*Department of Cell Biology, Kyoto University Faculty of Medicine, Kyoto, Japan; Department of Ophthalmology, Osaka University Medical School, Osaka, Japan; and Shirakawa Institute of Animal Genetics, Fukushima, Japan.
Correspondence to Dr. Shoichiro Tsukita, Department of Cell Biology, Kyoto University Faculty of Medicine, Konoe-Yoshida, Sakyo-ku, Kyoto 606, Japan. Phone: 81-75-753-4372; Fax: 81-75-753-4660; E-mail: htsukita{at}mfour.med.kyoto-u.ac.jp
ABSTRACT. As the first step in understanding the physiologicfunctions of claudins (tight junction integral membrane proteins)in nephrons, the expression of claudin-1 to -16 in mouse kidneyswas examined by Northern blotting. Among these claudins, onlyclaudin-6, -9, -13, and -14 were not detectable. Claudin-5 and-15 were detected only in endothelial cells. Polyclonal antibodiesspecific for claudin-7 and -12 were not available. Therefore,the distributions of claudin-1, -2, -3, -4, -8, -10, -11, and-16 in nephron segments were examined with immunofluorescencemicroscopy. For identification of individual segments, antibodiesspecific for segment markers were used. Immunofluorescence microscopicanalyses of serial frozen sections of mouse kidneys with polyclonalantibodies for claudins and segment markers revealed that claudinsdemonstrated very complicated, segment-specific, expressionpatterns in nephrons, i.e., claudin-1 and -2 in Bowman’scapsule, claudin-2, -10, and -11 in the proximal tubule, claudin-2in the thin descending limb of Henle, claudin-3, -4, and -8in the thin ascending limb of Henle, claudin-3, -10, -11, and-16 in the thick ascending limb of Henle, claudin-3 and -8 inthe distal tubule, and claudin-3, -4, and -8 in the collectingduct. These segment-specific expression patterns of claudinsare discussed, with special reference to the physiologic functionsof tight junctions in nephrons.
In multicellular organisms, epithelial cellular sheets not onlyfunction as diffusion barriers, to establish compositionallydistinct fluid compartments, but also are involved in activetransport of materials across the barrier, to dynamically maintainthe internal environment of each compartment. For cellular sheetsto exert these physiologic functions, there must be some sealto the diffusion of solutes through the paracellular pathway.Tight junctions (TJ) have been demonstrated to be responsiblefor this intercellular sealing (1–3). Morphologic andphysiologic studies, however, have revealed that TJ are notsimple barriers; they demonstrate ion and size selectivity,and their barrier function varies significantly in tightness,depending on the cell type and physiologic requirements (4,5).Such regulated diversified permeability of TJ is thought tobe required for dynamic maintenance of the interior environmentof each compartment.
With ultrathin-section electron microscopy, TJ appear as a seriesof discrete sites of apparent fusion, involving the outer leafletof the plasma membranes of adjacent cells (6). With freeze-fractureelectron microscopy, TJ appear as a set of continuous, anastomosing,intramembranous particle strands (TJ strands) (7,8). These morphologicfindings led to the following structural model for TJ: withinthe lipid bilayer of each membrane, the TJ strands, which areprobably composed of linearly aggregated integral membrane proteins,form networks through their ramifications (3). TJ strands laterallyand tightly associate with strands in the apposing membranesof adjacent cells to form paired strands, where the intercellulardistance becomes almost zero.
Two distinct types of integral membrane proteins have been identifiedas components of TJ strands, namely occludin and claudins (9,10).Both occludin and claudins have four transmembrane domains,but they demonstrate no sequence similarity with each other.Claudins, with molecular masses of approximately 23 kD, comprisea multigene family consisting of >20 members (3,10–12).When each claudin species or occludin was overexpressed in mouseL fibroblasts, claudin molecules, but not occludin, were polymerizedwithin the plasma membranes to reconstitute paired TJ strands,indicating that claudins are major structural components ofTJ strands (13). It was recently demonstrated that heterogeneousclaudin species are copolymerized to form individual TJ strandsas heteropolymers and that, between adjacent TJ strands, claudinmolecules adhere to each other in both homotypic and heterotypicmanners, except in some combinations (14). Importantly, detailedanalyses suggested that variations in the tightness of individualpaired TJ strands are determined by the combinations and mixingratios of claudin species (3,15–17).
The kidney is composed of numerous nephrons, which are delineatedby epithelial cellular sheets. Each nephron has several segments,which differ in histologic structure and function. Nephronsare directly involved in the production of urine via collectionof the blood filtrate, addition of metabolic waste, and reabsorptionof components that need to be conserved. Therefore, these compartments(i.e., nephrons) are essential for the physiologic functionsof the kidney. TJ play a central role in this compartmentalization,and the tightness of TJ varies significantly, depending on thenephron segment. As mentioned above, claudins have been identifiedas structural as well as functional components of TJ strands.Therefore, as the first step in understanding the functionsof TJ in individual nephron segments in molecular terms, itis necessary to determine which types of claudins are expressedin each segment. Interestingly, mutations in the gene encodingclaudin-16/paracellin-1, which is expressed in the thick ascendinglimb of Henle, were reported to affect the resorption of magnesiumions from the urine, resulting in human hereditary hypomagnesemia(16).
In this study, we systematically examined the expression patternsof claudins (claudin-1 to -16) in each mouse nephron segment.Although the expression and distribution of several claudinspecies remain undetermined because of the lack of availableantibodies, this study demonstrated the complicated patternsof segment-specific expression of claudin species in nephrons.The results presented here are important for future physiologicstudies of nephron functions.
Antibodies
Rabbit anti-mouse polyclonal antibodies (pAb) to claudin-2,-3, -4, -5, -8, and -11 and rat anti-mouse occludin monoclonalantibody (mAb) were raised and characterized as described previously(11,14,18–20). Rabbit anti-rat aquaporin-2 (AQP2) pAbwas kindly provided by Dr. Sei Sasaki (Tokyo Medical and DentalUniversity, Tokyo, Japan). Rabbit anti-claudin-1 pAb, rabbitanti-Tamm-Horsfall glycoprotein (THP) pAb, and rabbit anti-chloridechannel-K (ClC-K) pAb were purchased from Zymed Laboratories(South San Francisco, CA), Biomedical Technologies (Stoughton,MA), and Alomone Laboratories (Jerusalem, Israel), respectively.Rabbit anti-AQP1 pAb was purchased from Chemicon International(Temecula, CA).
For generation of anti-claudin-10 pAb in rabbits, a glutathioneS-transferase (GST) fusion protein with the carboxy-terminalcytoplasmic domain of mouse claudin-10, which was produced inEscherichia coli (strain DH5a) and purified with glutathione-Sepharose4B beads (Amersham-Pharmacia Biotech., Bucks, UK), was usedas an antigen. For generation of anti-claudin-7, -12, -15, and-16 pAb, polypeptides corresponding to the respective carboxy-terminalcytoplasmic domains of mouse claudin-7, -12, and -15 and bovineclaudin-16 (21) were synthesized and coupled, via cysteine,to keyhole limpet hemocyanin. These conjugated peptides wereused as antigens in rabbits. The pAb were affinity-purifiedon nitrocellulose membranes with the corresponding GST fusionproteins. During the generation of pAb, animal experiments wereconducted in accordance with the National Institutes of HealthGuide for the Care and Use of Laboratory Animals.
Northern Blotting
Total RNA from mouse kidneys was isolated according to the methoddescribed by Chomczynski and Sacchi (22). Poly(A)+ RNA was obtainedfrom total RNA by using oligo(dT)-cellulose beads (New EnglandBioLabs, Beverly, MA). Aliquots of approximately 10 µgof poly(A)+ RNA were separated on 1% formaldehyde/agarose gels,transferred to positively charged nylon membranes, and crosslinkedwith ultraviolet light. Hybridization with digoxigenin (DIG)-labeledRNA probes was performed according to the protocol describedby the manufacturer (Roche, Mannheim, Germany). RNA probes wereDIG-labeled using a DIG RNA labeling kit (Roche).
Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis and Immunoblotting
Lysates of E. coli expressing a maltose-binding protein fusionprotein with the cytoplasmic portion of mouse claudin-10 orGST fusion proteins with the cytoplasmic portions of the othermouse claudin species were subjected to one-dimensional sodiumdodecyl sulfate-polyacrylamide gel electrophoresis (12.5%),according to the method described by Laemmli (23), and gelswere stained with Coomassie Brilliant Blue R-250. For immunoblotting,proteins were electrophoretically transferred from gels to nitrocellulosemembranes, which were then incubated with the first antibody.Bound antibodies were detected with biotinylated second antibodiesand streptavidin-conjugated alkaline phosphatase (Amersham Corp.,Arlington Heights, IL). Nitroblue tetrazolium and bromochloroindolylphosphate were used as substrates for detection of alkalinephosphatase activity.
Immunofluorescence Microscopy
Kidneys were removed from mice and frozen with liquid nitrogen.For staining with anti-claudin-1 pAb, kidneys were fixed forat least 2 h with 4% paraformaldehyde in phosphate-bufferedsaline (PBS) (pH 7.2), immersed for 48 h in PBS containing 15%sucrose, and then frozen with liquid nitrogen. For TCA fixation(24), small pieces of kidney were soaked in ice-cold 10% TCAfor 1 h, washed three times with PBS, and then frozen with liquidnitrogen. Pairs of serial frozen sections (approximately 10µm thick) were cut with a cryostat, mounted on singleglass slides, and air-dried. Sections were then fixed with 95%ethanol at 4°C for 30 min, followed by 100% acetone at roomtemperature for 1 min. After soaking in PBS containing 1% bovineserum albumin (and 0.2% Triton X-100 for claudin-1 stainingonly), one of the sections was incubated with one of the anti-claudinpAb (or preimmune serum) and the other was treated with oneof the segment marker-specific pAb, in a moist chamber, for30 min. For control experiments, anti-claudin pAb were preincubatedwith excess amounts of GST fusion proteins with the cytoplasmicdomains of the respective claudins. Sections were then washedthree times with PBS, followed by a 30-min incubation with secondaryantibodies. Cy3-conjugated and FITC-conjugated secondary antibodieswere used for the anti-claudin pAb-treated and anti-segmentmarker pAb-treated sections, respectively. After being washedwith PBS, sections were embedded in 95% glycerol/PBS containing0.1% p-phenylendiamine and 1% n-propyl gallate and were observedwith a fluorescence microscope (Axiophot photomicroscope; CarlZeis, Inc., Thornwood, NY) equipped with a cooled, charge-coupleddevice camera system.
Expression of Claudin Isotypes in the Kidney
We first examined the expression of claudin-1 to -16 in mousekidneys by Northern blotting. When the poly(A)+ RNA isolatedfrom the kidneys was probed with DIG-labeled mouse claudin-1to -16 RNA, the expression of most claudin species was clearlydetected, whereas claudin-6, -9, -13, and -14 were not detected(Figure 1A). Because claudin-6, -9, -13, and -14 mRNA were clearlydetected with the same probes in other tissues, we concludedthat the expression levels of claudin-6, -9, -13, and -14 werefairly low in mouse kidneys.
Figure 1. (A) Northern blots of mouse claudin-1 to -16 (Cld-1 to -16) expression in the kidney. Claudin-6, -9, -13, and -14 were undetectable. Arrows, positions of individual claudin mRNA expected from Northern blotting analyses of other tissues, such as the liver. (B) Specificity of newly generated anti-claudin-10 and -16 polyclonal antibodies (pAb). Immunoblotting of total lysates of Escherichia coli expressing glutathione S-transferase (GST) (or maltose-binding protein [MAL]) fusion proteins with cytoplasmic domains of claudin-1 to -16 confirmed the specificities. CBB, Coomassie Brilliant Blue staining; anti-Cld-10 pAb, immunoblotting with anti-claudin-10 pAb; anti-Cld-16 pAb, immunoblotting with anti-claudin-16 pAb.
Next, to examine the distributions of individual claudins expressedin the kidney, we prepared antibodies specific for all of theseclaudins. Anti-claudin-1 pAb was purchased from Zymed Laboratories.pAb to claudin-2, -3, -4, -5, -8, and -11 were raised and characterizedpreviously (11,14,18,19). We then produced GST fusion proteinsor synthesized polypeptides from the cytoplasmic tails of theother claudins (claudin-7, -10, -12, -15, and -16) and raisedpAb using those proteins or polypeptides as antigens. The pAbfor claudin-10, -15, and -16 worked well for both immunoblottingand immunofluorescence microscopic analyses but, despite intensiveefforts, we failed to obtain pAb for claudin-7 or -12 that couldbe used for immunofluorescence microscopy. Therefore, we haveno information regarding the distributions of claudin-7 and-12 in nephrons. Furthermore, because claudin-5 and -15 wereexpressed specifically in endothelial cells of blood vessels,we do not discuss the expression or distribution of these claudinsin the kidney in detail in this report. The specificities ofnewly generated anti-claudin-10 and -16 pAb were demonstratedby Western blotting analyses of total lysates of E. coli expressingGST fusion proteins with the cytoplasmic domains of claudin-1to -16 (Figure 1B). As previously demonstrated (11,25), it wastechnically difficult to evaluate the specificities of anti-claudinpAb in Western blotting analyses of total lysates of whole organs,mainly because the levels of expressed claudins were fairlylow.
Segment-Specific Expression of Claudin-8 in Nephrons
To identify individual nephron segments with immunofluorescencemicroscopy, we used antibodies to several segment markers (Figure 2).The proximal tubules and the thin descending limb of Henlewere identified as AQP1-positive tubules in the cortex and themedulla, respectively (26). The thin and thick ascending limbsof Henle in the medulla were distinguished by the expressionof ClC-K (27,28) and THP, respectively (29). The anti-ClC-KpAb used in this study recognized both ClC-K1 and ClC-K2, butin the medulla only the thin ascending limb of Henle was reportedto express ClC-K1 (not ClC-K2) (27,28). Because the collectingtubules were reported to be positive for AQP2 (30), the distaltubules were identified as AQP1/2-double-negative tubules locatedaround glomeruli.
Figure 2. Identification of nephron segments with fluorescence microscopy. (A) Nomenclature for nephron segments used in this study. (B) Segment markers. Frozen sections of mouse kidneys were immunofluorescently stained with pAb for each segment marker. The proximal tubules and the thin descending limbs of Henle were identified as aquaporin-1 (AQP1)-positive tubules in the cortex and medulla, respectively. The thin and thick ascending limbs of Henle in the medulla were distinguished by the expression of chloride channel-K1 (ClC-K1) and Tamm-Horsfall glycoprotein (THP), respectively. The distal tubules were identified as AQP1/2-double-negative tubules located around glomeruli (asterisk), and the collecting tubules were identified as AQP2-positive tubules. Arrows, tubules identified by segment markers. Bars, 20 µm.
We then immunofluorescently stained serial frozen sections fromthe mouse kidneys with pAb for each claudin species or withpAb for the segment markers. This method indicated that individualclaudin species exhibit specific patterns of expression in nephronsegments. The segment-specific expression of claudin-8 is demonstratedin Figure 3, as an example. In the cortex, claudin-8 was expressedand concentrated at junctional regions (probably TJ) in somebut not all tubules. The AQP1-positive tubules lacked claudin-8expression, whereas all of the AQP2-positive tubules expressedclaudin-8, indicating that claudin-8 is expressed in collectingducts but not in proximal tubules. The TJ-specific concentrationof claudin-8 was also detected in the AQP1/2-double-negativetubules located around glomeruli, indicating the expressionof claudin-8 in distal tubules. In the medulla, some tubulesexhibited a concentration of claudin-8 at TJ. These tubuleswere ClC-K-positive but AQP1/THP-negative, leading to the conclusionthat claudin-8 was expressed exclusively in the thin ascendinglimb of Henle in the medulla.
Figure 3. Segment-specific expression pattern of claudin-8 in nephrons. One of each pair of serial frozen sections was stained with pAb for segment markers in green (markers), and the other was stained with anti-claudin-8 pAb in red (claudin-8). These two images were then merged (merge). This analysis revealed that claudin-8 was concentrated at tight junctions (TJ) in epithelial cells of the thin ascending limb of Henle, the distal tubule, and the collecting duct. Arrows, tubules identified with segment markers; arrowhead, proximal tubule; double arrowhead, collecting duct; asterisks, glomeruli. Bars, 20 µm.
To evaluate these segment-specific staining patterns, we performedthe following control staining experiments (Figure 4). First,frozen sections were double-stained with anti-occludin mAb andanti-claudin-8 pAb, to confirm that claudin-8 signals were derivedfrom TJ. Second, sections were stained with preimmune serum.Third, sections were stained with anti-claudin-8 pAb in thepresence of GST-claudin-8 or GST-claudin-5, to evaluate thespecificity of the staining with anti-claudin-8 pAb. Using thistype of analysis, with similar control staining experiments,we examined in detail the segment-specific expression of otherclaudin species in nephrons, as described below.
Figure 4. Specificities of anti-claudin-8 (anti-cld-8) pAb in immunofluorescence microscopic analyses. When the frozen sections were double-stained with anti-occludin monoclonal antibody (mAb) and anti-claudin-8 pAb, in some types of tubules (Figure 3) claudin-8 appeared to be concentrated in lines, which precisely coincided with the occludin-positive TJ. These claudin-8-positive lines were undetectable with preimmune serum or with anti-claudin-8 pAb pretreated with GST-claudin-8. Pretreatment with GST-claudin-5 did not affect the staining ability of anti-claudin-8 pAb. Bars, 20 µm.
Distinct Patterns of Expression of Claudins in Nephron Segments Bowman’s Capsule.
To examine whether certain claudin species are expressed inBowman’s capsule, we compared phase-contrast images offrozen sections with the corresponding immunofluorescence microscopicimages obtained by using specific pAb. As demonstrated in Figure 5A,claudin-1 and -2 were concentrated at the borders of epithelialcells of Bowman’s capsule. We noted that the claudin-1pAb (Zymed Laboratories) recognized not only claudin-1 but alsoclaudin-3. To evaluate the staining of Bowman’s capsulewith anti-claudin-1 pAb, we stained the kidneys of claudin-1-deficientmice (Furuse M, Hata M, Tsukita SH; unpublished data) with thisantibody. In those mice, no signal was detected for Bowman’scapsule, indicating that claudin-1 is specifically expressed,together with claudin-2, in the capsule.
Figure 5. (A) Claudins expressed in Bowman’s capsule. Claudin-1 and -2 were detected in Bowman’s capsule (arrows), which was identified by comparison of an immunofluorescence image (left) with the corresponding phase-contrast image (right). Asterisks, glomeruli. Bars, 20 µm. (B) Claudins expressed in the proximal tubule. Claudin-2, -10, and -11 were concentrated at TJ of epithelial cells in proximal tubules (red), which were identified as AQP1-positive tubules in the cortex (green). Bars, 20 µm.
Proximal Tubule.
Claudin-2, -10, and -11 were clearly concentrated at TJ of epithelialcells of AQP1-positive tubules in the cortex, i.e., the proximaltubules (Figure 5B). Among these, only claudin-2 seemed to bedistributed along basal plasma membranes in addition to TJ.This staining was specific for claudin-2, because the kidneysof claudin-2-deficient mice demonstrated no such staining (FuruseM, Hata M, Tsukita SH; unpublished data).
Thin Descending Limb of Henle.
Only claudin-2 was detected in most of the AQP-1-positive tubulesin the medulla, i.e., the thin descending limb of Henle (Figure 6).Under the fixation conditions used in this study, claudin-2did not seem to be concentrated in TJ-like thin lines but wasdiffusely distributed in broad bands. This characteristic stainingwas absent with preimmune serum; very strangely, when sectionswere doubly stained with anti-occludin mAb and anti-claudin-2pAb, not only claudin-2 but also occludin seemed to be diffuselydistributed in broad bands. This staining became undetectablewhen anti-claudin-2 pAb was pretreated with GST-claudin-2, andstaining was not observed in claudin-2-deficient mouse kidneys(data not shown). These findings suggested that the diffusedistribution of claudin-2 in the thin descending limb of Henlewas specific.
Figure 6. Claudins expressed in the thin descending limb of Henle. Only claudin-2 was detected in the thin descending limbs of Henle (red), which were identified as AQP1-positive tubules in the medulla (green). In these images, claudin-2 did not seem to be concentrated in TJ-like thin lines but was diffusely distributed in broad bands. Double staining with anti-occludin mAb and anti-claudin-2 pAb revealed that not only claudin-2 but also occludin seemed to be diffusely distributed in broad bands in the thin descending limb of Henle (arrows). This staining became undetectable when anti-claudin-2 pAb was pretreated with GST-claudin-2 (anti-cld-2 pAb + GST-cld-2, arrows). When the sections were fixed with TCA, similar diffuse claudin-2-staining was detected at low magnification (left), but close inspection (right, representing rectangle at left) revealed that individual sharp lines were intermingled within the claudin-2-positive zones (arrows). These lines were also Zomula Occludens-1-positive (occludin-positive) (arrows). Bars, 20 µm.
Then, to improve the preservation of fixed tubules, we appliedthe newly developed TCA fixation method (24) (Figure 6). Similardiffuse claudin-2 staining was detected at low magnification,but close inspection revealed that individual sharp lines wereintermingled within the claudin-2-positive bands. These lineswere also Zomula Occludens-1-positive (occludin-positive). Becausethe constituent cells in the thin descending limb of Henle arevery flat and are interdigitated in a very complex manner (31),it is safe to conclude that claudin-2 was concentrated specificallyat TJ in this segment.
Thin Ascending Limb of Henle.
In addition to claudin-8 (Figure 3), claudin-3 and -4 were concentratedat TJ of epithelial cells of ClC-K-positive tubules in the medulla,i.e., the thin ascending limb of Henle (Figure 7).
Figure 7. Claudins expressed in the thin ascending limb of Henle. In addition to claudin-8 (Figure 3), claudin-3 and -4 were concentrated at TJ of epithelial cells in the thin ascending limbs of Henle (red), which were identified as ClC-K-positive tubules (green). Bars, 20 µm.
Thick Ascending Limb of Henle.
Four distinct species of claudins, namely claudin-3, -10, -11,and -16, were detected at TJ of epithelial cells of THP-positivetubules in the medulla, i.e., the thick ascending limb of Henle(Figure 8). In the cortex of some species, including mice, THPhas been reported to be expressed also in the distal tubules(32); however, among claudin-3, -10, -11, and -16, only claudin-3staining was positive in the distal tubules, as demonstratedbelow. Because the frozen sections were not very thin, theseclaudin-positive lines frequently seemed to correspond to apicalsurfaces, but detailed double staining of transversely sectionedtubules with anti-occludin mAb clearly revealed that these linescorresponded to TJ, not to apical surfaces (Figure 8, insets).
Figure 8. Claudins expressed in the thick ascending limb of Henle. Four distinct claudin species, namely claudin-3, -10, -11, and -16, were concentrated at TJ of epithelial cells in the thick ascending limbs of Henle (red), which were identified as THP-positive tubules (green). Insets, double immunostaining of transversely sectioned tubules with anti-occludin (occ) mAb and anti-claudin-10 (-11 or -16) (cld-10) pAb. The basal regions of these renal tubules were occasionally stained with anti-claudin-10 and -11 pAb but, as indicated by several control observations, this staining did not seem to be specific (insets). Bars, 20 µm.
Distal Tubule.
As demonstrated above, the distal tubules were identified asAQP1/2-double-negative tubules around glomeruli. In this typeof tubule, claudin-3 (Figure 9A) and -8 (Figure 3) were expressedand localized at TJ.
Figure 9. (A) Claudins expressed in the distal tubule. In addition to claudin-8 (Figure 3), claudin-3 was detected in TJ of distal tubules (red), which were identified as AQP1/2-double-negative tubules (arrows) located around glomeruli (asterisk). Double staining with anti-occludin mAb confirmed that these claudin-8 signals were derived from TJ (data not shown). Some signals seemed to be detected for glomeruli, including parietal epithelium, but control staining experiments (Figure 4) revealed that they were nonspecific. Bar, 20 µm. (B) Claudins expressed in the collecting duct. In addition to claudin-8 (Figure 3), claudin-3 and -4 were concentrated at TJ of epithelial cells in collecting ducts (red), which were identified as AQP2-positive tubules (green). Bars, 20 µm.
Collecting Duct.
In addition to claudin-8 (Figure 3), claudin-3 and -4 were clearlydetected at TJ of epithelial cells of AQP2-positive tubules,i.e., collecting tubules (Figure 9B). These segment-specificexpression patterns of claudins in nephrons are summarized inFigure 10.
Figure 10. Summary of the segment-specific expression patterns of claudins in mouse nephrons. In the thin descending limb of Henle, some, but not all, AQP1-positive tubules were positive for claudin-2 (Cld-2). Similarly, from the thin ascending limb of Henle to the collecting duct, claudin-3 was detected in some, but not all, marker-positive tubules. These findings may be consistent with previous reports that the distal tubules, as well as the collecting tubules, are composed of several functionally distinct subsegments. In other cases, claudins were detected in all of the respective marker-positive tubules.
To date, >20 members of the claudin family have been identified,but our knowledge regarding the expression patterns of theseclaudin species in individual organs is still fragmentary. Becausethe kidney has a complicated epithelial cell architecture, weexpected to observe the coexpression of several claudin speciesin this organ. Surprisingly, Northern blotting revealed that,of the 16 species of claudins investigated (claudin-1 to -16),12 distinct species were expressed in the kidney. More importantly,immunofluorescence microscopy revealed that these claudins demonstratedsegment-specific expression patterns in nephrons, except forclaudin-5 and -15, which were expressed in endothelial cells;antibodies specific for claudin-7 and -12 were not available.Therefore, questions have naturally arisen regarding the physiologicrelevance of such complicated patterns of segment-specific expressionof various claudin species in nephrons.
Several distinct types of claudins have been reported to becoexpressed in other types of cells, such as hepatocytes (14)and intestinal epithelial cells (33). In those cells, heterogeneousclaudin species are thought to be copolymerized into individualTJ strands; within paired strands, claudins adhere to each otherin homotypic as well as heterotypic manners (14). Several linesof evidence recently suggested that the combinations and mixingratios of claudins within individual paired strands determinetheir tightness (3,17). Therefore, the distinct patterns ofexpression of claudins in nephron segments may be involved inthe diversity of the paracellular flux in various segments.The following two recent observations seem to favor this suggestion.
There are two strains of the cultured epithelial cell line establishedfrom dog kidneys, i.e., MDCK I and II cells. MDCK I cells demonstratemuch higher transepithelial electric resistance than do MDCKII cells, although the strains bear similar numbers of TJ strands(34). The expression patterns of claudins in these cells wererecently reported (15). Claudin-1 and -4 were expressed in bothMDCK I and II cells, whereas the expression of claudin-2 wasrestricted to MDCK II cells. Dog claudin-2 cDNA was then introducedinto MDCK I cells, to mimic the claudin expression pattern ofMDCK II cells. Interestingly, the transepithelial electric resistancevalues of MDCK I clones stably expressing claudin-2 decreasedto the levels of MDCK II cells (>20-fold decrease). Thesefindings indicated that the addition of claudin-2 markedly decreasedthe tightness of individual TJ strands. As summarized in Figure 10,claudin-2 was specifically concentrated in TJ of the proximaltubules and Bowman’s capsule in the kidney, and it iswell known that TJ in proximal tubules are fairly leaky (35,36).Therefore, it is tempting to speculate that claudin-2 playsan important role in keeping TJ of proximal tubules leaky, althoughit is also known that the number of TJ strands in proximal tubulesis less than that in distal tubules (37).
The second observation was made among patients with hereditaryhypomagnesemia (16). The renal resorption of Mg2+ occurs predominantlyvia the paracellular pathway in the thick ascending limb ofHenle, but in these patients this paracellular flux is blocked,resulting in severe hypomagnesemia. Positional cloning identifiedclaudin-16/paracellin-1 as the gene responsible for this disease.Interestingly, claudin-16 was demonstrated to be exclusivelyexpressed in the thick ascending limb of Henle in human kidneys,which was confirmed in mouse kidneys in this study. This findingled to a very intriguing conclusion, namely that claudin-16is directly involved in the selective paracellular conductanceof Mg2+ ions in claudin-based TJ strands, with claudin-16 formingaqueous pores that function as paracellular Mg2+ channels withinpaired strands. It is possible that claudin-16 forms such poresvia homotypic adhesion within paired strands. However, as wediscussed previously (17), it is also possible that the weakheterotypic adhesion of claudin-16 to other claudin speciesresults in the formation of aqueous pores with high conductancefor Mg2+ ions within the paired strands. As demonstrated inFigure 10, together with claudin-16, claudin-3, -10, and -11constituted TJ strands in the thick ascending limb of Henle.Therefore, whether claudin-3, -10, or -11 is involved in theparacellular Mg2+ channels in collaboration with claudin-16should be examined in the near future.
This study provides the first detailed description of the expressionpatterns of claudins in nephrons, but several questions remainunanswered. Notably, information regarding the distributionsof claudin-7 and -12 in nephrons is still lacking. It is alsopossible that some of claudin-17 to -24 are expressed in nephrons.Indeed, although two to four distinct claudin species were coexpressedin most nephron segments, only claudin-2 was detected in thethin descending limb of Henle. It is possible that we overlookedthe expression of other types of claudins in this segment.
The molecular mechanisms underlying material transport acrossrenal epithelial cells via the transcellular pathway have beenintensively analyzed. However, lack of information regardingthe TJ-specific integral membrane proteins has hampered directassessment, at the molecular level, of the molecular mechanismunderlying paracellular flux across the renal epithelium. Claudinshave been identified, and their patterns of expression in mousenephron segments have been clarified. The results of this studycan facilitate understanding not only of the physiologic functionsof nephron segments but also of various pathologic states anddiseases caused by alterations in the tightness of TJ in eachnephron segment, in molecular terms.
Acknowledgments
We thank Dr. S. Sasaki (Tokyo Medical and Dental University,Tokyo, Japan) for generously donating anti-AQP2 pAb. Our thanksare also due to all of the members of our laboratory (Departmentof Cell Biology, Faculty of Medicine, Kyoto University) forhelpful discussions. This study was supported in part by a Grant-in-Aidfor Cancer Research and a Grant-in-Aid for Scientific Research(A) from the Ministry of Education, Science, and Culture ofJapan (to Dr. Tsukita) and by a Japan Society for the Promotionof Science Research for the Future Program grant (to Dr. Furuse).
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Received for publication August 7, 2001.
Accepted for publication November 22, 2001.
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