Claudins are a family of integral membrane proteins of the tight junction that are thought to participate in the permeation of solutes across epithelia via the paracellular pathway. Claudin-8 is expressed in the distal renal tubule, which has a characteristically low passive permeability to monovalent cations. To test the hypothesis that claudin-8 plays a role in forming a tight paracellular barrier to cations, stably transfected MadinDarby canine kidney II cell lines with inducible expression of claudin-8 were generated. Induction of claudin-8 expression was associated with down-regulation of endogenous claudin-2 protein. Other tight junction proteins were expressed and targeted normally, and the number of junctional strands was minimally altered. By Ussing chamber and radiotracer flux studies, claudin-8 expression was found to reduce paracellular permeability to monovalent inorganic and organic cations and to divalent cations but not to anions or neutral solutes. The size selectivity, charge dependence, and activation energy of paracellular cation permeation were all unchanged. These observations are consistent with a model in which claudin-2 encodes a highly cationpermeable channel, whereas claudin-8 acts primarily as a cation barrier. When exogenous claudin-8 is expressed, it replaces endogenous claudin-2, inserting in its place into existing tight junction strands, thereby reducing the apparent number of functional cation pores. Our findings suggest that claudin-8 plays an important role in the paracellular cation barrier of the distal renal tubule.

The proximal nephron possesses a leaky epithelium with unique paracellular permeability properties that underlie its high rate of passive NaCl and water reabsorption, but the molecular basis is unknown. The claudins are a large family of transmembrane proteins that are part of the tight junction complex and likely form structural components of a paracellular pore. To localize claudin-2 in the mouse kidney, we performed in situ hybridization using an isoform-specific riboprobe and immunohistochemistry using a polyclonal antibody directed against a COOH-terminal peptide. Claudin-2 mRNA and protein were found throughout the proximal tubule and in the contiguous early segment of the thin descending limb of long-looped nephrons. The level of expression demonstrated an axial increase from proximal to distal segments. In confocal images, the subcellular localization of claudin-2 protein coincided with that of the tight junction protein ZO-1. Our findings suggest that claudin-2 is a component of the paracellular pathway of the most proximal segments of the nephron and that it may be responsible for their uniquely leaky permeability properties.

The SLC12A6 gene encoding the K(+)-Cl(-) cotransporter KCC3 is expressed in multiple tissues, including kidney. Here, we report the molecular characterization of several NH(2)-terminal isoforms of human and mouse KCC3, along with intrarenal localization and functional characterization in Xenopus laevis oocytes. Two major isoforms, KCC3a and KCC3b, are generated by transcriptional initiation 5' of two distinct first coding exons. Northern blot analysis of mouse tissues indicates that KCC3b expression is particularly robust in the kidney, which also expresses KCC3a. Western blotting of mouse tissue using an exon 3-specific antibody reveals that the kidney is also unique in expressing immunoreactive protein of a lower mass, suggestive evidence that the shorter KCC3b protein predominates in kidney. Immunofluorescence reveals basolateral expression of KCC3 protein along the entire length of the proximal tubule, in both the mouse and rat. Removal of the 15-residue exon 2 by alternative splicing generates the KCC3a-x2M and KCC3b-x2M isoforms; other splicing events at an alternative acceptor site within exon 1a generate the KCC3a-S isoform, which is 60 residues shorter than KCC3a. This variation in sequence of NH(2)-terminal cytoplasmic domains occurs proximal to a stretch of highly conserved residues and affects the content of putative phosphorylation sites. Kinetic characterization of KCC3a in X. laevis oocytes reveals apparent K(m)s for Rb(+) and Cl(-) of 10.7 +/- 2.5 and 7.3 +/- 1.2 mM, respectively, with an anion selectivity of Br(-) > Cl(-) > PO(4) = I(-) = SCN(-) = gluconate. All five NH(2)-terminal isoforms are activated by cell swelling (hypotonic conditions), with no activity under isotonic conditions. Although the isoforms do not differ in the osmotic set point of swelling activation, this activation is more rapid for the KCC3a-x2M and KCC3a-S proteins. In summary, there is significant NH(2)-terminal heterogeneity of KCC3, with particularly robust expression of KCC3b in the kidney. Basolateral swelling-activated K(+)-Cl(-) cotransport mediated by KCC3 likely functions in cell volume regulation during the transepithelial transport of both salt and solutes by the proximal tubule.

The neuron-specific K ؉ -Cl ؊ cotransporter KCC2 plays a crucial role in determining intracellular chloride activity and thus the neuronal response to ␥-aminobutyric acid and glycine.

The proximal nephron possesses a leaky epithelium with unique paracellular permeability properties that underlie its high rate of passive NaCl and water reabsorption, but the molecular basis is unknown. The claudins are a large family of transmembrane proteins that are part of the tight junction complex and likely form structural components of a paracellular pore. To localize claudin-2 in the mouse kidney, we performed in situ hybridization using an isoform-specific riboprobe and immunohistochemistry using a polyclonal antibody directed against a COOH-terminal peptide. Claudin-2 mRNA and protein were found throughout the proximal tubule and in the contiguous early segment of the thin descending limb of long-looped nephrons. The level of expression demonstrated an axial increase from proximal to distal segments. In confocal images, the subcellular localization of claudin-2 protein coincided with that of the tight junction protein ZO-1. Our findings suggest that claudin-2 is a component of the paracellular pathway of the most proximal segments of the nephron and that it may be responsible for their uniquely leaky permeability properties.

CLC5 is an intracellular chloride channel of unknown function, expressed in the renal proximal tubule. The subcellular localization and function of CLC5 were investigated in the LLC-PK1 porcine proximal tubule cell line. We cloned a cDNA for the porcine CLC5 ortholog (pCLC5) that is predicted to encode an 83-kDa protein with 97% amino acid sequence identity to rat and human CLC5. By immunofluorescence, pCLC5 was localized to early endosomes of the apical membrane fluid-phase endocytotic pathway and to the Golgi complex. Xenopus oocytes injected with pCLC5 cRNA exhibited outwardly rectifying whole cell currents with a relative conductance profile (nitrate > > Cl ؊ Ϸ Br ؊ > I ؊ > acetate > gluconate) different from that of control oocytes. Acidification of the extracellular medium reversibly inhibited this outward current with a pK a of 6.0 and a Hill coefficient of 1. Overexpression of CLC5 in LLC-PK1 cells resulted in morphological changes, including loss of cell-cell contacts and the appearance of multiple prominent vesicles. These findings are consistent with a potential role for CLC5 in the acidification of membrane compartments of both the endocytic and the exocytic pathway and suggest that its function may be important for normal intercellular adhesion and vesicular trafficking.

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