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.

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.

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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 Mg2+ and Ca2+ 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 Mg2+ 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.

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.Sheets of polarized epithelia constitute barriers that separate fluid compartments of different chemical composition and mediate exchange of solutes and ions via transcellular and paracellular pathways. A large body of evidence suggests that transport via the paracellular pathway occurs through pores in the tight junctions that are formed by tetraspan membrane proteins, known as claudins (1-3).Our current understanding of paracellular pores is that they are size-and charge-selective water-filled channels that, in contrast to channels for transmembrane transport, are oriented parallel instead of perpendicular to the lipid layer of the cell membrane. Size exclusion experiments suggest a pore diameter of 6.4 -8 Å (4, 5). Furthermore, site-directed mutagenesis and overexpression of claudins in epithelial cells identified the first extracellular domain as playing an important role in the charge selectivity of paracellular transport (6 -8). The first extracellular domain of claudins contains various basic and acidic amino acids, some of which are conserved in different claudin isoforms, and these could be involved in the mechanism of ion permeation. Several studies have demonstrated homo-or heterotypic interaction of claudins, suggesting that paracellular pores are formed by oligomers of claudins (9 -11). Taken together, significant progress has been made in uncovering the nature of the paracellular pathway and mechanisms of selectivity ...

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. Gα12 (GNA12) binds the TJ protein ZO-1 (TJP1), and Gα12 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 Gα12 is necessary for signaling. TJ integrity is disrupted by Gα12-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 Gα12-stimulated Src activation and phosphorylation, but does not affect protein levels or the Gα12–ZO-1 interaction. Using the calcium-switch model of TJ assembly and GST-TPR (GST-fused TPR domain of PP5) pull-downs of activated Gα12, we demonstrate that switching to normal calcium medium activates endogenous Gα12 during TJ assembly. Thrombin increases permeability and delays TJ assembly by activating Gα12, but not Gα13, signaling pathways. These findings reveal an important role for Gα12, Src and Hsp90 in regulating the TJ in established epithelia and during TJ assembly.

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.

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). Effects of calcium chelation (EGTA), energy depletion (sodium azide (NaN 3 ) or decreased temperature), and endocytosis inhibitors methyl-β-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 clathrinmediated endocytosis and (2) is dependent upon PNP physicochemical properties. We conclude that uptake/trafficking of nanoparticles into/across epithelia is dependent both on properties of the nanoparticles and the specific epithelial cell type.