Claudin-8 Expression in Madin-Darby Canine Kidney Cells Augments the Paracellular Barrier to Cation Permeation

Abstract

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 Madin-Darby 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 cation-permeable 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. 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 Madin-Darby 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 cation-permeable 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. Claudin-8 expression in Madin-Darby canine kidney cells augments the paracellular barrier to cation permeation. Vol. 278 (2003) 17350-17359Journal of Biological ChemistryVol. 279Issue 49PreviewPage 17356, Fig. 7: The permeability measurements shown in the original figure were 10-fold too high. The corrected figure is shown below: Full-Text PDF Open Access Madin-Darby canine kidney cells transepithelial resistance doxycycline Vectorial transport across epithelia can occur via the transcellular or paracellular route. The molecular basis of transcellular transport, mediated by proteins that catalyze transmembrane movement of solutes and water at the apical and basolateral surfaces, is well understood. By contrast, little is known about the mechanisms of paracellular transport. The tight junction, which is the most apical of the intercellular junctional complexes at the lateral membrane, is generally believed to be the rate-limiting step in paracellular transport. The tight junction is composed of a complex of multiple proteins, many of uncertain function (1Denker B.M. Nigam S.K. Am. J. Physiol. 1998; 274: F1-F9Crossref PubMed Google Scholar, 2Fanning A.S. Mitic L.L. Anderson J.M. J. Am. Soc. Nephrol. 1999; 10: 1337-1345PubMed Google Scholar). Most of these proteins are cytoplasmic in location, and several are associated with the cytoskeleton via the perijunctional actin ring. In recent years, a number of integral membrane proteins associated with the tight junction have been identified, including occludin (3Furuse M. Hirase T. Itoh M. Nagafuchi A. Yonemura S. Tsukita S. Tsukita S. J. Cell Biol. 1993; 123: 1777-1788Crossref PubMed Scopus (2141) Google Scholar), junction-associated membrane proteins 1 and 2 (4Martin-Padura I. Lostaglio S. Schneemann M. Williams L. Romano M. Fruscella P. Panzeri C. Stoppacciaro A. Ruco L. Villa A. Simmons D. Dejana E. J. Cell Biol. 1998; 142: 117-127Crossref PubMed Scopus (1157) Google Scholar, 5Cunningham S.A. Arrate M.P. Rodriguez J.M. Bjercke R.J. Vanderslice P. Morris A.P. Brock T.A. J. Biol. Chem. 2000; 275: 34750-34756Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar), Coxsackie- and adenovirus-associated receptor (6Cohen C.J. Shieh J.T. Pickles R.J. Okegawa T. Hsieh J.T. Bergelson J.M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 15191-15196Crossref PubMed Scopus (558) Google Scholar), 1G8 antigen (7Nasdala I. Wolburg-Buchholz K. Wolburg H. Kuhn A. Ebnet K. Brachtendorf G. Samulowitz U. Kuster B. Engelhardt B. Vestweber D. Butz S. J. Biol. Chem. 2002; 277: 16294-16303Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar), and the claudins (8Furuse M. Sasaki H. Fujimoto K. Tsukita S. J. Cell Biol. 1998; 143: 391-401Crossref PubMed Scopus (794) Google Scholar). Integral membrane proteins are of particular interest because, by definition, their extracellular domains must protrude into the lateral intercellular space and, therefore, may potentially have direct contact with solutes as they permeate through the paracellular pathway. Claudins are the most heterogeneous of these integral membrane proteins and consist of a family of at least 20 homologous isoforms (9Tsukita S. Furuse M. J. Cell Biol. 2000; 149: 13-16Crossref PubMed Scopus (407) Google Scholar). Moreover, each isoform clearly exhibits a tissue-specific and segment-specific pattern of distribution in epithelia such as those of the gastrointestinal tract (10Rahner C. Mitic L.L. Anderson J.M. Gastroenterology. 2001; 120: 411-422Abstract Full Text Full Text PDF PubMed Scopus (449) Google Scholar) and renal tubule (11Yu A. Enck A. J. Am. Soc. Nephrol. 2001; 12 (abstr.): 45Google Scholar, 12Kiuchi-Saishin Y. Gotoh S. Furuse M. Takasuga A. Tano Y. Tsukita S. J. Am. Soc. Nephrol. 2002; 13: 875-886Crossref PubMed Google Scholar, 13Reyes J.L. Lamas M. Martin D. Del Carmen Namorado M. Islas S. Luna J. Tauc M. Gonzalez-Mariscal L. Kidney Int. 2002; 62: 476-487Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). Claudins are therefore leading candidates for the molecular determinants responsible for the variety of different paracellular permeability properties found in different epithelia. Several recent reports provide convincing evidence that claudins regulate paracellular permeability. Human mutations in claudin-16 cause failure of paracellular reabsorption of divalent cations in the thick ascending limb of the renal tubule, leading to familial hypercalciuric hypomagnesemia (14Simon D.B. Lu Y. Choate K.A. Velazquez H. Al-Sabban E. Praga M. G. A. G. J. D. D. S. 1999; PubMed Scopus Google Scholar), and a that claudin-16 may have a Y. Choate K.A. T. J. Am. Soc. Nephrol. 2001; 12 (abstr.): Scholar). M. M. Furuse K. Y. A. Y. T. A. Tsukita S. J. Cell Biol. 2002; PubMed Scopus Google Scholar) and K. 2002; Google Scholar) permeability to of claudins and in cells all transepithelial resistance T. J. Y. J. Cell Biol. 1999; PubMed Scopus Google Scholar, S.A. J.M. J. J. Cell Sci. 2000; Google Scholar, C. C. Anderson J.M. J. 2001; PubMed Scopus Google Scholar, C. Anderson J.M. Am. J. Physiol. Cell Physiol. 2002; PubMed Scopus Google Scholar), whereas of claudin-2 M. Furuse K. 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In such a claudins a as a barrier by the and a through that barrier the apical to the lateral intercellular may the of a isoform can have In the of in tight junction proteins, in the and the paracellular of the tight junction and in for the the paracellular may may the of The is complex and the properties of the expressed and of endogenous tight junction In a model with of to claudin-8 in (11Yu A. Enck A. J. Am. Soc. Nephrol. 2001; 12 (abstr.): 45Google Scholar) and Y. Gotoh S. Furuse M. Takasuga A. Tano Y. Tsukita S. J. Am. Soc. Nephrol. 2002; 13: 875-886Crossref PubMed Google Scholar) that it is expressed primarily in the distal In these of the renal tubule, the paracellular barrier is tight to monovalent cations and to for for and for therefore the hypothesis that claudin-8 plays a role in paracellular cation permeation. that the of and a to the of such to the findings of such and that that claudin-8 acts as a cation barrier. the claudin-8 for the was by a and into a Enck B. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar) that the claudin-8 was with a is by a The was and into the to the was transfected by into the cell a of and the II cells the a were with in the of were by in cell lines were and and with of claudin-8 expression for were in with and 20 doxycycline To claudin-8 doxycycline was the the of were generally To tight junction expression by cells were and by at as Enck B. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). claudin-8 was in the The of of and cells was and the of membrane was in of of membrane were and to membrane, and were the Claudin-8 expression was with the at a to and claudins were and at the by the and adenovirus-associated receptor of of was at were with a were to and were To the of claudin-8 cell lines were with at for was with the at or the shown in Fig. with a at all in the of Enck B. J. Biol. 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PubMed Scopus Google Scholar) the 2 is the is the and is the of the permeability of to that of as by the The of each monovalent was to be the as that of and the and cation in each were to have the To the role of claudin-8 in paracellular cell lines the claudin-8 protein. by low that the canine renal tubule cell not claudin-8 not by II cell lines with expression of claudin-8 the of the Claudin-8 expression was in these cells by doxycycline the and by doxycycline By and by the claudin-8 was found to be expressed in cells but was in cells and By claudin-8 in cell was expressed at the apical of the lateral cell membrane, the tight junction is and with the tight junction of the number of tight junction strands and a but in number in in junction was strands in cells that were found in By that claudin-8 is into the tight junction strands and that claudin-8 was targeted to the tight junction and that the of an at the not with To the of the tight junction, the and of proteins known to be in tight of II cells were by with and by The expression of and adenovirus-associated and claudins and were not different and and their of to the lateral membrane were all by claudin-2 expression in cells was found to be by different with cells and for claudin-2 at the tight junction was to be II cells are with of M. 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PubMed Scopus Google Scholar) a of in which has a paracellular permeability that of II In the paracellular as a of or by with for to cations. The of is that of claudin-8 to a in paracellular permeability to cations but not to anions or neutral solutes. In are in which a in permeability can be the size of the paracellular may be or its in such a as to permeation. the and the may be altered. may occur with that charge or with of or with and charge and direct the the of the may be but the number of may be of a in the number of or of the is Our that the size of the to organic cations in size to is the Our that the permeability different is and that permeability to monovalent and divalent cations is the Moreover, of the are by that the activation energy for permeation is unchanged. are to the that claudin-8 must reduce the number of pores. The of the properties of claudins the of transfected cell lines in the has been for a variety of it is uncertain the into existing tight junction strands or to the paracellular barrier. In of permeability M. Furuse K. Sasaki H. Tsukita S. J. Cell Biol. 2001; PubMed Scopus Google Scholar, S. T. J. M. J. Cell Sci. 2002; PubMed Scopus Google Scholar), whereas in it it T. J. Y. J. Cell Biol. 1999; PubMed Scopus Google Scholar, S.A. J.M. J. J. Cell Sci. 2000; Google Scholar, C. C. Anderson J.M. J. 2001; PubMed Scopus Google Scholar). in is that it is not known the is to existing tight junction or for a that of the of of it is not to all of the claudins that are and of claudins or tight junction membrane proteins be for at the tight junction permeability may be not the or of a particular but the or of of the it is not to that the permeability of a tight junction be different that of an but occludin found by S.A. J.M. J. J. Cell Sci. 2000; Google each may not be by the of a Tsukita and M. 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