In Calu-3 cells, PGE₂ stimulated Cl^- secretion is mediated by CFTR and Ca²⁺-activated Cl^- channels.

<p><b>A.</b> Representative <i>I</i>_<i>sc</i> trace with vertical deflections indicating the change in <i>I</i>_<i>sc</i> after a 1 mV pulse was applied (every 1 minute). Calu-3 cells were exposed to serosal to mucosal Cl^- gradient with equivalent bilateral HCO₃^-. PGE₂ (1 μM, serosal) was added to Calu-3 cells after a baseline period of ≥ 10 minutes, with CFTR_inh-172 (20 μM, mucosal) added after 30 minutes. <b>B.</b> Representative <i>I</i>_<i>sc</i> trace of Calu-3 cells incubated in CFTR_inh-172 (20 μM, mucosal) for at least 30 minutes prior to PGE₂ (1 μM, serosal) stimulation. After 30 minutes, niflumic acid (100 μM, mucosal) was added. <b>C.</b> Change in PGE₂-stimulated <i>I</i>_<i>sc</i> (mean ± SEM, n ≥ 4) in Calu-3 cells, with comparisons between no inhibition, CFTR inhibition, or CFTR and Ca²⁺-activated Cl^- inhibition. Asterisks denote significance by Student’s t-test (*, P < 0.05). Mean percent inhibition compared to PGE₂ stimulation alone noted.</p

Simplified working model of PGE₂-stimulated Cl^- and HCO₃^- secretion and mucociliary clearance in non-CF and CF airway.

<p><b>A.</b> In the airway, microbial infections cause an increase in PGE₂ through release from infiltrating inflammatory cells (not pictured) and production by airway epithelia <i>via</i> COX-2 activation. H₂O₂ produced by DUOX activates COX-2 and HVCN1 channels provide the H⁺ shunt from H₂O₂ production. PGE₂ exits the cell and activates PGE₂ (EP) receptors. In the current study we did not examine specific EP receptor involvement, however, we propose that EP₄ is the predominant mediator of serosal PGE₂ stimulation in bronchial epithelial cells. Submucosal gland cells may also utilize the EP₃ receptor, or Ca²⁺-activated Cl^- channels (CaCC) may get activated <i>via</i> EP₄-mediated cAMP-Ca²⁺ crosstalk. In bronchial epithelial cells, PGE₂ stimulates Cl^- and HCO₃^- secretion <i>via</i> CFTR, whereas in submucosal glands, both CFTR and CaCC are activated. Cl^- and HCO₃^- secretion will then influence airway pH, mucus properties, hydration, and ultimately, mucociliary clearance. <b>B.</b> In CF airway, lack of CFTR-dependent Cl^- and HCO₃^- secretion in bronchial epithelial cells, coupled with no HCO₃^- secretion and decreased Cl^- secretion from submucosal glands, leads to an acidic airway pH, thick-adherent mucus, and decreased mucociliary clearance. This results in increased microbial infection and rampant inflammation, in part by increased PGE₂ production.</p

PGE₂-stimulated mucociliary transport in ferret trachea.

<p><b>A.</b> PGE₂ stimulates a dose-dependent increase in MCC in ferret trachea. Each tissue was exposed to 2–3 doses of PGE₂ for 30 minutes each (n = 3 each dose). Data are shown as the mean PGE₂-stimulated increase in MCC over baseline ± SEM. The half-maximal effective concentration (EC₅₀) is noted in lower right corner. <b>B.</b> Timecourse of PGE₂-stimulated MCC with and without CFTR inhibition (n ≥ 6 each). For CFTR inhibition, tissues were bathed in apical and serosal solution for 30 minutes with CFTR_inh-172 (20 μM) prior to the 15-minute period and kept in the serosal bath for the length of the experiment. PGE₂ (1 μM) was added to the serosal bath. Circles represent means with bars indicating SEM. Asterisks represent P < 0.05 by ANOVA.</p

In ferret trachea, PGE₂ stimulated <i>I</i>_<i>sc</i> is mediated by CFTR and Ca²⁺-activated Cl^- channels.

<p><b>A.</b> Representative <i>I</i>_<i>sc</i> trace with vertical deflections indicating the change in <i>I</i>_<i>sc</i> after a 1 mV pulse was applied (every 1 minute). Ferret trachea was exposed to serosal to mucosal Cl^- gradient with equivalent bilateral HCO₃^-. PGE₂ (1 μM, serosal) was added to ferret trachea after a baseline period of ≥ 10 minutes, with CFTR_inh-172 (20 μM, mucosal) added after 30 minutes. <b>B.</b> Representative <i>I</i>_<i>sc</i> trace of ferret trachea incubated in CFTR_inh-172 (20 μM, mucosal) for at least 30 minutes prior to PGE₂ (1 μM, serosal) stimulation. After 30 minutes, niflumic acid (100 μM, mucosal) was added. <b>C.</b> Change in PGE₂-stimulated <i>I</i>_<i>sc</i> (mean ± SEM, n ≥ 5) in ferret trachea, with comparisons between no inhibition, CFTR inhibition, or CFTR and Ca²⁺-activated Cl^- inhibition. Asterisks denote significance by Student’s t-test (*, P < 0.05, **, P < 0.01). Mean percent inhibition compared to PGE₂ stimulation alone noted.</p

In CFBE41 cells, PGE₂ stimulated HCO₃^- secretion is completely CFTR dependent.

<p><b>A.</b> Representative <i>I</i>_<i>sc</i> trace with vertical deflections indicating the change in <i>I</i>_<i>sc</i> after a 1 mV pulse was applied (every 1 minute). CFBE41 WT cells were exposed to serosal to mucosal HCO₃^- gradient with equivalent bilateral Cl^-. PGE₂ (1 μM, serosal) was added to CFBE41 WT cells after a baseline period of ≥ 10 minutes, with CFTR_inh-172 (20 μM, mucosal) added afterwards. <b>B.</b> Representative <i>I</i>_<i>sc</i> trace from a similar experiment with CFBE41 CF cells. To verify cell viability, ATP (500 μM, mucosal) was added. <b>C.</b> Representative <i>I</i>_<i>sc</i> trace from a similar experiment as Fig 5A with CFBE41 WT cells, except experiments were performed in HCO₃^--free conditions. <b>D.</b> Change in PGE₂-stimulated <i>I</i>_<i>sc</i> (mean ± SEM, n = 3) in CFBE41 WT and CF cells in HCO₃^- containing and HCO₃^--free conditions. Asterisks denote significance by Student’s t-test (**, P < 0.01). Mean percent inhibition compared to CFBE41 WT noted.</p

In Calu-3 cells, PGE₂ stimulated HCO₃^- secretion is completely CFTR dependent.

<p><b>A.</b> Representative <i>I</i>_<i>sc</i> trace with vertical deflections indicating the change in <i>I</i>_<i>sc</i> after a 1 mV pulse was applied (every 1 minute). Calu-3 cells were exposed to serosal to mucosal HCO₃^- gradient with equivalent bilateral Cl^-. PGE₂ (1 μM, serosal) was added to Calu-3 cells after a baseline period of ≥ 10 minutes, with CFTR_inh-172 (20 μM, mucosal) added 30 minutes after. <b>B.</b> Representative <i>I</i>_<i>sc</i> trace from a similar experiment with Calu-3 cells in HCO₃^--free conditions. <b>C.</b> Change in PGE₂-stimulated <i>I</i>_<i>sc</i> (mean ± SEM, n = 3) in Calu-3 cells, with comparisons between no inhibition, CFTR inhibition, and HCO₃^--free conditions. Asterisks denote significance by Student’s t-test (**, P < 0.01, ***, P < 0.001). Mean percent inhibition compared to Calu-3 cells under control conditions. <b>D.</b> Timecourse of HCO₃^- secretion measured by pH-stat. The serosal solution was bathed with 95% O₂/5% CO₂ (similar to experiments in A-C), but the mucosal solution was bathed with 100% O₂ to prevent base formation from carbonhic anhydrase conversion of CO₂. Calu-3 cells were incubated in DMSO (5 μL; 1:1000 with bath; n = 10) or CFTR_inh-172 (20 μM, mucosal; n = 6) for 30–60 minutes prior to PGE₂ stimulation (1 μM, serosal). Circles represent means with bars indicating SEM. Asterisks represent P < 0.05 by ANOVA. <b>E.</b> Timecourse of <i>I</i>_<i>sc</i> measured by pH-stat measured simultaneously as pH-stat. Circles represent means with bars indicating SEM. Asterisks represent P < 0.05 by ANOVA.</p

In Calu-3 cells, PGE₂ stimulated HCO₃^- secretion is not affected by apical oubain, an inhibitor of the non-gastric H⁺/K⁺ ATPase.

<p>Experiments were performed to determine the potential role of ATP12A in measured PGE₂-stimulated HCO₃^- secretion in normal and CF conditions. Calu-3 experiments were performed similar to that in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0189894#pone.0189894.g006" target="_blank">Fig 6</a>, with the exception that an additional set of experiments were done with oubain (10 μM, mucosal) pre-treatment for ≥ 40 minutes prior to PGE₂ stimulation. Bars represent change in PGE₂-stimulated <i>I</i>_<i>sc</i> (mean ± SEM, n ≥ 5) in Calu-3 cells. Statistical comparisons were done between PGE₂ with and without oubain and PGE₂ with CFTR inhibition with and without oubain. No statistical difference was noted in either case (P > 0.05 by Student’s t-test).</p

Dataset for: Diabetic rats present higher urinary loss of proteins and lower renal expression of megalin, cubilin, ClC-5 and CFTR

Diabetic nephropathy (DN) occurs in around 40% of those with diabetes. Proteinuria is the main characteristic of DN and develops as a result of increased permeability of the glomerulus capillary wall and/or decreased proximal tubule endocytosis. The goal of this work was to evaluate renal function and the expression of megalin, cubilin, CFTR (cystic fibrosis transmembrane conductance regulator) and ClC-5 in the proximal tubule and renal cortex of rats with type 1 diabetes. Male Wistar rats were randomly assigned to control (CTRL) and diabetic (DM) groups for 4 weeks. Renal function was assessed in 24-h urine by calculating clearance and fractional excretion of solutes. The RNA and protein content of ClC-5, CFTR, megalin and cubilin was determined in the renal proximal tubule and cortex using real-time polymerase chain reaction and western blotting techniques, respectively. The results showed higher creatinine clearance and higher urinary excretion of proteins, albumin and transferrin in the DM group than in the CTRL group. Furthermore, the renal cortex and proximal tubule of diabetic animals showed downregulation of megalin, cubilin, ClC-5 and CFTR, critical components of the endocytic apparatus. These data suggest dysfunction in proximal tubule low-molecular-weight endocytosis and protein glomerulus filtration in the kidney of diabetic rats