Claudin-2–deficient mice are defective in the leaky and cation-selective paracellular permeability properties of renal proximal tubules Shigeaki Mutoa,1,2, Masaki Hatab,1, Junichi Taniguchic, Shuichi Tsuruokad, Kazumasa Moriwakie, Mitinori Saitouf, Kyoko Furuseb, Hiroyuki Sasakib, Akio Fujimurad, Masashi Imaic, Eiji Kusanoa, Shoichiro Tsukitaf,3, and Mikio Furuseg Departments of aNephrology, cPharmacology, and dClinical Pharmacology, Jichi Medical University, Shimotsuke, Tochigi 329-0498, Japan; bKAN Research Institute, Inc., Kobe MI R&D Center, Kobe, Hyogo 650-0047, Japan; Divisions of eVascular Biology and gCell Biology, Department of Physiology and Cell Biology, Kobe University Graduate School of Medicine, Kobe, Hyogo 650-0017, Japan; and fDepartment of Cell Biology, Kyoto University Faculty of Medicine, Kyoto 606-8501, Japan Edited* by Gerhard Giebisch, Yale University School of Medicine, New Haven, CT, and approved March 18, 2010 (received for review November 10, 2009) Claudin-2 is highly expressed in tight junctions of mouse renal prox- RT was attributed to an increase in the cation-selective permeability imal tubules, which possess a leaky epithelium whose unique perme- of TJs (10). Furthermore, the overexpression of human claudin-4 in ability properties underlie their high rate of NaCl reabsorption. To MDCK II cells increased RT by selectively decreasing the para- – investigate the role of claudin-2 in paracellular NaCl transport in this cellular permeability for Na+ without affecting that for Cl or an nephron segment, we generated knockout mice lacking claudin-2 uncharged solute (11). Similarly, overexpression of claudin-8 in −/− −/− (Cldn2 ).TheCldn2 mice displayed normal appearance, activity, MDCK II cells decreased paracellular permeability to cations but growth, and behavior. Light microscopy revealed no gross histolog- not to anions or neutral solutes (12). The combination and ratios of −/− ical abnormalitiesin the Cldn2 kidney. Ultrathin sectionand freeze- claudins may determine the tightness and charge selectivity of indi- fracture replica electron microscopy revealed that, similar to those of −/− vidual TJ strands, and some claudin species may constitute charge- wild types, the proximal tubules of Cldn2 mice were characterized selective paracellular channels within TJ strands (9, 11, 12). This by poorly developed tight junctions with one or two continuous tight hypothesis was also proposed through analysis of human hereditary junction strands. In contrast, studiesin isolated, perfused S2 segments hypomagnesemia caused by mutations in claudin-16 (13). However, of proximal tubules showed that net transepithelial reabsorption of the results obtained from exogenous expression of claudins in cul- + – fi Cldn2−/− Na ,Cl, and water was signi cantly decreased in mice and tured epithelial cells are unclear: claudin function must be inves- that there was an increase in paracellular shunt resistance without tigated without knowing the exact combination and ratios of en- affecting the apical or basolateral membrane resistances. Moreover, dogenous claudins, and it is not assured whether exogenous claudins deletion of claudin-2 caused a loss of cation (Na+) selectivity and – form TJ strands correctly without affecting endogenous claudins. therefore relative anion (Cl ) selectivity in the proximal tubule para- Mouse lines lacking the expression of several claudin species have cellular pathway. With free access to water and food, fractional Na+ – −/− been generated (14–17), but the barrier functions of their TJs have and Cl excretions in Cldn2 mice were similar to those in wild types, Cldn2−/− not always been evaluated by electrophysiology. but both were greater in mice after i.v. administration of We focused on the function of claudin-2 in the kidney. Claudin-2 2% NaCl. We conclude that claudin-2 constitutes leaky and cation + – is highly expressed, together with other claudin isoforms such as (Na ) selective paracellular channels within tight junctions of mouse claudin-10, in the proximal tubule of the kidney (18, 19), which is proximal tubules. composed of a leaky epithelium (20). In the proximal tubule, approximately one-third of the NaCl reabsorption is passive via mouse proximal tubule | tight junction | paracellular transport | Na/Cl the paracellular pathway; the remainder is active via the trans- transport | water transport cellular pathway. The movement of NaCl therefore results in passive water reabsorption. In this tubule, the molecular mecha- ight junctions (TJs) are circumferential seals around cells that nisms behind the transcellular transport of NaCl and water have Tselectively modulate paracellular permeability between extrac- been extensively evaluated, but it remains totally elusive how these – ellular compartments (1 3). On ultrathin-section electron micro- molecules are transported across TJs. In this study, we generated scopy, TJs appear as foci where the plasma membranes of neighboring claudin-2–deficient mice and examined whether claudin-2 is cells make complete contact (4). On freeze-fracture electron micro- involved in the paracellular transport of NaCl and water in vivo. scopy, TJs appear as a continuous and anastomosing network of intramembranous particle strands (TJ strands) (5). These strands are Results and Discussion mainly composed of linearly polymerized integral membrane proteins We produced mice unable to express claudin-2. Nucleotide called claudins with molecular masses of ∼23 kDa (2, 3, 6). The sequencing and restriction mapping identified one exon that cov- claudin gene family contains more than 20 members in humans and in ers the whole ORF of claudin-2. We constructed a targeting vector mice (2, 3, 7). The expression pattern of claudins varies considerably; to disrupt the claudin-2 gene by replacing part of the ORF (a.a. 1– most cell types express more than two claudins in various combina- tions to constitute mosaic TJ strands. Through the formation of TJ strands, claudins are directly Author contributions: S.M., S. Tsukita, and M.F. designed research; S.M., M.H., J.T., involved in creating a primary barrier to the paracellular diffusion of S. Tsuruoka, K.M., M.S., K.F., H.S., and M.F. performed research; S.M., A.F., M.I., E.K., solutes and water across epithelia (8). However, TJs are not a simple S. Tsukita, and M.F. analyzed data; and S.M., S. Tsukita, and M.F. wrote the paper. barrier: the barrier varies in tightness, measured by the trans- The authors declare no conflict of interest. epithelial electrical resistance (RT), and charge selectivity. Furuse *This Direct Submission article had a prearranged editor. et al. (9) reported that, when canine claudin-2 cDNA was transfected 1S.M. and M.H. contributed equally to this work. into high-resistance Madin-Darby canine kidney (MDCK) I cells 2To whom correspondence should be addressed. E-mail: [email protected]. primarily expressing claudins-1 and -4, the RT decreased to a level 3Deceased December 11th, 2005. PHYSIOLOGY similar to that of low-resistance MDCK II cells expressing endoge- This article contains supporting information online at www.pnas.org/cgi/content/full/ nous claudins-1, -2, and -4. A similar claudin-2–induced decrease in 0912901107/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.0912901107 PNAS | April 27, 2010 | vol. 107 | no. 17 | 8011–8016 Downloaded by guest on September 25, 2021 − − 111) of claudin-2 with the neomycin resistance gene (Fig. S1A). proximal tubules in Cldn2 / kidneys, but the distribution of clau- Two lines of mice were generated from distinct ES cell clones in din-10 was not significantly altered (Fig. 2A). To investigate whether which the claudin-2 gene was disrupted by homologous recombi- other claudins were up-regulated to compensate for the lack of − − nation. Southern blotting confirmed the expected disruption of the claudin-2 in Cldn2 / proximal tubules, we performed quantitative claudin-2 gene (Fig. S1B). real-time PCR using mRNA from isolated proximal tubules. The − − Claudin-2 null [Cldn2 / ] mice were born in the expected amount of claudin-10a and -10b mRNA was not statistically dif- − − Mendelian ratios. Their growth rate, appearance, activity, and ferent between the two groups (Fig. 2B). In Cldn2 / proximal behavior were normal. This enabled an examination of the struc- tubules, we did not find any significant mRNA up-regulation of any − − ture and function of proximal tubules using male adult Cldn2 / claudin expressed in nephron segments apart from the proximal mice and their wild-type littermates [Cldn2+/+]. The two lines of tubule, including -4,-8, and -16 (Fig. 2B). Furthermore, by immu- − − − − Cldn2 / mice showed the same phenotype, so we will present data nofluorescence in Cldn2 / kidneys, the claudins that were report- obtained from one line of the two. edly expressed in other nephron segments, including claudins-4, -8, We first compared light microscopic images of H&E-stained and -16, were still undetectable at TJs of their proximal tubules (Fig. sections of paraffin-embedded kidneys from 8-week-old Cldn2+/+ S4). In addition, ZO-1 and cingulin, proteins localized in the cyto- and Cldn2−/− mice (Fig. S2). No gross morphological malformations plasmic region of TJs, colocalized with Lotus tetragonolobus agglu- − − − − were observed in the Cldn2 / kidney. Ultrathin section electron tinin (LTA), a proximal tubule marker (19, 22), in Cldn2 / kidneys microscopy also identified no significant differences between (Fig. S5). Therefore, claudin-2 appeared to be simply absent from − − − − − − Cldn2+/+