Amniotic fluid stem cells prevent ß-cell injury.

BACKGROUND AIMS: The contribution of amniotic fluid stem cells (AFSC) to tissue protection and regeneration in models of acute and chronic kidney injuries and lung failure has been shown in recent years. In the present study, we used a chemically induced mouse model of type 1 diabetes to determine whether AFSC could play a role in modulating ß-cell injury and restoring ß-cell function. METHODS: Streptozotocin-induced diabetic mice were given intracardial injection of AFSC; morphological and physiological parameters and gene expression profile for the insulin pathway were evaluated after cell transplantation. RESULTS: AFSC injection resulted in protection from ß-cell damage and increased ß-cell regeneration in a subset of mice as indicated by glucose and insulin levels, increased islet mass and preservation of islet structure. Moreover, ß-cell preservation/regeneration correlated with activation of the insulin receptor/Pi3K/Akt signaling pathway and vascular endothelial growth factor-A expression involved in maintaining ß-cell mass and function. CONCLUSIONS: Our results suggest a therapeutic role for AFSC in preserving and promoting endogenous ß-cell functionality and proliferation. The protective role of AFSC is evident when stem cell transplantation is performed before severe hyperglycemia occurs, which suggests the importance of early intervention. The present study demonstrates the possible benefits of the application of a non-genetically engineered stem cell population derived from amniotic fluid for the treatment of type 1 diabetes mellitus and gives new insight on the mechanism by which the beneficial effect is achieved.

Claudin-2 pore function requires an intramolecular disulfide bond between two conserved extracellular cysteines.

Claudins constitute a family of tight junction transmembrane proteins whose first extracellular loop (ECL1) determines the paracellular permeability and ion selectivity in epithelia. There are two cysteines in the ECL1 that are conserved among all claudins. We hypothesized that these extracellular cysteines are linked by an intramolecular disulfide bond that is necessary for correct pore folding and function. To test this, we mutated C54 and C64 in claudin-2, either individually or together to alanine or serine, and generated stable Madin-Darby canine kidney (MDCK) I Tet-off cell lines. Immunoblotting showed a higher molecular mass band in the mutants with a single cysteine mutation, consistent with a claudin-2 dimer, suggesting that the two conserved cysteines normally form an intramolecular disulfide bond in wild-type claudin-2. By immunofluorescent staining, the alanine mutants were mislocalized intracellularly, while the serine mutants were expressed at the tight junction. Thus dimerization of both C54A and C64A did not require tight junction expression, suggesting that C54 and C64 are located near an intermolecular interface involved in cis-interaction. The conductance and Na(+) permeability of the serine mutants were markedly lower than the wild type, but there was no difference between the single mutants and the double mutant. We conclude that the disulfide bond between the conserved extracellular cysteines in claudin-2 is necessary for pore formation, probably by stabilizing the ECL1 fold, but is not required for correct protein trafficking. We further speculate that this role is generalizable to other claudin family members.

Stem cells as a therapeutic approach to chronic kidney diseases.

Much attention recently has been focused on stem cell technology as a possible alternative modality of treatment of a variety of diseases. Chronic kidney disease is a serious health problem and most chronic kidney diseases share in common the presence of interstitial and glomerular fibrosis, regardless of the underlying cause. To date there are no specific therapies aimed at treating fibrosis in the kidney. In a novel effort to address the underlying pathology in kidney disease, researchers are demonstrating that stem cell therapy can attenuate fibrosis in chronic kidney disease in animal models. This review will focus on the recent developments in stem cell research and their possible implications to treat chronic kidney disease.

Polystyrene nanoparticle trafficking across MDCK-II.

Polystyrene nanoparticles (PNP) cross rat alveolar epithelial cell monolayers via non-endocytic transcellular pathways. To evaluate epithelial cell type-specificity of PNP trafficking, we studied PNP flux across Madin Darby canine kidney cell II monolayers (MDCK-II). The effects of calcium chelation (EGTA), energy depletion (sodium azide (NaN(3)) or decreased temperature), and endocytosis inhibitors methyl-ß-cyclodextrin (MBC), monodansylcadaverine and dynasore were determined. Amidine-modified PNP cross MDCK-II 500 times faster than carboxylate-modified PNP. PNP flux did not increase in the presence of EGTA. PNP flux at 4 °C and after treatment with NaN(3) decreased 75% and 80%, respectively. MBC exposure did not decrease PNP flux, whereas dansylcadaverine- or dynasore-treated MDCK-II exhibited ∼80% decreases in PNP flux. Confocal laser scanning microscopy revealed intracellular colocalization of PNP with clathrin heavy chain. These data indicate that PNP translocation across MDCK-II (1) occurs via clathrin-mediated endocytosis and (2) is dependent on PNP physicochemical properties. We conclude that uptake/trafficking of nanoparticles (NPs) into/across epithelia depends both on properties of the NPs and on the specific epithelial cell type.

Structure-function studies of claudin extracellular domains by cysteine-scanning mutagenesis.

Claudins form size- and charge-selective pores in the tight junction that control the paracellular flux of inorganic ions and small molecules. However, the structural basis for ion selectivity of paracellular pores is poorly understood. Here we applied cysteine scanning to map the paracellular pathway of ion permeation across claudin-2-transfected Madin-Darby canine kidney type I cells. Four potential pore-lining amino acid residues in the first extracellular loop were mutated to cysteine and screened for their accessibility to thiol-reactive reagents. All mutants were functional except D65C, which formed dimers by intermolecular disulfide bonding, leading to a loss of charge and size selectivity. This suggests that claudin-2 pores are multimeric and that Asp(65) lies close to a protein-protein interface. Methanethiosulfonate reagents of different size and charge and the organic mercury derivate, p-(chloromercuri)benzenesulfonic acid, significantly decreased paracellular ion permeation across I66C-transfected cells by a mechanism that suggests steric blocking of the pore. The conductance of wild-type claudin-2 and the other cysteine mutants was only weakly affected. The rate of reaction with I66C decreased dramatically with increasing size of the reagent, suggesting that Ile(66) is buried deep within a narrow segment of the pore with its side group facing into the lumen. Furthermore, labeling with N-biotinoylaminoethyl methanethiosulfonate showed that I66C was weakly reactive, whereas Y35C was strongly reactive, suggesting that Tyr(35) is located at the protein surface outside of the pore.

Phorbol ester induced short- and long-term permeabilization of the blood-CSF barrier in vitro.

Interconnected by tight junctions, the epithelial cells of the choroid plexus form a barrier separating the cerebrospinal fluid (CSF) from blood. Using an in vitro model based on porcine choroid plexus epithelial cells (PCPEC), we investigated the influence of PKC activating phorbol 12-myristate 13-acetate (PMA) on barrier properties and analyzed mechanisms involved in the regulation of barrier tightness. Applied in concentrations of 5-25 nM, PMA induced a fast and lasting decrease of the transepithelial electrical resistance (TER), which could be blocked by rottlerin, indicating the involvement of PKCdelta in signal transduction. The immediate impairment of barrier integrity was accompanied by dephosphorylation of occludin and formation of actin bundles. Moreover, in the presence of at least 25 nM PMA, changes of cell shape as well as discontinuities of tight junction strands were observed, suggesting the disruption of cell-cell contacts. Exposure to PMA for 1-2 days additionally induced down-regulation of claudin-2 and up-regulation of barrier modulating matrix metalloproteinase (MMP)-9, respectively. The results show that different interconnected mechanisms directly and indirectly targeting at the tight junctions are released by PMA contributing to the short-term and long-term decrease of TER and opening of the blood-CSF barrier in vitro.

Usefulness and limitation of primary cultured porcine choroid plexus epithelial cells as an in vitro model to study drug transport at the blood-CSF barrier.

The epithelial cells of the choroid plexus form the anatomical structure responsible for the blood-cerebrospinal fluid (CSF) barrier. Here we present our recent progress in the application of porcine choroid plexus epithelial cells in the investigation of permeability and transport properties of this tissue in vitro. Isolated cells are seeded on permeable supports where they grow to confluent monolayers. The cell differentiation is significantly increased under serum-free culture conditions, verifiable by an improvement of barrier properties and enhanced characteristics of the epithelial phenotype. We underline the importance of a tight model system for the investigation of transport processes by showing that permeability and transport properties critically depend on the electrical tightness of the monolayer. The mechanisms of vectorial transfer of micronutrients across the epithelial layer have been investigated in detail for ascorbic acid and myo-inositol transport. Additionally, we describe the transfer of organic anions and the expression of the corresponding transport proteins in vitro. The model system was applied to determine permeation rates of various drugs into the CSF. In conclusion our porcine in vitro model of the blood-CSF barrier provides a reliable system to study the transport characteristics of the choroid plexus epithelium and to probe the passage of drugs.

Functional characterisation of the active ascorbic acid transport into cerebrospinal fluid using primary cultured choroid plexus cells.

Crossing the blood-CSF barrier is an important pathway for certain nutrients to enter the CNS. Cultured choroid plexus epithelial cells are a potent model system to study active transport properties of this tissue in vitro. In the present study this in vitro model was used to analyse ascorbic acid transport across the blood-CSF barrier that is supposedly mediated by the Na(+)-dependent transporter SVCT2. The expression of SVCT2 in the cultured cells was proven by RT-PCR. Active transport across the cell monolayer resulted in ascorbic acid enrichment at the CSF mimicking side. Ascorbic acid transport and uptake were decreased to 13 and 27%, respectively, in the presence of 200 microM phloretin. Inhibition of both transepithelial substrate transport (to 7.5%) and cytoplasmatic uptake (to 20%) was observed in Na(+)-free medium indicating that a basolaterally located and Na(+)-dependent transporter mediates ascorbic acid uptake. Substituting Cl(-) by either iodide or D-gluconate increased ascorbic acid uptake by factors of 3.7 or 2.5, respectively. Similar observations were made when Na(+)-dependent myo-inositol transport was analysed. Additionally, in presence of 100 microM bumetanide, an inhibitor of Na(+)-Cl(-)-cotransport, indirectly increased ascorbic acid and myo-inositol transport rates were observed showing that ascorbic acid-Na(+)-cotransport might balance low intracellular Na(+) concentration.

Cysteine mutagenesis to study the structure of claudin-2 paracellular pores.

The structure and transport mechanism of paracellular pores are only poorly understood. Here we describe for the first time how the substituted cysteine accessibility method (SCAM), previously developed to study transmembrane transport, can be applied to analyze the pathway of paracellular ion permeation. Using stable transfected Madin Darby canine kidney type I cells, induced to express claudin-2, we show that paracellular cation transport can be blocked by sulfhydryl-specific methanethiosulfonate (MTS) and that SCAM can be used to identify residues that line paracellular pores.

Molecular basis for cation selectivity in claudin-2-based paracellular pores: identification of an electrostatic interaction site.

Paracellular ion transport in epithelia is mediated by pores formed by members of the claudin family. The degree of selectivity and the molecular mechanism of ion permeation through claudin pores are poorly understood. By expressing a high-conductance claudin isoform, claudin-2, in high-resistance Madin-Darby canine kidney cells under the control of an inducible promoter, we were able to quantitate claudin pore permeability. Claudin-2 pores were found to be narrow, fluid filled, and cation selective. Charge selectivity was mediated by the electrostatic interaction of partially dehydrated permeating cations with a negatively charged site within the pore that is formed by the side chain carboxyl group of aspartate-65. Thus, paracellular pores use intrapore electrostatic binding sites to achieve a high conductance with a high degree of charge selectivity.

Biology of claudins.

Claudins are a family of tight junction membrane proteins that regulate paracellular permeability of epithelia, likely by forming the lining of the paracellular pore. Claudins are expressed throughout the renal tubule, and mutations in two claudin genes are now known to cause familial hypercalciuric hypomagnesemia with nephrocalcinosis. In this review, we discuss recent advances in our understanding of the physiological role of various claudins in normal kidney function, and in understanding the fundamental biology of claudins, including the molecular basis for selectivity of permeation, claudin interactions in tight junction formation, and regulation of claudins by protein kinases and other intracellular signals.

Galpha12 regulates protein interactions within the MDCK cell tight junction and inhibits tight-junction assembly.

The polarized functions of epithelia require an intact tight junction (TJ) to restrict paracellular movement and to separate membrane proteins into specific domains. TJs contain scaffolding, integral membrane and signaling proteins, but the mechanisms that regulate TJs and their assembly are not well defined. Galpha12 (GNA12) binds the TJ protein ZO-1 (TJP1), and Galpha12 activates Src to increase paracellular permeability via unknown mechanisms. Herein, we identify Src as a component of the TJ and find that recruitment of Hsp90 to activated Galpha12 is necessary for signaling. TJ integrity is disrupted by Galpha12-stimulated Src phosphorylation of ZO-1 and ZO-2 (TJP2); this phosphorylation leads to dissociation of occludin and claudin 1 from the ZO-1 protein complex. Inhibiting Hsp90 with geldanamycin blocks Galpha12-stimulated Src activation and phosphorylation, but does not affect protein levels or the Galpha12-ZO-1 interaction. Using the calcium-switch model of TJ assembly and GST-TPR (GST-fused TPR domain of PP5) pull-downs of activated Galpha12, we demonstrate that switching to normal calcium medium activates endogenous Galpha12 during TJ assembly. Thrombin increases permeability and delays TJ assembly by activating Galpha12, but not Galpha13, signaling pathways. These findings reveal an important role for Galpha12, Src and Hsp90 in regulating the TJ in established epithelia and during TJ assembly.

Claudins and paracellular transport: an update.

PURPOSE OF REVIEW: Claudins are tight junction proteins that form paracellular barriers and pores. The purpose of this timely review is to provide an update on the exciting new advances in our understanding of claudin biology and their relevance to renal physiology and pathophysiology. RECENT FINDINGS: Accumulating evidence from numerous studies indicates that the primary role of claudins is to determine the permeability and charge selectivity of the paracellular pathway to small ions. Studies in which claudins are overexpressed in cell lines have potential limitations and need to be interpreted cautiously. Ribonucleic acid interference is a novel approach to functional characterization. Claudins are believed to assemble into multimers by homophilic and heterophilic side-by-side and head-to-head interaction; however, there is still limited evidence for this. The roles of a few claudins in the renal tubule, including claudins 2, 8, 10, 16 and 19, have now been elucidated. SUMMARY: These findings reveal tantalizing clues to claudin biology and function. Much remains unknown, however, and these findings will hopefully encourage further research in this important area.

Claudin-8 expression in renal epithelial cells augments the paracellular barrier by replacing endogenous claudin-2.

Claudins are transmembrane proteins of the tight junction that determine and regulate paracellular ion permeability. We previously reported that claudin-8 reduces paracellular cation permeability when expressed in low-resistance Madin-Darby canine kidney (MDCK) II cells. Here, we address how the interaction of heterologously expressed claudin-8 with endogenous claudin isoforms impacts epithelial barrier properties. In MDCK II cells, barrier improvement by claudin-8 is accompanied by a reduction of endogenous claudin-2 protein at the tight junction. Here, we show that this is not because of relocalization of claudin-2 into the cytosolic pool but primarily due to a decrease in gene expression. Claudin-8 also affects the trafficking of claudin-2, which was displaced specifically from the junctions at which claudin-8 was inserted. To test whether replacement of cation-permeable claudin-2 mediates the effect of claudin-8 on the electrophysiological phenotype of the host cell line, we expressed claudin-8 in high-resistance MDCK I cells, which lack endogenous claudin-2. Unlike in MDCK II cells, induction of claudin-8 in MDCK I cells (which did not affect levels of endogenous claudins) did not alter paracellular ion permeability. Furthermore, when endogenous claudin-2 in MDCK II cells was downregulated by epidermal growth factor to create a cell model with low transepithelial resistance and low levels of claudin-2, the permeability effects of claudin-8 were also abolished. Our findings demonstrate that claudin overexpression studies measure the combined effect of alterations in both endogenous and exogenous claudins, thus explaining the dependence of the phenotype on the host cell line.

Renal localization and function of the tight junction protein, claudin-19.

Claudins form a family of transmembrane tight junction proteins that play a key role in control and selectivity of paracellular transport. Mutations in claudin-19, which is expressed in kidney, retina, and myelinated peripheral neurons, were identified in familial hypomagnesemia with hypercalciuria and nephrocalcinosis, a hereditary disease causing renal Mg(2+) and Ca(2+) wasting. Here, we studied the distribution and possible functional role of claudin-19 in the renal tubule. By immunofluorescence staining of mouse kidney, claudin-19 was found to be expressed at the tight junction of the thick ascending limb of Henle, the major site of paracellular Mg(2+) reabsorption, where it colocalized with claudin-16, as well as in the thin ascending limb. The role of claudin-19 in paracellular transport was tested by stable transfection into Madin Darby canine kidney II TetOff cells to generate inducible cell lines. Claudin-19 increased the transepithelial electrical resistance and decreased permeability to monovalent and divalent cations, while anion and urea permeability were not affected. Our data suggest that claudin-19 acts as a selective cation barrier at the tight junction. This would be consistent with its physiological role to electrically seal myelinated peripheral neurons. The normal role of claudin-19 in renal tubule function remains to be determined.

Claudin-8 modulates paracellular permeability to acidic and basic ions in MDCK II cells.

Renal net acid excretion requires tubular reabsorption of filtered bicarbonate, followed by secretion of protons and ammonium in the collecting duct, generating steep transtubular gradients for these ions. To prevent passive backleak of these ions, the tight junctions in the collecting duct must be highly impermeable to these ions. We previously generated a Madin-Darby canine kidney (MDCK II) cell line with inducible expression of claudin-8, a tight junction protein expressed in the collecting duct. In these cells, claudin-8 was shown to function as a paracellular barrier to alkali metal and divalent cations. We have now used this model to test the hypothesis that claudin-8 also functions as a paracellular barrier to acidic or basic ions involved in renal acid excretion. We developed a series of precise and unbiased methods, based on a combination of diffusion potential, short-circuit current, and pH stat measurements, to estimate paracellular permeability to protons, ammonium and bicarbonate in MDCK II cells. We found that under control conditions (i.e. in the absence of claudin-8), these cells are highly permeable to the acidic and basic ions tested. Interestingly, proton permeation exhibited an unusually low activation energy similar to that in bulk solution. This suggests that paracellular proton transfer may occur by a Grotthuss mechanism, implying that the paracellular pores are sufficiently wide to accommodate water molecules in a freely mobile state. Induction of claudin-8 expression reduces permeability not only to protons, but also to ammonium and bicarbonate. We conclude that claudin-8 probably functions to limit the passive leak of these three ions via paracellular routes, thereby playing a permissive role in urinary net acid excretion.

Porcine choroid plexus epithelial cells induce Streptococcus suis bacteriostasis in vitro.

The involvement of the choroid plexus in host defense during bacterial meningitis is unclear. Aiming to elucidate possible antibacterial mechanisms, we stimulated primary porcine choroid plexus epithelial cells (pCPEC) with proinflammatory cytokines and challenged them with various Streptococcus suis strains. In the supernatant of gamma interferon (IFN-gamma)-stimulated pCPEC, streptococcal growth was markedly suppressed. Costimulation with tumor necrosis factor alpha enhanced this bacteriostatic effect, while supplementation of L-tryptophan completely eliminated it. We also demonstrate that an activation of indoleamine 2,3-dioxygenase in the pCPEC seems to be responsible for the IFN-gamma-induced bacteriostasis. This supports the hypothesis of an active role of the choroid plexus in host defense against bacterial meningitis.