Abstract To reveal the role of cadherin complex in pod-ocyte differentiation, the present study describes the lo-calization of the cadherin complex, including p120 cat-enin (p120ctn), in the developing and in the aminonucle-oside nephrosis (PAN nephrosis) rat kidney, by immuno-fluorescence microscopy and immunogold electron mi-croscopy. p120ctn and β-catenin were co-localized at theapical part of lateral cell membranes in presumptive po-docytes of the S-shaped body, and their localizationshifted to the basal margin of lateral cell membranes inthe capillary loop stage. There was no expression of thecadherin complex at the slit diaphragm, the intercellularjunction of mature podocytes. After the regression of thepodocyte junctional structure in PAN nephrosis, the cad-herin complex was not re-expressed. The dynamicchanges in the localization of the cadherin complex sug-gest that the it plays an important role during podocytedifferentiation, including the rearrangement of the inter-cellular junction and the formation of the slit diaphragm.

Keywords Podocyte · p120 Catenin · β-Catenin · Cadherin complex · Aminonucleoside nephrosis · Rat

Introduction

Visceral epithelial cells of renal glomeruli, generallytermed podocytes, are known to organize the ultrafiltra-tion barrier with terminal differentiation and contributeto glomerular permeability (for a review, see Kriz et al.1998). They differentiate from metanephric mesenchy-mal cells in concert with morphological steps of glomer-ular differentiation (for a review, see Lechner and Dressler 1997). Morphological studies have so far re-vealed that the transition of the junctional complex isclosely associated with podocyte differentiation (Aoki1967; Humbert et al. 1976; Reeves et al. 1978; Nouwenet al. 1993; Tassin et al. 1994; Cho et al. 1998; Goto etal. 1998). During the process of podocyte differentiation,the intercellular junction first appears in the vesicles(Reeves et al. 1978; Garrod and Fleming 1990). In the S-shaped body stage, presumptive podocytes show ajunctional complex along the lateral cell membrane be-tween neighboring cells (Aoki 1967; Humbert et al.1976; Reeves et al. 1978; Garrod and Fleming 1990). Inthe capillary loop stage, the intercellular junction shiftsfrom the apical toward the basal part of the lateral cellmembrane and remains as the slit diaphragm at the basalpart of the lateral membrane at the stage of terminal dif-ferentiation. These findings suggest that the slit dia-phragm is a modified intercellular junction (Humbert et al. 1976; Reeves et al. 1978; Schnabel et al. 1989,1990; Kawachi and Shimizu 2000; Reiser et al. 2000).Although previous studies of the junctional complex in podocytes have largely described it as a tight junc-tion based on its ultrastructure and ZO-1 expression(Humbert et al. 1976; Reeves et al. 1978; Schnabel et al.1989, 1990; Kurihara et al. 1992), the presence of the adherens junction during podocyte differentiation has notbeen well described.

Cadherins are the chief molecular components of theadherens junction (Boller et al. 1985; Takeichi 1988).Cadherins are transmembrane calcium-dependent homo-philic cell-adhesion molecules that play important rolesin pattern formation during morphogenesis, tumor me-

J. Usui · Y. Shu · S. Tomari · K. Kanemoto · M. Nagata (✉)Department of Pathology, Institute of Clinical Medicine,University of Tsukuba, 1-1-1 Ten-nodai, Tsukuba, 305-8575, Ibaraki, Japane-mail: nagatam@md.tsukuba.ac.jpTel.: +81-298-533171, Fax: +81-298-533171

J. Usui · A. KoyamaDepartment of Internal Medicine, Institute of Clinical Medicine,University of Tsukuba, Tsukuba, Ibaraki, Japan

H. Kurihara · T. SakaiDepartment of Anatomy, Juntendo University School of Medicine,Tokyo, Japan

T. TakahashiDivision of Nephrology, Departments of Medicine and Cell Biology,Vanderbilt University, Nashville, Tenn., USA

Anat Embryol (2003) 206:175–184DOI 10.1007/s00429-002-0298-x

O R I G I N A L A RT I C L E

Joichi Usui · Hidetake Kurihara · Yujing ShuShinsuke Tomari · Katsuyoshi KanemotoAkio Koyama · Tatsuo Sakai · Takamune TakahashiMichio Nagata

Localization of intercellular adherens junction protein p120 cateninduring podocyte differentiationAccepted: 12 November 2002 / Published online: 17 January 2003© Springer-Verlag 2003

tastasis, and signal transduction (for reviews, see Gumbiner 2000; Nollet et al. 2000). The cadherin molecule forms complexes with various catenins, viz.,α-catenin, β-catenin, plakoglobin, and p120 catenin(p120ctn), and its function is modulated by the catenins.These catenins interact with the cytoplasmic tail of cad-herin at the catenin-binding domain (CBD), anchor it tothe actin cytoskeleton, and transduce intercellular signals(Gottardi and Gumbiner 2001). It is noteworthy thatp120ctn has different functions from those of other cat-enins. For instance, p120ctn binds to the juxtamembranedomain rather than the CBD. Additionally, p120ctn doesnot interact with α-catenin or tumor suppressor adeno-matous polyposis coli protein (Reynolds et al. 1992; Anastasiadis and Reynolds 2000). More interestingly,transient overexpression of p120ctn in mouse fibroblastsinduces the branching of cellular processes, resulting in aso-called dendritic phenotype (Reynolds et al. 1996).p120ctn promotes this process formation through theregulation of actin cytoskeletal dynamics via the inhibi-tion of RhoA (Anastasiadis et al. 2000). Rho familysmall GTPases including RhoA are involved in processformation in mouse cultured podocytes (Kobayashi2002), and process formation is also the most character-istic feature of differentiation in podocytes. Previousstudies have demonstrated the presence of cadherin-catenin complexes in presumptive podocytes (Nouwen etal. 1993; Tassin et al. 1994; Cho et al. 1998; Goto et al.1998; Shimazui et al. 2000). Nonetheless, the precise lo-calization of these molecules has not been fully deter-mined, and the presence of cadherin-catenin complexesin mature podocytes remains controversial (Nouwen etal. 1993; Tassin et al. 1994; Piepenhagen and Nelson1995; Cho et al. 1998; Goto et al. 1998; Kwon et al.1998; Reiser et al. 2000; Ruotsalainen et al. 2000; Yaoitaet al. 2001).

In this study, we examined the expression of the cad-herin complex, including p120ctn, in podocytes during ratglomerulogenesis and after PAN nephrosis. We found thatthe accumulation and disappearance of the cadherin com-plex during process formation were associated with thedynamism of the differentiation of glomerular podocytes.

Materials and methods

Animals and antibodies

Wistar rats were purchased from Japan SLC (Hamamatsu, Japan)for histological experiments. Male Sprague-Dawley rats (SD rats,Japan SLC) were used for the study of PAN nephrosis. Rats weremaintained in the Laboratory Animal Research Center in the Uni-versity of Tsukuba. All experiments were performed according tothe Guide for the Care and Use of Laboratory Animals at the Uni-versity of Tsukuba. Mouse monoclonal antibodies for immunohis-tochemistry included anti-p120ctn antibody (Transduction Labora-tories, Lexington, Ky., USA), anti-β-catenin antibody (Trans-duction Laboratories), and anti-pan cadherin antibody (Sigma-Aldrich, Saint Louis, Mo., USA). The anti-pan cadherin antibodywas produced by immunizing a synthetic peptide corresponding tothe C-terminal amino acids of chicken N-cadherin with an extraN-terminal lysine residue (24 amino acids) and reacted with multi-

ple cadherins in a variety of tissues and species (Takeichi 1988;Geiger et al. 1990). Fluorescein-5-isothiocyanate (FITC)-conju-gated donkey anti-mouse IgG antibody (Jackson ImmunoResearchLaboratories, West Grove, Pa., USA) was used as a secondary antibody. Goat anti-mouse IgG antibody coupled to 10-nm gold(British BioCell, Cardiff, UK) was obtained for immunogold labeling.

Immunofluorescence microscopy

Fetal (E18) and newborn (PD 8 days) rat kidneys were used. Thekidneys were either unfixed or fixed with 1% paraformaldehydefixative for 1 h. The tissue was snap-frozen in a Tissue-Tek OCTcompound (Miles, Elkhart, Ind., USA) by hexane surrounded withcooled acetone. Sections (2–4 µm in thickness) were cut on acryostat and mounted on glass slides. The unfixed sections weretreated with acetone for 20 min at 4°C. In fixed sections, antigenwas occasionally retrieved with short-term autoclave oven heatingat 120°C in 10 mmol/l sodium citrate (pH 6.0). After nonspecificreactions had been blocked, the sections were incubated with either anti-p120ctn (1:100 dilution), anti-β-catenin (1:200 dilu-tion), or anti-pan cadherin antibodies (1:250 dilution) for 2 h atroom temperature. Secondary antibody (1:100 dilution) was thenapplied for 1 h at room temperature. Sections were subsequentlymounted with glycerin containing phosphate-buffered saline(PBS). Normal mouse IgG was used as a control. An immunofluo-rescence and phase-contrast microscope (AX-70, Olympus, Tokyo, Japan, or DM RXA2, Leica, Heidelberg, Germany) wasused for inspection.

Transmission electron microscopy

Fetal (E18) Wistar rat kidneys were removed and cut into smalltissue blocks (1 mm3), and the blocks were immersed in a 2.5%glutaraldehyde fixative with a 0.2 mol/l phosphate buffer (PB,pH 7.4) solution overnight at 4°C. The tissue blocks were thenpostfixed in 1% osmium tetroxide in a 0.2 mol/l PB solution for2 h at 4°C, dehydrated in a series of graded ethanol preparations,and embedded in epoxy resin (Poly/Bed 812 Embedding Media,Polysciences, Warrington, Pa., USA). Ultra-thin sections were cuton a Leica Ultracut S microtome (Vienna, Austria) and stainedwith uranyl acetate and lead citrate. Sections were observed bytransmission electron microscopy (TEM; H-7000, Hitachi, Tokyo,Japan) at 75 kV.

Immunogold electron microscopy

Immunogold electron microscopy was performed by the methoddescribed previously with some modifications (Inoue et al. 2001).Briefly, kidneys from newborn Wistar rats (1 or 2 days afterbirth) were perfused with a paraformaldehyde-lysine-periodatefixative buffer (containing 2% paraformaldehyde). The kidneyswere removed and cut into small tissue blocks (1 mm3), whichwere reimmersed in the same fixative for 30 min at 4°C. Thereaf-ter, the tissue blocks were immersed in 30% polyvinylpyrrolidonein 2.3 mol/l sucrose buffered with a 0.1 mol/l PB solution, over-night at 4°C. The samples were embedded on nails and frozen inliquid nitrogen. Ultra-thin cryosections were cut on a Leica Ultra-cut UCT equipped with the Leica EM FCS cryoattachment at -110°C and then transferred to nickel grids (150 mesh) and coatedwith formvar and carbon. Subsequent incubation steps were car-ried out by floating the grids on droplets of the relevant filteredsolution. After quenching free aldehyde groups with 0.01 mol/Lglycine in PBS, we incubated the sections overnight with eithermouse monoclonal anti-p120ctn (1:50 dilution in PBS containing10% fetal bovine serum) or anti-β-catenin antibodies (1:100 dilu-tion). The sections were then incubated with goat anti-mouse IgGcoupled to 10-nm gold (1:200 dilution) for 2 h at room tempera-ture. After being immunostained, they were fixed with 2.5% glu-

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taraldehyde buffered with a 0.1 mol/l PB solution. The sectionswere then contrasted with a 2% uranyl acetate solution for 20 minand absorption-stained with 2% polyvinyl alcohol containing0.3% uranyl acetate for 20 min. All sections were observed byelectron microscopy (1200-EX electron microscope, JEOL, To-kyo, Japan).

Expression of p120ctn and β-catenin on the slit diaphragm inthe capillary loop stage was quantitatively estimated by the ratioof slit membranes having gold particles to all the slit membranesencountered.

Experimental aminonucleoside nephrosis

Three male SD rats (body weight approximately 150 g) receivedsingle intraperitoneal injections of puromycin aminonucleoside(PAN, 15 mg per 100 g per rat, Sigma-Aldrich) as described pre-viously (Ryan and Karnovsky 1975). Animals were killed on the10th day after injection. All animals exhibited massive proteinur-ia and ascites at the time of sacrifice. The same rat strain was in-jected with buffered saline and served as the control. Kidneyswere processed for immunofluorescence and TEM as describedabove.

Results

Cadherin complex in metanephric mesenchyme and ureteric bud

Immunofluorescence for the pan cadherin antibody wasweakly detected in the mesenchymal condensate aroundthe ureteric bud (Fig. 1a). Anti-pan cadherin antibodydid not react with uncondensed mesenchymal cells orureteric buds. By contrast, expression of p120ctn and β-catenin was weakly detected in both the mesenchymalcondensate and the uncondensed mesenchyme (Fig. 1b, c).In the ureteric bud, both catenins were localized alongthe lateral cell borders.

Cadherin complex in the renal vesicle

In the renal vesicle, immunoreaction with anti-pan cad-herin, p120ctn, and β-catenin antibodies showed similarlocalization pattern and intensity. Expression was foundas dots in the apical part of the lateral membranes and asdiscrete lines along the lateral membrane between vesic-ular cells (Fig. 1a–c).

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Fig. 1a–c Cadherin complex in the rat nephrogenic zone (E18) la-beled with anti-pan cadherin (a), anti-p120ctn (b), and anti-β-cat-enin antibodies (c). a Pan cadherin staining is detected in the mes-enchymal condensate (MC) around the ureteric bud (UB). The ure-teric bud and the uncondensed mesenchyme are negative. In therenal vesicle (V), expression is found as dots in the apical part oflateral membranes and weak discrete lines along the lateral cellmargins. b p120ctn expression is weakly detected between cells inthe mesenchymal condensate. Intense staining is seen in the inter-cellular boundaries of the ureteric bud cells. Staining in the uncon-densed mesenchyme is positive. The staining pattern in the renalvesicle is similar to that with the anti-pan cadherin antibody. c Theβ-catenin expression pattern is similar to that of p120ctn expres-sion. These sections are from a series. ×200

p120ctn and β-catenin in podocyte differentiation

The signals for p120ctn and β-catenin showed similarexpression patterns in podocytes from the S-shaped bodystage to the maturing stage. In the early S-shaped bodystage, before the urinary space was formed, p120ctn waslocalized between the apical part of the presumptive po-docytes and of Bowman’s epithelial cells (Fig. 2a, b).The expression of p120ctn at the apical cell membranedisappeared together with the development of the urinaryspace in the later stages of the S-shaped body (Fig. 2c,d). During the S-shaped body stage, tight cell-cell con-tact remained between presumptive podocytes, whichformed a single cuboidal cell layer. Immunofluorescence

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Fig. 2a–h p120ctn in rat podocyte differentiation (a–f E18,g, h PD8). a p120ctn localizes as intense dots at the apical parts oflateral membranes in presumptive podocytes (white arrows) andof Bowman’s epithelial cells (white arrowheads) in the early S-shaped body stage. Between presumptive podocytes, stainingoccurs as weak discrete lines in the lateral membranes. ×1800.c During the late S-shaped body stage, p120ctn is observed as in-tense fluorescent dots (white arrows) at the apical points of the lat-eral membrane and as dots or discrete lines along the lateral cellborders. ×2000. e In the capillary loop stage, bright immunofluo-rescence is localized along the basal margin of presumptive po-docytes (white arrows). Significant p120ctn expression is simulta-neously observed in endothelial cells (white arrowheads). ×2800.g In the maturing stage, p120ctn expression is not detected in po-docytes . On the other hand, immunofluorescence of Bowman’sepithelial cells is extant (white arrows). p120ctn staining of endo-thelial (white arrowheads) and mesangial cells is also positive.×1800. b, d, f, h Lower magnification phase-contrast images cor-responding to the fluorescent images left (squares). ×1000

Fig. 3a–f β-Catenin in rat podocyte differentiation (a–d E18,e, f PD8). a β-Catenin immunostaining is shown as intense fluo-rescent dots at the apical parts of lateral membranes in presump-tive podocytes (white arrows) and of Bowman’s epithelial cells(white arrowheads) in the early S-shaped body stage. ×2300. c Inthe capillary loop stage, bright immunofluorescence is localizedalong the basal margin of presumptive podocytes (white arrows).Distinct β-catenin expression is simultaneously observed in endo-thelial cells (white arrowheads). ×2500. e β-catenin is absent frommature podocytes. On the other hand, β-catenin staining of Bowman’s epithelial cells (white arrows), endothelial cells (whitearrowheads), and mesangial cells is positive. ×2800. b, d, f Lowermagnification phase-contrast images corresponding to the fluores-cent images left (squares). ×1000

Fig. 4a–c Pan cadherin staining in rat podocyte differentiation (a,b E18, c PD8). a Pan cadherin staining is observed as intense fluo-rescent dots at the apical parts of presumptive podocytes (whitearrows) and of Bowman’s epithelial cells (white arrowheads) inthe early S-shaped body stage. b In the early capillary loop stage,bright immunofluorescence is localized along the basal margin ofpresumptive podocytes (white arrows). Staining as dots or discretelines is partly seen in the adhesive lateral regions (white arrow-heads). c Pan cadherin labeling is absent from mature podocytesand endothelial cells. On the other hand, fluorescence of Bow-man’s epithelial cells (white arrows) and mesangial cells (whitearrowheads) is positive. ×1300

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for p120ctn was most intense at the apical part and lessintense at the basal part of the lateral membranes(Fig. 2a–d). The expression took the form of a weaklydiscrete line in the lateral part. In addition, weak immu-nofluorescence for p120ctn was found in the cell-cellcontact area between Bowman’s epithelial cells.

In the capillary loop stage, the intensity of the fluores-cence with p120ctn antibody reached its peak at the bas-al margin of podocytes, and no distinct fluorescence wasobserved at the apical side (Fig. 2e, f). At the beginningof this stage, as the contact region of adjacent presump-tive podocytes lowered toward the base, intense fluores-cent dots were seen in the apical part of the contiguouslateral cell membranes, with dots or discrete lines of flu-orescence in the adhesive lateral part (Fig. 2c, d). Con-commitantly with the maturation of presumptive podocy-tes, p120ctn expression was localized along the basalmargin (Fig. 2e, f). Simultaneously, significant p120ctnexpression was observed in endothelial and mesangialcells beneath the glomerular basement membrane(GBM). In the mature glomerulus, p120ctn expressionultimately disappeared in podocytes (Fig. 2g, h). On theother hand, immunofluorescence for p120ctn remainedpersistent and extant in Bowman’s epithelial cells. Theexpression of β-catenin showed similar patterns as thatof p120ctn in podocyte differentiation (Fig. 3a–f).

Pan cadherin in podocyte differentiation

The signals detected with pan cadherin antibody fromthe S-shaped body stage to the maturing stage in podocy-tes were similar to those found for p120ctn and β-catenin

(Fig. 4a–c). Immunofluorescence for pan cadherin wasalso detected in Bowman’s epithelial cells as catenins,whereas glomerular endothelial cells were not stained bypan cadherin antibody during any stage of glomerulardifferentiation.

Ultrastructural localization of p120ctn and β-catenin in podocyte differentiation

In the late S-shaped body stage, a few gold particles la-beling p120ctn were localized along the cytoplasmic sideof the plasma membrane on the apical part of the lateralmembrane of presumptive podocytes (Fig. 5a). Gold par-ticles were rarely seen along the lateral membrane. Weobserved p120ctn at the cell-cell contacts of Bowman’sepithelial cells (data not shown). In the capillary loopstage, an accumulation of p120ctn gold particles wasidentified at the intercellular junction between the majorprocesses of presumptive podocytes (Fig. 5b). A few par-ticles were often seen at the junction between neighbor-ing foot processes (Fig. 5c), whereas p120ctn particleswere absent when the processes acquired slit diaphragms.Quantitatively, 55.4% (62/112) of intercellular junctionson foot processes (before slit diaphragms were formed)had immunoreaction of gold particles, whereas gold parti-cles were present in only 1.30% (3/231) of slit dia-phragms. We saw accumulated gold particles near Bow-man’s epithelial cells in the mature glomerulus (data notshown). The spatiotemporal localization of β-catenin wassimilar to that of p120ctn during podocyte differentiation,as shown by immunoelectron microscopy (Fig. 5d-f).Similarly, quantitative assessments of β-catenin immuno-reaction in cellular junctions revealed that 50.0%(64/128) were positive, whereas only 1.97% (3/152) ofslit diaphragms had gold particles. The accumulation ofgold particles labeling p120ctn and β-catenin appeared inthe intercellular junction between endothelial cells in thecapillary loop stage and maturing stage (Fig. 5f).

Intercellular junction in glomerulogenesis

In the early S-shaped body stage, before the urinaryspace was formed, presumptive podocytes and Bow-

man’s epithelial cells were often attached to one another.Adherens junctions were observed scattered not only be-tween presumptive podocytes and between Bowman’sepithelial cells, but also between the apical part of pre-sumptive podocytes and that of Bowman’s epithelialcells (Fig. 6a, b). At the late S-shaped body stage, tightjunctions and adherens junctions were seen at the apicalpart of the lateral membrane (Fig. 6c, d). However, eachjunction was hard to specify as being either a tight or ad-

herens junction and was often recognized as a complexof these two junctions. As podocytes differentiated, bothtight and adherens junctions were found partway downon the lateral cell membranes between the apical and thebasal regions. These junctions seemed to migrate alongthe lateral cell surface from the apical to basal part. Atthe capillary loop stage, tight and adherens junctions stillremained between major processes in podocytes, al-though they appeared to be fused and became indistin-

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Fig. 5a–f Immunogold elec-tron microscopy for p120ctn(a–c) and β-catenin (d–f) dur-ing podocyte differentiation(asterisks glomerular basementmembrane). a In the S-shapedbody stage, a few gold particlesfor p120ctn are localized alongthe cytoplasmic side of the api-cal part of the lateral membranebetween presumptive podocy-tes. ×25,000. b In the capillaryloop stage, the gold particlesfor p120ctn accumulate at theintercellular junctions betweenmajor processes of neighboringpresumptive podocytes. ×12,000.c With the maturation of theglomerulus, the gold particlesfor p120ctn disappear fromfoot processes with slit dia-phragms (arrowheads). A fewparticles are often seen on thecytoplasmic side of the imma-ture junctions between footprocesses (arrows). ×30,000.d–f Immunogold particles forβ-catenin exhibit a similar pat-tern to that of p120ctn. d A fewparticles for β-catenin are lo-calized on the apical part of thelateral membrane in presump-tive podocytes in the S-shapedbody stage. ×25,000. e Manyparticles labelling β-catenin are localized on major process-es in the capillary loop stage.×15,000. f The particles for β-catenin remain at the imma-ture intercellular junctions be-tween foot processes (arrow)but not on mature foot process-es with slit diaphragms (arrow-head). An accumulation ofsome gold particles is seen inthe cell-cell contacts betweenadjacent endothelial cells(white arrow). ×25,000

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Fig. 6a–f Transmission electron microscopic findings of intercel-lular junctions during rat glomerulogenesis (VEC visceral epithe-lial cell, PEC parietal epithelial cell, EC endothelial cell, RBC redblood cell). a Intercellular junctions are frequently seen in pre-sumptive podocytes (VEC) and Bowman’s epithelial cells (PEC)of the early S-shaped body. ×6000. b Higher magnification of a.Adherens junctions are observed between podocytes and PECs(arrows), between adjacent PECs (arrowheads), and between ad-

jacent podocytes (white arrowhead). ×20,000. c In the S-shapedbody stage, both tight junctions and adherens junctions (arrow)are seen at the apical parts of the lateral membranes between pre-sumptive podocytes. ×5000. d Higher magnification of c. Thejunction structurally shows atypical features characterized by thecoupling of tight and adherens junctions. ×30,000. e In the capil-lary loop stage, similar junctions are observed between major pro-cesses. ×5000. f Higher magnification of e. ×30,000

guishable (Fig. 6e, f). Finally, mature glomerulus losttheir typical tight and adherens junctions concurrentlywith the widening of the intercellular space between ad-jacent podocytes, accompanied by slit diaphragm forma-tion.

Cadherin complex in aminonucleoside nephrosis

Next, we tested whether adherens junction moleculeswere re-expressed after remodeling of the junctionalcomplex in the podocyte foot process by using a ratnephrosis model. By TEM, junctional structures wereseen to re-appear at the lateral cell membrane of the footprocesses with a dislocation of the slit diaphragms(Fig. 7a). Immunofluorescence for p120ctn, β-catenin,and pan cadherin was not detected at the basal part of thepodocytes; we observed the same staining pattern as thatfound in the normal mature glomerulus (Fig. 7b–d).

Discussion

In the present study, we demonstrated the close associa-tion of cadherins, p120ctn, and β-catenin during podo-cyte differentiation. Accumulation of these moleculeswas noted by the dot-like features at the apical part ofthe lateral cell membranes in presumptive podocytes,and the localization of these molecules shifted to the bas-al sites of major processes as glomerulogenesis proceed-ed. Finally, their expression disappeared from podocytesin the maturing glomerulus. These findings suggest thatcadherins, p120ctn, and β-catenin form a complex, andthat the complex shifts during podocyte process forma-tion.

The presence of adherens junctions in podocyte dif-ferentiation has been previously shown by electron mi-croscopy. Earlier studies demonstrated the ultrastructureof adherens junctions among the epithelial cells of therenal vesicle, presumptive podocytes in the S-shapedbody stage, and major processes of podocytes in the cap-illary loop stage (Aoki 1967; Reeves et al. 1978; Garrodand Fleming 1990; Goto et al. 1998), whereas these stud-ies failed to identify adherens junctions in mature po-docytes.

Adherens junctions in podocytes were identified byimmunofluorescence microscopy with antibodies againsttheir molecular components. Cadherins, including P-, N-,R-cadherin, and cadherin-6, were expressed in the pre-sumptive podocytes (Nouwen et al. 1993; Tassin et al.

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Fig. 7a–d Cadherin complex in aminonucleoside nephrosis. a Bytransmission electron microscopy, the displacement of slit dia-phragms and reappearance of intercellular junctions (arrows) arefrequently observed between the adjacent podocytes. ×20,000.b By immunofluorescence microscopy, p120ctn expression occurswith the same pattern as that in the normal glomerulus and is notfound in the podocyte along the outer surface of the glomerularbasement membrane (white asterisks). ×2400. c β-Catenin expres-sion exhibits a similar pattern to that of p120ctn; it does not showthe typical “podocyte pattern” (white asterisks). ×2400. d Pan cad-herin staining is not detected in podocytes (white asterisks). ×2400

1994; Cho et al. 1998; Goto et al. 1998; Shimazui et al.2000). Although the molecular species of cadherins testedin each study varied, these findings collectively indicatedthe presence of some cadherins in the presumptive po-docytes. Nonetheless, the precise localization of thesemolecules has not been fully determined, and the pres-ence of cadherins in mature podocytes remains controver-sial. Two reports have suggested that P-cadherin is local-ized in mature podocytes as a component of slit dia-phragms (Tassin et al. 1994; Reiser et al. 2000). By con-trast, several reports have claimed that antibodies againstclassical cadherins, including P-, N-, E-, R-cadherin, cad-herin-6, and pan cadherin, did not react with mature po-docytes, as revealed by immunofluorescence microscopy(Nouwen et al. 1993; Tassin et al. 1994; Cho et al. 1998;Goto et al. 1998; Ruotsalainen et al. 2000; Yaoita et al.2001). In addition, catenin expression was shown in presumptive podocytes by using antibodies against α-catenin, β-catenin, and plakoglobin (Tassin et al. 1994;Goto et al. 1998; Shimazui et al. 2000). As is the case of cadherin, catenin expression in mature po-docytes remains uncertain. One report has shown that α-catenin and β-catenin are present in mature podocytes (Piepenhagen and Nelson 1995), whereas others havesuggested that α-catenin, β-catenin, and plakoglobin areabsent from mature podocytes (Tassin et al. 1994; Goto etal. 1998; Kwon et al. 1998). In the present study, we havedemonstrated found that p120ctn and β-catenin are pres-ent in presumptive podocytes, and that their localizationshifts from the apical to the basal part of lateral cell mem-branes as differentiation occurs. The expression pattern ofp120ctn and β-catenin is closely associated with the ultra-structural observations. We have not found distinct im-munoreaction of these antibodies in mature podocytes.We have also established the absence of cadherin com-plexes in mature podocytes in human kidneys (unpub-lished). These inconsistent findings of cadherin-cateninexpression in mature podocytes may be attributable todifferences in the animal species or antibodies used.

In the present study, we have found that the cadherincomplex appears at the apical part of lateral membranesin presumptive podocytes, and that its localization shiftsto the basal margin during the S-shaped body and thecapillary loop stages. The localization of cadherin com-plex in our result is similar to that of ZO-1 demonstratedin previous studies (Schnabel et al. 1990; Ruotsalainen etal. 2000). However, we have demonstrated a more prom-inent accumulation of cadherin complexes at the basalmargin compared with the apical part of lateral mem-brane. Thus, it is still uncertain whether cadherin com-plexes at the basal margin are newly formed in situ, orwhether they migrated from the apical to basal part alongthe lateral membrane.

Another significant finding of the present study is theexpression of cadherin complexes in PAN nephrosis. Ourultrastructural studies have revealed the regression of in-tercellular junctions at the cell-cell contacts in podocy-tes. Previously, we have shown the presence of ZO-1 atthe cellular junctions in podocytes in PAN nephrosis

(Kurihara et al. 1992). However, in the present study, wehave not been able to find cadherin complex during theregression of intercellular junctions in podocytes. Thisimplies that the cadherin complex is not necessarily as-sociated with ZO-1 and has a distinct function in the pro-cess of podocyte differentiation.

The identical localization of p120ctn and β-catenin inour results suggests that components of cadherin com-plexes are synergistically involved in podocyte differen-tiation. Recent reports have shown that p120ctn and β-catenin are localized not only on the cell membrane butalso in the cytoplasm and the nucleus (Anastasiadis et al.2000; Gottardi and Gumbiner 2001; Aho et al. 2002). In-terestingly, a prominent cytoplasmic pool of cadherin-uncoupled p120ctn inhibits RhoA and induces branchingof cytoplasmic processes (Anastasiadis et al. 2000). Inaddition, this branching phenotype is associated with thenuclear localization of some specific p120ctn isoforms(Aho et al. 2002). Likewise, β-catenin is bound to T-cellfactor-type transcription factors in the nucleus and con-trols the expression of genes that are targets of the Wntsignaling pathway (Gottardi and Gumbiner 2001). Al-though, in the present study, we have not observed eitherthe cytoplasmic or the nuclear localization of p120ctnand β-catenin during podocyte differentiation, it remainspossible that histologically undetectable levels ofp120ctn and β-catenin in the cytoplasm or the nucleusare involved in the final differentiation of podocytes.

In summary, we have demonstrated that the localiza-tion of the cadherin complex, including p120ctn, chang-es during podocyte differentiation. We suggest that themigration, accumulation, and disappearance of the cad-herin complex play an important role in the differentia-tion of glomerular podocytes.

Acknowledgments This work was supported in part by a HealthScience Research Grant from the Ministry of Health, Labour andWelfare, Japan, a Tsukuba Research Project Grant, and a ResearchGrant from Showa University. The authors greatly appreciatehelpful discussion with Dr. Eishin Yaoita (Niigata, Japan), ToruShimazui (Tsukuba, Japan), and Albert B. Reynolds (Nashville,Tenn., USA). They also wish to thank Dr. Keigyou Yoh and Dr.Satoru Takahashi (Tsukuba, Japan) for technical support.

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