schmitz-Schumann M, et al. Inhalation of amiloride improves the mucociliary and the cough clearance in patients with cystic fibroses. Eur Respir J Dis Suppl 1986; 146:319 –326 Knowles M, Church N, Waltner W, et al. A pilot study of aerosolized amiloride for the treatment of lung disease in cystic fibrosis. N Engl J Med 1990; 322:1189 –1194 Graham A, Hasani A, Alton E, et al. No added benefit from nebulized amiloride in patients with cystic fibrosis. Eur Respir J 1993; 6:1243–1248 Hofmann T, Stutts MJ, Ziersch A, et al. Effects of topically delivered benzamil and amiloride on nasal potential difference in cystic fibrosis. Am J Respir Crit Care Med 1998; 157:1844 –1849 Hirsh AJ, Molino BF, Zhang J, et al. Design, synthesis, and structure-activity relationships of novel 2-substituted pyrazinoylguanidine epithelial sodium channel blockers: drugs for cystic fibrosis and chronic bronchitis. J Med Chem 2006; 49:4098 – 4115
Translating Basic Research Into Clinical Practice
The epithelial sodium channel (ENaC) represents the rate-limiting step of sodium absorption across airway epithelia and thereby constitutes the major pathway for volume absorption from the airway surface liquid compartment. ENaC dysregulation leads to dehydration of airway surfaces in patients with cystic fibrosis, which in turn disrupts the primary innate lung defense mechanism, mucus clearance. The development of treatment strategies that address this defect is a logical and promising means of preventing or delaying the onset of this lethal lung disease. (CHEST 2007; 132:1631–1636) Key words: cystic fibrosis; epithelial sodium channel Abbreviations: ASL ⫽ airway surface liquid; ATP ⫽ adenosine triphosphate; CACC ⫽ calcium-activated chloride channel; cAMP ⫽ cyclic adenosine monophosphate; CF ⫽ cystic fibrosis; CFTR ⫽ cystic fibrosis transmembrane conductance regulator; ENaC ⫽ epithelial sodium channel; MCC ⫽ mucociliary clearance; PCL ⫽ periciliary liquid
fibrosis (CF) is an autosomal recessive C ystic disorder that results from mutations in the CF transmembrane conductance regulator (CFTR) gene. Although CFTR is known to function as an apical epithelial chloride channel, dysregulated sodium transport is an additional, well-described phenomenon that is proposed to play a major role in the pathophysiology of CF lung disease. As a result, a significant effort has been made to better understand the role that the epithelial sodium channel (ENaC) plays in both lung health and CF lung disease. A number of in vitro studies,1–3 as well as in vivo observations in an animal model of sodium hyperabsorption, have established the link between ion transport activities, airway surface liquid (ASL) volume, and mucus clearance. ENaC, therefore, appears to play a critical role in normal airway defense *From the Cystic Fibrosis Research and Treatment Center, University of North Carolina at Chapel Hill, Chapel Hill, NC. Dr. Boucher holds equity and has served as a consultant for Inspire Pharmaceuticals, Parion Sciences, and Respirics. Dr. Donaldson has received research grants from Parion Sciences. Manuscript received January 31, 2007; revision accepted May 17, 2007. Reproduction of this article is prohibited without written permission from the American College of Chest Physicians (www.chestjournal. org/misc/reprints.shtml). Correspondence to: Scott H. Donaldson, MD, Assistant Professor of Medicine, 6007B Thurston Bowles Building, CB# 7248, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599; e-mail: [email protected] DOI: 10.1378/chest.07-0288 www.chestjournal.org
and the pathogenesis of CF lung disease. The development of specific, clinically effective inhibitors of ENaC provides hope that correction of this aspect of CF disease pathogenesis will prevent the cascade of impaired mucus clearance, chronic airways infection, and bronchiectasis that causes death in the majority of patients.
CF Ion Transport Defects Prior to the cloning of the CFTR gene in 1989, a number of investigators4 –7 recognized that CF epithelia possessed abnormal ion transport properties. Because the concentrations of Na⫹ and Cl⫺ were noted to be elevated in sweat from affected individuals, studies7 on isolated sweat glands were performed and revealed an abnormally low Cl⫺ permeability and reduced rates of Cl⫺ and Na⫹ reabsorption in CF glands. In the respiratory tract, in vivo measurements of ion transport revealed evidence for markedly elevated sodium channel activity and reduced chloride permeability.8 In fact, in vivo nasal potential difference measurements, which reveal heightened amiloride responses (owing to ENaC hyperactivity) and reduced responses to conditions that favor CFTR-mediated chloride transport, have become a useful tool for the diagnosis of CF when classic criteria are not met. A plethora of in vitro studies5,6 confirmed these findings in airway CHEST / 132 / 5 / NOVEMBER, 2007
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epithelia but left open the controversy over the primary CF defect (ie, reduced Cl⫺ permeability or increased Na⫹ absorption). After the CFTR gene was cloned in 1989, it was quickly established that this gene did indeed encode a cyclic adenosine monophosphate (cAMP)-regulated chloride channel. Interestingly, however, in vitro reconstitution of CFTR function in airway epithelia via adenovirusmediated expression of a normal CFTR gene resulted not only in restored chloride channel activity but also alleviated sodium hyperabsorption.9 Furthermore, CFTR expression was found to regulate the open probability of native epithelial sodium channels and to invert their response to phosphorylation by protein kinase A.10,11 These data, therefore, suggest that CFTR acted both as a chloride channel and as a regulator of other apical ion transport processes, including sodium transport. Ongoing research further supports the notion that both ion transport defects are important contributors to the development of CF lung disease.12
Structure and Regulation of the ENaC Cloning of the ␣, , and ␥ subunits of the ENaC via a functional complementation assay in Xenopus oocytes13 has allowed us to learn an enormous amount in the last decade about the structure and regulation of the ENaC. The channel is expressed in a variety of epithelial lined organs and has recognized functional importance in the kidney, colon, sweat duct, and lung. ENaC also plays important roles in sensory organs, including those mediating the perception of salt taste14 and mechanosensation.15 The channel is now known to be a heteromultimer that is likely composed of two ␣, one , and one ␥ subunits.16 Other combinations of these subunits (eg, ␣22, or ␣2␥2) or the inclusion of the more recently identified ␦ subunit are also possible and may underlie functionally important differences in channel activity/regulation in different lung regions.17 The regulation of the ENaC has been intensively studied and a theme of organ-level specificity for these regulatory pathways has emerged. Whereas ENaC activity in organs involved in total body volume homeostasis (ie, kidney, colon) is regulated in part by systemic levels of mineralocorticoids and their downstream effectors, ENaC regulation in airways is largely refractory to these “global” signals.18 Rather, ENaC in the airways appears to be regulated by local signals that reflect the status of the ASL compartment that bathes airway surfaces (Fig 1). One local signal that normal airway epithelia respond to by altering ENaC activity is the concentration of purine nucleotides and nucleo1632
Figure 1. Local regulators of ion transport in airway epithelia. ENaC activity is inhibited by extracellular ATP, through P2Y2 receptor-mediated signaling, and by CFTR activity in normal airways. Released ATP is also metabolized by ectonucleotidases to form adenosine (ADO), which activates CFTR by signaling through the A2B receptor. ENaC is activated by extracellular serine proteases, which may be membrane bound or released into the ASL. These channel activating proteases are, in turn, inhibited by endogenous Kunitz-type protease inhibitors.
sides (eg, adenosine triphosphate [ATP] and adenosine) in the ASL compartment. Extracellular ATP binds to P2Y2 and possibly P2X receptors,19 raises intracellular calcium levels, and induces transepithelial chloride secretion. An increase in local ATP concentration also inhibits ENaC activity.20,21 Importantly, both ATP release and extracellular metabolic pathways are present and responsive to various stimuli that may be encountered at the airway surface.22,23 Adenosine, a metabolic product of ATP released into the ASL, binds to A2B adenosine receptors, stimulates cAMP production, and activates CFTR in a protein kinase A-dependent fashion. In the presence of CFTR, adenosine also inhibits ENaC.10 However, in the absence of CFTR, cAMP stimulation actually increases ENaC activity and may worsen ASL volume depletion.5,11 Unfortunately, however, it remains unclear how CFTR regulates ENaC activity, and a number of mechanisms that range from altered cellular trafficking of ENaC to direct protein/ protein interactions remain under investigation.24 –27 A third ASL signal used by normal airways epithelia is the local concentration/activity of specific “channelactivating proteases.” These extracellular serine proteases activate ENaC by converting a “silent” channel at the apical membrane into a channel that is actively gating between open and closed states.28 Interestingly, a variety of proteases that are expressed under normal conditions (eg, prostasin) or various disease states (eg, neutrophil elastase) may be involved in this regulatory pathway.29 –31 Clearly, the level of endogenous antiproteases (eg, hepatocyte growth factor activator inhibitors 1 and 2) will also be an important factor that is involved in setting ENaC activity levels as well.32 Of note, there Translating Basic Research Into Clinical Practice
is growing evidence that the balance between channelactivating protease activities and their inhibitors may in fact be altered in CF, favoring heightened ENaC activity.30,33 Relationship Between Na⫹ Hyperabsorption and CF Lung Disease The volume of liquid on airway surfaces is vanishingly small (roughly 10 mL) relative to the large surface area (approximately 2 m2) it covers, yet its precise regulation has proven to be vitally important. The ASL compartment, comprised of a thin (7 to 30 m) liquid layer surrounding cilial shafts and an overlying mucus layer, fulfills important functions that are critical to the maintenance of mucociliary and cough clearance.34 First, the periciliary liquid (PCL) layer provides a low-viscosity environment in which cilia can beat effectively and, therefore, propel the overlying mucus layer toward the mouth. The PCL may also prevent the mucus layer from having direct contact with the epithelial surface, thereby preventing the development of mucus adhesion and formation of mucus plaques/plugs.1,3 Finally, the mucus layer itself traps inhaled pathogens/particles, allowing their removal via mucociliary clearance without the need to trigger a potentially injurious inflammatory response. The hydration status of the mucus layer is a critical determinant of the viscoelastic properties of the mucus gel required for efficient clearance. Because ENaC and CFTR control both PCL and mucus hydration, these channels are primary determinants of mucociliary clearance and careful regulation of their activities is necessary.35 Confocal imaging of airway epithelia that are cultured under conditions that mirror the growth/ differentiation/function of in vivo airway epithelia have been central to our ability to link changes in ENaC and CFTR activity to alterations in ASL volume and mucus clearance. In normal airway epithelia, the activity of ENaC and CFTR are reciprocally regulated such that the demand for increased or decreased ASL volume is met through coordinated changes in ENaC and CFTR activities.23 The well-documented inhibitory effect of CFTR over ENaC is likely central to the ability to up-regulate and down-regulate ENaC activity in response to local ASL conditions. Calcium-activated chloride channels (CACCs) are also available to rapidly respond to local stimuli (ie, extracellular ATP) with increased chloride and water secretion.23 As a result, normal airways epithelia are able to maintain a relatively constant volume of ASL and thereby maintain mucociliary clearance (MCC) under a variety of physiologic conditions (Fig 2, top left, A, and top right, B). In the setting of CF lung disease, the absence of CFTR-mediated chloride secretion and, hence, the www.chestjournal.org
ability to add volume to the ASL layer in response to stimuli that signal through intracellular cAMP levels, is lost. Fortunately, CACCs partially buffer against the loss of CFTR chloride channel activity. Equally important, the inhibitory effect that CFTR exerts on ENaC is lost, and sodium/water absorption proceeds at an unregulated rate, predisposing the airway to further dehydration. The reserve capacity to secrete chloride/water afforded by ATP signaling is vulnerable to insults (eg, viral infection) that reduce the capacity to balance sodium absorption and CACCmediated chloride secretion.23 During these insults, ion transport abnormalities manifest as PCL volume depletion and mucus layer dehydration.1 Both of these phenomena contribute to reductions in mucociliary and cough clearance, as cilial movement is restricted in PCL that is more shallow than the height of the cilia themselves, and because the mucus layer is less transportable when dehydrated and ultimately adheres to the airway surface (Fig 2, bottom left, C, and bottom right, D). Mucus stasis results, and adherent mucus is the nidus for the onset of first intermittent, and then chronic bacterial airway infection. In some models of CF pathogenesis, inherent glandular defects or inflammatory responses are believed to be key determinants of lung pathology. In any event, secondary steps of CF lung disease pathogenesis then result from the ensuing robust inflammatory response that, while failing to clear the underlying infection, destroys airway walls and lung parenchyma, ultimately leading to bronchiectasis and respiratory failure in the majority of patients. Powerful additional proof that excessive ENaC activity will not only deplete ASL volume but also reduce mucus clearance and cause a CF-like lung disease comes from a mouse model.3 In this mouse, ENaC subunits were overexpressed specifically in airway surface epithelia using a cell-specific promoter. The expected increase in amiloride-sensitive sodium currents was observed, and indeed this change translated into a more shallow ASL layer (revealed by electron microscopy), slowed mucociliary clearance (revealed by an in vivo microdialysis technique), and caused the development of mucus plugging in airways, spontaneous airway inflammation, and early death of these animals.3 Therapeutic Importance of ENaC in CF With the realization that altered ion transport and ASL volume depletion constitute initiating events of CF lung disease pathogenesis comes the opportunity to devise treatment strategies aimed at the cause of this disease, rather than downstream events. Toward this end, multiple strategies are being devised to improve airway surface hydration and thereby deCHEST / 132 / 5 / NOVEMBER, 2007
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Figure 2. The relationship between ion transport, ASL volume, and mucus transport in normal and CF epithelia. Top left, A: In normal epithelia, sodium absorption (through ENaC) and chloride secretion (via CFTR and CACC) constitute the major apical membrane ion transport pathways. Top right, B: When confronted with an excess of ASL volume, normal airway epithelia actively absorb this liquid until an approximately 7 m height is reach, approximating that of an outstretched cilium. This ASL height is then maintained through the coordinated activities of ENaC and CFTR, thus preserving an environment that is conducive for MCC. Bottom left, C: CF epithelia demonstrate accelerated sodium absorption through ENaC and lack CFTR-mediated chloride transport. A reduced ability to add volume to the ASL layer is maintained through the activation of CACCs. Bottom right, D: When challenged with an excess volume of ASL experimentally, CF epithelia rapidly absorb this liquid but are unable to appropriately adjust the rate of sodium transport to match the existing ASL conditions. As a result, the ASL volume becomes depleted and is, therefore, unable to normally support MCC.
fend against collapse of mucociliary clearance. Perhaps the most successful approach to date is the use of inhaled hypertonic saline solution to osmotically draw water into the ASL compartment. This approach has been shown to increase rates of mucociliary clearance in a sustained fashion, improve lung function, reduce pulmonary exacerbations, and improve quality of life.36,37 Other major approaches being developed include the use of agents that increase chloride secretion via non-CFTR pathways,38 restorers of CFTR function in the setting of mutant CFTR protein (pharmacologically39 or via gene transfer40), and inhibitors of ENaC. We will focus on the latter. The classical inhibitor of ENaC that has been used extensively in vitro and in human studies is amiloride. Despite some evidence41 that amiloride mod1634
estly improves mucociliary clearance, clinical trials42,43 of inhaled amiloride did not yield robust improvements in lung function. Further, when used prior to hypertonic saline solution in the attempt to prolong its duration of action, amiloride pretreatment actually prevented the accrual of benefits experienced by subjects treated with hypertonic saline solution alone.36 As a result, this agent has largely been abandoned as a CF therapeutic, and potential antagonistic interactions between ENaC inhibitors and osmotic airway hydrators are being carefully scrutinized. Closer examination of the pharmacokinetic profile of inhaled amiloride may explain its failure as a therapeutic for CF. This compound is rapidly absorbed from airway surfaces, leading to a very short duration of action.44 Therefore, longeracting and more potent amiloride analogs are being Translating Basic Research Into Clinical Practice
developed specifically for the treatment of CF lung disease. These agents (approximately 100 times more potent than amiloride) are designed to not only block ENaC more effectively and selectively, but also dissociate from the channel more slowly, increasing their duration of action.45 These agents are also designed to minimize the renal route of elimination, thus circumventing the potential problem of hyperkalemia that is typical of potassium-sparing diuretics. One such “third-generation” amiloride analog (PS55202; Parion Sciences; Durham, NC) has entered into phase 1 and 2 clinical trials in normal and CF subjects. Although conceptually exciting, the efficacy of these ENaC blockers with improved pharmacodynamic profiles remains to be determined. Conclusions Intense study of the pathogenesis of CF lung disease has elucidated the role that ENaC plays in this process. This knowledge is important because it provides a therapeutic target in CF with the potential for intervening early in the disease cascade. In addition, our understanding of CF lung disease pathogenesis, including ASL depletion as a consequence of dysregulated ENaC and CFTR pathways, may have unexpected relevance to other lung diseases, including chronic bronchitis exacerbations and other syndromes associated with reductions in mucociliary clearance, thereby creating the possibility for novel treatment strategies. Ultimately, it is hoped that therapies targeted at ENaC will substantially alter the trajectory of lung disease progression in CF. References 1 Matsui H, Grubb B, Tarran R, et al. Evidence for periciliary liquid layer depletion, not abnormal ion composition, in the pathogenesis of cystic fibrosis airways disease. Cell 1998; 95:1005–1015 2 Tarran R. Regulation of ariway surface liquid volume and mucus transport by active ion transport. Proc Am Thorac Soc 2004; 1:42– 46 3 Mall M, Grubb BR, Harkema JR, et al. Increased airway epithelial Na⫹ absorption produces cystic fibrosis-like lung disease in mice. Nat Med 2004; 10:487– 493 4 Boucher RC, Cheng EH, Paradiso AM, et al. Chloride secretory response of cystic fibrosis human airway epithelia: preservation of calcium but not protein kinase C- and A-dependent mechanism. J Clin Invest 1989; 84:1424 –1431 5 Boucher RC, Stutts MJ, Knowles MR, et al. Na⫹ transport in cystic fibrosis respiratory epithelia: abnormal basal rate and response to adenylate cyclase activation. J Clin Invest 1986; 78:1245–1252 6 Knowles MR, Stutts MJ, Spock A, et al. Abnormal ion permeation through cystic fibrosis respiratory epithelium. Science 1983; 221:1067–1070 7 Quinton PM. Chloride impermeability in cystic fibrosis. Nature 1983; 301:421– 422 www.chestjournal.org
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the - and ␥-ENaC subunits are involved in the functional interactions between cystic fibrosis transmembrane conductance regulator and epithelial sodium channel. J Biol Chem 2000; 275:27947–27956 Caldwell RA, Boucher RC, Stutts MJ. Serine protease activation of near-silent epithelial Na⫹ channels. Am J Physiol Cell Physiol 2004; 286:C190 –C194 Tong Z, Illek B, Bhagwandin VJ, et al. Prostasin, a membraneanchored serine peptidase, regulates sodium currents in JME/ CF15 cells, a cystic fibrosis airway epithelial cell line. Am J Physiol Lung Cell Mol Physiol 2004; 287:L928 –L935 Tarran R, Trout L, Donaldson SH, et al. Soluble mediators, not cilia, determine airway surface liquid volume in normal and cystic fibrosis superficial airway epithelia. J Gen Physiol 2006; 127:591– 604 Donaldson SH, Hirsh A, Li DC, et al. Regulation of the epithelial sodium channel by serine proteases in human airways. J Biol Chem 2002; 277:8338 – 8345 Bridges RJ, Newton BB, Pilewski JM, et al. Na⫹ transport in normal and CF human bronchial epithelial cells is inhibited by BAY 39 –9437. Am J Physiol Lung Cell Mol Physiol 2001; 281:L16 –L23 Myerburg MM, Butterworth MB, McKenna EE, et al. Airway surface liquid volume regulates ENaC by altering the serine protease-protease inhibitor balance: a mechanism for sodium hyperabsorption in cystic fibrosis. J Biol Chem 2006; 281: 27942–27949 Knowles MR, Boucher RC. Mucus clearance as a primary innate defense mechanism for mammalian airways. J Clin Invest 2002; 109:571–577 Tarran R, Grubb BR, Gatzy JT, et al. The relative roles of passive surface forces and active ion transport in the modulation of airway surface liquid volume and composition. J Gen Physiol 2001; 118:223–236 Donaldson SH, Bennett WD, Zeman KL, et al. Mucus
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