Activation of a polyvalent cation-sensing receptor decreases magnesium transport via claudin-16

Renal magnesium is mainly reabsorbed by a paracellular pathway in the thick ascending limb of Henle. The expression of claudin-16 increased magnesium transport in Madin-Darby canine kidney (MDCK) cells. Little is known about the regulatory mechanism of magnesium transport via claudin-16. Here we examined the effect of a polyvalent cation-sensing receptor (CaSR) on the intracellular distribution of and transport of magnesium by claudin-16. FLAG-tagged claudin-16 was stably expressed in MDCK cells using a Tet-OFF system. The activation of CaSR by magnesium, calcium, neomycin, and gadolinium did not affect the expression of FLAG-tagged claudin-16, CaSR, or ZO-1, a tight junctional scaffolding protein. These activators decreased the phosphoserine level of FLAG-tagged claudin-16 and the association of FLAG-tagged claudin-16 with ZO-1. The activation of CaSR induced a decrease in PKA activity. Immunofluorescence microscopy revealed that FLAG-tagged claudin-16 is distributed at the cell-cell border under unstimulated conditions, whereas it translocates to the intracellular compartment, mainly lysosome, with the activation of CaSR. In contrast, the distribution of ZO-1 was unaffected by the activation. The expression of FLAG-tagged claudin-16 increased transepithelial electrical resistance (TER) and transepithelial magnesium transport without affecting FITC-dextran (MW 4000) flux. The activation of CaSR decreased TER and magnesium transport, which were recovered by co-treatment with dibutyryl cAMP, a membrane-permeable cAMP analogue. Taken together, CaSR activation may decrease PKA activity, resulting in a decrease in phosphorylated claudin-16, the translocation of claudin-16 to lysosome and a decrease in magnesium reabsorption.     

Claudin-16 is directly phosphorylated by protein kinase A independently of a vasodilator-stimulated phosphoprotein-mediated pathway

Claudin-16 (CLDN-16) is involved in the paracellular reabsorption of Mg(2+) in the thick ascending limb of Henle. The tight junctional localization and Mg(2+) transport of CLDN-16 are regulated by cAMP/PKA-dependent phosphorylation. Here, we examined whether PKA phosphorylates CLDN-16 in a direct or indirect manner. CLDN-16 was stably expressed in Madin-Darby canine kidney (MDCK) cells using a Tet-OFF system. The phosphorylation of CLDN-16 is upregulated by fetal calf serum (FCS). This phosphorylation was completely inhibited by a PKA inhibitor, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride. Without FCS, dibutyryl cAMP (DBcAMP) increased the phosphoserine level of CLDN-16 in a concentration-dependent manner. The phosphorylated CLDN-16 elicited increases of transepithelial electrical resistance (TER) and transepithelial transport of Mg(2+). Vasodilator-stimulated phosphoprotein (VASP) was also phosphorylated in the presence of FCS or DBcAMP. In the glutathione-S-transferase (GST) pull down assay, a cytosolic carboxyl domain of CLDN-16 was associated with PKA, but not with VASP. Furthermore, PKA was immunoprecipitated with CLDN-16 in MDCK cells, but VASP was not. In cells expressing a dephosphorylated mutant (Ser160Ala) of VASP, CLDN-16 was phosphorylated by DBcAMP and was associated with ZO-1, a tight junctional-scaffolding protein, without integral cell-cell junctions. We suggest that PKA directly phosphorylates CLDN-16, resulting in the localization to tight junctions (TJs) and the maintenance of Mg(2+) reabsorption.     

Claudin-1 contributes to the epithelial barrier function in MDCK cells

Tight junctions (TJs) create a paracellular permeability barrier and also act as a fence preventing intermixing of proteins and lipids between the apical and basolateral plasma membranes. Recently, claudin-1 has been identified as an integral membrane protein localizing at TJs, and introduced claudin-1 can form TJ-like networks in fibroblasts. To investigate the function of claudin-1, MDCK cells were transfected with a mammalian expression vector containing myc-tagged mouse claudin-1, and four stable clones were obtained. The myc-tagged claudin-1 precisely colocalized with both occludin and ZO-1 at cell-cell contact sites, indicating that exogenous claudin-1 was properly targeted to the TJs. Immunoblot analysis revealed that overexpression of claudin-1 increased expression of ZO-1 but not of occludin or ZO-2. The barrier functions of these cells were evaluated by transepithelial electrical resistance (TER) and paracellular flux. Claudin-1-expressing cells exhibited about four times higher TER than wild-type MDCK cells. Consistent with the increase of TER, the cells overexpressing claudin-1 showed reduced paracellular flux, estimated at 4 and 40 kD FITC-dextrans. These results suggest that claudin-1 is involved in the barrier function at TJs.     

Conversion of zonulae occludentes from tight to leaky strand type by introducing claudin-2 into Madin-Darby canine kidney I cells

There are two strains of MDCK cells, MDCK I and II. MDCK I cells show much higher transepithelial electric resistance (TER) than MDCK II cells, although they bear similar numbers of tight junction (TJ) strands. We examined the expression pattern of claudins, the major components of TJ strands, in these cells: claudin-1 and -4 were expressed both in MDCK I and II cells, whereas the expression of claudin-2 was restricted to MDCK II cells. The dog claudin-2 cDNA was then introduced into MDCK I cells to mimic the claudin expression pattern of MDCK II cells. Interestingly, the TER values of MDCK I clones stably expressing claudin-2 (dCL2-MDCK I) fell to the levels of MDCK II cells (>20-fold decrease). In contrast, when dog claudin-3 was introduced into MDCK I cells, no change was detected in their TER. Similar results were obtained in mouse epithelial cells, Eph4. Morphometric analyses identified no significant differences in the density of TJs or in the number of TJ strands between dCL2-MDCK I and control MDCK I cells. These findings indicated that the addition of claudin-2 markedly decreased the tightness of individual claudin-1/4-based TJ strands, leading to the speculation that the combination and mixing ratios of claudin species determine the barrier properties of individual TJ strands.     

Extracellular signal-regulated kinases 1/2 control claudin-2 expression in Madin-Darby canine kidney strain I and II cells

The tight junction of the epithelial cell determines the characteristics of paracellular permeability across epithelium. Recent work points toward the claudin family of tight junction proteins as leading candidates for the molecular components that regulate paracellular permeability properties in epithelial tissues. Madin-Darby canine kidney (MDCK) strain I and II cells are models for the study of tight junctions and based on transepithelial electrical resistance (TER) contain "tight" and "leaky" tight junctions, respectively. Overexpression studies suggest that tight junction leakiness in these two strains of MDCK cells is conferred by expression of the tight junction protein claudin-2. Extracellular signal-regulated kinase (ERK) 1/2 activation by hepatocyte growth factor treatment of MDCK strain II cells inhibited claudin-2 expression and transiently increased TER. This process was blocked by the ERK 1/2 inhibitor U0126. Transfection of constitutively active mitogen-activated protein kinase/extracellular signal-regulated kinase kinase into MDCK strain II cells also inhibited claudin-2 expression and increased TER. MDCK strain I cells have higher levels of active ERK 1/2 than do MDCK strain II cells. U0126 treatment of MDCK strain I cells decreased active ERK 1/2 levels, induced expression of claudin-2 protein, and decreased TER by approximately 20-fold. U0126 treatment also induced claudin-2 expression and decreased TER in a high resistance mouse cortical collecting duct cell line (94D). These data show for the first time that the ERK 1/2 signaling pathway negatively controls claudin-2 expression in mammalian renal epithelial cells and provide evidence for regulation of tight junction paracellular transport by alterations in claudin composition within tight junction complexes.     

Juxtacrine activation of EGFR regulates claudin expression and increases transepithelial resistance

Heparin-binding (HB)-EGF, a ligand for EGF receptors, is synthesized as a membrane-anchored precursor that is potentially capable of juxtacrine activation of EGF receptors. However, the physiological importance of such juxtacrine signaling remains poorly described, due to frequent inability to distinguish effects mediated by membrane-anchored HB-EGF vs. mature "secreted HB-EGF." In our studies, using stable expression of a noncleavable, membrane-anchored rat HB-EGF isoform (MDCK(rat5aa) cells) in Madin-Darby canine kidney (MDCK) II cells, we observed a significant increase in transepithelial resistance (TER). Similar significant increases in TER were observed on stable expression of an analogous, noncleavable, membrane-anchored human HB-EGF construct (MDCK(human5aa) cells). The presence of noncleavable, membrane-anchored HB-EGF led to alterations in the expression of selected claudin family members, including a marked decrease in claudin-2 in MDCK(rat5aa) cells compared with the control MDCK cells. Reexpression of claudin-2 in MDCK(rat5aa) cells largely prevented the increases in TER. Ion substitution studies indicated decreased paracellular ionic permeability of Na(+) in MDCK(rat5aa) cells, further indicating that the altered claudin-2 expression mediated the increased TER seen in these cells. In a Ca(2+)-switch model, increased phosphorylation of EGF receptor and Akt was observed in MDCK(rat5aa) cells compared with the control MDCK cells, and inhibition of these pathways inhibited TER changes specifically in MDCK(rat5aa) cells. Therefore, we hypothesize that juxtacrine activation of EGFR by membrane-anchored HB-EGF may play an important role in the regulation of tight junction proteins and TER.     

Hypoglycosylated E-cadherin promotes the assembly of tight junctions through the recruitment of PP2A to adherens junctions

Epithelial cell-cell adhesion is controlled by multiprotein complexes that include E-cadherin-mediated adherens junctions (AJs) and ZO-1-containing tight junctions (TJs). Previously, we reported that reduction of E-cadherin N-glycosylation in normal and cancer cells promoted stabilization of AJs through changes in the composition and cytoskeletal association of E-cadherin scaffolds. Here, we show that enhanced interaction of hypoglycosylated E-cadherin-containing AJs with protein phosphatase 2A (PP2A) represents a mechanism for promoting TJ assembly. In MDCK cells, attenuation of cellular N-glycosylation with siRNA to DPAGT1, the first gene in the N-glycosylation pathway, reduced N-glycosylation of surface E-cadherin and resulted in increased recruitment of stabilizing proteins γ-catenin, α-catenin, vinculin and PP2A to AJs. Greater association of PP2A with AJs correlated with diminished binding of PP2A to ZO-1 and claudin-1 and with increased pools of serine-phosphorylated ZO-1 and claudin-1. More ZO-1 was found in complexes with occludin and claudin-1, and this corresponded to enhanced transepithelial resistance (TER), indicating physiological assembly of TJs. Similar maturation of AJs and TJs was detected after transfection of MDCK cells with the hypoglycosylated E-cadherin variant, V13. Our data indicate that E-cadherin N-glycans coordinate the maturity of AJs with the assembly of TJs by affecting the association of PP2A with these junctional complexes.     

Extracellular Mg regulates the tight junctional localization of claudin-16 mediated by ERK-dependent phosphorylation

Claudin-16 is involved in the paracellular reabsorption of Mg²⁺ in the thick ascending limb of Henle. Little is known about the mechanism regulating the tight junctional localization of claudin-16. Here, we examined the effect of Mg²⁺ deprivation on the distribution and function of claudin-16 using Madin-Darby canine kidney (MDCK) cells expressing FLAG-tagged claudin-16. Mg²⁺ deprivation inhibited the localization of claudin-16 at tight junctions, but did not affect the localization of other claudins. Re-addition of Mg²⁺ induced the tight junctional localization of claudin-16, which was inhibited by U0126, a MEK inhibitor. Transepithelial permeability to Mg²⁺ was also inhibited by U0126. The phosphorylation of ERK was reduced by Mg²⁺ deprivation, and recovered by re-addition of Mg²⁺. These results suggest that the MEK/ERK-dependent phosphorylation of claudin-16 affects the tight junctional localization and function of claudin-16. Mg²⁺ deprivation decreased the phosphothreonine levels of claudin-16. The phosphothreonine levels of T225A and T233A claudin-16 were decreased in the presence of Mg²⁺ and these mutants were widely distributed in the plasma membrane. Furthermore, TER and transepithelial Mg²⁺ permeability were decreased in the mutants. We suggest that the tight junctional localization of claudin-16 requires a physiological Mg²⁺ concentration and the phosphorylation of threonine residues via a MEK/ERK-dependent pathway.     

Expression and function of tight junction associated molecules in human breast tumor cells is not affected by the Ras-MEK1 pathway.

Constitutive activation of Ras or Ras-mediated signaling pathways is one of the initial steps during tumorigenesis that promotes neoplastic transformation. Recently it was reported that in Ha-Ras overexpressing MDCK cells the tight junction proteins claudin-1, occludin and ZO-1 were absent at cell-cell contact sites but present in the cytoplasm. Inhibition of MEK1 activity recruited all three proteins to the cell membrane leading to a restoration of the tight junction barrier function in MDCK cells. In order to evaluate the relevance of the MEK1 pathway in tight junction regulation in breast cancer cells, we investigated the effect ofMEK1 inhibition on expression of claudin-1, occludin and ZO-1 in natively claudin-1 expressing T47-D cells (low Ras activity), claudin-1 negative MCF-7 cells (elevated Ras activity) as well as two retroviral claudin-1 transduced MCF-7 daughter cell lines with prominent membrane and cytoplasmic claudin-1 dominant homing, respectively. Although we effectively blocked phosphorylation of MAPKs ERK-1 and ERK-2 using the selective MEK1 inhibitor PD98059, no quantitative changes of mRNA or protein levels of claudin-1, occludin and ZO-1 could be detected in all cell lines investigated. Furthermore, immnfluorescence analysis of claudin-1 revealed that inhibition of the MAPK pathway did not alter th e subcellular cytoplasmic distribution of claudin-1 to be more membrane specific. Finally, the diffusion barrier properties of tight junctions as analyzed by transepithelial resistance (TER) or paracellular flux analysis of 3 and 40 kDa dextran of tight junctions were not altered in the claudin-1 positive T47-D and the MCF-7 cell lines. Our findings indicate that the proposed involvement of the Ras-MEK-ERK pathway is likely not involved in the dysregulated tight junction formation in breast tumor cells and indicates that elevated activity of Ras might not be of general importance for the disruption of tight junction structures in breast tumors.     

Actin depolymerization disrupts tight junctions via caveolae-mediated endocytosis

The tight junction (TJ) determines epithelial barrier function. Actin depolymerization disrupts TJ structure and barrier function, but the mechanisms of this effect remain poorly understood. The goal of this study was to define these mechanisms. Madin-Darby canine kidney (MDCK) cells expressing enhanced green fluorescent protein-, enhanced yellow fluorescent protein-, or monomeric red fluorescent protein 1-fusion proteins of beta-actin, occludin, claudin-1, ZO-1, clathrin light chain A1, and caveolin-1 were imaged by time-lapse multidimensional fluorescence microscopy with simultaneous measurement of transepithelial electrical resistance (TER). Actin depolymerization was induced with latrunculin A (LatA). Within minutes of LatA addition TER began to fall. This coincided with occludin redistribution and internalization. In contrast, ZO-1 and claudin-1 redistribution occurred well after maximal TER loss. Occludin internalization and TER loss, but not actin depolymerization, were blocked at 14 degrees C, suggesting that membrane traffic is required for both events. Inhibition of membrane traffic with 0.4 M sucrose also blocked occludin internalization and TER loss. Internalized occludin colocalized with caveolin-1 and dynamin II, but not with clathrin, and internalization was blocked by dominant negative dynamin II (K44A), but not by Eps15Delta95-295 expression. Inhibition of caveolae-mediated endocytosis by cholesterol extraction prevented both LatA-induced TER loss and occludin internalization. Thus, LatA-induced actin depolymerization causes TJ structural and functional disruption by mechanisms that include caveolae-mediated endocytosis of TJ components.     

TFF3-peptide increases transepithelial resistance in epithelial cells by modulating claudin-1 and -2 expression

TFF3 plays an important role in the protection and repair of the gastrointestinal mucosa. The molecular mechanisms of TFF function, however, are still largely unknown. Increasing evidence indicates that apart from stabilizing mucosal mucins TFF3 induces cellular signals that modulate cell–cell junctions of epithelia. In transfected HT29/B6 and MDCK cells stably expressing FLAG-tagged human TFF3 we have recently shown that TFF3 down-regulates E-cadherin, impairs the function of adherens junctions and thus facilitates cell migration in wounded epithelial cell layers. Here we investigate TFF3-induced effects on the composition and function of tight junctions in these cells. TFF3 increased the cellular level of tightening claudin-1 and decreased the amount of claudin-2 known to form cation-selective channels. Expression of ZO-1, ZO-2 and occludin was not altered. The change in claudin-1 and -2 expression in TFF3-expressing HT29/B6 cells was accompanied by an increase in the transepithelial resistance in confluent monolayers of these cells. These data suggest that TFF3 plays a role in the regulation of intestinal barrier function by altering the claudin composition within tight junctions thus decreasing paracellular permeability of the intestinal mucosa.     

Developmental regulation of claudin localization by fetal alveolar epithelial cells

Tight junction proteins in the claudin family regulate epithelial barrier function. We examined claudin expression by human fetal lung (HFL) alveolar epithelial cells cultured in medium containing dexamethasone, 8-bromo-cAMP, and isobutylmethylxanthanine (DCI), which promotes alveolar epithelial cell differentiation to a type II phenotype. At the protein level, HFL cells expressed claudin-1, claudin-3, claudin-4, claudin-5, claudin-7, and claudin-18, where levels of expression varied with culture conditions. DCI-treated differentiated HFL cells cultured on permeable supports formed tight transepithelial barriers, with transepithelial resistance (TER) >1,700 ohm/cm(2). In contrast, HFL cells cultured in control medium without DCI did not form tight barriers (TER <250 ohm/cm(2)). Consistent with this difference in barrier function, claudins expressed by HFL cells cultured in DCI medium were tightly localized to the plasma membrane; however, claudins expressed by HFL cells cultured in control medium accumulated in an intracellular compartment and showed discontinuities in claudin plasma membrane localization. In contrast to claudins, localization of other tight junction proteins, zonula occludens (ZO)-1, ZO-2, and occludin, was not sensitive to HFL cell phenotype. Intracellular claudins expressed by undifferentiated HFL cells were localized to a compartment containing early endosome antigen-1, and treatment of HFL cells with the endocytosis inhibitor monodansylcadaverine increased barrier function. This suggests that during differentiation to a type II cell phenotype, fetal alveolar epithelial cells use differential claudin expression and localization to the plasma membrane to help regulate tight junction permeability.     

Cyclic AMP induces phosphorylation of claudin-5 immunoprecipitates and expression of claudin-5 gene in blood-brain-barrier endothelial cells via protein kinase A-dependent and -independent pathways

Cyclic AMP (cAMP) promotes functions of tight junctions in endothelial cells, although its target remains unknown. We showed here that cAMP increased gene expression of claudin-5 and decreased that of claudin-1 in porcine blood-brain-barrier endothelial cells via protein kinase A (PKA)-independent and -dependent pathways, respectively. cAMP also enhanced immunoreactivity of claudin-5 along cell borders and in the cytoplasm, reorganized actin filaments, and altered signals of claudin-5, occludin, ZO-1, and ZO-2 along cell boundaries from zipperlike to linear patterns. In contrast, claudin-1 was detected only in the cytoplasm in a dotlike pattern, and its immunolabeling was reduced by cAMP. Interestingly, 31- and 62-kDa claudin-5 immunoprecipitates in the NP-40-soluble and -insoluble fractions, respectively, were highly phosphorylated on threonine residue(s) upon cAMP treatment. All these changes induced by cAMP, except for claudin-5 expression and its signals in the cytoplasm, were reversed by an inhibitor of PKA, H-89. We also demonstrated that cAMP elevated the barrier function of tight junctions in porcine blood-brain-barrier endothelial cells in PKA-dependent and -independent manners. These findings indicate that both PKA-induced phosphorylation of claudin-5 immunoprecipitates and cAMP-dependent but PKA-independent induction of claudin-5 expression could be involved in promotion of tight-junction function in endothelial cells.     

LXA4 stimulates ZO-1 expression and transepithelial electrical resistance in human airway epithelial (16HBE14o-) cells

Lipoxin A(4) (LXA(4)) is a biologically active eicosanoid produced in human airways that displays anti-inflammatory properties. In cystic fibrosis and severe asthma, LXA(4) production has been reported to be decreased, and, in such diseases, one of the consequences of airway inflammation is disruption of the tight junctions. In the present study, we investigated the possible role of LXA(4) on tight junction formation, using transepithelial electrical resistance (TER) measurements, Western blotting, and immunofluorescence. We observed that exposure to LXA(4) (100 nM) for 2 days significantly increased zonula occludens-1 (ZO-1), claudin-1, and occludin expression at the plasma membrane of confluent human bronchial epithelial 16HBE14o- cells. LXA(4) (100 nM) stimulated the daily increase of the 16HBE14o- cell monolayer TER, and this effect was inhibited by boc-2 (LXA(4) receptor antagonist). LXA(4) also had a rapid effect on ZO-1 immunofluorescence at the plasma membrane and increased TER within 10 min. In conclusion, our experiments provide evidence that LXA(4) plays certainly a new role for the regulation of tight junction formation and stimulation of the localization and expression of ZO-1 at the plasma membrane through a mechanism involving the LXA(4) receptor.     

Compromised E-cadherin adhesion and epithelial barrier function with activation of G protein-coupled receptors is rescued by Y-to-F mutations in beta-catenin

Activation of the type 1 histamine (H1) or the type 2 protease-activated (PAR-2) G protein-coupled receptors interrupts E-cadherin adhesion and decreases the transepithelial resistance (TER) of epithelium. Several reports suggest that cadherin adhesive function depends on the association of cadherin with beta-catenin and that this association is regulated by phosphorylation of tyrosines in beta-catenin. We tested the hypothesis that loss of cadherin adhesion and compromise of TER on activation of the H1 or PAR-2 receptor is due to phosphorylation of tyrosines in beta-catenin. L cells were stably transfected to express E-cadherin (L-E-cad cells) and H1 (L-H1-E-cad cells). L cells and Madin-Darby canine kidney (MDCK) cells constitutively express PAR-2. Stably transfected L-E-cad, L-H1-E-cad, and MDCK cells were also stably transfected with FLAG-tagged wild-type (WT) or mutant beta-catenin, converting tyrosine 142, 489, or 654 to the nonphosphorylatable mimetic, phenylalanine (WT, Y142F, Y489F, or Y654F). Activation of H1 or PAR-2 interrupted adhesion to an immobilized E-cadherin-Fc fusion protein of L-H1-E-cad, L-E-cad, and MDCK cells expressing WT or Y142F beta-catenin but did not interrupt adhesion of L-H1-E-cad, L-E-cad, and MDCK cells expressing the Y489F or Y654F mutant beta-catenins. PAR-2 activation decreased the TER of monolayers of MDCK cells expressing WT or Y142F beta-catenin 40-45%. However, PAR-2 activation did not decrease the TER of monolayers of MDCK cells expressing Y489F or Y654F beta-catenin. The protein tyrosine phosphatase PTP1B binds to the cadherin cytoplasmic domain and dephosphorylates beta-catenin. Inhibition of PTP1B interrupted adhesion to E-cadherin-Fc of MDCK cells expressing WT beta-catenin but did not affect the adhesion of MDCK cells expressing Y489F or Y654F beta-catenin. Similarly, inhibition of PTP1B compromised the TER of MDCK cells expressing WT beta-catenin but did not affect the TER of MDCK cells expressing Y489F or Y654F beta-catenin. We conclude that phosphorylation of tyrosines 489 and 654 in beta-catenin is a necessary step in the process by which G protein-coupled H1 and PAR-2 receptors interrupt E-cadherin adhesion. We also conclude that activation of PAR-2 has no effect on the TER without first interrupting E-cadherin adhesion.     

Reorganization of ZO-1 by sodium-dependent glucose transporter activation after heat stress in LLC-PK1 cells

Heat stress (HS) induces activation of high-affinity sodium-dependent glucose transporter (SGLT1) in porcine renal LLC-PK(1) cells. In this study, we investigated the roles of SGLT1 activation in reorganization of zonula occludens-1 (ZO-1), a cytosolic tight junction (TJ) protein, after HS. HS (42 degrees C, 3 h) caused decrease in transepithelial electrical resistance (TER). Subsequent incubation at 37 degrees C for 12 h increased TER above pre-HS level. The treatment of phloridzin, a potent SGLT1 inhibitor, or the replacement of glucose with a nonmetabolizable glucose analog blocked the recovery of TER and increased the transepithelial flux of FITC-dextran (4,000 Da). Immunofluorescent staining of ZO-1 showed that HS diffused ZO-1 from cell contact to cytosolic sites. Furthermore, the fraction of ZO-1 was distributed from the Triton X-100 insoluble to the Triton X-100 soluble pool. After incubation at 37 degrees C for 12 h, cell contact and ZO-1 extractability with Triton X-100 returned to pre-HS conditions, but the recovery was completely prevented by phloridzin. Tyrosine kinases activity was increased by HS that was inhibited by phloridzin. Genistein and CGP77675, tyrosine kinases inhibitors, blocked the recovery of TER and increased the transepithelial flux of FITC-dextran. Furthermore, these inhibitors prevented the recovery of cell contact and ZO-1 extractability with Triton X-100 as same as phloridzin. These findings suggested that the activation of SGLT1 reorganized ZO-1 mediated by elevation of tyrosine kinases activity after heat injury.     

Sodium-dependent glucose transporter reduces peroxynitrite and cell injury caused by cisplatin in renal tubular epithelial cells

Cisplatin causes nephropathy accompanied by two types of cell death, necrosis and apoptosis, according to its dosage. The mechanisms of necrosis are still unclear. In this study, we examined how high doses of cisplatin induce cell injury and whether a high affinity sodium-dependent glucose transporter (SGLT1) has a cytoprotective function in renal epithelial LLC-PK₁ cells. Cisplatin decreased in transepithelial electrical resistance (TER) and increased in the number of necrotic dead cells in a time dependent manner. Phloridzin, a potent SGLT1 inhibitor, enhanced both TER decrease and increase of necrotic dead cells caused by cisplatin. Cisplatin increased in the intracellular nitric oxide, superoxide anion and peroxynitrite productions. Phloridzin enhanced the peroxynitrite production caused by cisplatin. The intracellular diffusion of ZO-1 and TER decrease caused by cisplatin were inhibited by N-nitro-l-arginine methyl ester, a nitric oxide synthase inhibitor. Protein kinase C was not involved in the cisplatin-induced injury. 5,10,15,20-tetrakis-(4-sulfonatophenyl)-porphyrinato iron (III) and reduced glutathione, peroxynitrite scavengers, inhibited the cisplatin-induced ZO-1 diffusion, TER decrease, and increase of necrotic dead cells. These results suggest that peroxynitrite is a key mediator in the nephrotoxicity caused by high doses of cisplatin. SGLT1 endogenously carries out the cytoprotective function by the reduction of peroxynitrite production.     

RhoA and Rho kinase mediate cyclosporine A and sirolimus-induced barrier tightening in renal proximal tubular cells

The regulation and maintenance of the paracellular transport in renal tubular epithelia is vital for kidney functions. Combination of the immunosuppressant drugs cyclosporine A (CsA) and sirolimus (SRL) exerts powerful immunosuppression, but also causes nephrotoxicity. We have previously shown that CsA and SRL elevate transepithelial resistance (TER) in kidney tubular cells partly through MEK/ERK1/2. In this work we examined the hypothesis that the RhoA pathway may also be mediating effects of CsA and SRL. We show that CsA and the CsA/SRL combination activated RhoA, induced cofilin phosphorylation and promoted stress fiber generation. The Rho kinase (ROK) inhibitor, Y27632, prevented CsA and CsA/SRL-induced cofilin phosphorylation and actin remodelling, reduced the TER increase and prevented the rise in claudin-7 levels caused by the drugs. Expression of the exchange factor GEF-H1/lfc was elevated in cells treated with CsA and CsA/SRL. GEF-H1 silencing inhibited RhoA activation by ≈50%, and potently reduced cofilin phosphorylation and stress fiber formation induced by CsA and CsA/SRL. However, GEF-H1 downregulation did not prevent the TER change. Thus the Rho/Rho kinase pathway was involved in mediating CsA and CsA/SRL-induced cytoskeleton rearrangement and TER changes via claudin-7 expression. Our data however point to differential regulation of Rho activation involved in central cytoskeleton remodelling, that is GEF-H1-dependent and junctional permeability that does not require GEF-H1.     

Functional linkage between the transport characteristics of the MDCK1 cell monolayer and their actin cytoskeleton organization

The dynamics of the actin cytoskeleton spatial organization and transepithelial electric resistance (TEER) in the MDCK1 cell monolayer exposed to arginine–vasopressin (AVP) and forskolin, a protein kinase A (PKA) activator, have been studied. These physiologically active substances are shown to depolymerize filamentous actin in MDCK1 cells (in both the apical and basal cytoplasm) and, concurrently, to considerably decrease the TEER of the cell monolayer. A decrease in TEER suggests an increase in the ion current through the cell monolayer. Correspondingly, the created ion gradient stimulates AVP-sensitive water flow. To clarify the routes of ions and water in MDCK monolayer, the localization of claudin-1 and -2 in tight junctions of ATCC (American Type Culture Collection) MDCK (a low TEER) and MDCK1 (a high TEER) cells was studied by immunofluorescence assay. Claudin-1 and -2 are detectable in the tight junctions of ATCC MDCK cells; however, the tight junctions of MDCK1 cells contain only claudin-1, whereas poreforming claudin-2 is absent. The exposure of MDCK1 cells to forskolin fails to change the distribution of the studied claudins, thereby suggesting that a decrease in TEER caused by forskolin is associated with a change in transcellular, rather than paracellular, permeability of the monolayer