epithelial na channels ( enac ) are of importance in na - absorptive epithelium , such as in the airway , the alveolus , the kidney , and the distal colon , and control the overall rate of transapical na transport .
enac proteins are mainly located in the apical membrane of polarized epithelial cells and have four homologous subunits ( i.e. , the  ,  ,  and  subunits ) 1 , 2 . the  or  enac subunit , which is essential for acting as a sodium channel , forms the channel pore , whereas the  and  enac subunits are critical for amplifying the efficiency of na influx . in mouse ,
the gene scnn1d , which encodes  enac , is assumed to be a pseudogene 2 . in the lungs ,
alveolar lining fluid is critical for efficient gas exchange , and enac complexes play a crucial role in alveolar fluid clearance to maintain homeostasis of the luminal liquid . pulmonary diseases , including acute lung injury , cystic fibrosis , chronic obstructive pulmonary disease , and asthma result from or are associated with the dysfunction or dysregulation of enac , and the regeneration of epithelial cells and the restoration of ion transport are two key steps in recovery from those diseases . in this article , we review the progress of research on enac - mediated lung injury repair , in particular the role of enac proteins in re - epithelialization by endogenous and allogeneic stem / progenitor cells .
the mammalian pulmonary epithelium , a multilevel , branched network , can be functionally divided into the proximal conducting airways and the distal gas - exchange domain .
the development of the airways and the lung is the result of specification and separation of a group of progenitor cells from the ventral region of the anterior foregut . in humans , the development of the airways and alveoli from fetal to adult stage
, the airway epithelium consists of undifferentiated columnar cells , and then ciliated cells , secretory cells , club cells , alveolar type i ( ati ) and type ii ( atii ) cells differentiate from these progenitor cells at different stages of gestation ( figure 1 ) .
a recent study indicated that both ati and atii cells are derived from a lineage of bipotential progenitors during embryonic development and that atii cells undergo a switch that functions both to self - renew and to generate ati cells 4 .
lineage hierarchies constructed using single - cell rna - seq unveiled the differentiation steps of these bipotential progenitors 5 . in adults , the lung not only undergoes a slow turnover , but also repairs itself rapidly , indicating the existence of a subpopulation of stem cells or progenitors with preserved differentiation potential .
the stem / progenitor cells able to repair injured lungs include club cells , bronchioalveolar stem cells ( bascs ) , and atii cells .
in addition , submucosal gland duct stem cells and neuroendocrine cells have the potential to differentiate into club , basal , serous , or ciliated cells , as well as into distal airway epithelium . club cells , bascs , and atii cells are dominant in the pulmonary epithelium and have been generally well studied .
club cells are able to self - renew , differentiate into ciliated epithelial cells , and contribute to the replenishment of both ati and atii cells in lungs injured by bleomycin or infection with h1n1 influenza 6 - 8 .
bascs are located in bronchioalveolar duct junctions , and following catastrophic alveolar epithelial injury , can replenish cell lineages in the alveolus 8 .
in addition to club cells , distal airway stem cells ( dascs ) and basal cells contribute to replenishing other pulmonary epithelial cells .
atii cells are involved in the regeneration of alveoli , maintaining a slow self - renewal in normal lungs and then differentiating into ati cells during lung injury repair . additionally , a subpopulation of alveolar epithelial cells expressing integrin 6/4 and dascs has the potential to differentiate into atii and club cells 9 , 10 .
recently , a new type of basally located dascs that express trp63 and keratin 5 was also reported to be crucial for epithelium regeneration in airways and alveoli 11 - 13 .
the expression of enac in the human respiratory system was confirmed in a development - dependent manner 3 . at the early stage of embryonic development (  16 wk of gestation ) , the - and -enac subunits were not detected in human airways 3 . near birth , increased enac activity was present on the apical surface of lung epithelial cells , and active na transport was promoted 14 . in adult airways ,
the expression pattern of enac was similar to that in the canalicular period ( 17 - 24 wk ) ( figure 1 ) .
transcripts of -enac were expressed in club cells throughout fetal lung development 15 , and all four subunits (  ,  ,  and  ) were detected in adult club cells 1 , 16 .
enac mrna was distributed in atii cells after 28 wk of gestation 17 , and enac subunits were also expressed in atii cells ( figure 1 ) 2 , 18 , 19 .
in postnatal pulmonary epithelium , na ions flow into epithelial cells via apically located enac proteins and are actively pumped out of the cells by na - k - atpase at the basolateral membrane .
apical fluid volume regulated the activity and abundance of enac in h441 cells originally derived from human club cells .
dexamethasone , a corticosteroid , regulated enac activity in club- and atii - like cell cultures , by promoting the expression of enac 20 subsequent to activating the sgk1 , pi3k and camp / pka signaling pathways 21 .
treatment with dexamethasone and camp - elevating agents can lead to the differentiation of the aforementioned native stem cells to club cells or alveolar cells 22 , 23 .
sgk1 and pka can phosphorylate the ubiquitin ligase nedd4 - 2 , which mediates the internalisation and degradation of enac by binding to the proline - rich domains of enac 24 .
moreover , a number of signaling molecules , such as hydrogen sulfide , nitric oxide , utp , and cpt - cgmp regulate enac activity in h441 cells 25 - 27 , and respiratory syncytial virus can inhibit enac - mediated alveolar fluid clearance by upregulating the synthesis of utp and nitric oxide 28 .
phosphorylation of erk1/2 results in a decrease in the expression and function of enac in atii cells 29 .
nedd4 - 2 facilitates the effects of pkc on enac activity in atii 29 and club cells 24 , and pkc may also play a role in enac - pip2-marcks complexes , which regulate the open probability of enac and can be stabilized by binding with tnf 18 , another factor that may also enhance enac activity in atii cells 30 , 31 .
pkc , camp / pi3k , pka and cgmp mediate the regulation of enac by lps in atii and club cells 32 , 33 , and pkc was shown to mediate wnt signaling , which regulates the differentiation of mesenchymal stem cells ( mscs ) to atii cells and of atii to ati cells 34 .
additionally , cgmp levels were inversely related with the expression of atii markers when undifferentiated lung epithelial cells were treated with inhaled nitric oxide 35 .
therefore , regulation of enac by erk , pkc , camp , pka , and tnf may play a role in the differentiation of endogenous progenitor / stem cells .
whenever injury occurs , the epithelial stem / progenitor cells go through essentially the same process , including migrating to the injured region to cover the denuded airway and alveolar sac , and proliferating vigorously to provide enough cells for epithelium repair , differentiation , and remodeling , and finally the normal airways and lungs are regenerated structurally and functionally .
for example , in naphthalene - induced airway epithelial injury , bascs exhibit highly proliferative activity in response to the injury of club cells 36 .
methylation of enac is an important event while aldosterone promotes the wound healing in bewo cells and other epithelial cells .
mechanistically , migration of cultured epithelial and nonepithelial cells occurs in an enac - dependent manner 37 , 38 , and serum- and glucocorticoid - induced kinase 1 ( sgk1 ) regulates cell proliferation through an enac - associated process .
cell migration generally undergoes several processes , including depolarization , membrane elongation , adhesion , contraction , and de - adhesion , that are regulated by interactions between cells , and between cells and the extracellular matrix .
chifflet et al . reported that actin reorganization and membrane depolarization depend on enac - regulated extracellular na ions during wound healing of bovine corneal endothelial cells 39 .
enac proteins are the central part of a complex that links the cytoskeleton with the extracellular matrix 40 , and the binding of the c - termini of - and -enac with filamins exerts an inhibitory effect on enac function 41 .
found that a deficiency in enac caused more d54-mg cells to arrest in g0/g1 phase , with fewer cells accumulating in the s and g2/m phases 43 ; it was suggested that cell division is depressed when asic1 and enac are inhibited and that phosphorylation of erk1/2 may be an underlying mechanism 43 .
this group also found that interactions between amiloride - sensitive cation channels ( asic1 and enac ) and integrin-1 , mediated by -actinin , could in part regulate the proliferation and migration of glioma cells 44 .
in addition , enac is involved in the proliferation and migration of various cancer cells 38 .
the effects of enac on osteoblast differentiation have been studied by several groups who found that the expression of enac mrna accompanied the osteoclastogenesis of rat osteoblasts .
stimulation of osteoblast differentiation by 8-pcpt - cgmp is also dependent on the expression of enac 45 and therefore , enac activity is apparently required for the differentiation of both osteoblasts and osteoclasts . in club and atii cells
, the aberrance of enac expression usually leads to na absorption disorder , hydropic degeneration and necrosis of club cells , goblet cell metaplasia , failure of airway mucus clearance , susceptibility to spontaneous bacterial infection , airway inflammation , and even death caused by airway obstruction and asphyxiation 24 .
enac proteins are electrically detectable as functional biomarkers of differentiated epithelial cells . because of the role of enac in the migration and proliferation of stem / progenitor cells
, it can be speculated that normal enac function would be critical in the repair of injured lungs by mscs 46 , 47 .
goolaerts and colleagues reported that impaired enac activity under hypoxic and cytomixic conditions was restored by co - cultured mscs and paracrine kgf 48 .
moreover , impaired enac function of alveolar fluid clearance was recovered by human mscs delivered intratracheally in a clinically related , human lung injury model 49 .
the improvement of enac function and the contribution of enac to re - epithelialization potentially explain the promising results of clinical trials that show a significant reduction in lung injury score in acute respiratory distress syndrome ( ards ) and other lung injury treated with stem cells 50 - 53 .
lung injury is associated with defective epithelium and dysfunctional ion transport , and stem cell therapy to repair injured tissue has broadened the prospects for treatment beyond supportive approaches .
the functional consequences of normalizing injured epithelium are usually evaluated by detecting the expression and activity of enac . however , our understanding of the mechanisms by which enac regulates differentiation of lung stem / progenitor cells is incomplete , for example , whether enac contributes to the release of paracrines from allogeneic mscs , and what are the roles of enac in the re - epithelialization mediated by these paracrines .
 regeneration of the epithelium of mammalian lungs is essential for restoring normal function following injury , and various cells and mechanisms contribute to this regeneration and repair . 
 club cells , bronchioalveolar stem cells ( bascs ) , and alveolar type ii epithelial cells ( atii ) are dominant stem / progenitor cells for maintaining epithelial turnover and repair . 
 epithelial na+ channels ( enac ) , a critical pathway for transapical salt and fluid transport , are expressed in lung epithelial progenitors , including club and atii cells . 
 since enac activity and expression are development- and differentiation - dependent , apically located enac activity has therefore been used as a functional biomarker of lung injury repair . 
 enac activity may be involved in the migration and differentiation of local and circulating stem / progenitor cells with diverse functions , eventually benefiting stem cells spreading to re - epithelialize injured lungs . 
 this review summarizes the potential roles of enac expressed in native progenitor and stem cells in the development and regeneration of the respiratory epithelium . 
Introduction
Stem/progenitor cells for pulmonary epithelium
Expression of ENaC in pulmonary epithelial stem/progenitor cells
Regulation of ENaC in pulmonary epithelial stem/progenitor cells
ENaC-mediated epithelial repair
ENaC as a biomarker for injury repair
Future Perspective