AMPK Agonists Ameliorate Sodium and Fluid Transport and Inflammation in Cystic Fibrosis Airway Epithelial Cells

Abstract

The metabolic sensor AMP-activated kinase (AMPK) inhibits both the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) Cl -channel and epithelial Na 1 channel (ENaC), and may inhibit secretion of proinflammatory cytokines in epithelia. Here we have tested in primary polarized CF and non-CF human bronchial epithelial (HBE) cells the effects of AMPK activators, metformin and 5-aminoimidazole-4-carboxamide-1-b-D-riboside (AICAR), on various parameters that contribute to CF lung disease: ENaC-dependent short-circuit currents (I sc ), airway surface liquid (ASL) height, and proinflammatory cytokine secretion. AMPK activation after overnight treatment with either metformin (2-5 mM) or AICAR (1 mM) substantially inhibited ENaC-dependent I sc in both CF and non-CF airway cultures. Live-cell confocal images acquired 60 minutes after apical addition of Texas Red-dextran-containing fluid revealed significantly greater ASL heights after AICAR and metformin treatment relative to controls, suggesting that AMPK-dependent ENaC inhibition slows apical fluid reabsorption. Both metformin and AICAR decreased secretion of various proinflammatory cytokines, both with and without prior LPS stimulation. Finally, prolonged exposure to more physiologically relevant concentrations of metformin (0.03-1 mM) inhibited ENaC currents and decreased proinflammatory cytokine levels in CF HBE cells in a dose-dependent manner. These findings suggest that novel therapies to activate AMPK in the CF airway may be beneficial by blunting excessive sodium and ASL absorption and by reducing excessive airway inflammation, which are major contributors to CF lung disease.Keywords: metformin; cystic fibrosis transmembrane conductance regulator; ENaC; airway surface liquid; inflammationThe serious genetic disease cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) Cl -channel expressed at the apical membrane in a variety of epithelial tissues, including the lung, where CF lung disease causes considerable morbidity and mortality (1). Defective CFTR function prevents airway epithelium from adequately regulating the airway surface liquid (ASL) volume, which has an adverse effect on mucus clearance from the lung. The CF airway invariably becomes colonized by bacteria such as Pseudomonas aeruginosa and develops excessive inflammation, which can cause extensive damage to lung architecture and eventuate in respiratory failure (2). There is growing evidence that a lack of functional CFTR promotes an exaggerated or prolonged inflammatory response to bacterial and other insults, although the underlying pathophysiologic mechanisms are unclear (3-5). Both up-regulation of proinflammatory mediators (e.g., IL-8, IL-6, TNF-a, and GM-CSF) and down-regulation of anti-inflammatory mediators (e.g., IL-10 and inducible nitric oxide synthase) in the CF airway may play an important role in this process (6-8).Excessive activity of the epithelial sodium channel (ENaC) on the luminal airway surface is a key factor involved in th...

Does the ΔF508-CFTR mutation induce a proinflammatory response in human airway epithelial cells?

“…We report that P. aeruginosa elicited a more robust increase in cytokine and chemokine expression (e.g., IL-8, CXCL1, CXCL2, and TNF-␣) in CFBE-wt-CFTR cells compared with CFBE-⌬F508-CFTR cells. These data add to a growing body of evidence revealing that the inflammatory response of human airway epithelial cells expressing wt-CFTR and ⌬F508-CFTR to P. aeruginosa is model dependent, even when considering matched cell lines and human airway epithelial cells in primary culture (4,6,8,16,18,22,26,28,31,37,39,41). Taken together with other published studies, our data demonstrate that there is no compelling evidence to support the view that mutations in CFTR induce a hyperinflammatory response in human airway epithelial cells in vivo.…”

“…Although it is generally assumed that CF airway epithelial cells are hyperinflammatory in response to P. aeruginosa compared with non-CF airway epithelial cells, human CF airway cells in culture often fail to show a hyperinflammatory phenotype compared with airway epithelial cells expressing wild-type (wt)-CFTR (16,21,35). Indeed, one-third of published studies reveal that CF airway cells elaborate a more robust increase in IL-8 production than non-CF cells in response to P. aeruginosa (28,37,39,41), one-third report no difference (4,6,8,16,26), and one-third actually report that wt-CFTR cells release more IL-8 than CF cells in response to P. aeruginosa (18,22,31). Moreover, because the lungs during infection with P. aeruginosa contain immune cells that, like airway epithelial cells, produce cytokines and chemokines, it is not possible to determine whether the cytokines and chemokines in vivo originate from immune cells and/or airway epithelial cells.…”

High salt diet and caffeine: food for thought

“…In order to mechanistically characterize the observed phenomena, the authors assayed the activity of three kinases that might be involved in regulation of ENaC activity and found that caffeine did not change the abundance of SGK1, ERK1/2 or PKCα, but rather dosedependently decreased phosphorylation and abundance of AMPKα. Earlier findings by others also revealed that AMPK activation can decrease ENaC-mediated sodium current in mouse collecting ducts and airway epithelial cell lines (15,16). The reported data is an excellent confirmation of the idea that downregulation of ENaC is beneficial during SS hypertension, and chronic caffeine consumption may be one of the factors facilitating a reduction in ENaC function and/or expression.…”

Anti-inflammatory Effects of Thiazolidinediones in Human Airway Smooth Muscle Cells

“…Consequently, we examined the hypothesis that adenosine monophosphate-activated kinase (AMPK), a key cellular energy sensor and regulator, may contribute to the anti-inflammatory effects of TZDs. We considered a role for AMPK because (1) TZDs can activate AMPK (26,27), and (2) the activation of AMPK by 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) or by metformin inhibits inflammatory cytokine production from other cell types (28)(29)(30)(31)(32)(33)(34). In vivo, AICAR is also effective at alleviating inflammation in experimental murine colitis (35) and models of asthma (36).…”