Proc. Nadl. Acad. Sci. USAVol. 89, pp. 7075-7079, August 1992Cell Biology

Diversity among tight junctions in rat kidney: Glomerular slitdiaphragms and endothelial junctions express only oneisoform of the tight junction protein ZO-1HIDETAKE KURIHARA*, JAMES MELVIN ANDERSONt, AND MARILYN GIST FARQUHAR*t*Division of Cellular and Molecular Medicine and tCenter for Molecular Genetics, University of California, San Diego, La Jolla, CA; and tDepartment ofMedicine, Yale University School of Medicine, New Haven, CT 06510

Contributed by Marilyn Gist Farquhar, April 30, 1992

ABSTRACT ZO-1 is a 225-kDa peripheral membraneprotein present in all tight junctions. It was recently shown toconsist of two isoforms that differ in the presence of an internal80-amino acid domain termed motif-a. To obtain informationon their distribution and potential functional sgn e wehave localized the two isoforms in rat kidney by using anti-bodies that recognize either both ZO-1 isoforms or the larger,motif-a-containing isoform. By immfluorescence, stainingwith both antibodies was demonstrated at all tight junctions oftubular epithelial cells and the epithelial cells of Bowman'scapsule. In contrast, the motif-a-containing isoform was ab-sent from the slit diaphrgms of the glomeular epithelium andthe tight junctions of glomerular and peritubular capillaryendothelial cells. This restricted isoform expresson was con-firmed by immunoblot analysis comparing proteins from pu-rified glomeruli with those from kidney cortex or medulla.Thus, while both isoforms are expressed in typical epitheltight junctions, only a single isoform, lacking motif-a, isexpressed in the highly s d slit dI s, where theintercellular spaces are normally open, and in endothelialjunctions, which are readily opened by physiologic signals. Thedifferential expression of ZO-1 isoforms in s lly andfunctionally distinct junctions in the kidney a that theymay contribute to defning the variable fuctional properties,in particular the lability of these intercellular junctions.

The tight or occluding junctions of kidney epithelia varyconsiderably in their morphologic and functional properties(1-3). They range from those of the distal tubule and collect-ing duct with the most extensive junctions, which are verytight and maintain a very high transepithelial potential dif-ference, to those of the proximal tubule, which are shallowand leaky and have a low transepithelial potential difference.Previously we have shown that ZO-1, a 225-kDa tight junc-tion marker protein (4), is located on the cytoplasmic side ofall these junctions (5). Additionally, we have shown that theslit diaphragms, intercellular junctions between the epithelialcells of the renal glomerulus, are modified tightjunctions (5).This conclusion is based on the observations that the slitdiaphragm (i) arises during development from a tightjunction(6), (ii) defines the boundary ofthe polarized basal and lateralplasma membrane domains of the podocyte (6), and (iii)contains the tight junction protein ZO-1 (5). Thus the slitdiaphragm represents another, extreme functional variant ofthe tight junction where the intercellular spaces are main-tained wide open to allow passage of the glomerular filtrate.

Recently, ZO-1 cDNA sequencing disclosed alternativeRNA splicing that results in the expression of two isoformsof ZO-1 differing in the presence of an 80-amino acid regionreferred to as "motif-a" (7). Both transcripts as well as the

protein isoforms were found in several human epithelial celllines (Caco-2, T84, Hep G2). The existence of the two(ZO-la' and ZO-lal) isoforms raises the question ofwhether these isoforms contribute to tight junction diversityin different epithelia. To gain information about this possi-bility we have localized ZO-1 in glomerular and tubularepithelia and in glomerular and peritubular capillary endo-thelial cells, using antibodies that recognize both isoforms orexclusively recognize the larger isoform, containing motif-a.We here demonstrate diversity among renal junctions in thedistribution of motif-a.

MATERIALS AND METHODSMaterials. Polyvinyl alcohol (Mr 10,000), polyvinylpyrroli-

done (Mr 10,000), antipain, pepstatin A, leupeptin, benzami-dine, diisopropyl fluorophosphate, and phenylmethylsulfo-nyl fluoride were obtained from Sigma. F(ab')2 fragnents ofgoat anti-rabbit IgG conjugated to fluorescein isothiocyanateand goat anti-rabbit IgG (heavy- and light-chain-specific)were from Zymed Laboratories. Rabbit anti-goat IgG cou-pled to 5-nm colloidal gold was from Amersham, iodinatedprotein A from ICN, methylcellulose from Fisher, and nitro-cellulose from Schleicher & Schuell.

Animals. Male Sprague-Dawley rats (200-300 g) and new-born (2-day-old) rats were obtained from Harlan-Sprague-Dawley.

Induction of Nephrosis. Rats were treated with puromycinaminonucleoside (Sigma) for 10 days or perfused with prot-amine sulfate (Merck) as described (8).

Antibody. Polyclonal rabbit anti-ZO-1 (no. 7445) was raisedagainst a recombinant glutathione S-transferase fusion pro-tein containing 38 kDa of rat ZO-1 and was affinity purifiedas described (9). This antibody recognizes both ZO-1a+ andZO-la-. Polyclonal rabbit anti-ZO-1 motif-a (no. 8040) wasraised against a fusion protein containing 54 amino acidresidues of ZO-1 motif-a in fusion with glutathione S-trans-ferase (7). It recognizes only ZO-la+.

Preparation of Glomerular Fractions. Glomeruli from nor-mal rats were isolated by graded sieving at 4°C in the presenceof protease inhibitors (2.5 mM benzamidine, 0.2 mM phe-nylmethylsulfonyl fluoride, and 1 mM each antipain, pepsta-tin A, leupeptin, and diisopropyl fluorophosphate) as de-scribed (10). The resultant fractions consisted of >90%oglomeruli.Immunoblot Analysis. Rat kidney cortex, medulla, or iso-

lated glomeruli were solubilized in SDS/PAGE samplebuffer, and the proteins were separated by SDS/PAGE,transferred to nitrocellulose membranes, and subjected toimmunoblot analysis (9, 10).

Preparation of Kidney Tissue for Electron Microscopy. Ratswere perfused via the abdominal aorta with aldehyde fixative(3% paraformaldehyde/0.05% glutaraldehyde/0.1 M sodiumphosphate buffer, pH 7.4) at room temperature, and kidneytissue was removed and cut into small blocks. Tissue blocks

7075

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7076 Cell Biology: Kurihara et al.

M C G

FIG. 1. Identification of ZO-1 isoforms in rat kidney by immu-noblot analysis. Similar amounts ofprotein from medulla (M), cortex(C), and isolated glomeruli (G) were separated by SDS/PAGE andprobed with ZO-1 antibodies. Two bands (225 and 235 kDa) weredetected in all fractions with antibody 7445, which recognizes bothZO-1 isoforms (ZO-la+ and ZO-la-). The lower band, correspond-ing to ZO-la-, predominates in the cortex whereas the higher band,corresponding to ZO-la+, is most prominent in the medulla. ZO-la-is highly enriched in the glomerular lysate. Antibody 8040, specificfor motif-a, recognizes only the higher molecular weight band.

were then immersed in 2% glutaraldehyde buffered with 0.1M sodium cacodylate (pH 7.4) for 1 hr at room temperature,postfixed with 2% OsO4 in 0.1 M sodium cacodylate (pH 7.4)for 1 hr at 40C, and stained with 2% uranyl acetate for 1 hr atroom temperature prior to dehydration and embedding inEpon epoxy resin. Ultrathin sections, doubly stained with

uranyl acetate and lead citrate, were examined with a PhilipsCM10 electron microscope.

Preparation of Kidney Thsne for ImmRat kidneys were flushed with Dulbecco's modified Eagle'smedium through the abdominal aorta and fixed in situ byperfusion of 3% paraformaldehyde/0.05% glutaraldehyde/0.1 M sodium phosphate buffer, pH 7.4, or 2% paraformal-dehyde/75 mM lysine/10 mM NaIO4/37.5 mM sodium phos-phate (11). Two-day-old rats were perfused with the samefixative via the left ventricles. Kidneys were then cut intosmall pieces and immersed in the same fixative for anadditional hour. Samples were immersed in 20% polyvinylalcohol/2.3M sucrose/0.1 M phosphate, pH 7.4, mounted onnails, frozen, and stored in liquid nitrogen.Immunoflorescence. Semithin cryosections (1-2 ,m) were

prepared from aldehyde-fixed rat kidneys by using a ReichertUltracut E equipped with an FC-4E low-temperature sec-tioning system and were transferred to gelatin-coated glassslides. The sections were incubated with affinity-purifiedpolyclonal anti-ZO-1 IgG (no. 7445, 4 ng/ml) or affinity-purified polyclonal anti-motif-a IgG (no. 8040, 10 ng/ml) for2 hr at room temperature or overnight at 4°C, followed byfluorescein-conjugated goat anti-rabbit F(ab')2 (diluted 1:100)

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FIG. 2. Immunofluorescence labeling of semithin cryosections (1-2 ttm) of rat kidney cortex for ZO-1 isoforms. Phase-contrast (Left) andfluorescence (Right) micrographs of each section are shown. (A and B) Immunoreactivity for antibody 7445, which recognizes both ZO-1isoforms, is detected in the foot-process layer along the outer aspect of the glomerular basement membrane. This signal has previously beenshown to be due to staining of the filtration slit diaphragms, which represent a variant of the tightjunction (8). (C and D) Antibody 8040, specificfor motif-a, stains celljunctions ofthe tubular epithelium and Bowman's capsule (arrows), but the glomerular epithelial cells and the glomerularand peritubular endothelial cells are not stained. G, glomeruli; D, distal tubule; P, proximal tubule. (x370.)

Proc. Nad. Acad Sci. USA 89 (1992)

Proc. Natl. Acad. Sci. USA 89 (1992) 7077

for 1 hr at room temperature. They were mounted in 90%glycerol in phosphate-buffered saline containing phenylene-diamine (1 mg/ml), examined by epifluorescence, and pho-tographed using a Zeiss Axiophot and Kodak Tri-X Pan (ASA400) film.Immunogold Labeling on Ultrathin Cryosections. Ultrathin

cryosections were cut with an Ultracut E equipped with theFC-4E cryoattachment at - 110'C following the techniques ofTokuyasu (12, 13). Sections were transferred to nickel grids(300 mesh) which had been coated with Formvar and carbon.After free aldehyde groups were quenched with 0.01 Mglycine in phosphate-buffered saline, sections were incu-bated overnight with affinity-purified antibody 8040 IgG (20ng/ml), followed by goat anti-rabbit IgG (diluted 1:200 withphosphate-buffered saline containing 10%o fetal bovine se-rum) for 2 hr and rabbit anti-goat IgG coupled to 5-nm gold

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FIG. 3. Immunogold staining for ZO-la+ in normal kidney cor-

tex. Gold particles are located along the tight junctions (arrows)between epithelial cells of the distal tubule (A), Bowman's capsule(B), and proximal tubule (C). No labeling is seen along the slitmembranes (arrows) between the foot processes (fp) of the glomer-ular epithelium. In epithelia of the renal tubule and Bowman'scapsule the gold is concentrated along the cytoplasmic surface oftight junctions, which is in keeping with the fact that ZO-1 is a

peripheral membrane protein (14). This is particularly evident in C,in which a tight junction (TJ) of a proximal tubule is cut in grazingsection. mv, Microvilli; B, basement membrane. (A, x85,000; B,x106,000; C, x55,000.)

(diluted 1:50 with phosphate-buffered saline containing 10%fetal bovine serum) for 1 hr at room temperature. They werethen fixed with 2% glutaraldehyde/0.1 M phosphate buffer,pH 7.4, contrasted with 2% neutral uranyl acetate (20 min),and absorption-stained with 3% polyvinyl alcohol containing0.2% methylcellulose and 0.4% uranyl acetate (5 min).

RESULTSZO-1 Isoforms Are Detected by Immunoblotting in Rat

Kidney. When immunoblotting was carried out on extracts ofkidney cortex and medulla with antibody 7445 (which reactswith both ZO-1 isoforms), two closely spaced bands, 235 and225 kDa, were detected. The lower band, previously shownto represent ZO-1 lacking motif-a (7), was much moreprominent than the upper one. With antibody 8040, specificfor motif-a, only the upper band was detected (data notshown). When equal amounts of protein from the cortex,medulla, and glomeruli were loaded and probed with anti-body 7445, motif-a was seen in all three fractions but was

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FIG. 4. Immunofluorescence labeling of semithin cryosections ofdeveloping (1-2 days old) rat kidneys for ZO-1a+ (antibody 8040). (Aand B) The earliest stage in glomerular development is the renalvesicle (v), which consists of a cluster of epithelial cells. ZO-1a+ isnot expressed in the renal vesicle, but the tight junctions of theterminal ampulla (a), from which the distal tubules and collectingducts develop, are stained. (C and D) Staining for motif-a is first seenbetween presumptive proximal tubule epithelial cells (P) at theS-shaped-body stage. T, distal tubule. (E and F) Motif-a appears injunctions between epithelial cells (arrows) of Bowman's capsuleduring the capillary loop stage, when the primitive capillaries andpolarized glomerular epithelial cells are formed. Motif-a is notexpressed in glomerular (G) epithelial and endothelial cells at anystage during glomerular development. (x310.)

Cell Biology: Kurihara et al.

7078 Cell Biology: Kurihara et al.

most prominent in the medulla. The ratio of ZO-la+ toZO-la- varied among the three fractions. The highest signalwas obtained in the glomerular fractions, which appeared tobe highly enriched in ZO-1, primarily in ZO-la& (Fig. 1).ZO-la+ Is Not Detected in Junctions of Glomenerar Epithe-

fla Cells by Immunofluorescence. Results obtained with an-tibody 7445, which recognizes both ZO-1 isoforms, werecomparable to those previously reported (5): all renal junc-tions, including those of the glomeruli, the renal tubule,Bowman's capsule, and peritubular capillary endothelium,were reactive for ZO-1 (Fig. 2B). In glomeruli staining wasconcentrated in the foot-process layer due to its concentra-tion along the slit membranes between adjacent foot pro-cesses. When staining was done with antibody 8040, which isspecific for ZO-la+, thejunctions between the epithelial cellsofBowman's capsule and epithelial cell junctions of the renaltubule were prominently stained throughout the medulla andcortex (Fig. 2D). By contrast the glomeruli and the endothe-

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Proc. Natl. Acad. Sci. USA 89 (1992)

lial cell junctions of the peritubular capillaries did not reactwith the antibody, demonstrating that these two types ofjunctions express only the ZO-la- isoform.

ZO-1a4 Is Not Expressed in Glomeruli of Nephrotlc Rats. Inglomeruli from puromycin aminonucleoside- or protaminesulfate-treated nephrotic rats, the epithelial slits becomemodified and converted into typical tight junctions withextensive areas of contact between the membranes of adja-cent foot processes that have ZO-1 on their cytoplasmicsurfaces (8). It was therefore of interest to determine whetherboth isoforms of ZO-1 were expressed in these experimentalanimals. When renal cortical tissue from puromycin amino-nucleoside- or protamine sulfate-treated nephrotic rats wereincubated with the two ZO-1 antibodies, the results were thesame as in normal controls; i.e., ZO-la+ was not detected inglomeruli or in peritubular capillary endothelial cells (data notshown). Immunoblot analysis with antibody 7445 also sug-gested that the ratio between ZO-la+ and ZO-la- was

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FIG. 5. Expression of ZO-1a+ in the developing (1-2 days old) rat kidney stained with antibody 8040 (recognizes ZO-la+). Staining forZO-1a+ is observed in cell junctions of the proximal (P), distal (D), and collecting duct (C) epithelium and those of the Bowman's capsular (BC)epithelium (arrow). Staining for motif-a on the proximal tubule epithelial cells is more prominent than in the adult kidney. No staining ofpodocytes or the endothelium of the glomerular (G) or peritubular capillaries is seen. Ar, artery. (X480.)

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Proc. Natl. Acad. Sci. USA 89 (1992) 7079

unchanged in nephrotic rats. We conclude that ZO-la' is notexpressed in glomeruli from either normal or diseased (ne-phrotic) rats.ZO-la+ Is Detected Along Tight Junctions of Renal Tubular

Epithelial Cells by Immunogold Labeling. Previous studieshave established that ZO-1 is a peripheral membrane protein.By immunogold labeling ZO-1a+ (recognized by antibody8040) was detected along the cytoplasmic surfaces of alltubularjunctions ofthe renal cortex and medulla (Fig. 3). Fig.3C, showing a shallow junction between two proximal-tubuleepithelial cells, demonstrates that in grazing sections goldparticles for motif-a were concentrated along the areas offusion between the neighboring cell membranes. Some par-ticles were also distributed in the cytoplasm. The foot pro-cesses of glomerular epithelial cells and the glomerular andperitubular capillary endothelial cells in the same sectionswere not labeled for motif-a (Fig. 3D).

Expression ofZO-1 Isoforms in Developing Rat Kidney. Thekidneys of newborn rats (1-2 days old) are not fully devel-oped and contain immature glomeruli and tubular epithelialcells. Therefore, they are very convenient for checking theexpression of ZO-1 during renal development. By using anantibody that detects both isoforms, it was previously shownthat ZO-1 was first detected in developing kidney during theS-shaped-body stage, where it was associated with immatureepithelial cells of glomeruli, tubules, and Bowman's capsule(5). With antibody 8040, immunofluorescence staining formotif-a was seen in the epithelial cells of the presumptiveproximal tubule at the S-shaped-body stage and was detectedin those of Bowman's capsule during the capillary-loop stage(Fig. 4). Expression of motif-a was not observed in glomer-ular epithelial cells at any stage of development (Figs. 4 and5). The signal for motif-a on the proximal tubule epithelialcells was evident as soon as these cells could be recognizedand was more prominent than that on epithelial cells ofproximal tubules in the adult rat kidney (Fig. 5). It could beused as a marker to distinguish between cells destined todifferentiate into glomerular epithelial cells and those thatwill differentiate into proximal tubule epithelial cells.

DISCUSSIONWe have shown that renal tubule epithelia express bothisoforms of ZO-1 but only the ZO-la- isoform is expressedin the slit membranes of the glomerulus and in the junctionsbetween glomerular and peritubular capillary endothelia.This conclusion is based on (i) the lack of staining of thesejunctions by immunofluorescence and immunogold labelingwith antibodies that recognize motif-a and (ii) the results ofimmunoblotting on glomerular lysates, where the ZO-la-predominated. The presence ofa small amount ofthe ZO-1a+isoform in glomerular fractions is in all likelihood due topresence of Bowman's capsule epithelia and contaminationwith tubule fragments (estimated to be 5-10% of the total).

Willott et al. (7) demonstrated the existence of two ZO-1isoforms and presented evidence that they arise by alterna-tive splicing. They also examined the expression of the twoZO-1 isoforms and found that their relative expression dif-fered markedly in the cell lines and tissues studied. The ratiosfor ZO-lca to ZO-la- were 0.5:1, 3:1, and 20:1 in Hep G2,Caco-2, and T84 cells, respectively. Yet the location of theZO-1 protein isoforms within the cell appeared to be identi-cal. Thus no clues were obtained as to the functional signif-icance of the two isoforms.The question then arises as to what are the possible

functional implications of the absence of the motif-a expres-sion in the glomerular filtration slits and in endothelial

junctions. Tight junctions are known to function as a seal torestrict passage of proteins, ions, and water through theintercellular spaces, as fences to prevent mixing of apical andbasolateral plasmalemmal domains, and as channels to reg-ulate the passage of ions and water through the intercellularspaces (14, 15). These junctions are known to vary widely intheir relative leakiness, and this is reflected in the number oftight junction strands seen by freeze-fracture (1, 3). Onepossibility is that the two isoforms could reflect the relativeleakiness of the junctions, with ZO-1a+ being found only intighter junctions, and therefore they would be expected to bemissing from endothelial junctions and slit membranes,which are very leaky. However, this does not appear to be thecase, as by immunocytochemistry motif-a is found in junc-tions of all tubule epithelia, including both the very leakyproximal tubule junctions (containing few interrupted tightjunction strands) and the distal and collecting duct junctions,which are among the tightest of epithelia (containing multiplestrands) (1).What features do the slit diaphragms and the endothelial

cell junctions have in common that are not shared by thoseof tubule epithelia? One such feature is their lability-theyboth must be able to open rather than to remain firmlyattached. Endothelial cell junctions must open and close toallow migration of leukocytes from the blood to the intersti-tial space, and glomerular junctions are normally maintainedopen but close partially under pathologic conditions. Thusboth these junctions need to be able to dissociate andreassociate in response to physiologic or pathophysiologicstimuli, whereas the remaining renal tight junctions mustremain tightly attached to one another. If this is the case, ourfindings imply that the 80-amino acid, motif-a peptide may beinvolved in the binding of ZO-1 to other proteins associatedwith the tight junction in order to maintain their attachment.Conversely, the absence of this sequence permits dissocia-tion. Examination of ZO-1 isoforms in other tissues wherejunctions are known to be labile should provide furtherinformation on this point.

This research was supported by National Institutes of HealthResearch Grant DK17724 (to M.G.F.). J.M.A. is a Lucille MarkeyScholar.

1. Claude, P. & Goodenough, D. A. (1973) J. Cell Biol. 58,390-400.

2. Farquhar, M. G. & Palade, G. E. (1%3) J. Cell Biol. 17,375-412.

3. Friend, D. S. & Gilula, N. B. (1972) J. Cell Biol. 53, 758-776.4. Stevenson, B. R., Siliciano, J. D., Mooseker, M. S. & Good-

enough, D. A. (1986) J. Cell Biol. 103, 755-766.5. Schnabel, E., Anderson, J. M. & Farquhar, M. G. (1990) J.

Cell Biol. 111, 1255-1263.6. Schnabel, E., Dekan, G., Miettinen, A. & Farquhar, M. G.

(1989) Eur. J. Cell Biol. 48, 313-326.7. Willott, E., Balda, M. S., Heintzelman, M., Jameson, B. &

Anderson, J. M. (1992) Am. J. Physiol. 262, C1119-C1124.8. Kurihara, H., Anderson, J. M., Kerjaschki, D. & Farquhar,

M. G. (1992) Am. J. Pathol., in press.9. Anderson, J. M., Van Itallie, C. M., Peterson, M. D., Steven-

son, B. R., Carew, E. A. & Mooseker, M. S. (1989) J. CellBiol. 109, 1047-1056.

10. Keraschki, D., Sharkey, D. J. & Farquhar, M. G. (1984) J.Cell Biol. 98, 1591-15%.

11. McLean, W. & Nakane, P. F. (1974) J. Histochem. Cytochem.22, 1077-1083.

12. Tokuyasu, K. T. (1986) J. Microsc. (Oxford) 143, 139-149.13. Tokuyasu, K. T. (1989) Histochem. J. 21, 163-171.14. Stevenson, B. R., Anderson, J. M. & Bullivant, S. (1988) Mol.

Cell. Biochem. 83, 129-145.15. Gumbiner, B. (1987) Am. J. Physiol. 253, C749-C758.

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