Enhanced antibiotic resistance of Pseudomonas aeruginosa in the cystic fibrosis (CF) lung is thought to be due to the formation of biofilms. However, there is no information on the antibiotic resistance of P. aeruginosa biofilms grown on human airway epithelial cells or on the effects of airway cells on biofilm formation by P. aeruginosa. Thus we developed a coculture model and report that airway cells increase the resistance of P. aeruginosa to tobramycin (Tb) by >25-fold compared with P. aeruginosa grown on abiotic surfaces. Therefore, the concentration of Tb required to kill P. aeruginosa biofilms on airway cells is 10-fold higher than the concentration achievable in the lungs of CF patients. In addition, CF airway cells expressing DeltaF508-CFTR significantly enhanced P. aeruginosa biofilm formation, and DeltaF508 rescue with wild-type CFTR reduced biofilm formation. Iron (Fe) content of the airway in CF is elevated, and Fe is known to enhance P. aeruginosa growth. Thus we investigated whether enhanced biofilm formation on DeltaF508-CFTR cells was due to increased Fe release by airway cells. We found that airway cells expressing DeltaF508-CFTR released more Fe than cells rescued with WT-CFTR. Moreover, Fe chelation reduced biofilm formation on airway cells, whereas Fe supplementation enhanced biofilm formation on airway cells expressing WT-CFTR. These data demonstrate that human airway epithelial cells promote the formation of P. aeruginosa biofilms with a dramatically increased antibiotic resistance. The DeltaF508-CFTR mutation enhances biofilm formation, in part, by increasing Fe release into the apical medium.

The most common mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene in individuals with cystic fibrosis, ⌬F508, causes retention of ⌬F508-CFTR in the endoplasmic reticulum and leads to the absence of CFTR Cl ؊ channels in the apical plasma membrane. Rescue of ⌬F508-CFTR by reduced temperature or chemical means reveals that the ⌬F508 mutation reduces the half-life of ⌬F508-CFTR in the apical plasma membrane. Because ⌬F508-CFTR retains some Cl ؊ channel activity, increased expression of ⌬F508-CFTR in the apical membrane could serve as a potential therapeutic approach for cystic fibrosis. However, little is known about the mechanisms responsible for the short apical membrane half-life of ⌬F508-CFTR in polarized human airway epithelial cells. Accordingly, the goal of this study was to determine the cellular defects in the trafficking of rescued ⌬F508-CFTR that lead to the decreased apical membrane half-life of ⌬F508-CFTR in polarized human airway epithelial cells. We report that in polarized human airway epithelial cells (CFBE41o؊) the ⌬F508 mutation increased endocytosis of CFTR from the apical membrane without causing a global endocytic defect or affecting the endocytic recycling of CFTR in the Rab11a-specific apical recycling compartment.The cystic fibrosis transmembrane conductance regulator (CFTR) 2 is an ATP binding cassette (ABC) transporter and a cAMP-regulated Cl Ϫ channel that mediates transepithelial Cl Ϫ transport in the airways, intestine, pancreas, testis, and other tissues (1-3). Cystic fibrosis (CF), a lethal genetic disease, is caused by mutations in the CFTR gene (1, 2). The most common mutation in CFTR is ⌬F508 (4, 5). ⌬F508-CFTR does not fold properly, and most of the protein is retained within the endoplasmic reticulum (ER) where it is subsequently degraded (5, 6). Several studies suggest that the ER retention of ⌬F508-CFTR is not complete, and some ⌬F508-CFTR is constitutively expressed in the plasma membrane of primary epithelial cells from individuals homozygous for the ⌬F508 mutation (7-10). Because ⌬F508-CFTR retains some Cl Ϫ channel activity when expressed in the plasma membrane (5,6,(11)(12)(13)(14), it would be desirable to increase the expression of ⌬F508-CFTR in the plasma membrane to alleviate the symptoms in CF patients. The trafficking of ⌬F508-CFTR to the plasma membrane can be increased by chemical means or reduced temperature (15-21). Yet, functional and biochemical studies in heterologous cell lines demonstrate that rescued ⌬F508-CFTR has a greatly reduced stability or halflife in the post-ER compartments, including the plasma membrane (13,(22)(23)(24). Very little is known about the apical membrane half-life of rescued ⌬F508-CFTR in polarized human airway epithelial cells. A recent study demonstrates that the functional stability of ⌬F508-CFTR in the apical membrane of differentiated respiratory epithelial cells derived from nasal polyps from individuals homozygous for the ⌬F508 mutation is decreased compared with WT-CFTR (25). Furthermore, the bioc...

The C terminus of CFTR contains a PDZ interacting domain that is required for the polarized expression of cystic fibrosis transmembrane conductance regulator (CFTR) in the apical plasma membrane of polarized epithelial cells. To elucidate the mechanism whereby the PDZ interacting domain mediates the polarized expression of CFTR, Madin-Darby canine kidney cells were stably transfected with wild type (wt-CFTR) or C-terminally truncated human CFTR (CFTR-⌬TRL). We tested the hypothesis that the PDZ interacting domain regulates sorting of CFTR from the Golgi to the apical plasma membrane. Pulse-chase studies in combination with domain-selective cell surface biotinylation revealed that newly synthesized wt-CFTR and CFTR-⌬TRL were targeted equally to the apical and basolateral membranes in a nonpolarized fashion. Thus, the PDZ interacting domain is not an apical sorting motif. Deletion of the PDZ interacting domain reduced the half-life of CFTR in the apical membrane from ϳ24 to ϳ13 h but had no effect on the half-life of CFTR in the basolateral membrane. Thus, the PDZ interacting domain is an apical membrane retention motif. Next, we examined the hypothesis that the PDZ interacting domain affects the apical membrane half-life of CFTR by altering its endocytosis and/or endocytic recycling. Endocytosis of wt-CFTR and CFTR-⌬TRL did not differ. However, endocytic recycling of CFTR-⌬TRL was decreased when compared with wt-CFTR. Thus, deletion of the PDZ interacting domain reduced the half-life of CFTR in the apical membrane by decreasing CFTR endocytic recycling. Our results identify a new role for PDZ proteins in regulating the endocytic recycling of CFTR in polarized epithelial cells.

We previously reported that Pseudomonas aeruginosa PA14 secretes a protein that can reduce the apical membrane expression of the cystic fibrosis transmembrane conductance regulator (CFTR) protein. Here we report that we have used a proteomic approach to identify this secreted protein as PA2394, and we have named the gene cif, for CFTR inhibitory factor. We demonstrate that Cif is a secreted protein and is found associated with outer membrane-derived vesicles. Expression of Cif in Escherichia coli and purification of the C-terminal six-His-tagged Cif protein showed that Cif is necessary and sufficient to mediate the reduction in apical membrane expression of CFTR and a concomitant reduction in CFTR-mediated Cl ؊ ion secretion. Cif demonstrates epoxide hydrolase activity in vitro and requires a highly conserved histidine residue identified in ␣/␤ hydrolase family enzymes to catalyze this reaction. Mutating this histidine residue also abolishes the ability of Cif to reduce apical membrane CFTR expression. Finally, we demonstrate that the cif gene is expressed in the cystic fibrosis (CF) lung and that nonmucoid isolates of P. aeruginosa show greater expression of the gene than do mucoid isolates. We propose a model in which the Cif-mediated decrease in apical membrane expression of CFTR by environmental isolates of P. aeruginosa facilitates the colonization of the CF lung by this microbe.

Cystic fibrosis transmembrane conductance regulator (CFTR)-mediated Cl؊ secretion across fluid-transporting epithelia is regu-

Ischemia-reperfusion injury in CAD kidney transplants is associated with a duration-dependent increase in tubular cell apoptosis, mediated at least in part by activation of mitochondrial pathways.

The most common mutation in the CFTR gene in individuals with cystic fibrosis (CF), DeltaF508, leads to the absence of CFTR Cl(-) channels in the apical plasma membrane, which in turn results in impairment of mucociliary clearance, the first line of defense against inhaled bacteria. Pseudomonas aeruginosa is particularly successful at colonizing and chronically infecting the lungs and is responsible for the majority of morbidity and mortality in patients with CF. Rescue of DeltaF508-CFTR by reduced temperature or chemical means reveals that the protein is at least partially functional as a Cl(-) channel. Thus current research efforts have focused on identification of drugs that restore the presence of CFTR in the apical membrane to alleviate the symptoms of CF. Because little is known about the effects of P. aeruginosa on CFTR in the apical membrane, whether P. aeruginosa will affect the efficacy of new drugs designed to restore the plasma membrane expression of CFTR is unknown. Accordingly, the objective of the present study was to determine whether P. aeruginosa affects CFTR-mediated Cl(-) secretion in polarized human airway epithelial cells. We report herein that a cell-free filtrate of P. aeruginosa reduced CFTR-mediated transepithelial Cl(-) secretion by inhibiting the endocytic recycling of CFTR and thus the number of WT-CFTR and DeltaF508-CFTR Cl(-) channels in the apical membrane in polarized human airway epithelial cells. These data suggest that chronic infection with P. aeruginosa may interfere with therapeutic strategies aimed at increasing the apical membrane expression of DeltaF508-CFTR.

Tubulointerstitial nephritis (TIN) is a frequent cause of acute kidney injury (AKI) that can lead to chronic kidney disease (CKD). TIN is associated with an immune-mediated infiltration of the kidney interstitium by inflammatory cells, which may progress to fibrosis. Patients often present with non-specific symptoms, which can lead to delayed diagnosis and treatment of the disease. Etiology of TIN can be drug-induced, infectious, idiopathic, genetic, or related to a systemic inflammatory condition such as tubulointerstitial nephritis and uveitis (TINU) syndrome, inflammatory bowel disease, or IgG4-associated immune complex multiorgan autoimmune disease (MAD). It is imperative to have a high clinical suspicion for TIN in order to remove potential offending agents and treat any associated systemic diseases. Treatment is ultimately dependent on underlying etiology. While there are no randomized controlled clinical trials to assess treatment choice and efficacy in TIN, corticosteroids have been a mainstay of therapy and recent studies have suggested a possible role for mycophenolate mofetil. Urinary biomarkers such as alpha1-microglobulin and beta2-microglobulin may help diagnose and monitor disease activity in TIN. Screening for TIN should be implemented in children with inflammatory bowel disease, uveitis, or IgG4-associated MAD.