Short-circuit Current Measurements Research Articles

7. Lentiviral Vector-Mediated CFTR Gene Transfer to CF Pig Airways Corrects the Anion Transport Defect In Vivo

Cystic fibrosis (CF) is the most common autosomal recessive disorder in Caucasian populations and is caused by mutations in CFTR. Complementation of CFTR rescues the anion transport defect in vitro, making this monogenetic disease a candidate for CF gene therapy. Lentiviral vector-mediated CFTR gene transfer has long been discussed as a promising strategy for gene therapy. Lentiviral vectors can efficiently transduce non-dividing cells and persistently express a therapeutic transgene. We previously demonstrated that the non-primate lentiviral vector derived from feline immunodeficiency virus (FIV) and pseudotyped with a GP64 envelope confers gene transfer to porcine airway epithelia both in well-differentiated primary cultures in vitro and in wild-type pig lungs in vivo. The CF pig model recapitulates many features of human disease and provides a new platform for preclinical gene therapy studies. Here, we show that lentiviral-mediated delivery of CFTR to nasal and lung epithelia corrects the anion channel defect in the CF pig model. We delivered GP64-FIV-pigCFTR to the nose and lung of 2-week old gut-corrected CF pigs using a MADgic atomizer. Two weeks post-transduction, we harvested sinus, trachea, and bronchus tissue to assay for gene correction. In freshly excised trachea and bronchus tissue explants, we observed a short-circuit current response to forskolin and IBMX (F&I) and inhibition by GlyH-101, consistent with CFTR function. Compared to non-transduced animals, cultured ethmoid sinus epithelia also showed evidence of correction by short-circuit current measurements. Loss of CFTR results in reduced bicarbonate transport and a lower ASL pH. A reduction in ASL pH impairs bacterial killing. Following gene transfer, we observed an increase in nasal and tracheal pH and rescue of bacterial killing. Together, these exciting results demonstrate the first evidence of functional in vivo correction of CFTR by a lentivirus in the CF pig model. These findings support the utility of a lentivirus for CF gene therapy.

Calcium-sensing receptor stimulates Cl(-)- and SCFA-dependent but inhibits cAMP-dependent HCO3(-) secretion in colon.

Colonic bicarbonate (HCO3(-)) secretion is a well-established physiological process that is closely linked to overall fluid and electrolyte movement in the mammalian colon. These present studies show that extracellular calcium-sensing receptor (CaSR), a fundamental mechanism for sensing and regulating ionic and nutrient compositions of extracellular milieu in the small and large intestine, regulates HCO3(-) secretion. Basal and induced HCO3(-) secretory responses to CaSR agonists were determined by pH stat techniques used in conjunction with short-circuit current measurements in mucosa from rat distal colon mounted in Ussing chambers. R568, a specific CaSR activator, stimulated lumen Cl(-)- and short-chain fatty acid (SCFA)-dependent HCO3(-) secretion but inhibited cyclic nucleotide-activated HCO3(-) secretion. Consequently, at physiological conditions (either at basal or during lumen acid challenge) when electroneutral Cl(-)/HCO3(-) and SCFA/HCO3(-) exchangers dominate, CaSR stimulates HCO3(-) secretion; in contrast, in experimental conditions that stimulate fluid and HCO3(-) secretion, e.g., when forskolin activates electrogenic cystic fibrosis transmembrane conductance regulator-mediated HCO3(-) conductance, CaSR activation inhibits HCO3(-) secretion. Corresponding changes in JHCO3 (μeq·h(-1)·cm(-2), absence vs. presence of R568) were 0.18 ± 0.03 vs. 0.31 ± 0.08 under basal nonstimulated conditions and 1.85 ± 0.23 vs. 0.45 ± 0.06 under forskolin-stimulated conditions. Similarly, activation of CaSR by R568 stimulated Cl(-)- and SCFA-dependent HCO3(-) secretion and inhibited cAMP-dependent HCO3(-) secretion in colon mucosa of wild-type mice; such effects were abolished in CaSR-null mice. These results suggest a new paradigm for regulation of intestinal ion transport in which HCO3(-) secretion may be fine-tuned by CaSR in accordance with nutrient availability and state of digestion and absorption. The ability of CaSR agonists to inhibit secretagogue-induced intestinal HCO3(-) secretion suggests that modulation of CaSR activity may provide a new therapeutic approach to correct HCO3(-) deficit and metabolic acidosis, a primary cause of morbidity and mortality in acute infectious diarrheal illnesses.

Deleterious impact of Pseudomonas aeruginosa on cystic fibrosis transmembrane conductance regulator function and rescue in airway epithelial cells.

The epithelial response to bacterial airway infection, a common feature of lung diseases such as chronic obstructive pulmonary disease and cystic fibrosis, has been extensively studied. However, its impact on cystic fibrosis transmembrane conductance regulator (CFTR) channel function is not clearly defined. Our aims were, therefore, to evaluate the effect of Pseudomonas aeruginosa on CFTR function and expression in non-cystic fibrosis airway epithelial cells, and to investigate its impact on ΔF508-CFTR rescue by the VRT-325 corrector in cystic fibrosis cells. CFTR expression/maturation was evaluated by immunoblotting and its function by short-circuit current measurements. A 24-h exposure to P. aeruginosa diffusible material (PsaDM) reduced CFTR currents as well as total and membrane protein expression of the wildtype (wt) CFTR protein in CFBE-wt cells. In CFBE-ΔF508 cells, PsaDM severely reduced CFTR maturation and current rescue induced by VRT-325. We also confirmed a deleterious impact of PsaDM on wt-CFTR currents in non-cystic fibrosis primary airway cells as well as on the rescue of ΔF508-CFTR function induced by VRT-325 in primary cystic fibrosis cells. These findings show that CFTR function could be impaired in non-cystic fibrosis patients infected by P. aeruginosa. Our data also suggest that CFTR corrector efficiency may be affected by infectious components, which should be taken into account in screening assays of correctors.

Interaction of ultrashort laser pulses and silicon solar cells under short circuit conditions

Ultrashort pulse lasers are promising tools for numerous measurement purposes. Among other benefits their high peak powers allow for efficient generation of wavelengths in broad spectral ranges and at spectral powers that are orders of magnitude higher than in conventional light sources. Very recently this has been exploited for the establishment of sophisticated measurement facilities for electrical characterization of photovoltaic (PV) devices. As the high peak powers of ultrashort pulses promote nonlinear optical effects they might also give rise to nonlinear interactions with the devices under test that possibly manipulate the measurement outcome. In this paper, we present a comprehensive theoretical and experimental study of the nonlinearities affecting short circuit current (ISC) measurements of silicon (Si) solar cells. We derive a set of coupled differential equations describing the radiation-device interaction and discuss the nonlinearities incorporated in those. By a semi-analytical approach introducing a quasi-steady-state approximation and integrating a Green's function we solve the system of equations and obtain simulated ISC values. We validate the theoretical model by ISC ratios obtained from a double ring resonator setup capable for reproducible generation of various ultrashort pulse trains. Finally, we apply the model to conduct the most prominent comparison of ISC generated by ultrashort pulses versus continuous illumination. We conclude by the important finding that the nonlinearities induced by ultrashort pulses are negligible for the most common ISC measurements. However, we also find that more specialized measurements (e.g., of concentrating PV or Si-multijunction devices as well as highly localized electrical characterizations) will be biased by two-photon-absorption distorting the ISC measurement.

Light Scattering and Current Enhancement for Microcrystalline Silicon Thin-Film Solar Cells on Aluminium-Induced Texture Glass Superstrates with Double Texture

Microcrystalline silicon (μc-Si:H) thin-film solar cells are processed on glass superstrates having both micro- and nanoscale surface textures. The microscale texture is realised at the glass surface, using the aluminium-induced texturing (AIT) method, which is an industrially feasible process enabling a wide range of surface feature sizes (i.e., 700 nm–3 μm) of the textured glass. The nanoscale texture is made by conventional acid etching of the sputter-deposited transparent conductive oxide (TCO). The influence of the resulting “double texture” on the optical scattering is investigated by means of atomic force microscopy (AFM) (studying the surface topology), haze measurements (studying scattering into air), and short-circuit current enhancement measurements (studying scattering into silicon). A predicted enhanced optical scattering efficiency is experimentally proven by a short-circuit current enhancementΔIscof up to 1.6 mA/cm2(7.7% relative increase) compared to solar cells fabricated on a standard superstrate, that is, planar glass covered with nanotextured TCO. Enhancing the autocorrelation length (or feature size) of the AIT superstrates might have the large potential to improve theμc-Si:H thin-film solar cell efficiency, by reducing the shunting probability of the device while maintaining a high optical scattering performance.

An intelligent control strategy of fractional short circuit current maximum power point tracking technique for photovoltaic applications

This paper presents an improved Fractional Short Circuit Current (FSCC) Maximum Power Point Tracking (MPPT) technique in which an additional control loop is used to find the proper moment to measure the SCC. The target is to reduce the power losses in MPPT process that occur as a result of intermittent time based short circuit current measurements. The proposed modification enables the conventional FSCC MPPT to decide intelligently about the measurement of SCC thus reduces the number of times the photovoltaic (PV) module is isolated from the load. Although number of algorithms for tracking MPPT has been reported, the proposed method suits well for low cost PV applications. A Matlab/Simulink based model is employed to test the functional abilities of the proposed method. The comparison of the proposed method and conventional time based FSCC method is also presented in the simulation analysis. Finally, a 130 W prototype based on the dSPACE DS1104 controller and experimental results are presented to verify the effectiveness of the proposed method. The technique is verified under uniform shading conditions. The results show satisfactory performance against test conditions.

Measurements of Functional Responses in Human Primary Lung Cells as a Basis for Personalized Therapy for Cystic Fibrosis

BackgroundThe best investigational drug to treat cystic fibrosis (CF) patients with the most common CF-causing mutation (F508del) is VX-809 (lumacaftor) which recently succeeded in Phase III clinical trial in combination with ivacaftor. This corrector rescues F508del-CFTR from its abnormal intracellular localization to the cell surface, a traffic defect shared by all Class II CFTR mutants. Our goal here is to test the efficacy of lumacaftor in other Class II mutants in primary human bronchial epithelial (HBE) cells derived from CF patients. MethodsThe effect of lumacaftor was investigated in primary HBE cells from non-CF and CF patients with F508del/F508del, A561E/A561E, N1303K/G542X, F508del/G542X and F508del/Y1092X genotypes by measurements of Forskolin plus Genistein-inducible equivalent short-circuit current (Ieq-SC-Fsk+Gen) in perfused open-circuit Ussing chambers. Efficacy of corrector C18 was also assessed on A561E/A561E and F508del/F508del cells. ResultsOur data indicate that A561E (when present in both alleles) responds positively to lumacaftor treatment at equivalent efficacy of F508del in primary HBE cells. Similarly, lumacaftor has a positive impact on Y1092X, but not on N1303K. Our data also show that cells with only one copy of F508del-CFTR respond less to VX-809. Moreover, there is great variability in lumacaftor responses among F508del-homozygous cells from different donors. Compound C18 failed to rescue A561E-CFTR but not in F508del-CFTR, thus plausibly it has a different mechanism of action distinct from lumacaftor. ConclusionsCF patients with A561E (and likely also those with Y1029X) can potentially benefit from lumacaftor. Moreover, the methodology used here exemplifies how ex vivo approaches may apply personalized therapies to CF and possibly other respiratory diseases.

Norepinephrine stimulates the epithelial Na+ channel in cortical collecting duct cells via α2-adrenoceptors.

There is good evidence for a causal link between excessive sympathetic drive to the kidney and hypertension. We hypothesized that sympathetic regulation of tubular Na(+) absorption may occur in the aldosterone-sensitive distal nephron, where the fine tuning of renal Na(+) excretion takes place. Here, the appropriate regulation of transepithelial Na(+) transport, mediated by the amiloride-sensitive epithelial Na(+) channel (ENaC), is critical for blood pressure control. To explore a possible effect of the sympathetic transmitter norepinephrine on ENaC-mediated Na(+) transport, we performed short-circuit current (Isc) measurements on confluent mCCDcl1 murine cortical collecting duct cells. Norepinephrine caused a complex Isc response with a sustained increase of amiloride-sensitive Isc by ∼44%. This effect was concentration dependent and mediated via basolateral α2-adrenoceptors. In cells pretreated with aldosterone, the stimulatory effect of norepinephrine was reduced. Finally, we demonstrated that noradrenergic nerve fibers are present in close proximity to ENaC-expressing cells in murine kidney slices. We conclude that the sustained stimulatory effect of locally elevated norepinephrine on ENaC-mediated Na(+) absorption may contribute to the hypertensive effect of increased renal sympathetic activity.

Stimulation effect of wide type CFTR chloride channel by the naturally occurring flavonoid tangeretin

Cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-activated chloride channel expressed in the apical membrane of serous epithelial cells. Both deficiency and overactivation of CFTR may cause fluid and salt secretion related diseases. In the present study, we identified tangeretin from Pericarpium Citri Reticulatae Viride as a CFTR activator using high-throughput screening based on FRT cell-based fluorescence assay. The activation effect of tangeretin on CFTR chloride channel and the possible underlying mechanisms were investigated. Fluorescence quenching tests showed that tangeretin dose- and time-dependently activated CFTR chloride channel, the activity had rapid and reversible characteristics and the activation effect could be completely reversed by the CFTR specific blocker CFTRinh-172. Primary mechanism studies indicated that the activation effect of tangeretin on CFTR chloride channel was FSK dependent as well as had additional effect with FSK and IBMX suggesting that tangeretin activates CFTR by direct interacting with the protein. Ex-vivo tests revealed that tangeretin could accelerate the speed of the submucosal gland fluid secretion. Short-circuit current measurement demonstrated that tangeretin activated rat colonic mucosa chloride current. Thus, CFTR Cl− channel is a molecular target of natural compound tangeretin. Tangeretin may have potential use for the treatment of CFTR-related diseases like cystic fibrosis, bronchiectasis and habitual constipation.

Internal quantum efficiency analysis of plasmonic textured silicon solar cells: surface plasmon resonance and off-resonance effects

Silver nanoparticles (Ag NPs) of various sizes and concentration were integrated on textured silicon solar cells for further confinement of incident light, generated photocurrent modifications were investigated using spectrally resolved short-circuit current measurements. Internal quantum efficiency (IQE) spectra were used for quantifying the effective minority carrier diffusion lengths (Leff) of plasmonic cells in the long wavelength region (850 < λ < 1020 nm). The Leff of an optimized plasmonic solar cell enhanced to 431 µm compared to 338 µm of the bare cell, which is due to interacting Ag NPs' scattered fields, leading to enhanced light absorption in the plasmonic cell. Despite the enhanced Leff values, the overall generated photocurrent reduced with Ag NPs which is due to the significant losses near the surface plasmon resonant region. Reduced IQE of plasmonic cells near and below the surface plasmon resonant region is due to size-dependent parasitic absorption and enhanced back scattering of Ag NPs, and a modified surface recombination process due to Ag NPs' strong near-fields.

Human induced pluripotent stem cells (hiPSC)‐derived endothelial cells: new opportunity for corneal bioengineering

AbstractPurpose hiPSCs can self‐renew indefinitely, while maintaining the capacity to differentiate into somatic cells. As such, they represent an essentially inexhaustible source of committed corneal endothelial cells (CECs) of potential use in cell‐based corneal therapies. We present a method of differentiation of hiPSCs into CE‐like cells.Methods hiPSCs reprogrammed from adipocytes by retrovirus (SBI) were expanded in mTSR1 medium. Differentiation was induced by sequential treatment mimicking the normal embryonic endothelial development, using co‐cultures during 3 weeks (not detailed because of ongoing patent application). Cells were sorted using magnetic beads with negative selection with anti‐K3/K12. Sorted cells were subsequently cultivated in maintenance medium for 3‐4 weeks. Differentiation was assessed by immunolabeling of ZO‐1, Glypican‐4, CD200R, Na/K/ATPase, CLCN3, MiTF, K3/K12. Stemcellness was assessed using anti‐OCT‐4.Results hiPSCs were successfully and stably differentiated into a monolayer of tightly packed cells expression typical ECs makers and neither keratocytes nor epithelial markers. We observed the consecutive formation of embryoïd bodies after 4 days (D), neural crest cells after 7D, mesenchymal cells after 10D and cells with CEC phenotype after 15D. They lost expression of OCT‐4.Conclusion hiPSCs are a promising source of CECs. In addition to the short‐circuit current measurement using a Ussing chamber, their ability to deswell corneas is being studied using our innovative bioreactor. Safe sources of cornea‐derived hiPSCs are also under study to facilitate clinical translation. Grants: ABM2013, IUF2012‐17, UJM2013

Variations in epithelial Na(+) transport and epithelial sodium channel localisation in the vaginal cul-de-sac of the brushtail possum, Trichosurus vulpecula, during the oestrous cycle.

The fluid in the vaginal cul-de-sac of the brushtail possum, Trichosurus vulpecula, is copious at ovulation when it may be involved in sperm transport or maturation, but is rapidly reabsorbed following ovulation. We have used the Ussing short-circuit current (Isc) technique and measurements of transcript and protein expression of the epithelial Na(+) channel (ENaC) to determine if variations in electrogenic Na(+) transport are associated with this fluid absorption. Spontaneous Isc (<20µAcm(-2) during anoestrus, 60-80µAcm(-2) in cycling animals) was inhibited by serosal ouabain. Mucosal amiloride (10µmolL(-1)), an inhibitor of ENaC, had little effect on follicular Isc but reduced luteal Isc by ~35%. This amiloride-sensitive Isc was dependent on mucosal Na(+) and the half-maximal inhibitory concentration (IC50)-amiloride (0.95μmolL(-1)) was consistent with ENaC-mediated Na(+) absorption. Results from polymerase chain reaction with reverse transcription (RT-PCR) indicate that αENaC mRNA is expressed in anoestrous, follicular and luteal phases. However, in follicular animals αENaC immunoreactivity in epithelial cells was distributed throughout the cytoplasm, whereas immunoreactivity was restricted to the apical pole of cells from luteal animals. These data suggest that increased Na(+) absorption contributes to fluid absorption during the luteal phase and is regulated by insertion of ENaC into the apical membrane of cul-de-sac epithelial cells.

WS17.5 A new tool for CF diagnosis: short circuit current measurements in human nasal epithelial cells collected by nasal brushing

WS17.5 A new tool for CF diagnosis: short circuit current measurements in human nasal epithelial cells collected by nasal brushing

Cytochalasin E alters the cytoskeleton and decreases ENaC activity in Xenopus 2F3 cells.

Numerous reports have linked cytoskeleton-associated proteins with the regulation of epithelial Na(+) channel (ENaC) activity. The purpose of the present study was to determine the effect of actin cytoskeleton disruption by cytochalasin E on ENaC activity in Xenopus 2F3 cells. Here, we show that cytochalasin E treatment for 60 min can disrupt the integrity of the actin cytoskeleton in cultured Xenopus 2F3 cells. We show using single channel patch-clamp experiments and measurements of short-circuit current that ENaC activity, but not its density, is altered by cytochalasin E-induced disruption of the cytoskeleton. In nontreated cells, 8 of 33 patches (24%) had no measurable ENaC activity, whereas in cytochalasin E-treated cells, 17 of 32 patches (53%) had no activity. Analysis of those patches that did contain ENaC activity showed channel open probability significantly decreased from 0.081 ± 0.01 in nontreated cells to 0.043 ± 0.01 in cells treated with cytochalasin E. Transepithelial current from mpkCCD cells treated with cytochalasin E, cytochalasin D, or latrunculin B for 60 min was decreased compared with vehicle-treated cells. The subcellular expression of fodrin changed significantly, and several protein elements of the cytoskeleton decreased at least twofold after 60 min of cytochalasin E treatment. Cytochalasin E treatment disrupted the association between ENaC and myristoylated alanine-rich C-kinase substrate. The results presented here suggest disruption of the actin cytoskeleton by different compounds can attenuate ENaC activity through a mechanism involving changes in the subcellular expression of fodrin, several elements of the cytoskeleton, and destabilization of the ENaC-myristoylated alanine-rich C-kinase substrate complex.

Characterization of Defects in Ion Transport and Tissue Development in Cystic Fibrosis Transmembrane Conductance Regulator (CFTR)-Knockout Rats

Animal models for cystic fibrosis (CF) have contributed significantly to our understanding of disease pathogenesis. Here we describe development and characterization of the first cystic fibrosis rat, in which the cystic fibrosis transmembrane conductance regulator gene (CFTR) was knocked out using a pair of zinc finger endonucleases (ZFN). The disrupted Cftr gene carries a 16 base pair deletion in exon 3, resulting in loss of CFTR protein expression. Breeding of heterozygous (CFTR+/−) rats resulted in Mendelian distribution of wild-type, heterozygous, and homozygous (CFTR−/−) pups. Nasal potential difference and transepithelial short circuit current measurements established a robust CF bioelectric phenotype, similar in many respects to that seen in CF patients. Young CFTR−/− rats exhibited histological abnormalities in the ileum and increased intracellular mucus in the proximal nasal septa. By six weeks of age, CFTR−/− males lacked the vas deferens bilaterally. Airway surface liquid and periciliary liquid depth were reduced, and submucosal gland size was abnormal in CFTR−/− animals. Use of ZFN based gene disruption successfully generated a CF animal model that recapitulates many aspects of human disease, and may be useful for modeling other CF genotypes, including CFTR processing defects, premature truncation alleles, and channel gating abnormalities.

Antidiarrheal Efficacy and Cellular Mechanisms of a Thai Herbal Remedy

Screening of herbal remedies for Cl− channel inhibition identified Krisanaklan, a herbal extract used in Thailand for treatment of diarrhea, as an effective antidiarrheal in mouse models of secretory diarrheas with inhibition activity against three Cl− channel targets. Krisanaklan fully inhibited cholera toxin-induced intestinal fluid secretion in a closed-loop mouse model with ∼50% inhibition at a 1∶50 dilution of the extract. Orally administered Krisanaklan (5 µL/g) prevented rotavirus-induced diarrhea in neonatal mice. Short-circuit current measurements showed full inhibition of cAMP and Ca2+ agonist-induced Cl− conductance in human colonic epithelial T84 cells, with ∼50% inhibition at a 1∶5,000 dilution of the extract. Krisanaklan also strongly inhibited intestinal smooth muscle contraction in an ex vivo preparation. Together with measurements using specific inhibitors, we conclude that the antidiarrheal actions of Krisanaklan include inhibition of luminal CFTR and Ca2+-activated Cl− channels in enterocytes. HPLC fractionation indicated that the three Cl− inhibition actions of Krisanaklan are produced by different components in the herbal extract. Testing of individual herbs comprising Krisanaklan indicated that agarwood and clove extracts as primarily responsible for Cl− channel inhibition. The low cost, broad antidiarrheal efficacy, and defined cellular mechanisms of Krisanaklan suggests its potential application for antisecretory therapy of cholera and other enterotoxin-mediated secretory diarrheas in developing countries.

Amiloride-sensitive fluid resorption in NCI-H441 lung epithelia depends on an apical Cl(-) conductance.

Proper apical airway surface hydration is essential to maintain lung function. This hydration depends on well‐balanced water resorption and secretion. The mechanisms involved in resorption are still a matter of debate, especially as the measurement of transepithelial water transport remains challenging. In this study, we combined classical short circuit current (ISC) measurements with a novel D2O dilution method to correlate ion and water transport in order to reveal basic transport mechanisms in lung epithelia. D2O dilution method enabled precise analysis of water resorption with an unprecedented resolution. NCI‐H441 cells cultured at an air–liquid interface resorbed water at a rate of 1.5 ± 0.4 μL/(h cm2). Water resorption and ISC were reduced by almost 80% in the presence of the bulk Cl− channel inhibitor 5‐nitro‐2‐(3‐phenylpropylamino)benzoic acid (NPPB) or amiloride, a specific inhibitor of epithelial sodium channel (ENaC). However, water resorption and ISC were only moderately affected by forskolin or cystic fibrosis transmembrane regulator (CFTR) channel inhibitors (CFTRinh‐172 and glybenclamide). In line with previous studies, we demonstrate that water resorption depends on ENaC, and CFTR channels have only a minor but probably modulating effect on water resorption. However, the major ENaC‐mediated water resorption depends on an apical non‐CFTR Cl− conductance.

Pranlukast inhibits renal epithelial cyst progression via activation of AMP-activated protein kinase

Cysteinyl leukotriene receptor 1 (CysLT1 receptor) antagonists were found to inhibit chloride secretion in human airway epithelial cells. Since chloride secretion in renal epithelial cells, which shares common mechanisms with airway epithelial cells, plays important roles in renal cyst progression in polycystic kidney disease (PKD), this study was aimed to investigate effects of drugs acting as CysLT1 receptor antagonists on renal cyst progression and its underlying mechanisms. Effects of CysLT1 receptor antagonists on renal cyst growth and formation were determined using Madine Darby canine kidney (MDCK) cyst models. Mechanisms of actions of CysLT1 receptor antagonists were determined using short-circuit current measurement, assays of cell viability and cell proliferation, and immunoblot analysis of signaling proteins. Of the three drugs acting as CysLT1 receptor antagonists (montelukast, pranlukast and zafirlukast) tested, pranlukast was the most promising drug that inhibited MDCK cyst growth and formation without affecting cell viability. Its effect was independent of the inhibition of CysLT1 receptors. Instead, it reduced cAMP-activated chloride secretion and proliferation of MDCK cells in an AMP-activated protein kinase (AMPK)-dependent manner and had no effect on CFTR protein expression. Interestingly, pranlukast enhanced AMPK activation via calcium/calmodulin-dependent protein kinase kinase beta (CaMKKβ) with consequent activation of acetyl-CoA carboxylase (ACC) and suppression of mammalian target of rapamycin (mTOR) pathway. These results indicate that pranlukast retards renal epithelial cyst progression by inhibiting cAMP-activated chloride secretion and cell proliferation via CaMKKβ-AMPK-mTOR pathway. Therefore, pranlukast represents a class of known drugs that may have potential utility in PKD treatment.

Exocrine pancreas ER stress differentially induced by different fatty acids

Exocrine pancreas ER stress differentially induced by different fatty acids

37 Luminal Acid-Associated Post-Prandial Epithelial Hypoxia Maintains the Expression of Iron Absorptive DMT1 and DCYTB, and Cyclooxygenases

Background: We recently identified Slc26a9 as an anion conductance that is upregulated in airway inflammation and prevents bronchial mucus obstruction (Anagnostopoulou et al. JCI 2012). Slc26a9 variants were recently found associated with meconium ileus in cystic fibrosis infants (Sun et al. Nature 2012). Aim: The association with meconium ileus raises the question where Slc26a9 is expressed in the gastrointestinal tract and what is its function. Methods: Acid, HCO3, short circuit current (Isc) measurements were performed in isolated mucosa and acid, HCO3 , and fluid absorptive and secretory rates were measured by single pass perfusion and back titration in anesthetized Slc26a9 KO and WT mice by inhalation of 2.0% isoflurane. Slc26a9 cellular expression was studied by laser dissection and qPCR, and quantitative morphometry was performed in the different segments of the murine gastrointestinal tract. Results: Slc26a9 was found highly expressed in the mucosae of the upper gastrointestinal tract, with abrupt decrease of expression levels to virtually undectable levels beyond the duodenum. As previously reported, Slc26a9 KO mice had completely lost the ability to secrete acid in adulthood. However, Slc26a9 was found highly expressed along the whole gastric gland, even in areas without H+,K+-ATPase expression. Proximal duodenal bicarbonate and fluid secretory rates, which are higher in the proximal than the distal duodenum in WT mice, as well as the ability to stimulate these rates with forskolin, were reduced in the absence of Slc26a9 expression. The gastric antrum, as well as the fundus (after omeprazole treatment to rule out any residual acid secretory capacity) was studied to test whether Slc26a9 transports HCO3 itself. Gastric antrum, while expressing high Slc26a9 levels in WT mice, had lower basal and forskolin-stimulated HCO3 rate as well as lower Isc response in WT than KO mice, arguing against a role of Slc26a9 as a HCO3 transporter. Morphometry revealed strongly elongated fundic as well as antral glands, and slightly elongated proximal duodenal villi as well as crypts. Conclusions: Slc26a9 expression is necessary for normal gastric acid and proximal duodenal bicarbonate secretion, but it is not expressed inmore distal parts of the gastrointestinal mucosa. The increased risk for meconium ileus may be due to loss of digestive function of the stomach and proximal duodenum.