Carole Planès

Defective ENaC Processing and Function in Tissue Kallikrein-deficient Mice

An inverse relationship exists between urinary tissue kallikrein (TK) excretion and blood pressure in humans and rodents. In the kidney TK is synthesized in large amounts in the connecting tubule and is mainly released into the urinary fluid where its function remains unknown. In the present study mice with no functional gene coding for TK (TK–/–) were used to test whether the enzyme regulates apically expressed sodium transporters. Semiquantitative immunoblotting of the renal cortex revealed an absence of the 70-kDa form of γ-ENaC in TK–/– mice. Urinary Na+ excretion after amiloride injection was blunted in TK–/– mice, consistent with reduced renal ENaC activity. Amiloride-sensitive transepithelial potential difference in the colon, where TK is also expressed, was decreased in TK–/– mice, whereas amiloride-sensitive alveolar fluid clearance in the lung, where TK is not expressed, was unchanged. In mice lacking the B2 receptor for kinins, the abundance of the 70-kDa form of γ-ENaC was increased, indicating that its absence in TK–/– mice is not kinin-mediated. Incubation of membrane proteins from renal cortex of TK–/– mice with TK resulted in the appearance of the 70-kDa band of the γ-ENaC, indicating that TK was able to promote γ-ENaC cleavage in vitro. Finally, in mouse cortical collecting ducts isolated and microperfused in vitro, the addition of TK in the luminal fluid increased significantly intracellular Na+ concentration, consistent with an activation of the luminal entry of the cation. The results demonstrate that TK, like several other proteases, can activate ENaC in the kidney and the colon.

Regulation of the Epithelial Na+ Channel by Peptidases

Recent investigations point to an important role for peptidases in regulating transcellular ion transport by the epithelial Na(+) channel, ENaC. Several peptidases, including furins and proteasomal hydrolases, modulate ENaC maturation and disposal. More idiosyncratically, apical Na(+) transport by ENaC in polarized epithelia of kidney, airway, and gut is stimulated constitutively by one or more trypsin-family serine peptidases, as revealed by inhibition of amiloride-sensitive Na(+) transport by broad-spectrum antipeptidases, including aprotinin and bikunin/SPINT2. In vitro, the transporting activity of aprotinin-suppressed ENaC can be restored by exposure to trypsin. The prototypical channel-activating peptidase (CAP) is a type 1 membrane-anchored tryptic peptidase first identified in Xenopus kidney cells. Frog CAP1 strongly upregulates Na(+) transport when coexpressed with ENaC in oocytes. The amphibian enzymes apparent mammalian orthologue is prostasin, otherwise known as CAP1, which is coexpressed with ENaC in a variety of epithelia. In airway cells, prostasin is the major basal regulator of ENaC activity, as suggested by inhibition and knockdown experiments. Other candidate regulators of mature ENaC include CAP2/TMPRSS4 and CAP3/matriptase (also known as membrane-type serine protease 1/ST14). Mammalian CAPs are potential targets for treatment of ENaC-mediated Na(+) hyperabsorption by the airway in cystic fibrosis (CF) and by the kidney in hypertension. CAPs can be important for mammalian development, as indicated by embryonic lethality in mice with null mutations of CAP1/prostasin. Mice with selectively knocked out expression of CAP1/prostasin in the epidermis and mice with globally knocked out expression of CAP3/matriptase exhibit phenotypically similar defects in skin barrier function and neonatal death from dehydration. In rats, transgenic overexpression of human prostasin disturbs salt balance and causes hypertension. Thus, several converging lines of evidence indicate that ENaC function is regulated by peptidases, and that such regulation is critical for embryonic development and adult function of organs such as skin, kidney, and lung.

ENaC-mediated alveolar fluid clearance and lung fluid balance depend on the channel-activating protease 1

Sodium transport via epithelial sodium channels (ENaC) expressed in alveolar epithelial cells (AEC) provides the driving force for removal of fluid from the alveolar space. The membrane‐bound channel‐activating protease 1 (CAP1/Prss8) activates ENaC in vitro in various expression systems. To study the role of CAP1/Prss8 in alveolar sodium transport and lung fluid balance in vivo, we generated mice lacking CAP1/Prss8 in the alveolar epithelium using conditional Cre‐loxP‐mediated recombination. Deficiency of CAP1/Prss8 in AEC induced in vitro a 40% decrease in ENaC‐mediated sodium currents. Sodium‐driven alveolar fluid clearance (AFC) was reduced in CAP1/Prss8‐deficient mice, due to a 48% decrease in amiloride‐sensitive clearance, and was less sensitive to β2‐agonist treatment. Intra‐alveolar treatment with neutrophil elastase, a soluble serine protease activating ENaC at the cell surface, fully restored basal AFC and the stimulation by β2‐agonists. Finally, acute volume‐overload increased alveolar lining fluid volume in CAP1/Prss8‐deficient mice. This study reveals that CAP1 plays a crucial role in the regulation of ENaC‐mediated alveolar sodium and water transport and in mouse lung fluid balance.

Gene regulation in the adaptive process to hypoxia in lung epithelial cells

Lung alveolar epithelial cells are normally very well oxygenated but may be exposed to hypoxia in many pathological conditions such as pulmonary edema, acute respiratory distress syndrome, chronic obstructive pulmonary diseases, or in some environmental conditions such ascent to high altitude. The ability of alveolar epithelial cells to cope with low oxygen tensions is crucial to maintain the structural and functional integrity of the alveolar epithelium. Alveolar epithelial cells appear to be remarkably tolerant to oxygen deprivation as they are able to maintain adequate cellular ATP content during prolonged hypoxic exposure when mitochondrial oxidative phosphorylation is limited. This property mostly relies on the ability of the cells to rapidly modify their gene expression program, stimulating the expression of genes involved in anaerobic energy supply and repressing expression of genes involved in some ATP-consuming cellular processes. This adaptive strategy of the cells is mostly, but not entirely, dependent on the expression of hypoxia-inducible factors (HIFs), known to be responsible for orchestrating a large number of hypoxia-sensitive genes. This review focuses on the role of HIF isoforms expressed in alveolar epithelial cells exposed to hypoxia and on the specific hypoxic gene regulation that takes place in alveolar epithelial cells either through HIF-dependent or -independent pathways.

Importance of ENaC-mediated sodium transport in alveolar fluid clearance using genetically-engineered mice.

The lung possesses specific transport systems that intra- and extracellularly maintain salt and fluid balance necessary for its function. At birth, the lungs rapidly transform into a fluid (Na+)-absorbing organ to enable efficient gas exchange. Alveolar fluid clearance, which mainly depends on sodium transport in alveolar epithelial cells, is an important mechanism by which excess water in the alveoli is reabsorbed during the resolution of pulmonary edema. In this review, we will focus and summarize on the role of ENaC in alveolar lung liquid clearance and discuss recent data from mouse models with altered activity of epithelial sodium channel function in the lung, and more specifically in alveolar fluid clearance. Recent data studying mice with hyperactivity of ENaC or mice with reduced ENaC activity clearly illustrate the impaired lung fluid clearance in these adult mice. Further understanding of the physiological role of ENaC and its regulatory proteins implicated in salt and water balance in the alveolar cells may therefore help to develop new therapeutic strategies to improve gas exchange in pulmonary edema.

Conditioned media from mesenchymal stromal cells restore sodium transport and preserve epithelial permeability in an in vitro model of acute alveolar injury

Mesenchymal stromal cells (MSCs) or their media (MSC-M) were reported to reverse acute lung injury (ALI)-induced decrease of alveolar fluid clearance. To determine the mechanisms by which MSC-M exert their beneficial effects, an in vitro model of alveolar epithelial injury was created by exposing primary rat alveolar epithelial cells (AECs) to hypoxia (3% O2) plus cytomix, a combination of IL-1β, TNF-α, and IFN-γ. MSC-M were collected from human MSCs exposed for 12 h to either normoxia (MSC-M) or to hypoxia plus cytomix (HCYT-MSC-M). This latter condition was used to model the effect of alveolar inflammation and hypoxia on paracrine secretion of MSCs in the injured lung. Comparison of paracrine soluble factors in MSC media showed that the IL-1 receptor antagonist and prostaglandin E2 were markedly increased while keratinocyte growth factor (KGF) was twofold lower in HCYT-MSC-M compared with MSC-M. In AECs, hypoxia plus cytomix increased protein permeability, reduced amiloride-sensitive short-circuit current (AS-Isc), and also decreased the number of α-epithelial sodium channel (α-ENaC) subunits in the apical membrane. To test the effects of MSC media, MSC-M and HCYT-MSC-M were added for an additional 12 h to AECs exposed to hypoxia plus cytomix. MSC-M and HCYT-MSC-M completely restored epithelial permeability to normal. MSC-M, but not HCYT-MSC-M, significantly prevented the hypoxia plus cytomix-induced decrease of ENaC activity and restored apical α-ENaC channels. Interestingly, KGF-deprived MSC-M were unable to restore amiloride-sensitive sodium transport, indicating a possible role for KGF in the beneficial effect of MSC-M. These results indicate that MSC-M may be a preferable therapeutic option for ALI.

β‐Liddle mutation of the epithelial sodium channel increases alveolar fluid clearance and reduces the severity of hydrostatic pulmonary oedema in mice

Transepithelial sodium transport via alveolar epithelial Na+ channels and Na+,K+‐ATPase constitutes the driving force for removal of alveolar oedema fluid. Decreased activity of the amiloride‐sensitive epithelial Na+ channel (ENaC) in the apical membrane of alveolar epithelial cells impairs sodium‐driven alveolar fluid clearance (AFC) and predisposes to pulmonary oedema. We hypothesized that hyperactivity of ENaC in the distal lung could improve AFC and facilitate the resolution of pulmonary oedema. AFC and lung fluid balance were studied at baseline and under conditions of hydrostatic pulmonary oedema in the β‐Liddle (L) mouse strain harbouring a gain‐of‐function mutation (R566stop) within the Scnn1b gene. As compared with wild‐type (+/+), baseline AFC was increased by 2‐ and 3‐fold in heterozygous (+/L) and homozygous mutated (L/L) mice, respectively, mainly due to increased amiloride‐sensitive AFC. The β2‐agonist terbutaline stimulated AFC in +/+ and +/L mice, but not in L/L mice. Acute volume overload induced by saline infusion (40% of body weight over 2 h) significantly increased extravascular (i.e. interstitial and alveolar) lung water as assessed by the bloodless wet‐to‐dry lung weight ratio in +/+ and L/L mice, as compared with baseline. However, the increase was significantly larger in +/+ than in L/L groups (P= 0.01). Volume overload also increased the volume of the alveolar epithelial lining fluid in +/+ mice, indicating the presence of alveolar oedema, but not in L/L mice. Cardiac function as evaluated by echocardiography was comparable in both groups. These data show that constitutive ENaC activation improved sodium‐driven AFC in the mouse lung, and attenuated the severity of hydrostatic pulmonary oedema.

Mutations of the Serine Protease CAP1/Prss8 Lead to Reduced Embryonic Viability, Skin Defects, and Decreased ENaC Activity

CAP1/Prss8 is a membrane-bound serine protease involved in the regulation of several different effectors, such as the epithelial sodium channel ENaC, the protease-activated receptor PAR2, the tight junction proteins, and the profilaggrin polypeptide. Recently, the V170D and the G54-P57 deletion mutations within the CAP1/Prss8 gene, identified in mouse frizzy (fr) and rat hairless (fr(CR)) animals, respectively, have been proposed to be responsible for their skin phenotypes. In the present study, we analyzed those mutations, revealing a change in the protein structure, a modification of the glycosylation state, and an overall reduction in the activation of ENaC of the two mutant proteins. In vivo analyses demonstrated that both fr and fr(CR) mutant animals present analogous reduction of embryonic viability, similar histologic aberrations at the level of the skin, and a significant decrease in the activity of ENaC in the distal colon compared with their control littermates. Hairless rats additionally had dehydration defects in skin and intestine and significant reduction in the body weight. In conclusion, we provided molecular and functional evidence that CAP1/Prss8 mutations are accountable for the defects in fr and fr(CR) animals, and we furthermore demonstrate a decreased function of the CAP1/Prss8 mutant proteins. Therefore, fr and fr(CR) animals are suitable models to investigate the consequences of CAP1/Prss8 action on its target proteins in the whole organism.

Hypoxia-Induced Inhibition of Epithelial Na+ Channels in the Lung. Role of Nedd4-2 and the Ubiquitin-Proteasome Pathway

Transepithelial sodium transport via alveolar epithelial Na(+) channels (ENaC) and Na(+),K(+)-ATPase constitutes the driving force for removal of alveolar edema fluid. Alveolar hypoxia associated with pulmonary edema may impair ENaC activity and alveolar Na(+) absorption through a decrease of ENaC subunit expression at the apical membrane of alveolar epithelial cells (AECs). Here, we investigated the mechanism(s) involved in this process in vivo in the β-Liddle mouse strain mice carrying a truncation of β-ENaC C-terminus abolishing the interaction between β-ENaC and the ubiquitin protein-ligase Nedd4-2 that targets the channel for endocytosis and degradation and in vitro in rat AECs. Hypoxia (8% O2 for 24 h) reduced amiloride-sensitive alveolar fluid clearance by 69% in wild-type mice but had no effect in homozygous mutated β-Liddle littermates. In vitro, acute exposure of AECs to hypoxia (0.5-3% O2 for 1-6 h) rapidly decreased transepithelial Na(+) transport as assessed by equivalent short-circuit current Ieq and the amiloride-sensitive component of Na(+) current across the apical membrane, reflecting ENaC activity. Hypoxia induced a decrease of ENaC subunit expression in the apical membrane of AECs with no change in intracellular expression and induced a 2-fold increase in α-ENaC polyubiquitination. Hypoxic inhibition of amiloride-sensitive Ieq was fully prevented by preincubation with the proteasome inhibitors MG132 and lactacystin or with the antioxidant N-acetyl-cysteine. Our data strongly suggest that Nedd4-2-mediated ubiquitination of ENaC leading to endocytosis and degradation of apical Na(+) channels is a key feature of hypoxia-induced inhibition of transepithelial alveolar Na(+) transport.

Modulation of Epithelial Sodium Channel Trafficking and Function by Sodium 4-Phenylbutyrate in Human Nasal Epithelial Cells

Sodium 4-phenylbutyrate (4-PBA) has been shown to correct the cellular trafficking of several mutant or nonmutant plasma membrane proteins such as cystic fibrosis transmembrane conductance regulator through the expression of 70-kDa heat shock proteins. The objective of the study was to determine whether 4-PBA may influence the functional expression of epithelial sodium channels (ENaC) in human nasal epithelial cells (HNEC). Using primary cultures of HNEC, we demonstrate that 4-PBA (5 mm for 6 h) markedly stimulated amiloride-sensitive sodium channel activity and that this was related to an increased abundance of α-, β-, and γ-ENaC subunits in the apical membrane. The increase in ENaC cell surface expression (i) was due to insertion of newly ENaC subunits as determined by brefeldin A experiments and (ii) was not associated with cell surface retention of ENaC subunits because endocytosis of ENaC subunits was unchanged. In addition, we find that ENaC co-immunoprecipitated with the heat shock protein constituvely expressed Hsc70, that has been reported to modulate ENaC trafficking, and that 4-PBA decreased Hsc70 protein level. Finally, we report that in cystic fibrosis HNEC obtained from two cystic fibrosis patients, 4-PBA increased functional expression of ENaC as demonstrated by the increase in amiloride-sensitive sodium transport and in α-, β-, and γ-ENaC subunit expression in the apical membrane. Our results suggest that in HNEC, 4-PBA increases the functional expression of ENaC through the insertion of new α-, β-, and γ-ENaC subunits into the apical membrane and also suggest that 4-PBA could modify ENaC trafficking by reducing Hsc70 protein expression.