Regulation of sodium transport in mammalian collecting duct cells by aldosterone-induced kinase, SGK1: structure/function studies

Serum- and glucocorticoid-induced kinases (SGK) are members of the serine-threonine kinase family. SGK1, the isoform identified first, is rapidly induced by aldosterone. In this study, we determined that the two recently described isoforms, SGK2 and SGK3 are also expressed in renal cortical collecting duct (CCD) cells; however, their expression is not induced by aldosterone or glucocorticoids. SGK1 increases the activity of the epithelial sodium channel (ENaC) in oocytes but its cellular targets in native mineralocorticoid target cells and its mechanism of action are still unknown. We studied the role of SGK1 in corticosteroid-regulated Na transport in M-1 mouse CCD cell lines that stably over-express or down-regulate SGK1. Basal rates of transepithelial Na transport were significantly lower in CCD cells in which SGK1 expression or activity was down-regulated than in SGK1 overexpressing cells. Importantly, corticosteroid treatment failed to stimulate Na transport in cells with down-regulated SGK1 while it significantly increased Na transport in parent and SGK1 overexpressing M-1 cells. To determine if C-terminal PDZ interactions are important for SGK's effect on ENaC activity or trafficking, we examined the effects of mutant SGK1 in which the conserved PDZ binding domain has been eliminated. However, such mutations did not decrease its stimulatory effect on ENaC current in Xenopus oocytes. Fluorescence confocal microscopy revealed that the intracellular localization of full-length and PDZ binding mutated SGK1 was identical: they both localize to intracellular vesicular structures. On the other hand, N-terminally truncated (delta 60)-SGK1 did not increase ENaC activity. We conclude that SGK1 is a critical component in corticosteroid-regulated Na transport in mammalian CCD cells. Our data also indicate that the N-terminal of SGK1 is necessary for its stimulatory effect on Na transport while elimination of the C-terminal PDZ binding domain did not change its function.     

Collecting Duct Principal Cell Transport Processes and Their Regulation

The principal cell of the kidney collecting duct is one of the most highly regulated epithelial cell types in vertebrates. The effects of hormonal, autocrine, and paracrine factors to regulate principal cell transport processes are central to the maintenance of fluid and electrolyte balance in the face of wide variations in food and water intake. In marked contrast with the epithelial cells lining the proximal tubule, the collecting duct is electrically tight, and ion and osmotic gradients can be very high. The central role of principal cells in salt and water transport is reflected by their defining transporters—the epithelial Na⁺ channel (ENaC), the renal outer medullary K⁺ channel, and the aquaporin 2 (AQP2) water channel. The coordinated regulation of ENaC by aldosterone, and AQP2 by arginine vasopressin (AVP) in principal cells is essential for the control of plasma Na⁺ and K⁺ concentrations, extracellular fluid volume, and BP. In addition to these essential hormones, additional neuronal, physical, and chemical factors influence Na⁺, K⁺, and water homeostasis. Notably, a variety of secreted paracrine and autocrine agents such as bradykinin, ATP, endothelin, nitric oxide, and prostaglandin E₂ counterbalance and limit the natriferic effects of aldosterone and the water-retaining effects of AVP. Considerable recent progress has improved our understanding of the transporters, receptors, second messengers, and signaling events that mediate principal cell responses to changing environments in health and disease. This review primarily addresses the structure and function of the key transporters and the complex interplay of regulatory factors that modulate principal cell ion and water transport.     

Liddle's syndrome mutations increase Na+ transport through dual effects on epithelial Na+ channel surface expression and proteolytic cleavage

Liddle's syndrome, an inherited form of hypertension, is caused by mutations that delete or disrupt a C-terminal PY motif in the epithelial Na+ channel (ENaC). Previous work indicates that these mutations increase expression of ENaC at the cell surface by disrupting its binding to Nedd4-2, an E3 ubiquitin-protein ligase that targets ENaC for degradation. However, it remains uncertain whether this mechanism alone is responsible; increased activity of ENaC channels could also contribute to excessive Na+ transport in Liddle's syndrome. ENaC activity is controlled in part by its cleavage state; proteolytic cleavage produces channels with a high open-state probability, whereas uncleaved channels are inactive. Here, we found that Liddle's syndrome mutations have two distinct effects of ENaC surface expression, both of which contribute to increased Na+ transport. First, these mutations increased ENaC expression at the cell surface; second, they increased the fraction of ENaC at the cell surface that was cleaved (active). This disproportionate increase in cleavage was reproduced by expression of a dominant-negative Nedd4-2 or mutation of ENaC ubiquitination sites, interventions that disrupt ENaC endocytosis and lysosomal degradation. Conversely, overexpression of Nedd4-2 had the opposite effect, decreasing the fraction of cleaved ENaC at the cell surface. Thus, the data not only suggest that Nedd4-2 regulates epithelial Na+ transport in part by controlling the relative expression of cleaved and uncleaved ENaC at the cell surface but also provide a mechanism by which Liddle's syndrome mutations alter ENaC activity.     

The epithelial sodium channel in hypertension

Our understanding of Na⁺ transport defects has exploded in the past several years, and has provided unique insights into epithelial transport processes, and unusual clinical syndromes resulting from mutations of specific ion transporters. These genetic disorders affect Na⁺ balance, with both Na⁺ retaining and Na⁺ wasting conditions being the consequence. A major focus of these studies has been the epithelial sodium channel (ENaC), which can be directly affected by mutations (eg, Liddle syndrome, autosomal recessive pseudohypoaldosteronism, type I) or by changes in the response to (autosomal recessive pseudohypoaldosteronism, type I), or production of mineralocorticoids (apparent mineralocorticoid excess syndrome, glucocorticoid-remediable aldosteronism). As a result, we now have clearly defined syndromes in which ENaC activity is dysregulated with subsequent development of disorders of systemic blood pressure that can be attributed to a primary renal mechanism.     

TGF-β directs trafficking of the epithelial sodium channel ENaC which has implications for ion and fluid transport in acute lung injury

TGF-β is a pathogenic factor in patients with acute respiratory distress syndrome (ARDS), a condition characterized by alveolar edema. A unique TGF-β pathway is described, which rapidly promoted internalization of the αβγ epithelial sodium channel (ENaC) complex from the alveolar epithelial cell surface, leading to persistence of pulmonary edema. TGF-β applied to the alveolar airspaces of live rabbits or isolated rabbit lungs blocked sodium transport and caused fluid retention, which—together with patch-clamp and flow cytometry studies—identified ENaC as the target of TGF-β. TGF-β rapidly and sequentially activated phospholipase D1, phosphatidylinositol-4-phosphate 5-kinase 1α, and NADPH oxidase 4 (NOX4) to produce reactive oxygen species, driving internalization of βENaC, the subunit responsible for cell-surface stability of the αβγENaC complex. ENaC internalization was dependent on oxidation of βENaC Cys⁴³. Treatment of alveolar epithelial cells with bronchoalveolar lavage fluids from ARDS patients drove βENaC internalization, which was inhibited by a TGF-β neutralizing antibody and a Tgfbr1 inhibitor. Pharmacological inhibition of TGF-β signaling in vivo in mice, and genetic ablation of the nox4 gene in mice, protected against perturbed lung fluid balance in a bleomycin model of lung injury, highlighting a role for both proximal and distal components of this unique ENaC regulatory pathway in lung fluid balance. These data describe a unique TGF-β–dependent mechanism that regulates ion and fluid transport in the lung, which is not only relevant to the pathological mechanisms of ARDS, but might also represent a physiological means of acutely regulating ENaC activity in the lung and other organs.The acute respiratory distress syndrome (ARDS) is a devastating syndrome characterized by alveolar flooding (edema), which impairs gas exchange, leading to respiratory failure (1). The high mortality rate of 35–45% observed in patients with ARDS and the lack of any pharmacological therapy (1) underscores the need to better understand the pathomechanisms of this lethal disease, in the hope of facilitating improved clinical management of affected patients.Alveolar edema occurs as a consequence of increased fluid influx into the alveolar airspaces from the vasculature, across the thin alveolo-capillary barrier (2), as well as a failure of transepithelial Na⁺ and Cl⁻ ion transport, which drives fluid clearance from the alveolar airspaces. Transepithelial sodium transport is undertaken by the concerted action of several ion transporters, namely the Na⁺/K⁺-ATPase (3) and the epithelial sodium channel (ENaC) (4, 5), which actively transport Na⁺ out of the fluid lining the alveolar airspaces (epithelial lining fluid, ELF). This process generates an osmotic gradient that clears water from the alveolar airspaces (6). This fluid clearance process is defective in ARDS patients with compromised alveolo-capillary barrier function, and it is widely believed that edema fluid must be cleared for patients with ARDS to survive (7, 8).TGF-β is a key mediator of acute lung injury (ALI), where TGF-β is activated locally by integrin α_vβ₆ (9) in cooperation with protease-activated receptor-1 (10), to increase epithelial and endothelial permeability and promote alveolar flooding. In further support of a role for TGF-β in ALI, two studies have demonstrated increased TGF-β levels in lung fluids from patients with ALI/ARDS (11, 12), and in these patients lower TGF-β levels correlate with more ventilator-free and intensive care unit-free days (11). Some evidence has also implicated TGF-β in transepithelial ion transport in vitro, where TGF-β down-regulated gene expression of one of three ENaC subunits (13), temporally modulated gene expression of the Na⁺/K⁺-ATPase (14), and impacted Cl⁻ transport (15). In animal models of ALI/ARDS, administration of a soluble type II TGF-β receptor, which sequesters free TGF-β, attenuated the degree of pulmonary edema (9), confirming a role for TGF-β in disturbed lung fluid dynamics associated with experimental ALI/ARDS, however, a role for TGF-β in regulating alveolar fluid reabsorption has not been established.In this study, a unique TGF-β signaling pathway is described, whereby TGF-β—acting through the Tgfbr1/Smad2/3 axis (16)—recruits phosphoinositide-metabolizing enzymes and an NADPH oxidase to generate reactive oxygen species (ROS), which drive αβγENaC complex internalization from the lung epithelial cell surface and, hence, block the sodium-transporting capacity of alveolar epithelial cells. Using animal and isolated organ models of edema resolution, we demonstrate that TGF-β, applied at clinically relevant doses, rapidly blocked the transepithelial ion fluxes necessary for alveolar fluid reabsorption, and indeed alveolar fluid reabsorption itself. Given the rapid onset and progression of ARDS and the critical role played by ENaC-mediated alveolar fluid clearance in the survival of ARDS patients, the pathway described here has important implications for the understanding of the pathological mechanisms that promote formation or persistence of alveolar edema in ARDS patients. This idea is highlighted by the findings reported here that identify TGF-β, exclusively, as the factor in the lung fluids of ARDS patients responsible for promoting loss of ENaC from the lung epithelial cell surface. In addition to revealing an entirely unique TGF-β signaling pathway that regulates ion channel trafficking, these data point to a pathway that may be amenable to pharmacological manipulation in patients with ARDS, a devastating and lethal syndrome for which no pharmacological therapy currently exists.     

Src-family protein tyrosine kinase phosphorylates WNK4 and modulates its inhibitory effect on KCNJ1 (ROMK)

With-no-lysine kinase 4 (WNK4) inhibits the activity of the potassium channel KCNJ1 (ROMK) in the distal nephron, thereby contributing to the maintenance of potassium homeostasis. This effect is inhibited via phosphorylation at Ser1196 by serum/glucocorticoid-induced kinase 1 (SGK1), and this inhibition is attenuated by the Src-family protein tyrosine kinase (SFK). Using Western blot and mass spectrometry, we now identify three sites in WNK4 that are phosphorylated by c-Src: Tyr¹⁰⁹², Tyr¹⁰⁹⁴, and Tyr¹¹⁴³, and show that both c-Src and protein tyrosine phosphatase type 1D (PTP-1D) coimmunoprecipitate with WNK4. Mutation of Tyr¹⁰⁹² or Tyr¹¹⁴³ to phenylalanine decreased the association of c-Src or PTP-1D with WNK4, respectively. Moreover, the Tyr1092Phe mutation markedly reduced ROMK inhibition by WNK4; this inhibition was completely absent in the double mutant WNK4^Y1092/1094F. Similarly, c-Src prevented SGK1-induced phosphorylation of WNK4 at Ser¹¹⁹⁶, an effect that was abrogated in the double mutant. WNK4^Y1143F inhibited ROMK activity as potently as wild-type (WT) WNK4, but unlike WT, the inhibitory effect of WNK4^Y1143F could not be reversed by SGK1. The failure to reverse WNK4^Y1143F-induced inhibition of ROMK by SGK1 was possibly due to enhancing endogenous SFK effect on WNK4 by decreasing the WNK4–PTP-1D association because inhibition of SFK enabled SGK1 to reverse WNK4^Y1143F-induced inhibition of ROMK. We conclude that WNK4 is a substrate of SFKs and that the association of c-Src and PTP-1D with WNK4 at Tyr¹⁰⁹² and Tyr¹¹⁴³ plays an important role in modulating the inhibitory effect of WNK4 on ROMK.With-no-lysine kinase 4 (WNK4) is expressed in the connecting tubule (CNT) and cortical collecting duct (CCD) (1, 2) and plays an important role in modulating the balance between renal K secretion and Na reabsorption (3–8). The effect of WNK4 on renal K secretion is partially mediated through inhibition of KCNJ1 (ROMK) channels in the CNT and in the CCD. ROMK inhibition is achieved by a stimulation of clathrin-mediated endocytosis (1), an effect that is dependent on intersectin, a scaffold protein containing two Eps15 homology domains (9).Serum/glucocorticoid-induced kinase 1 (SGK1), a downstream mediator of aldosterone signaling, suppresses the inhibitory effect of WNK4 on ROMK channels through phosphorylation of WNK4 at Ser¹¹⁶⁹ (2) and Ser¹¹⁹⁶ (5). Both volume depletion and high K intake increase aldosterone and SGK1 levels (10). However, it is not clear why a high K intake or volume depletion modulates differently the effect of SGK1 on ROMK channels.Candidate regulators of differential ROMK expression in hyperkalemia and hypovolemia should be regulated in a potassium-dependent manner. One such protein is the protein tyrosine kinase c-Src, whose expression in renal cortex is reduced in states of high potassium intake (11). We have previously demonstrated a key role of c-Src in determining the effect of SGK1 on WNK4 (12). C-Src abolishes SGK1-induced phosphorylation of WNK4 and restores the inhibitory effect of WNK4 on ROMK channels in the presence of SGK1 (13). This effect may play a role in preventing K secretion in the absence of hyperkalemia. High potassium intake, in contrast, will diminish c-Src levels, restore SGK1-induced phosphorylation of WNK4, and lead to increased renal potassium secretion via ROMK.Whereas protein phosphatase activity has been shown to be involved in c-Src–mediated modulation of the interaction between SGK1 and WNK4 (13), the molecular mechanism of c-Src’s interaction with WNK4 has been elusive. We here identify previously undescribed tyrosine phosphorylation sites in WNK4 that are targets of c-Src. We characterize the effects of tyrosine phosphorylation on the SGK1–WNK4 interaction, as well as WNK4-mediated ROMK inhibition.     

Minireview: regulation of epithelial Na+ channel trafficking

The epithelial Na(+) channel (ENaC) is a pathway for Na(+) transport across epithelia, including the kidney collecting duct, lung, and distal colon. ENaC is critical for Na(+) homeostasis and blood pressure control; defects in ENaC function and regulation are responsible for inherited forms of hypertension and hypotension and may contribute to the pathogenesis of cystic fibrosis and other lung diseases. An emerging theme is that epithelial Na(+) transport is regulated in large part through trafficking mechanisms that control ENaC expression at the cell surface. ENaC trafficking is regulated at multiple steps. Delivery of channels to the cell surface is regulated by aldosterone (and corticosteroids) and vasopressin, which increase ENaC synthesis and exocytosis, respectively. Conversely, endocytosis and degradation is controlled by a sequence located in the C terminus of alpha, beta, and gammaENaC (PPPXYXXL). This sequence functions as an endocytosis motif and as a binding site for Nedd4-2, an E3 ubiquitin protein ligase that targets ENaC for degradation. Mutations that delete or disrupt this motif cause accumulation of channels at the cell surface, resulting in Liddle's syndrome, an inherited form of hypertension. Nedd4-2 is a central convergence point for ENaC regulation by aldosterone and vasopressin; both induce phosphorylation of a common set of three Nedd4-2 residues, which blocks Nedd4-2 binding to ENaC. Thus, aldosterone and vasopressin regulate epithelial Na(+) transport in part by altering ENaC trafficking to and from the cell surface.     

Nongenomic Actions of Thyroid Hormone and Intracellular Calcium Metabolism

Nongenomic actions of thyroid hormone include several that involve or require calcium. Actions of thyroid hormone at the plasma or intracellular membranes include stimulation of membrane glucose transport and of the Na⁺/H⁺ antiporter (exchanger) by mechanisms that require liberation of intracellular calcium and stimulation of the cell membrane and sarcoplasmic reticulum calcium pumps (Ca²⁺-ATPases). These pumps not only transport Ca²⁺, but also are regulated by the intracellular calmodulin-Ca²⁺ complex (plasma membrane/sarcolemma) or calmodulin-dependent protein kinase II phosphorylation of phospholamban (sarcoplasmic reticulum). Intracellular calcium ion concentration may also be subject to regulation by other nongenomic effects of iodothyronines, such as those on the Na⁺/H⁺ antiporter or sodium current, that secondarily affect the Na⁺/Ca²⁺ exchanger. Certain of these nongenomic actions of thyroid hormone, e.g., Na⁺/H⁺ exchanger, Ca²⁺-ATPase, are now recognized to begin at a recently described hormone receptor on a heterodimeric structural membrane protein, integrin αvβ3. The thyroid hormone signal at this receptor is further transduced by the mitogen-activated protein kinase (MAPK; extracellular regulated kinase1/2, ERK1/2) pathway.     

A mouse model for the renal salt-wasting syndrome pseudohypoaldosteronism

Aldosterone-dependent epithelial sodium transport in the distal nephron is mediated by the absorption of sodium through the highly selective, amiloride-sensitive epithelial sodium channel (ENaC) made of three homologous subunits (α, β, and γ). In human, autosomal recessive mutations of α, β, or γENaC subunits cause pseudohypoaldosteronism type 1 (PHA-1), a renal salt-wasting syndrome characterized by severe hypovolemia, high plasma aldosterone, hyponatremia, life-threatening hyperkaliemia, and metabolic acidosis. In the mouse, inactivation of αENaC results in failure to clear fetal lung liquid at birth and in early neonatal death, preventing the observation of a PHA-1 renal phenotype. Transgenic expression of αENaC driven by a cytomegalovirus promoter in αENaC(−/−) knockout mice [αENaC(−/−)Tg] rescued the perinatal lethal pulmonary phenotype and partially restored Na⁺ transport in renal, colonic, and pulmonary epithelia. At days 5–9, however, αENaC(−/−)Tg mice showed clinical features of severe PHA-1 with metabolic acidosis, urinary salt-wasting, growth retardation, and 50% mortality. Adult αENaC(−/−)Tg survivors exhibited a compensated PHA-1 with normal acid/base and electrolyte values but 6-fold elevation of plasma aldosterone compared with wild-type littermate controls. We conclude that partial restoration of ENaC-mediated Na⁺ absorption in this transgenic mouse results in a mouse model for PHA-1.     

From the Cover: PNAS Plus: Chloride transport-driven alveolar fluid secretion is a major contributor to cardiogenic lung edema

Alveolar fluid clearance driven by active epithelial Na⁺ and secondary Cl⁻ absorption counteracts edema formation in the intact lung. Recently, we showed that impairment of alveolar fluid clearance because of inhibition of epithelial Na⁺ channels (ENaCs) promotes cardiogenic lung edema. Concomitantly, we observed a reversal of alveolar fluid clearance, suggesting that reversed transepithelial ion transport may promote lung edema by driving active alveolar fluid secretion. We, therefore, hypothesized that alveolar ion and fluid secretion may constitute a pathomechanism in lung edema and aimed to identify underlying molecular pathways. In isolated perfused lungs, alveolar fluid clearance and secretion were determined by a double-indicator dilution technique. Transepithelial Cl⁻ secretion and alveolar Cl⁻ influx were quantified by radionuclide tracing and alveolar Cl⁻ imaging, respectively. Elevated hydrostatic pressure induced ouabain-sensitive alveolar fluid secretion that coincided with transepithelial Cl⁻ secretion and alveolar Cl⁻ influx. Inhibition of either cystic fibrosis transmembrane conductance regulator (CFTR) or Na⁺-K⁺-Cl⁻ cotransporters (NKCC) blocked alveolar fluid secretion, and lungs of CFTR^−/− mice were protected from hydrostatic edema. Inhibition of ENaC by amiloride reproduced alveolar fluid and Cl⁻ secretion that were again CFTR-, NKCC-, and Na⁺-K⁺-ATPase–dependent. Our findings show a reversal of transepithelial Cl⁻ and fluid flux from absorptive to secretory mode at hydrostatic stress. Alveolar Cl⁻ and fluid secretion are triggered by ENaC inhibition and mediated by NKCC and CFTR. Our results characterize an innovative mechanism of cardiogenic edema formation and identify NKCC1 as a unique therapeutic target in cardiogenic lung edema.Traditionally, the formation of cardiogenic pulmonary edema has been attributed to passive fluid filtration across an intact alveolocapillary barrier along an increased hydrostatic pressure gradient. However, recent studies show that cardiogenic edema is critically regulated by active signaling processes. Activation of mechanosensitive endothelial ion channels increases lung vascular permeability (1), whereas alveolar epithelial cells lose their physiological ability to clear the distal airspaces from excess fluid by their capacity to actively transport ions across the epithelial barrier (2–4).In the intact lung, the predominant force driving alveolar fluid clearance is an active transepithelial Na⁺ transport from the alveolar into the interstitial space. A major portion of the apical Na⁺ entry is mediated by the amiloride-inhibitable epithelial Na⁺ channel (ENaC), with basolateral Na⁺ extrusion through the Na⁺-K⁺-ATPase (5). Cl⁻ and water are considered to follow paracellularly for electroneutrality and osmotic balance. In cardiogenic lung edema, the physiological protection against alveolar flooding provided by an intact alveolar fluid clearance is largely attenuated (3, 4). Previously, we have outlined the signaling events at the alveolocapillary barrier that underlie this inhibition of alveolar fluid clearance by showing that hydrostatic stress increases endothelial NO production in lung capillaries (6), which in turn, blocks alveolar Na⁺ and liquid absorption by a cGMP-dependent inhibition of epithelial ENaC (2).Unexpectedly, however, we observed that increased hydrostatic pressure not only blocks alveolar fluid clearance but reverses transepithelial fluid transport, resulting in effective alveolar fluid secretion that accounts for up to 70% of the total alveolar fluid influx at elevated hydrostatic pressure (2). This effect is not explicable by impaired alveolar fluid clearance and/or passive fluid leakage, and thus, it points to a previously unrecognized and potentially therapeutically exploitable pathomechanism in cardiogenic lung edema, namely alveolar fluid secretion driven by active transepithelial ion transport.Here, we aimed to analyze alveolar fluid secretion and its underlying cellular mechanisms in cardiogenic lung edema. We considered the Cl⁻ channel cystic fibrosis transmembrane conductance regulator (CFTR) as a putative key ion channel in this scenario, because it permits bidirectional permeation of anions under physiologically relevant conditions (7). Hence, the direction of Cl⁻ flux by CFTR may reverse depending on actual electrochemical gradients, thus turning an absorptive into a secretory epithelium or vice versa. This notion is supported by reports describing CFTR as both an absorptive and secretory channel in the regulation of alveolar fluid homeostasis (8, 9). By a combination of indicator dilution, imaging, and radioactive tracer techniques for the measurement of alveolar ion and fluid fluxes in the isolated lung, we show a critical role for CFTR-mediated Cl⁻ secretion in cardiogenic lung edema and identify the Na⁺-K⁺-2Cl⁻ cotransporter 1 (NKCC1) as a therapeutic target in this pathology.     

Contribution of α7 nicotinic receptor to airway epithelium dysfunction under nicotine exposure

Loss or dysfunction of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) leads to impairment of airway mucus transport and to chronic lung diseases resulting in progressive respiratory failure. Nicotinic acetylcholine receptors (nAChRs) bind nicotine and nicotine-derived nitrosamines and thus mediate many of the tobacco-related deleterious effects in the lung. Here we identify α7 nAChR as a key regulator of CFTR in the airways. The airway epithelium in α7 knockout mice is characterized by a higher transepithelial potential difference, an increase of amiloride-sensitive apical Na⁺ absorption, a defective cAMP-dependent Cl⁻ conductance, higher concentrations of Na⁺, Cl⁻, K⁺, and Ca²⁺ in secretions, and a decreased mucus transport, all relevant to a deficient CFTR activity. Moreover, prolonged nicotine exposure mimics the absence of α7 nAChR in mice or its inactivation in vitro in human airway epithelial cell cultures. The functional coupling of α7 nAChR to CFTR occurs through Ca²⁺ entry and activation of adenylyl cyclases, protein kinase A, and PKC. α7 nAChR, CFTR, and adenylyl cyclase-1 are physically and functionally associated in a macromolecular complex within lipid rafts at the apical membrane of surface and glandular airway epithelium. This study establishes the potential role of α7 nAChR in the regulation of CFTR function and in the pathogenesis of smoking-related chronic lung diseases.     

SPLUNC1 regulates airway surface liquid volume by protecting ENaC from proteolytic cleavage

Many epithelia, including the superficial epithelia of the airways, are thought to secrete “volume sensors,” which regulate the volume of the mucosal lining fluid. The epithelial Na⁺ channel (ENaC) is often the rate limiting factor in fluid absorption, and must be cleaved by extracellular and/or intracellular proteases before it can conduct Na⁺ and absorb excess mucosal liquid, a process that can be blocked by proteases inhibitors. In the airways, airway surface liquid dilution or removal activates ENaC. Therefore, we hypothesized that endogenous proteases are membrane-anchored, whereas endogenous proteolysis inhibitors are soluble and can function as airway surface liquid volume sensors to inhibit ENaC activity. Using a proteomic approach, we identified short palate, lung, and nasal epithelial clone (SPLUNC)1 as a candidate volume sensor. Recombinant SPLUNC1 inhibited ENaC activity in both human bronchial epithelial cultures and Xenopus oocytes. Knockdown of SPLUNC1 by shRNA resulted in a failure of bronchial epithelial cultures to regulate ENaC activity and airway surface liquid volume, which was restored by adding recombinant SPLUNC1 to the airway surface liquid. Despite being able to inhibit ENaC, recombinant SPLUNC1 had little effect on extracellular serine protease activity. However, SPLUNC1 specifically bound to ENaC, preventing its cleavage and activation by serine proteases. SPLUNC1 is highly expressed in the airways, as well as in colon and kidney. Thus, we propose that SPLUNC1 is secreted onto mucosal surfaces as a soluble volume sensor whose concentration and dilution can regulate ENaC activity and mucosal volumes, including that of airway surface liquid.     

Molecular basis for pH-dependent mucosal dehydration in cystic fibrosis airways

The ability to maintain proper airway surface liquid (ASL) volume homeostasis is vital for mucus hydration and clearance, which are essential aspects of the mammalian lung’s innate defense system. In cystic fibrosis (CF), one of the most common life-threatening genetic disorders, ASL dehydration leads to mucus accumulation and chronic infection. In normal airways, the secreted protein short palate lung and nasal epithelial clone 1 (SPLUNC1) effectively inhibits epithelial Na⁺ channel (ENaC)-dependent Na⁺ absorption and preserves ASL volume. In CF airways, it has been hypothesized that increased ENaC-dependent Na⁺ absorption contributes to ASL depletion, and hence increased disease. However, this theory is controversial, and the mechanism for abnormal ENaC regulation in CF airways has remained elusive. Here, we show that SPLUNC1 is a pH-sensitive regulator of ENaC and is unable to inhibit ENaC in the acidic CF airway environment. Alkalinization of CF airway cultures prevented CF ASL hyperabsorption, and this effect was abolished when SPLUNC1 was stably knocked down. Accordingly, we resolved the crystal structure of SPLUNC1 to 2.8 Å. Notably, this structure revealed two pH-sensitive salt bridges that, when removed, rendered SPLUNC1 pH-insensitive and able to regulate ASL volume in acidic ASL. Thus, we conclude that ENaC hyperactivity is secondary to reduced CF ASL pH. Together, these data provide molecular insights into the mucosal dehydration associated with a range of pulmonary diseases, including CF, and suggest that future therapy be directed toward alkalinizing the pH of CF airways.The epithelial Na⁺ channel (ENaC) is the rate-determining step for Na⁺ absorption across the colon, kidney, and lung (1). ENaC is a heterotrimer consisting of α-, β-, and γ-subunits (2). The extracellular domains of the α- and γ-ENaC subunits must be proteolytically cleaved by serine proteases, such as trypsin or neutrophil elastase, or by intracellular furin-type convertases in order for the channel to become active and to conduct Na⁺ (2, 3). In contrast, the β-subunit is highly glycosylated and not cleaved but may form a regulatory subunit that governs ENaC surface densities (1, 4). In some cases, ENaC may bypass the steps required for proteolysis and be inserted into the plasma membrane as near-silent, inactive channels (5). Abnormal ENaC activity has been linked with the pathogenesis of several diseases, including cystic fibrosis (CF), Liddle syndrome, and salt-sensitive hypertension (6). In CF airways, the absence of functional cystic fibrosis transmembrane conductance regulator (CFTR) in the apical plasma membrane causes ENaC hyperactivity, and the resulting excessive Na⁺ absorption contributes to airway surface liquid (ASL) dehydration, mucus stasis, and bacterial infections (7). Similar lung disorders have been observed in transgenic mice either overexpressing β-ENaC or exhibiting altered regulation of ENaC by the ubiquitin protein ligase NEDD4L, thus linking Na⁺ hyperabsorption and ASL volume depletion to the development of pulmonary disease (4, 8). However, whether or not ENaC activity is up-regulated in CF airways is currently controversial (9). Part of this problem may lie in the lack of an identified mechanism for ENaC hyperactivity in CF airways. Thus, although ENaC has been shown to be up-regulated in vivo, in freshly isolated human airway tissues, and in cell culture, no mechanism for this up-regulation has been discovered (10–13).Normal but not CF airway cultures autoregulate ASL volume by coordinating CFTR and ENaC activity via soluble “reporter molecules” that, by virtue of their dilution and concentration, transmit information on ASL volume to the epithelia (14). The short palate lung and nasal epithelial clone 1 (SPLUNC1), the most abundant secreted protein in the airways, is one such reporter molecule and is absolutely required for limiting ENaC activity and maintaining normal ASL homeostasis (14, 15). We have previously shown that SPLUNC1 binds to ENaC, causing it to be internalized, thus protecting ENaC from proteolytic cleavage and activation (15). SPLUNC1 is also a secreted protein that has been proposed to share homology with the N-terminal domain of bacterial permeability-increasing protein (BPI) (16).Despite the presence of SPLUNC1 in CF ASL (17, 18), CF airway cultures are unable to regulate ENaC (19). Importantly, CF ENaC is not fully dysfunctional and is sensitive to exogenous protease inhibitors, suggesting that the defect lies elsewhere (19). CFTR conducts HCO₃⁻, which maintains ASL at near-neutral pH, and when CFTR is defective (20), the ASL becomes more acidic (21). Because SPLUNC1–ENaC interactions are extracellular, we hypothesized that an acidified ASL prevented SPLUNC1 from inhibiting ENaC. To test this hypothesis, we examined the relationship between SPLUNC1-dependent regulation of ENaC and the ASL pH. To explore this interaction further, we resolved the crystal structure of SPLUNC1 to ∼2.8 Å and used this structure to understand better how ENaC is regulated in CF airway cultures.     

Protein kinase Czeta mediated Raf-1/extracellular-regulated kinase activation by daunorubicin

In light of the emerging concept of a protective function of the mitogen-activated protein kinase (MAPK) pathway under stress conditions, we investigated the influence of the anthracycline daunorubicin (DNR) on MAPK signaling and its possible contribution to DNR-induced cytotoxicity. We show that DNR increased phosphorylation of extracellular-regulated kinases (ERKs) and stimulated activities of both Raf-1 and extracellular-regulated kinase 1 (ERK1) within 10 to 30 minutes in U937 cells. ERK1 stimulation was completely blocked by either the mitogen-induced extracellular kinase (MEK) inhibitor PD98059 or the Raf-1 inhibitor 8-bromo-cAMP (cyclic adenosine monophosphate). However, only partial inhibition of Raf-1 and ERK1 stimulation was observed with the antioxidant N-acetylcysteine (N-Ac). Moreover, the xanthogenate compound D609 that inhibits DNR-induced phosphatidylcholine (PC) hydrolysis and subsequent diacylglycerol (DAG) production, as well as wortmannin that blocks phosphoinositide-3 kinase (PI3K) stimulation, only partially inhibited Raf-1 and ERK1 stimulation. We also observed that DNR stimulated protein kinase C zeta (PKCzeta), an atypical PKC isoform, and that both D609 and wortmannin significantly inhibited DNR-triggered PKCzeta activation. Finally, we found that the expression of PKCzeta kinase-defective mutant resulted in the abrogation of DNR-induced ERK phosphorylation. Altogether, these results demonstrate that DNR activates the classical Raf-1/MEK/ERK pathway and that Raf-1 activation is mediated through complex signaling pathways that involve at least 2 contributors: PC-derived DAG and PI3K products that converge toward PKCzeta. Moreover, we show that both Raf-1 and MEK inhibitors, as well as PKCzeta inhibition, sensitized cells to DNR-induced cytotoxicity.     

SGK1 inhibits cellular apoptosis and promotes proliferation via the MEK/ERK/p53 pathway in colitis

AIM: To investigate the role of serum-and-glucocorticoid-inducible-kinase-1(SGK1) in colitis and its potential pathological mechanisms.METHODS: SGK1 expression in mucosal biopsies from patients with active Crohn's disease(CD) and normal controls was detected by immunohistochemistry. We established an acute colitis model in mice induced by 2,4,6-trinitrobenzene sulfonicacid, and demonstrated the presence of colitis using the disease activity index, the histologic activity index and hematoxylin and eosin staining. The cellular events and potential mechanisms were implemented with small interference RNA and an inhibitor of signaling molecule(i.e., U0126) in intestinal epithelial cells(IECs). The interaction between SGK1 and the signaling molecule was assessed by coimmunoprecipitation.RESULTS: SGK1 expression was significantly increased in the inflamed epithelia of patients with active CD and TNBS-induced colitis model(0.58 ± 0.055 vs 0.85 ± 0.06, P 0.01). At the cellular level, silencing of SGK1 by small interference RNA(si SGK1) significantly inhibited the phosphorylation of mitogen-activated protein kinase kinase 1(MEK1) and the downstream molecule extracellular signal regulated protein kinase(ERK) 1/2, which induced the upregulation of p53 and Bcl-2-associated X protein, mediating the subsequent cellular apoptosis and proliferation in IECs. Cells treated with MEK1 inhibitor(i.e., U0126) before si SGK1 transfection showed a reversal of the si SGK1-induced cellular apoptosis. CONCLUSION: Our data suggested that SGK1 may protect IECs in colitis from tumor necrosis factor-α-induced apoptosis partly by triggering MEK/ERK activation.