Paracellular Conductance Research Articles

Inflammatory Cytokines IL‐13 or IL‐17/TNFα Do Not Alter the Low Paracellular Bicarbonate Permeability of Cystic Fibrosis Airway Epithelia

The airway‐surface liquid, the thin aqueous layer on the apical side of airway epithelia, maintains a pH that is more acidic than the serosal pH. For human large airways, this acidic pH is the balance of HCO3− secretion by the cystic fibrosis transmembrane conductance regulator (CFTR) and H+ secretion by the H+/K+ pump ATP12A, both transcellular mechanisms. Newborn humans with cystic fibrosis (CF) have an acidic airway‐surface liquid pH compared to non‐CF newborns. With time, the pH difference between CF and non‐CF individuals disappears. However, adult CF epithelia cultured at the air‐liquid interface are more acidic than non‐CF epithelia. We hypothesized that CF‐related inflammation in vivo is responsible for the age‐dependent airway alkalinization. Here, we tested the hypothesis that cytokines increase paracellular movement of HCO3− into the airway‐surface liquid. Dilution potentials observed for cultured CF airway indicate that paracellular transport favored cations (PCl/PNa = 0.7). The paracellular conductance and selectivity were unaffected by changing the pH from 7.4 to 6.0. HCO3− was less permeable than Cl− through the paracellular pathway (PHCO3/PNa = 0.35). Treating cultures with cytokines (IL‐13 or IL‐17 and TNFa), which increase the airway‐surface liquid pH, did not affect paracellular PHCO3/PNa. These data suggest that differences in transcellular pH transport are responsible for cytokine‐mediated ASL pH changes. Similar transcellular mechanisms may cause the longitudinal ASL pH changes observed for CF patients.Support or Funding InformationMJW is an Investigator of the Howard Hughes Medical InstituteCystic Fibrosis Foundation Research Development ProgramNIH HL091842This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Evidence that Zinc Deficiency Impairs Gut Epithelial Barrier and intestinal Immunity

There is growing interest in dietary factors, in particular micronutrients, from the perspective of disease pathogenesis and potential for treatment. Diarrheal disease in infant results in abnormally low concentrations of serum zinc. Zinc has been tested for its ability to treat and prevent diarrheal diseases in many large field trials over a period of over 4 decades and has generally been found effective. The mechanism by which zinc deficiency causes diarrhea is not known. The present study investigated the impact of zinc deficiency on the host, particularly of intestinal tight junction (TJ) integrity and proteins that are involved in intestinal absorption and secretion along with the pathogenesis of enteric infection. We have used human colonic T84 cells to study barrier function and Shigella to test susceptibility of intestinal infection due to zinc deficiency. T84 grown onto transwell inserts in zinc deficient media showed a low transepithelial electrical resistance (TEER) compare to zinc sufficient media (1014 ± 165 Ω.cm2 vs 3663 ± 293 Ω.cm2) in confluent T84 monolayers. We furthermore analysed the correlation between TEER and paracellular ionic conductance. We found that zinc deficiency altered paracellular ionic conductance. Bacterial transmigration studies showed a significant increase in apico‐basolateral transmigration of Shigella (6.58 ± 0.13 vs 7.98 ± 0.23 log10 CFU/ml) which peaked at 6hrs post infection. Transmission electron microscopy data showed widened TJ complex due to few membrane fusion proteins and distorted TJ morphology, whereas the desmosomes were still intact. Electrophysiological studies with Ussing chamber and qPCR quantification demonstrated reduced cAMP dependent electrogenic Cl− secretion (34.5 ± 1.25 μA/cm2 vs 17.75 ± 2.3 μA/cm2) along with reduced expression of CFTR in zinc deficient cells, resulting dehydrated lumen thus aiding in bacterial colonization. Our results suggest that zinc deficiency caused (1) perturbed barrier function and integrity (2) altered Cl− secretion leading to susceptibility of infection. We conclude that adequate zinc is required to maintain intestinal barrier health to avoid risk against intestinal infection.Support or Funding InformationGovt of India National Funding AgencyThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Zinc deficiency as a codeterminant for airway epithelial barrier dysfunction in an ex vivo model of COPD.

There is now convincing evidence that the airway epithelium drives the pathogenesis of COPD. A major aspect of this is the disease-related reduction in barrier function that is potentiated by dysregulation of tight junction (TJ) protein complexes. However, a significant number of studies using in vitro smoke exposure models have not observed alterations in barrier permeability. We have previously shown that zinc (Zn) is an influential cytoprotective factor for the airway epithelium, and its depletion by cigarette smoke produces disease-related modifications consistent with inflammatory changes in COPD. We hypothesized that Zn deficiency is a significant co-stimulus with cigarette smoke extract (CSE) for potentiating the leaky barrier phenotype exhibited in COPD. We employed an ex vivo model of differentiated human airway epithelium exposed to Zn depletion and CSE to determine the contribution of Zn in maintaining normal epithelial permeability. Western blot analysis demonstrated a significant downregulation of the TJ proteins such as ZO-1 (−1.93-fold, P<0.05) and Claudin-1 (−3.37-fold, P<0.01) with the combination exposure. Assessment of barrier function via paracellular ionic conductance and tracer permeability also showed that Zn depletion was an important factor, which potentiated an increase in epithelial permeability (P<0.001 for both) compared to Zn depletion or CSE exposures in isolation. Visual inspection of the epithelium using transmission electron microscopy revealed a marked reduction in junction complexes between the adjacent airway epithelial cells treated with a combination of Zn depletion and CSE. These observations identify Zn deficiency as a significant codeterminant with CSE as a factor leading to an increase in airway epithelial permeability. Hence, as Zn dyshomeostasis has been reported in the airway epithelium exposed to chronic cigarette smoke and inflammation, targeting these phenomena may represent a promising strategy to ameliorate the leaky barrier phenotype that is synonymous with COPD.

Glucose transport across lagomorph jejunum epithelium is modulated by AMP-activated protein kinase under hypoxia.

The gastrointestinal epithelium possesses adaptation mechanisms to cope with huge variations in blood flow and subsequently oxygenation. Since sufficient energy supply is crucial under hypoxic conditions, glucose uptake especially must be regulated by these adaptation mechanisms. Therefore, we investigated glucose transport under hypoxic conditions. Jejunal epithelia of rabbits were incubated in Ussing chambers under short-circuit current conditions. Hypoxia was simulated by gassing with 1% O2 instead of 100% O2. The activity of sodium-coupled glucose transporter-1 (SGLT-1) was assessed by measuring the increase of short circuit current ( Isc) after the addition of 2 mM glucose to the mucosal buffer solution. We observed decreased activity of SGLT-1 after hypoxia compared with control conditions. To investigate underlying mechanisms, epithelia were exposed to agonists and antagonists of AMP-activated protein kinase (AMPK) before assessment of SGLT-1-mediated transport and the pAMPK/AMPK protein ratio. Preincubation with the antagonist restored SGLT-1 activity under hypoxic conditions to the level of control conditions, indicating an involvement of AMPK in the downregulation of SGLT-1 activity under hypoxia, which was confirmed in Western blot analysis of pAMPK/AMPK. Transepithelial flux studies using radioactively labeled glucose, ortho-methyl-glucose, fructose, and mannitol revealed no changes after hypoxic incubation. Therefore, we could exclude a decreased transepithelial glucose transport rate and increased paracellular conductance under hypoxia. In conclusion, our study hints at a decreased activity of SGLT-1 under hypoxic conditions in an AMPK-dependent manner. However, transepithelial transport of glucose is maintained. Therefore, we suggest other transport mechanisms, especially glucose transporter 1 and/or 2 to substitute SGLT-1 under hypoxia. NEW & NOTEWORTHY To our knowledge, this is the first approach to simulate hypoxia and study its effects in the jejunal epithelium using the Ussing chamber technique. We were able show that AMPK plays a role in the downregulation of SGLT-1 and that there seems to be an upregulation of other glucose transport mechanisms in the apical membrane of lagomorph jejunum epithelium under hypoxia, securing the epithelial energy supply and thus integrity.

Contribution of the tricellular tight junction to paracellular permeability in leaky and tight epithelia.

The tricellular tight junction (tTJ) is a potential weak point of the paracellular barrier. For solving the proportional contribution of the tTJ, ion conductances and macromolecule permeabilities were analyzed in cell lines of different leakiness. MDCK II, Caco-2, and HT-29/B6 cells were subjected to two-path impedance spectroscopy and morphological analyses in order to calculate the contribution of the tTJ to paracellular and total ion conductivity. The contribution to macromolecule permeability was evaluated by tricellulin overexpression or knockdown. Tricellulin-dependent macromolecule passage was comparably regulated in leaky and tight epithelia, but relative and absolute ion permeabilities of the tTJs were different. Assuming a minimal (50 pS) and maximal (146 pS) conductivity per single tTJ, the possible range of contribution of the tTJ to paracellular ion conductance amounted to only 0.3-1.1% in the leaky cell line MDCK II, but 3-25% in the moderately tight cell line Caco-2, and not less than 29% in the tight cell line HT-29/B6. In these cells, this resulted in a contribution to total epithelial conductance of 9-32%. In conclusion, in leaky epithelia the bicellular TJ accounts for nearly the entire paracellular ion conductance, whereas in tight epithelia the low bicellular TJ conductance has large impact on the tTJ.

Placental claudin expression and its regulation by endogenous sex steroid hormones

Tight junctions (TJs) form continuous intercellular contacts controlling the paracellular transportation across the cell-to-cell junction. TJ components include the peripheral protein zonula occludens-1 (ZO-1), junctional adhesion molecules (JAMs), and integral proteins such as occludin and claudins. Among the junction proteins, claudins play a major role in regulation of paracellular electrolyte transportation. This study explores the expression and distribution of tight junctions and their regulation during pregnancy. To study the regulation of claudin family, we examined expression of mouse placental tight junction proteins, including claudin-1 to -24, with real-time PCR and Western blotting and distribution of tight junction proteins with immunohistochemistry. Pregnant C57/BL6 mice were used in this study. The pregnant mice were divided into three groups depending on pregnant day (on days 12, 16, and 20 of gestation). Regarding the transcription levels, claudin-1, claudin-2, claudin-4, and claudin-5 expression levels were relatively high compared to other claudin family in all periods of pregnancy. Claudin-4 and 5 expressions, which reduce ion permeability, were increased over a period of time. However, claudin-2 expression, that is the responsive protein for a decrease in paracellular conductance, was decreased. Following this modulation of expression during mid-term pregnancy, we identified endogenous hormonal modulation of claudin family using estrogen receptor antagonist ICI 182,780 and progesterone receptor antagonist RU-486. After administration of ICI and RU-486, expression of claudin-4 mRNA and protein was increased. In addition, immunohistochemistry was performed to identify their localization for inferring permeability in placenta. Due to the function of claudins as effectors of ion transport at the end of regulatory pathways, they must be transducing proteins that modulate the function of claudins and thus link the physiologic inputs to the final effectors. This study will provide the claudin expressions and their localization in the mouse placenta, and their regulation by endogenous hormones. Taken together, the results of this study may contribute to assuming the roles and regulatory mechanism of these tight junction genes regarding maternal-fetal ion transportation in the placenta.

Paracellular pathway remodeling enhances sodium secretion by teleost fish in hypersaline environments.

In vertebrate salt-secreting epithelia, Na(+) moves passively down an electrochemical gradient via a paracellular pathway. We assessed how this pathway is modified to allow Na(+) secretion in hypersaline environments. Mummichogs (Fundulus heteroclitus) acclimated to hypersaline [2× seawater (2SW), 64‰] for 30 days developed invasive projections of accessory cells with an increased area of tight junctions, detected by punctate distribution of CFTR (cystic fibrosis transmembrane conductance regulator) immunofluorescence and transmission electron miscroscopy of the opercular epithelia, which form a gill-like tissue rich in ionocytes. Distribution of CFTR was not explained by membrane raft organization, because chlorpromazine (50 μmol l(-1)) and filipin (1.5 μmol l(-1)) did not affect opercular epithelia electrophysiology. Isolated opercular epithelia bathed in SW on the mucosal side had a transepithelial potential (Vt) of +40.1±0.9 mV (N=24), sufficient for passive Na(+) secretion (Nernst equilibrium voltage≡ENa=+24.11 mV). Opercular epithelia from fish acclimated to 2SW and bathed in 2SW had higher Vt of +45.1±1.2 mV (N=24), sufficient for passive Na(+) secretion (ENa=+40.74 mV), but with diminished net driving force. Bumetanide block of Cl(-) secretion reduced Vt by 45% and 29% in SW and 2SW, respectively, a decrease in the driving force for Na(+) extrusion. Estimates of shunt conductance from epithelial conductance (Gt) versus short-circuit current (Isc) plots (extrapolation to zero Isc) suggested a reduction in total epithelial shunt conductance in 2SW-acclimated fish. In contrast, the morphological elaboration of tight junctions, leading to an increase in accessory-cell-ionocyte contact points, suggests an increase in local paracellular conductance, compensating for the diminished net driving force for Na(+) and allowing salt secretion, even in extreme salinities.

216 HORMONAL REGULATION OF CLAUDIN-4 TIGHT JUNCTION MOLECULE IN MOUSE PLACENTA

Tight junctions (TJ) are composed of a branching network of sealing strands. TJ regulate paracellular conductance and ionic selectivity. TJ components include the peripheral protein ZO-1, junctional adhesion molecules (JAM) and the integral proteins such as occludin and claudin. Claudins are a family of proteins that are the most important components in the tight junctions. The established paracellular transport barriers that control transportation of molecules within intercellular space. The present study focused on the expression of claudin, suggesting as major working molecules in the paracellular transport system. To study the regulation and roles of claudin family, we examined expression of mouse placental claudin family. Fifteen pregnant C57/BL6 mice were used in this study and TJ proteins including Claudin-1 to Claudin-24 expressions by real-time RT–PCR and Western blotting. The mice were divided into 3 groups depending on the gestational day (on Days 12, 16, and 20 of gestation).The localization of TJ proteins were examined by immunohistochemistry. After we identified the fluctuation of claudin expression during pregnancy, we assumed that the hormones are one regulator for claudin family. Therefore, we performed an in vivo study with hormone receptor antagonists (ICI 182, 780, and RU-486) for examining hormonal effect on claudin expression in the placenta. Forty-nine mice were divided into 7 groups. The changes of claudin expression were examined with real-time RT-PCR and Western blotting. In the transcription levels, Claudin-1, claudin-2, claudin-4, and Claudin-5 expression levels were relatively high compared to others in the claudin family in all periods of the pregnancy. The claudin-4 expression, which reduces permeability of ions, increased over a period of time. However, caludin-5 expression that is the responsive protein for a decrease in paracellular conductance, were decreased. Claudin-1 and -4 have been known as responsive genes for a decrease in paracellular conductance. On the other hand, claudin 2 and 5 have been known as increasing paracellular conductance. In addition, immunohistochemistry was performed to identify their localization for inferring permeability in placenta. In summary, we analysed the claudin expressions and presented possible important claudins among its family. Furthermore, their localization was also examined in the mouse placenta. In addition, the regulation of critically expressed claudins by pregnancy-associated hormones, E2 and P4, was examined. These results may provide functional and structural roles of claudins and their involvement in the maternal-fetal interaction and in the transportation of placental materials.

The claudin-16 channel gene is transcriptionally inhibited by 1,25-dihydroxyvitaminD.

What is the central question of this study? In the kidney, the bulk of the filtered Mg(2+) is reabsorbed in the thick ascending limb by paracellular conductance, mediated by the tight junction protein, claudin-16, which is encoded by the gene CLDN16. The role of 1,25-dihydroxyvitamin D [1,25(OH)2 VitD] in renal Mg(2+) handling is unclear. We aimed to explore the molecular mechanisms underlying the effect of 1,25(OH)2 VitD on claudin-16-mediated Mg(2+) transport. What is the main finding and its importance? Paracellular, claudin-16-mediated Mg(2+) transport is transcriptionally repressed by 1,25(OH)2 VitD, probably via a Ca(2+)-sensing receptor-dependent mechanism. This renal effect of 1,25(OH)2 VitD may serve as an adaptive mechanism to the 1,25(OH)2 VitD-induced enteric hyperabsorption of dietary Mg(2+). Magnesium is reabsorbed in the thick ascending limb by paracellular conductance, mediated by the CLDN16-encoded tight junction protein, claudin-16. However, the role of 1,25-dihydroxyvitamin D [1,25(OH)2 VitD] in renal Mg(2+) handling is unclear. We have shown that Mg(2+) depletion increases and 1,25(OH)2 VitD inhibits CLDN16 transcription. We have now explored further the molecular mechanisms underlying the effect of 1,25(OH)2 VitD on claudin-16-mediated Mg(2+) transport. Adult mice received parenteral 1,25(OH)2 VitD or 1,25(OH)2 VitD combined with either high-Mg(2+) or low-Mg(2+) diets. Administration of 1,25(OH)2 VitD enhanced urinary excretion of Mg(2+) and Ca(2+). The 1,25(OH)2 VitD also increased renal Ca(2+)-sensing receptor (CaSR) mRNA and decreased renal claudin-16 and claudin-19 mRNA and claudin-16 protein, but did not affect renal claudin-2 mRNA. The 1,25(OH)2 VitD reversed the expected increase in claudin-16 mRNA in Mg(2+)-depleted animals. Comparably treated HEK 293 cells showed similar changes in claudin-16 mRNA, but 1,25(OH)2 VitD did not alter mRNA of the TRPM6 Mg(2+) channel. A luciferase reporter vector containing 2.5 kb of 5'-flanking DNA sequence from human CLDN16 (hCLDN16) was transfected into HEK 293 and OK cells. The hCLDN16 promoter activity was modestly decreased by 1,25(OH)2 VitD, but markedly inhibited in HEK 293 cells coexpressing CaSR. Coexpression in OK cells of dominant-negative CaSR completely abolished inhibition of hCLDN16 promoter activity by 1,25(OH)2 VitD. The 1,25(OH)2 VitD-induced decrease in hCLDN16 promoter activity was attenuated in Mg(2+)-depleted HEK 293 cells. In conclusion, 1,25(OH)2 VitD transcriptionally inhibits claudin-16 expression by a mechanism sensitive to CaSR and Mg(2+). This renal effect of 1,25(OH)2 VitD may serve as an adaptive response to the 1,25(OH)2 VitD-induced increase in intestinal Mg(2+) absorption.

Epigenetic regulation of microRNAs controlling CLDN14 expression as a mechanism for renal calcium handling.

The kidney has a major role in extracellular calcium homeostasis. Multiple genetic linkage and association studies identified three tight junction genes from the kidney--claudin-14, -16, and -19--as critical for calcium imbalance diseases. Despite the compelling biologic evidence that the claudin-14/16/19 proteins form a regulated paracellular pathway for calcium reabsorption, approaches to regulate this transport pathway are largely unavailable, hindering the development of therapies to correct calcium transport abnormalities. Here, we report that treatment with histone deacetylase (HDAC) inhibitors downregulates renal CLDN14 mRNA and dramatically reduces urinary calcium excretion in mice. Furthermore, treatment of mice with HDAC inhibitors stimulated the transcription of renal microRNA-9 (miR-9) and miR-374 genes, which have been shown to repress the expression of claudin-14, the negative regulator of the paracellular pathway. With renal clearance and tubule perfusion techniques, we showed that HDAC inhibitors transiently increase the paracellular cation conductance in the thick ascending limb. Genetic ablation of claudin-14 or the use of a loop diuretic in mice abrogated HDAC inhibitor-induced hypocalciuria. The genetic mutations in the calcium-sensing receptor from patients with autosomal dominant hypocalcemia (ADH) repressed the transcription of miR-9 and miR-374 genes, and treatment with an HDAC inhibitor rescued the phenotypes of cell and animal models of ADH. Furthermore, systemic treatment of mice with antagomiRs against these miRs relieved claudin-14 gene silencing and caused an ADH-like phenotype. Together, our findings provide proof of concept for a novel therapeutic principle on the basis of epigenetic regulation of renal miRs to treat hypercalciuric diseases.

Mo1959 Basolateral IKCA Channel Inhibition by Octreotide Prevents the Increase in Mucosal Permeability Induced by Chemical Hypoxia in Human Colon: Implications for Preventing Sepsis After Major Abdominal Surgery

Background: Intestinal ischemia occurs frequently during major abdominal surgery, causing increasedmucosal permeability and bacterial translocation, thus increasing the risk of postoperative sepsis.We previously showed that chemical hypoxia stimulated basolateral, intermediate conductance, Ca2+-sensitive K+ (IKCa) channels and doubled paracellular shunt conductance (GS) (which reflects mucosal permeability) in human colon in vitro, changes which were completely inhibited by the serosal addition of IKCa channel blockers TRAM-34 and clotrimazole (Am J Physiol Gastrointest Liver Physiol 300:G146-G153,2011). Aim: To determine whether inhibition of basolateral IKCa channels by somatostatin (SOM) and its synthetic analog octreotide (OCT) (Am J Physiol Gastrointest Liver Physiol 277:G967-G975,1999) prevents the increase in GS induced by chemical hypoxia in human colon. Methods: With informed consent, sheets of muscle-stripped normal human distal colon (from cancer patients) were mounted in an Ussing chamber, and bathed with high-K+ gluconate solution (mucosal side) and high-Na+ gluconate solution (serosal side) at 37oC. While passing 2-sec rectangular current pulses (I; 50 μA), transepithelial voltage (VT), total tissue conductance (GT; = I/ΔVT) and calculated short-circuit current (Isc; = VT.GT) were monitored until constant values were obtained. Tissues acting as ‘controls' were gassed throughout with 95%O2/5%CO2. Other tissues gassed with 95%O2/5%CO2 were switched to 95%N2/5%CO2 when 100 μM dinitrophenol (DNP) was added mucosally and serosally to induce chemical hypoxia over 5-min. 2 μM SOM was added to hypoxic tissues either 10-min before or immediately after addition of DNP. Nystatin (500 U/ml) was then added mucosally to permeabilize the apical membrane, and VT, GT and Isc monitored at 10-sec intervals to steady-state. Similar experiments in hypoxic tissues established the dose response to OCT. GS was estimated from the y-axis intercept of the linear relationship between GT and Isc (J Membr Biol 45:81108,1979). Results: GS was significantly greater in hypoxic tissues (5.52±1.22 mS/cm2; n= 10) than in controls (3.65±1.34 mS/cm2; n=11, P<0.005). GS was similar in hypoxic tissues irrespective of whether SOM was added before (3.28±1.56 mS/cm2; n=6) or after (3.48±1.21 mS/cm2; n=6) the application of DNP. GS in both SOM groups was significantly lower than in tissues treated with DNP alone (P<0.05), but similar to GS in controls. Dose response data indicated that 0.2 μM OCT and 2 μM OCT/SOM had the same protective effect on GS. Conclusion: Our results suggest that perioperative administration of OCT (a long-acting synthetic analog of SOM) during major abdominal surgery may prevent the increase in intestinal mucosal permeability associated with intestinal ischemia, thereby decreasing the likelihood of bacterial translocation and post-operative sepsis.

128 EXPRESSIONS OF MOUSE TIGHT JUNCTION MOLECULES IN PLACENTA – CLAUDINS AND OTHER PARACELLULAR TRANSPORT MOLECULES

Tight junctions (TJ) are composed of a branching network of sealing strands. Tight junctions regulate paracellular conductance and ionic selectivity. The TJ components include the peripheral protein ZO-1, junctional adhesion molecules (JAM), and integral proteins, such as occludin and claudin. Claudins, a family of proteins, are the most important components in TJ. They establish paracellular transport barriers, which control transportation of molecules within intercellular space. The current study focused on expression of claudin, suggesting claudin as a major working molecule in the paracellular transport system. In order to study the mechanisms and roles of the claudin family, the expression levels of claudin family in various organs should be determined. In this study, we examined expression of the mouse placental claudin family. Pregnant C57/BL6 mice (n = 5) were used in this study, and compared with β-actin, expression of TJ proteins, including Claudin-1 to Claudin-24, JAM-a, Zo-1, and occludin, tricellulin, and MarvelD3 was examined. In the transcription levels, Claudin-1 and -4 proteins were predominantly expressed. Claudin-1 and -4 have been known as a responsive gene for a direct decrease in paracellular conductance by selective reduction of the permeability of Na+ ions. On the other hand, Claudin-2, Claudin-3, Claudin-5, Cludin-11, and Claudin-12 showed ~33% expression compared with Claudin-1. Claudin-6 and Claudin-7 showed ~20% relative expression compared to Claudin 1. Claudin-10a and Claudin-16 were not detectable by real-time RT-PCR under our experimental conditions. Other claudin proteins showed low expression. Because the transcriptional levels of TJ genes were expressed variously, protein levels and their localization in the placenta will be further evaluated by immunostaining and Western blot assay. This study will provide data on expression of TJ genes and their localization in the mouse placenta, which may contribute to assuming the roles of these TJ genes regarding maternal-fetal ion transportation in the placenta.

CD36 and Fyn Kinase Mediate Malaria-Induced Lung Endothelial Barrier Dysfunction in Mice Infected with Plasmodium berghei

Severe malaria can trigger acute lung injury characterized by pulmonary edema resulting from increased endothelial permeability. However, the mechanism through which lung fluid conductance is altered during malaria remains unclear. To define the role that the scavenger receptor CD36 may play in mediating this response, C57BL/6J (WT) and CD36−/− mice were infected with P. berghei ANKA and monitored for changes in pulmonary endothelial barrier function employing an isolated perfused lung system. WT lungs demonstrated a >10-fold increase in two measures of paracellular fluid conductance and a decrease in the albumin reflection coefficient (σalb) compared to control lungs indicating a loss of barrier function. In contrast, malaria-infected CD36−/− mice had near normal fluid conductance but a similar reduction in σalb. In WT mice, lung sequestered iRBCs demonstrated production of reactive oxygen species (ROS). To determine whether knockout of CD36 could protect against ROS-induced endothelial barrier dysfunction, mouse lung microvascular endothelial monolayers (MLMVEC) from WT and CD36−/− mice were exposed to H2O2. Unlike WT monolayers, which showed dose-dependent decreases in transendothelial electrical resistance (TER) from H2O2 indicating loss of barrier function, CD36−/− MLMVEC demonstrated dose-dependent increases in TER. The differences between responses in WT and CD36−/− endothelial cells correlated with important differences in the intracellular compartmentalization of the CD36-associated Fyn kinase. Malaria infection increased total lung Fyn levels in CD36−/− lungs compared to WT, but this increase was due to elevated production of the inactive form of Fyn further suggesting a dysregulation of Fyn-mediated signaling. The importance of Fyn in CD36-dependent endothelial signaling was confirmed using in vitro Fyn knockdown as well as Fyn−/− mice, which were also protected from H2O2- and malaria-induced lung endothelial leak, respectively. Our results demonstrate that CD36 and Fyn kinase are critical mediators of the increased lung endothelial fluid conductance caused by malaria infection.

Identification of unitary tight junction pore opening/closing events in MDCK monolayers

Analyses of epithelial barrier function have led to the hypothesis that tight junctions are populated by claudin‐dependent pores that define paracellular ion conductance and charge selectivity. Consistent with this claudin‐2 expression increases paracellular Na+ permeability of MDCK I monolayers. However, available measures cannot resolve function of individual pores. To overcome this limitation, we have used patch clamp techniques to define local tight junction function. Local tight junction recordings reveal transient 63±8 pS openings (0.004±0.001 Hz). Claudin‐2 expression reduces global TER by a 71±1% and increases the frequency of individual openings to 0.023±0.007 Hz without changing their size (57±3 pS). Ion substitution analyses demonstrate that the openings are selective for cations (PNa/PCl = 4.9 ± 0.9) with radii &lt;2.8Å. Global analyses suggest that flux across claudin‐2I66C pores is blocked by cysteine derivatization. Consistent with this, while claudin‐2I66C expression had effects similar to claudin‐2 expression, openings in MDCK I expressing claudin‐2I66C were acutely terminated upon cysteine derivatization. These data show that claudin‐2 creates local, transient tight junction flux events that define global epithelial barrier function. We suggest that opening and closing of paracellular claudin pores forms the molecular basis of tight junction size and charge selectivity.

Improving mucosal function mitigates radiation‐induced gastrointestinal toxicity

Our objective was to determine the optimal amino acid (aa) formula to mitigate the effect of radiation on the electrolyte and nutrient absorption and mucosal barrier functions of the gastrointestinal (GI) tract.Studies were conducted on small intestinal mucosa of irradiated NIH Swiss mice (0, 1, 3, 5 or 7 Gy). The mucosal barrier mechanism was studied for dilution potential, conductance, and plasma endotoxin and IL‐1β; electrolyte, glucose, and aa absorption were studied with Ussing chamber flux studies and transepithelial electrical measurements. Amino acids that improved GI functions were used to formulate a drink that was tested in survival studies.Radiation (0 vs 7 Gy) decreased anion selectivity (0.48±0.05 vs 0.91±0.07) and increased paracellular conductance (30.5±4.1 vs 54.0±10.1). Plasma endotoxin levels were elevated at 7 Gy (20 vs 120). Radiation (0 vs 7 Gy) decreased Na absorption (9.8±0.8 vs − 0.6±0.3) and increased electrogenic Cl secretion (10.1±1.5 vs 0.1±0.01). Glucose absorption exhibited a dose‐dependent decrease and was abolished in 7‐Gy mice (140.7±32.2 vs 3.2±0.8). Lysine, aspartic acid, glycine, isoleucine, threonine, tyrosine, valine, serine, and tryptophan improved GI functions. Survival studies using the aa mixture showed increased survival for lethally irradiated mice.The aa mixture improved GI mucosal function and mitigated radiotoxicity.Support provided by Enterade, LLC.

Scanning ion conductance microscopy measurement of paracellular channel conductance in tight junctions.

Elucidation of epithelial transport across transcellular or paracellular pathways promises to advance the present understanding of ion transport and enables regulation of cell junctions critical to the cell and molecular biology of the epithelium. Here, we demonstrate a new instrumental technique, potentiometric scanning ion conductance microscopy (P-SICM), that utilizes a nanoscale pipet to differentiate paracellular and transcellular transport processes at high spatial resolution. The technique is validated for well-defined polymer membranes and then employed to study wild type and claudin-deficient mutants of Madin-Darby Canine Kidney strain II (MDCKII) cells. Paracellular permeabilities conferred by claudin-2 are captured by P-SICM which demonstrates the utility to monitor apparent conductance at subcellular levels.

Cytokines induce tight junction disassembly in airway cells via an EGFR-dependent MAPK/ERK1/2-pathway

Epithelial barrier permeability is altered in inflammatory respiratory disorders by a variety of noxious agents through modifications of the epithelial cell structure that possibly involve tight junction (TJ) organization. To evaluate in vitro whether pro-inflammatory cytokines involved in the pathogenesis of respiratory disorders could alter TJ organization and epithelial barrier integrity, and to characterize the signal transduction pathway involved Calu-3 airway epithelial cells were exposed to TNF-α, IL-4 and IFN-γ to assess changes in: (a) TJ assembly, that is, occludin and zonula occludens (ZO)-1 expression and localization, evaluated by confocal microscopy; (b) apoptotic activity, quantified using terminal transferase deoxyuridine triphosphate nick-end labeling staining; (c) epithelial barrier integrity, detected as transmembrane electrical resistance and expressed as GT values; (d) epidermal growth factor receptor (EGFR)-dependent mitogen-activated protein (MAP) kinase (MAPK)/extracellular signal-regulated kinases (ERK)1/2 phosphorylation, assessed by western blotting. Exposure to cytokines for 48 h induced a noticeable downregulation of the TJ transmembrane proteins. The degree ZO-1 and occludin colocalization was 62±2% in control cultures and significantly decreased in the presence of TNF-α (47±3%), IL-4 (43±1%) and INF-γ (35±3%). Although no apoptosis induction was detected following exposure to cytokines, changes in the epithelial barrier integrity were observed, with a significant enhancement in paracellular conductance. GT values were, respectively, 1.030±0.0, 1.300±0.04, 1.260±0.020 and 2.220±0.015 (mS/cm2) × 1000 in control cultures and in those exposed to TNF-α, IFN-γ and IL-4. The involvement of EGFR-dependent MAPK/ERK1/2 signaling pathway in cytokine-induced damage was demonstrated by a significant increase in threonine/tyrosine phosphorylation of ERK1/2, already detectable after 5 min incubation. All these cytokine-induced changes were markedly prevented when Calu-3 cells were cultured in the presence of an EGFR inhibitor (AG1478, 1 μM) or a MAP kinase inhibitor (U0126, 25 μM). In conclusion, cytokine-induced epithelial injury includes TJ disassembly and epithelial barrier permeability alteration and involves the EGFR-dependent MAPK/ERK1/2 signaling pathway.

Claudin-4 Levels Are Associated with Intact Alveolar Fluid Clearance in Human Lungs

The removal of edema from the air spaces is a critical function of the alveolar barrier requiring intact tight junctions. Alveolar fluid clearance contributes to graft function after transplantation and is associated with survival in patients with acute lung injury. Claudin-4 concentrations are known to increase during lung injury and the loss of claudin-4 decreases alveolar fluid clearance in mice. This study was therefore undertaken to evaluate whether differences in lung expression of the tight junction protein claudin-4 are associated with alveolar fluid clearance or clinical measures of lung function. Alveolar fluid clearance rates were measured in ex vivo perfused human lungs not used for transplantation and were compared with histological lung injury and clinical measures of lung injury in the donors. Claudin-4 staining demonstrated a positive correlation with alveolar fluid clearance (Spearman rank correlation [r(s)] = 0.71; P < 0.003); however, claudin-4 staining was not strongly associated with histological measures of lung injury. The expression of other tight junction proteins (including ZO-1) was not associated with alveolar fluid clearance or claudin-4 levels. Claudin-4 staining was lower in lungs from donors with greater impairment in respiratory physiology. These data suggest that claudin-4 may promote alveolar fluid clearance and demonstrate that the amount of claudin-4 expressed may provide specific information regarding alveolar epithelial barrier function that strengthens the link between histological changes and physiological impairment.

Calcium Inhibits Paracellular Sodium Conductance through Claudin-2 by Competitive Binding

Claudins form paracellular pores at the tight junction in epithelial cells. Profound depletion of extracellular calcium is well known to cause loosening of the tight junction with loss of transepithelial resistance. However, moderate variations in calcium concentrations within the physiological range can also regulate transepithelial permeability. To investigate the underlying molecular mechanisms, we studied the effects of calcium on the permeability of claudin-2, expressed in an inducible MDCK I cell line. We found that in the physiological range, calcium acts as a reversible inhibitor of the total conductance and Na(+) permeability of claudin-2, without causing changes in tight junction structure. The effect of calcium is enhanced at low Na(+) concentrations, consistent with a competitive effect. Furthermore, mutation of an intrapore negatively charged binding site, Asp-65, to asparagine partially abrogated the inhibitory effect of calcium. This suggests that calcium competes with Na(+) for binding to Asp-65. Other polyvalent cations had similar effects, including La(3+), which caused severe and irreversible inhibition of conductance. Brownian dynamics simulations demonstrated that such inhibition can be explained if Asp-65 has a relatively high charge density, thus favoring binding of Ca(2+) over that of Na(+), reducing Ca(2+) permeation by inhibiting its dissociation from this site, and decreasing Na(+) conductance through repulsive electrostatic interaction with Ca(2+). These findings may explain why hypercalcemia inhibits Na(+) reabsorption in the proximal tubule of the kidney.

Claudin-4 forms paracellular chloride channel in the kidney and requires claudin-8 for tight junction localization.

Tight junctions (TJs) play a key role in mediating paracellular ion reabsorption in the kidney. The paracellular pathway in the collecting duct of the kidney is a predominant route for transepithelial chloride reabsorption that determines the extracellular NaCl content and the blood pressure. However, the molecular mechanisms underlying the paracellular chloride reabsorption in the collecting duct are not understood. Here we showed that in mouse kidney collecting duct cells, claudin-4 functioned as a Cl(-) channel. A positively charged lysine residue at position 65 of claudin-4 was critical for its anion selectivity. Claudin-4 was observed to interact with claudin-8 using several criteria. In the collecting duct cells, the assembly of claudin-4 into TJ strands required its interaction with claudin-8. Depletion of claudin-8 resulted in the loss of paracellular chloride conductance, through a mechanism involving its recruitment of claudin-4 during TJ assembly. Together, our data show that claudin-4 interacts with claudin-8 and that their association is required for the anion-selective paracellular pathway in the collecting duct, suggesting a mechanism for coupling chloride reabsorption with sodium reabsorption in the collecting duct.