Slit diaphragm-reactive nephritogenic MAb 5-1-6 alters expression of ZO-1 in rat podocytes

Hiroshi Kawachi1,2, Hidetake Kurihara3, Peter S. Topham1, Dennis Brown4, Michael A. Shia1, Michiaki Orikasa2, Fujio Shimizu2, and David J. Salant1

1 Evans Memorial Department of Clinical Research and Department of Medicine, Boston University Medical Center, Boston, Massachusetts 02118; 2 Department of Cell Biology, Institute of Nephrology, Niigata University, School of Medicine, Niigata 951; 3 Division of Cell Biology, Shionogi Research Laboratories, Osaka 553, Japan; and 4 Renal Unit, Massachusetts General Hospital, Charlestown, Massachusetts 02129

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Monoclonal antibody (MAb) 5-1-6 identifies a 51-kDa protein (p51) on rat podocyte foot processes and causes severe complement- and leukocyte-independent proteinuria when injected into rats. In the studies reported here, we used various immunohistological techniques to define the precise location of p51 and its relationship to ZO-1, a known component of the podocyte slit diaphragm in adult rat glomeruli. Our results demonstrate that p51 and ZO-1 lie close to each other on opposite sides of the podocyte plasma membrane at the point of insertion of the slit diaphragm: ZO-1 on the cytoplasmic face and p51 on the slit diaphragm and adjoining outer leaflet of the plasma membrane bordering the filtration slits. In addition to their geographic proximity, there appears to be a relationship between p51 and ZO-1. After MAb 5-1-6 injection, there was a progressive decline in stainable ZO-1 in the podocytes of heavily proteinuric rats. In addition, Western blot analysis of glomerular lysates showed that the decline in staining was due to a loss of immunoreactive ZO-1 rather than redistribution or diffusion of the protein. Simultaneously, the distribution of glomerular-bound MAb 5-1-6 became more clumped, apparently because of partial endocytosis into a lysosomal compartment, while the slit diaphragms remained morphologically intact. These findings suggest that MAb 5-1-6 alters the molecular composition of the slit diaphragm and thereby affects the glomerular permeability barrier.

glomerular epithelial cell; foot process; tight junction; proteinuria; 51-kilodalton protein

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

MURINE MONOCLONAL ANTIBODY (MAb) 5-1-6 causes severe proteinuria when injected into rats (21). The target antigen is precipitated from solubilized glomeruli by MAb 5-1-6 and runs as a 51-kDa band (p51) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions (21). Proteinuria induced by MAb 5-1-6 is complement independent (18), and there is no evidence of inflammatory cell infiltration at any time after MAb 5-1-6 injection (21). Because severe proteinuria is induced immediately by MAb 5-1-6 binding to its antigen, p51, and because quantitative analysis with 125I-labeled MAb 5-1-6 indicated that the amount of p51 was decreased at the peak of proteinuria (8), p51 has been considered to be an essential component of the filtration barrier.

In studies reported elsewhere, we described the basolateral localization of p51 on developing and mature glomerular epithelial cells (GECs; podocytes) by immunoperoxidase electron microscopy and identified p51 as a likely component of the podocyte slit diaphragm (4, 7, 21). The slit diaphragm is a continuous bridgelike structure that spans the filtration slits between adjacent foot processes of mature GECs and forms the final barrier to glomerular ultrafiltration. Although its ultrastructural features have been described in detail (3, 22, 24), the molecular composition of the slit diaphragm is still unknown. ZO-1, a protein described by Stevenson et al. (30) on the cytoplasmic face of tight junctions, is also expressed on the cytoplasmic surface of podocyte foot processes at the point of insertion of the slit diaphragm (27). On the basis of this feature as well as the well-documented sequential dissolution of an apical embryonic GEC tight junction and subsequent appearance of the more basally situated slit diaphragm, it has been argued that the cytoplasmic leaflet of the slit diaphragm is derived from the primordial tight junction (28). The origin of the external domain of the slit diaphragm itself has yet to be established. Our developmental studies suggest that at least a component of the slit diaphragm arises from the basal surface (7). Several podocyte antigens have been isolated and characterized (6, 9, 14-16, 19, 23, 25, 31), and several other proteins have been identified as interacting cytoplasmic components of tight junctions (2, 5, 33). However, to our knowledge, of the tight junction-associated proteins, only ZO-1 has been found to be a component of the slit diaphragm (17).

On the premise that p51 is a component of the slit diaphragm, we hypothesized that MAb 5-1-6 binding to p51 causes a molecular rearrangement of the slit diaphragm and thereby alters glomerular permeability. To verify this hypothesis and to better understand the molecular structure of the slit diaphragm, we have studied the fate of two major slit diaphragm proteins, p51 and ZO-1, after the injection of MAb 5-1-6. Our results document the close apposition of ZO-1 and p51 on opposite sides of the podocyte plasma membrane at the point of insertion of the slit diaphragm and demonstrate their coordinate redistribution during the development of proteinuria induced by MAb 5-1-6.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Antibodies. Ascitic fluid containing MAb 5-1-6 was produced in mice primed with 2,6,10,14-tetramethylpentadecane (Sigma Chemical, St. Louis, MO) and injected intraperitoneally with a mouse immunoglobulin G1 (IgG1) hybridoma prepared as previously described (21). This fluid was subjected to 50% ammonium sulfate precipitation, and the immunoglobulin-rich fraction thus obtained was dialyzed against phosphate-buffered saline (PBS; 0.9% NaCl in 10 mM sodium phosphate buffer, pH 7.4) for 2 days and stored at -80°C. A stock solution at 40 mg/ml was diluted 1:50 or 1:100 for immunohistology. An irrelevant mouse monoclonal IgG1 antibody, RVG1, was used as a control at the same concentration. Rabbit antibody to ZO-1 was purchased from Zymed Laboratories (South San Francisco, CA) and used at a dilution of 1:100. This antiserum was raised against a 69-kDa fusion protein corresponding to amino acids 463-1109 of human ZO-1 (32). It recognizes both the motif-a+ and motif-a- isoforms of ZO-1, the latter being the one present in podocytes (10). Goat anti-mouse immunoglobulin G (IgG) coupled to 5 nm colloidal gold and goat anti-rabbit IgG coupled to 10 nm colloidal gold were from Amersham (Arlington Heights, IL). Rabbit polyclonal and murine monoclonal (IgG2b, PY20) antibodies to phosphotyrosine were purchased from UBI (Lake Placid, NY) and Chemicon International (Temecula, CA), respectively. For immunohistology, all antibodies were diluted in PBS containing 0.02% sodium azide (PBS-azide); 1% bovine serum albumin (BSA) or 10% fetal calf serum was added to the diluent for immunoelectron microscopy.

Animals. Normal adult Wistar rats weighing 150-175 g were purchased from Charles River Laboratories (Wilmington, MA) or from Charles River Japan (Atsugi, Japan). Sprague-Dawley rats were from Japan CLEA (Tokyo, Japan).

Experimental protocol. Wistar rats were injected intravenously with 2 mg of MAb 5-1-6 or RVG1. After 1, 2, 12, and 24 h and 3, 5, and 8 days, the urine was tested for albuminuria and the kidneys were removed under anesthesia. Albuminuria, as measured by qualitative dipstick analysis, was absent at 1 h but was present (>100 mg/dl) in specimens obtained from MAb 5-1-6-injected rats at 24 h and thereafter. In several experiments, quantitative proteinuria was measured on timed overnight urine collections as previously described (21).

Tissue preparation. The kidneys of normal adult rats and those of rats injected with MAb 5-1-6 or RVG1 were perfused via the heart with 1 mM phenylmethylsulfonyl fluoride (PMSF) in PBS under anesthesia. One kidney was removed for glomerular isolation, and the other was fixed in situ for immunohistology and electron microscopy. Glomeruli were promptly isolated from one kidney by differential sieving (26). Those from antibody-injected rats were fixed with periodate-lysine-paraformaldehyde (PLP) (13) or with 3% paraformaldehyde-0.05% glutaraldehyde for 1 h. The normal glomeruli were lightly digested with bacterial collagenase (Sigma Chemical; 1 mg/ml in PBS for 30 min at 37°C), blocked with 1% BSA, and then incubated with MAb 5-1-6 or RVG1, 500 µg/ml for 1.5 h at 4°C, before washing and fixation with PLP as above. After incubation and fixation, the glomeruli were washed thoroughly with PBS-azide at 4°C and then were pelleted in 0.5 ml of 6% gelatin in PBS-azide in a microcentrifuge tube. The gelled pellet was infiltrated with 2.3 M sucrose, embedded in Tissue-Tek OCT Compound (Miles, Elkhart, IN), snap frozen in liquid nitrogen, and sectioned in a Reichert FC4D ultracryomicrotome (Leica, Malvern, PA) at 70 nm for immunogold electron microscopy. The remaining kidney was perfusion fixed with PLP followed by overnight incubation of 2- to 3-mm sagittal slices in the same fixative. After being washed with PBS, the kidney slices were infiltrated with 30% sucrose and cryosectioned at 5 µm for conventional immunofluorescence microscopy or embedded in Epon, sectioned, and stained for transmission electron microscopy (1). For double-label, indirect immunogold electron microscopy, normal Sprague-Dawley rat kidneys were perfusion fixed with 4% paraformaldehyde in PBS, rinsed, and infiltrated with 40% polyvinyl alcohol-2.3 M sucrose in PBS before ultracryosectioning. In some studies, frozen sections were used for immunofluorescence.

Immunofluorescence microscopy. For indirect immunofluorescence, 5-µm cryostat sections of PLP-fixed normal rat kidney were blocked with 1% BSA in PBS; incubated with primary antibody, MAb 5-1-6, or rabbit anti-ZO-1 or antiphosphotyrosine (1:100; UBI); and then stained with CY3-conjugated donkey anti-mouse IgG (1:600; Jackson Immunoresearch Laboratories, West Grove, PA) or fluorescein isothiocyanate (FITC)-conjugated anti-rabbit IgG (1:60; Cappel-Organon Teknika, Durham, NC). For double-labeling immunofluorescence of MAb 5-1-6-injected and control kidneys, 5-µm cryosections of PLP-fixed kidney slices were blocked with 1% BSA in PBS and stained with rabbit anti-ZO-1 or antiphosphotyrosine antibody and FITC-goat anti-rabbit IgG, followed by CY3-conjugated donkey anti-mouse IgG. The donkey anti-mouse IgG-CY3 was absorbed with rabbit serum and was confirmed to have no cross-reactivity to rabbit IgG before use. In some reactions, the secondary antibodies were donkey anti-rabbit IgG-CY3 (1:600; Jackson Immunoresearch Laboratories) and goat anti-mouse IgG-FITC (1:60; Cappel-Organon Teknika). Specificity and lack of spectral overlap was confirmed by excluding the primary antibody (by injecting an irrelevant control MAb or by excluding anti-ZO-1 in vitro). Some sections of MAb 5-1-6-injected kidneys were also stained with PY20 antiphosphotyrosine MAb followed by FITC-conjugated anti-mouse IgG2b (1:20; Zymed laboratories). The sections were examined by epifluorescence microscopy (Optiphot; Nikon, Tokyo, Japan) and photographed with T-Max 400 film (Eastman Kodak, Rochester, NY) at ASA 1600. Double-stained images were also digitally captured, using an Optronix color charge-coupled device camera attached to a Macintosh 8500 Power PC running IP Lab Spectrum image analysis software. Separate images from the same field were captured, using FITC or CY3 filter combinations. Images were imported into Adobe Photoshop 3.04 and merged, using the "apply image" command. After construction of a composite plate, color output was generated, using a Kodak Phaser dye sublimation printer.

Immunoelectron microscopy. For double-label immunogold staining with MAb 5-1-6 and anti-ZO-1, ultracryosections of normal rat kidney were mounted on Formvar/carbon-coated nickel grids (150 mesh). Aldehyde groups were quenched with 0.01 M glycine in PBS and incubated overnight at 4°C with MAb 5-1-6 and anti-ZO-1 (both diluted 1:50 in PBS containing 10% fetal calf serum). They were then incubated with goat anti-mouse IgG coupled to 5 nm gold and goat anti-rabbit IgG coupled to 10 nm gold (both diluted 1:100 in PBS containing 10% fetal calf serum) for 2 h at room temperature. After immunostaining, they were washed with PBS, fixed with 2.5% glutaraldehyde, contrasted with 2% uranyl acetate for 20 min, absorption stained with 3% polyvinyl alcohol containing 0.2% uranyl acetate for 10 min, and viewed with a JEOL 100-CX electron microscope.

Immunogold staining of glomeruli from MAb 5-1-6-injected rats was performed as described (1). In brief, ultracryosections of isolated glomeruli were mounted on copper-coated grids and, after blocking with 1% BSA, were incubated sequentially for 1 h with rabbit anti-mouse IgG (Calbiochem, San Diego, CA) or rabbit anti-ZO-1 followed by protein A conjugated, as described, to 15-nm colloidal gold particles (29), each diluted 1:100 in 1% BSA-PBS. After incubation, the grids were washed once with high-salt PBS and twice with 0.1% BSA-PBS, fixed with 1% glutaraldehyde in PBS for 10 min, washed again, embedded in 2% methylcellulose-0.2% uranyl acetate, and examined and photographed with a Philips CM10 electron microscope (Philips Electronic Instruments, Mahwah, NJ).

Western blot analysis. Glomeruli were isolated from normal or MAb 5-1-6-injected rat kidneys by differential sieving and washed in PBS, and 100 µl of the glomerular pellet were solubilized by vortexing in 200 µl SDS-PAGE sample buffer [0.12 M tris(hydroxymethyl)aminomethane (Tris) hydrochloride, 2% SDS, and 26% glycerol, pH 6.8] without bromphenol blue. Protease inhibitors (0.2 mM PMSF and 1 mM each of antipain, pepstatin A, and diisopropyl fluorophosphate) and the tyrosine phosphatase inhibitor, sodium orthovanadate (1 mM), were included in all buffers. Insoluble material was removed by centrifugation at 15,000 g for 10 min. The protein concentration was measured by the bicinchoninic acid method (Pierce Chemical, Rockford, IL), and 25 µg were electrophoresed on 5% polyacrylamide gels and transferred to nitrocellulose membranes. The membranes were blocked with 5% skim milk in Tris-buffered saline and incubated with rabbit anti-ZO-1 (1:1,000) or antiphosphotyrosine (1:1,000), followed by incubation with alkaline phosphatase-conjugated anti-rabbit IgG (1:5,000; Bio Source International, Tago Immunologicals, Camarillo, CA), or with PY20 (1:1,000), followed by alkaline phosphatase-conjugated anti-mouse IgG2b (1:1,000; Zymed). The reaction was developed with an alkaline phosphatase chromogen kit (5-bromo-4-chloro-3-indolyl phosphate p-toluidine salt/nitro blue tetrazolium; Biomedica, Foster City, CA), and the density of the positive bands was quantified by Densitograph (ATTO, Tokyo, Japan).

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Localization of p51 and ZO-1. The patterns of glomerular staining of MAb 5-1-6 and anti-ZO-1 are similar on immunofluorescence of 5-µm cryosections of normal adult kidneys, exhibiting an interrupted peripheral capillary loop pattern (Fig. 1). The close proximity of the two antigens identified by these antibodies is further demonstrated by dual staining for mouse IgG and ZO-1 of 5-µm sections of glomeruli from MAb 5-1-6-injected rats 1 h after antibody injection (Fig. 2). Most of the staining for ZO-1 in these sections corresponds very closely to that of MAb 5-1-6 and occurs in an interrupted linear pattern along the capillary loops. In addition, anti-ZO-1 is known to stain the parietal epithelial cell membranes of Bowman's capsule and glomerular endothelial cell membranes (10, 27). At this time, there did not appear to be any change in the distribution of ZO-1, as demonstrated by an identical pattern of staining in glomeruli from rats injected with the control antibody, RVG1 (not shown); however, the intensity of staining was moderately reduced (see below). Furthermore, the distribution of mouse IgG in the glomeruli from MAb 5-1-6-injected rats after 1 h is the same as that seen in normal glomeruli incubated with MAb 5-1-6 in vitro after fixation with PLP (Fig. 1).


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Fig. 1.   Distribution of 51-kDa protein and ZO-1 in normal adult rat glomeruli. Indirect immunofluorescence of 5-µm cryosections of periodate-lysine-paraformaldehyde (PLP)-fixed normal rat kidney stained with monoclonal antibody (MAb) 5-1-6 (A) and rabbit anti-ZO-1 (B). Secondary antibodies were donkey anti-mouse-CY3 (A) and goat anti-rabbit-fluorescein isothiocyanate (B). Both antibodies decorate peripheral glomerular capillary wall in an interrupted linear pattern. Magnification, ×400.


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Fig. 2.   Localization of MAb 5-1-6 and ZO-1. Dual-label immunofluorescence for mouse IgG (A) and ZO-1 (B) in a 5-µm PLP-fixed section of a representative glomerulus from a rat 1 h after MAb 5-1-6 injection. Secondary antibodies were goat anti-mouse IgG-FITC (A and C) and donkey anti-rabbit IgG-CY3 (B and C). Close proximity of ZO-1 (B) and MAb 5-1-6 localization (A) is shown by interrupted linear yellow staining in merged figure (C). Arrows are to help align the 3 figures, which are best viewed under direct light. Magnification, ×600.

The precise localization of ZO-1 and MAb 5-1-6 was also examined by immunogold electron microscopy of normal glomeruli. As illustrated in Fig. 3, indirect immunogold electron microscopy shows that MAb 5-1-6 localizes exclusively on the external surface of the podocyte foot processes, predominantly on the slit diaphragms, whereas ZO-1 is seen to reside on the cytoplasmic face, close to the point of attachment of the slit diaphragm, as previously reported (27). The location of ZO-1 and its relationship to MAb 5-1-6 are further illustrated in Fig. 4. In these tangential sections of glomeruli isolated 1 h after injecting the monoclonal antibody, MAb 5-1-6 is seen within the filtration slits, on the outer surface of the foot processes, and on the slit diaphragms (Fig. 4A). In contrast, gold particles representing anti-ZO-1 are seen on the cytoplasmic face of the podocyte foot processes bordering the filtration slits (Fig. 4B). In addition, clusters of mouse IgG were frequently noted in podocyte lysosomes as early as 1 h after MAb 5-1-6 injection (Fig. 5) but not after RVG1 injection (not shown). From these results, it is apparent that p51, the antigen of MAb 5-1-6, and ZO-1 are closely juxtaposed but lie on opposite sides of the podocyte plasma membrane at the site of insertion of the slit diaphragm and that MAb 5-1-6 is rapidly and specifically endocytosed after binding the antigen in vivo.


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Fig. 3.   Localization of MAb 5-1-6 and ZO-1 in normal glomeruli. Indirect immunogold electron microscopy of an ultracryosection of a paraformaldehyde-fixed normal rat glomerulus. Small (5 nm) gold particles (arrowheads) represent MAb 5-1-6 and are distributed on the slit diaphragm bridging 2 adjacent foot processes. Large (10 nm) gold particles indicate location of ZO-1 (arrows) on cytoplasmic face of foot processes, close to insertion of slit diaphragm. GBM, glomerular basement membrane. Magnification, ×77,000.


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Fig. 4.   Immunogold localization of MAb 5-1-6 and ZO-1 after MAb 5-1-6 injection. Tangential ultracryosections through glomerular filtration slits (fs) of isolated glomeruli from a kidney obtained 1 h after MAb 5-1-6 injection and stained for mouse IgG (A) and ZO-1 (B). Note presence of MAb 5-1-6 in filtration slits, on adjacent plasma membrane, and on slit diaphragms themselves (arrows; A). ZO-1 is confined to cytoplasmic surface of podocyte plasma membranes bordering the filtration slits (arrowheads; B). fp, Foot process. Magnification, ×18,500.


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Fig. 5.   Endocytosis of MAb 5-1-6. Immunogold electron microscopy of a glomerulus isolated 1 h after injection of MAb 5-1-6. MAb 5-1-6 localizes exclusively on external surface of podocyte foot processes, predominantly in filtration slits (small arrows). This area of detail also shows gold labeling of mouse IgG in an intrapodocyte compartment, representing uptake of injected MAb 5-1-6 (large arrow). Magnification, ×13,500.

Concurrent alterations in p51 and ZO-1 distribution after MAb 5-1-6 injection. As previously shown (21), the distribution of staining for mouse IgG shifts from an interrupted linear pattern to a less intense coarsely granular pattern over several days after MAb 5-1-6 injection. In these studies, adult male Wistar rats injected with MAb 5-1-6 in doses from 2 to 5 mg developed proteinuria ranging from 27.5 to 189.3 mg/24 h (normal <5 mg/24 h). Over the course of 5 days, staining for MAb 5-1-6 shifted from a pseudolinear to a more granular pattern of less intensity. Simultaneously, there was a striking reduction in the staining for ZO-1, such that the only detectable ZO-1 in the glomerular tuft appeared to be that on the endothelial cell surface (Fig. 6). This coordinate change in distribution is most clearly seen on dual-labeling immunofluorescence (Fig. 7). The normal distribution of glomerular ZO-1 is seen in a section from a rat injected with the control antibody, RVG1 (Fig. 7, A and B). With the development of proteinuria after 24 h, staining with anti-ZO-1 was seen to diminish, and with the continuation of proteinuria at 5 days, MAb 5-1-6 partly redistributed into clumps and ZO-1 staining of podocytes was almost completely absent (Fig. 7, C-F). This reduction in ZO-1 staining was seen in all rats in which proteinuria exceeded 100 mg/24 h but was less evident in those with 30 mg/24 h or less. By day 10, when proteinuria has resolved and p51 staining has recovered (21), ZO-1 staining was again similar to normal controls (data not shown). The clumping of MAb 5-1-6 seen on immunofluorescence likely represents endocytosis and accumulation of the antibody in lysosomes as shown in Fig. 5.


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Fig. 6.   Immunofluorescence micrographs showing distribution of ZO-1 in glomeruli from normal (A) and MAb 5-1-6-injected rats 1 h (B), 1 day (C), and 5 days (D) after antibody injection. Signal for ZO-1 is discontinuously distributed along glomerular capillary wall in normal rat glomeruli. Staining intensity for ZO-1 is moderately decreased at 1 h or 1 day after MAb 5-1-6 injection. After 5 days, staining for ZO-1 is markedly decreased. Focal areas of residual staining are present in distribution of glomerular endothelial cells. Magnification, ×400.


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Fig. 7.   Effect of a nephritogenic dose of MAb 5-1-6 on ZO-1 distribution. Dual-labeling immunofluorescence for mouse IgG and ZO-1 was carried out as described in MATERIALS AND METHODS on control (A and B) and proteinuric rat kidneys 24 h (C and D) and 5 days (E and F) after MAb 5-1-6 injection. In absence of deposited mouse IgG (A), ZO-1 has a normal distribution on peripheral glomerular capillaries (B). Micrographs C and E show progressive clustering and decreasing intensity of MAb 5-1-6 24 h and 5 days after antibody injection, respectively. Simultaneously, there is a decline in intensity of podocyte staining for ZO-1 (D and F). Arrows (C-E) point to regions of residual MAb 5-1-6 staining of podocytes (C and E) with corresponding loss of staining for ZO-1 (D and F). Magnification, ×400.

Despite the development of proteinuria and these changes in MAb 5-1-6 and ZO-1 distribution, the slit diaphragms of MAb 5-1-6-injected rats remained morphologically normal at all times (Fig. 8, A and B) and indistinguishable from RVG1 controls (Fig. 8C). Rare foci of podocyte foot process effacement was the only abnormality noted (Fig. 8A).


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Fig. 8.   Transmission electron micrographs of representative glomeruli from rats examined 1 h (A) and 5 days (B) after MAb 5-1-6 injection or 5 days after control RVG1 injection (C). Slit diaphragms (arrows) appear morphologically intact at all times after MAb 5-1-6. Note also that podocyte (Podo) foot processes are well preserved after MAb 5-1-6, except for focal areas of effacement (arrowheads) seen at 1 h (A). US, urinary space; CL, capillary lumen. Magnification, ×14,000.

Western blot analysis of ZO-1 in MAb 5-1-6-injected rat glomeruli. To determine whether the reduced staining for ZO-1 in MAb 5-1-6-injected rat glomeruli represents a loss of immunoreactive protein or simply redistribution, Western blot analysis was performed on lysates of normal glomeruli and glomeruli isolated at various times after MAb 5-1-6 injection. As shown in Fig. 9, there was a substantial reduction in immunoreactive ZO-1 on day 1 and day 5 after MAb 5-1-6 injection. Glomeruli from two rats were used at each of the time points shown in Fig. 9. Corresponding levels of proteinuria in the MAb-injected rats were 63 and 110 mg/24 h on day 1 and 114 and 160 mg/24 h on day 5. Quantitative densitometry of the ZO-1 bands showed a significant reduction in band density in MAb 5-1-6-injected rat glomeruli compared with normal controls (1 day: 18.9 ± 6.1%; 5 days: 7.2 ± 2.9%; n = 4, P < 0.005). The ZO-1 band of these same membranes after stripping and of parallel blots of the same samples was consistently negative on Western blot analysis with polyclonal and monoclonal antiphosphotyrosine antibodies, despite inclusion of tyrosine phosphatase inhibitors.


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Fig. 9.   Representative Western blot analysis of ZO-1 in glomerular lysates from normal and MAb 5-1-6-injected rats. Isolated glomeruli from normal and MAb 5-1-6-injected rats were solubilized with SDS sample buffer, and 25 µg protein from each sample were loaded on a 5% polyacrylamide gel, electrophoresed, and transferred to nitrocellulose. Membrane was incubated with rabbit anti-ZO-1, followed by alkaline phosphatase-conjugated anti-rabbit IgG. ZO-1 was detected as a 225-kDa protein in glomerular lysate from normal rats (C). Fainter 225-kDa bands were detected 24 h (1d) and 5 days (5d) after MAb 5-1-6 injection.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Localization of p51 and ZO-1 on the podocyte slit diaphragm. Our present studies demonstrate that p51 and ZO-1 have a similar distribution in normal adult rat glomeruli. They lie in close proximity to each other on opposite sides of the podocyte plasma membrane at the point of insertion of the slit diaphragm: ZO-1 on the cytoplasmic face as previously reported (27) and p51 on the slit diaphragm and adjoining outer plasma membrane leaflet of the filtration slits. Whereas p51 is confined to the podocytes, ZO-1 is also present on glomerular endothelial and parietal epithelial cells.

The location of p51 and ZO-1 on the podocyte foot processes raises interesting questions about the origin and composition of the slit diaphragm. On the basis of a series of morphological changes and the eventual expression of ZO-1 on the cytoplasmic face of the mature slit diaphragm, Schnabel et al. (27) have proposed that the slit diaphragm arises by modification of an embryonic subapical tight junction in developing visceral glomerular epithelial cells. On the other hand, in a developmental study of p51, we showed that p51 is present along the basal and lateral surface below the occluding junctions of primitive podocytes (7). Once interdigitating foot processes are formed, we found that p51 becomes concentrated in the filtration slits (7). This implies that p51 and ZO-1 arrive at their final position on the podocyte slit diaphragm independently and from opposite directions. Moreover, using p51 as a marker, Fujugaki et al. (4) recently showed that the slit diaphragm is continuous with the outer leaflet of the podocyte plasma membrane. Together, these findings suggest that the slit diaphragm is a hybrid structure composed of elements from the basal plasma membrane as well as from the tight junction. Alternatively, it is possible that the slit diaphragm develops anew and independently of the embryonic tight junction and that the presence of ZO-1 on the cytoplasmic face of the slit diaphragm is coincidental.

Loss of immunoreactive ZO-1 during MAb 5-1-6-induced proteinuria. In addition to their geographic proximity, there does appear to be a relationship between p51 and ZO-1. Our immunofluorescence results showed a progressive decline in stainable ZO-1 in podocytes as early as 1 h after MAb 5-1-6 injection, such that ZO-1 was virtually undetectable after 5 days in heavily proteinuric rats. The only glomerular ZO-1 remaining at that time appeared to be in the distribution of endothelial cells. Interestingly, Western blot analysis showed that the decline in staining was evidently due to a loss of immunoreactive ZO-1 rather than redistribution or diffusion of the protein. Simultaneously, the distribution of glomerular-bound MAb 5-1-6 became more clumped, apparently because of partial endocytosis into a lysosomal compartment, and the amount of detectable p51 declined, as shown by quantitative 125I-MAb 5-1-6 binding (8), while the slit diaphragms remained morphologically intact. This differs from the findings of Kurihara et al. (11, 12) in rats injected with protamine sulfate or purine aminonucleoside. In those studies, the slit diaphragms dislocated and ZO-1 was tyrosine phosphorylated and partly redistributed to newly formed occluding type junctions; however, immunoreactive ZO-1 was still readily detectable (11, 12). Staining for p51 was also altered in the proteinuric phase of aminonucleoside nephrosis (20). These findings indicate that the loss of ZO-1 staining induced by MAb 5-1-6 is unique and is not simply a function of podocyte injury or proteinuria. Not surprisingly, given the prominent decay of immunoreactive ZO-1 in response to MAb 5-1-6, we were unable to detect tyrosine phosphorylation at any time, from 1 h up to 5 days after antibody injection. Nor did we see any glomerular staining for tyrosine phosphoproteins, despite the use of several polyclonal and monoclonal antisera. The fate of ZO-1 and p51 is presently uncertain. However, the rapid appearance of MAb 5-1-6 in podocyte lysosomes suggests that its antigen, p51, might have a similar fate. What about ZO-1? If p51 is somehow associated with ZO-1, and if their retention at the cell surface is dependent on this association, it is conceivable that the loss of p51 would cause ZO-1 to cycle from the plasma membrane into a degradative pathway. Unfortunately, the loss of ZO-1 immunoreactivity precluded following its fate by immunoelectron microscopy, and the lack of a suitable cell culture system does not permit further analysis of this phenomenon at this time.

p51 and ZO-1 are not essential for the maintenance of slit diaphragm structural integrity. It is remarkable that the morphology of the podocytes, including the structural integrity of the slit diaphragms, appeared largely unaffected by MAb 5-1-6, despite the loss of ZO-1 and the development of substantial proteinuria. This tends to suggest that a molecular, rather than structural, impairment is responsible for the altered permeability to serum albumin. Alterations in charge selectivity should be entertained as a possibility; however, the nature of the proteinuria induced by MAb 5-1-6 tends to suggest a size-selective defect (21). There are, of course, several other rational mechanisms whereby a monoclonal antibody directed at a cell membrane protein of the podocyte might cause proteinuria. For example, cell activation by antibody acting as a false ligand for a receptor-like protein might stimulate cytoskeleton-mediated changes in the slit pore that are not evident on electron microscopy. Alternatively, cell activation might induce the production of lipid or peptide mediators or the release of matrix-degrading proteases into the microenvironment between the basement membrane and basal surface of the foot processes. Whatever the mechanism of proteinuria in this model, it appears that p51 and ZO-1 are not essential for maintaining the shape of the podocyte foot processes or the location and structure of the slit diaphragms.

In conclusion, we have shown that p51 and ZO-1 are colocalized on opposite surfaces of the podocyte plasma membrane at the point of insertion of the slit diaphragm and that there is a loss of immunoreactive ZO-1 and redistribution of p51 coincident with the development of proteinuria in rats injected with MAb 5-1-6, despite preservation of slit diaphragm morphology. These findings suggest that a molecular interaction between p51 and ZO-1 may contribute to the normal glomerular permeability barrier.

    ACKNOWLEDGEMENTS

We thank Chihiro Tomita, Robert Tyszkowski, and Peg McLaughlin for technical assistance.

    FOOTNOTES

This study was supported by research Grants DK-48236 (to D. J. Salant) and DK-42596 (to D. Brown) from the National Institute of Diabetes and Digestive and Kidney Diseases and research Grants 08770878 (to H. Kawachi) and 08457286 (to F. Shimizu) from the Ministry of Education, Science, and Culture, Japan (1996).

Address for reprint requests: D. J. Salant, Renal Section, Boston Medical Center, 88 E. Newton St., Boston, MA 02118.

Received 12 November 1996; accepted in final form 6 August 1997.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

1.   Brown, D. Analysis of protein expression by immunocytochemistry. In: Gene Probes 2: A Practical Approach, edited by B. D. Hames, and S. J. Higgins. Oxford, UK: Oxford Univ. Press, 1995, p. 267-311.

2.   Citi, S., H. Sabanay, R. Jakes, B. Geiger, and J. Kendrick-Jones. Cingilin, a new peripheral component of tight junctions. Nature 333: 272-276, 1988[Medline].

3.   Farquhar, M. G., S. L. Wissig, and G. E. Palade. Glomerular permeability. I. Ferritin transfer across the normal glomerular capillary wall. J. Exp. Med. 113: 47-66, 1961.

4.   Fujugaki, Y., T. Morioka, K. Matsui, H. Kawachi, M. Orikasa, T. Oite, F. Shimizu, S. R. Batsford, and A. Vogt. Structual continuity of filtration slit (slit diaphragm) to plasma membrane of podocyte. Kidney Int. 50: 54-62, 1996[Medline].

5.   Gumbiner, B., T. Lowenkopf, and D. Apatira. Identification of a 160 kDa polypeptide that binds to the tight junction protein ZO-1. Proc. Natl. Acad. Sci. USA 88: 3460-3464, 1991[Abstract].

6.   Huang, T. W., and J. C. Langlois. Podoendin: a new cell surface protein of the podocyte and endothelium. J. Exp. Med 162: 245-267, 1985[Abstract].

7.   Kawachi, H., D. R. Abrahamson, P. L. St. John, D. J. Goldstein, M. A. Shia, K. Matsui, F. Shimizu, and D. J. Salant. Developmental expression of the nephritogenic antigen of monoclonal antibody 5-1-6. Am. J. Pathol. 147: 823-833, 1995[Abstract].

8.   Kawachi, H., K. Matsui, M. Orikasa, T. Morioka, T. Oite, and F. Shimizu. Quantitative studies of monoclonal antibody 5-1-6-induced proteinuric state in rats. Clin. Exp. Immunol. 87: 215-219, 1992[Medline].

9.   Kerjaschki, D., and M. G. Farquhar. Immunocytochemical localization of the Heymann nephritis antigen (gp330) in glomerular epithelial cells of normal Lewis rats. J. Exp. Med. 157: 667-686, 1983[Abstract/Free Full Text].

10.   Kurihara, H., J. M. Anderson, and M. G. Farquhar. Diversity among tight junctions in rat kidneys: glomerular slit diaphragms and endothelial junctions express only one isoform of the tight junction protein ZO-1. Proc. Natl. Acad. Sci. USA 89: 7075-7079, 1992[Abstract].

11.   Kurihara, H., J. M. Anderson, and M. G. Farquhar. Increased Tyr phosphorylation of ZO-1 during modification of tight junctions between glomerular foot processes. Am. J. Physiol. 268 (Renal Fluid Electrolyte Physiol. 37): F514-F524, 1995[Abstract/Free Full Text].

12.   Kurihara, H., J. M. Anderson, D. Kerjaschki, and M. G. Farquhar. The altered glomerular filtration slits seen in puromycin aminonucleoside nephrosis and protamine sulfate-treated rats contain the tight junction protein ZO-1. Am. J. Pathol. 141: 805-816, 1992[Abstract].

13.   McLean, I. W., and P. F. Nakane. Periodate-lysine-paraformaldehyde fixative. A new fixation for immunoelectron microscopy. J. Histochem. Cytochem. 22: 1077-1083, 1974[Medline].

14.   Mendrick, D. L., and H. G. Rennke. I. Induction of proteinuria in the rat by a monoclonal antibody against SGP-115/107. Kidney Int. 33: 818-830, 1988[Medline].

15.   Miettinen, A., G. Dekan, and M. G. Farquhar. Monoclonal antibodies against membrane proteins of the rat glomerulus: immunochemical specificity and immunofluorescence distribution of the antigens. Am. J. Pathol. 137: 929-944, 1990[Abstract].

16.   Mundel, P., P. Gilbert, and W. Kriz. Podocytes in glomerulus of rat kidney express a characteristic 44 kd protein. J. Histochem. Cytochem. 39: 1047-1056, 1991[Abstract].

17.   Mundel, P., and W. Kriz. Structure and function of podocytes: an update. Anat. Embryol. (Berl.) 192: 385-397, 1995[Medline].

18.   Narisawa, M., H. Kawachi, T. Oite, and F. Shimizu. Divalency of the monoclonal antibody 5-1-6 is required for induction of proteinuria in rats. Clin. Exp. Immunol. 92: 522-526, 1993[Medline].

19.   Natori, Y., I. Hayakawa, and S. Shibata. Identification of gp108, a pathogenic antigen of passive Heymann nephritis, as dipeptidyl peptidase IV. Clin. Exp. Immunol. 70: 434-439, 1987[Medline].

20.   Okasora, T., M. Nagase, H. Kawachi, K. Matsui, M. Orikasa, T. Morioka, I. Yamazaki, T. Oite, and F. Shimizu. Altered localization of antigen recognized by proteinuria-inducing monoclonal antibody in experimental nephrosis. Virchows Arch. 60: 41-46, 1991.

21.   Orikasa, M., M. Matsui, T. Oite, and F. Shimizu. Massive proteinuria induced in rats by a single intravenous injection of a monoclonal antibody. J. Immunol. 141: 807-814, 1988[Abstract/Free Full Text].

22.   Reeves, W., J. P. Caulfield, and M. G. Farquhar. Differentiation of epithelial foot processes and filtration slits: sequential appearance of occluding junctions, epithelial polyanion, and slit membranes in developing glomeruli. Lab. Invest. 39: 90-100, 1978[Medline].

23.   Reivinen, J., H. Holthofer, and A. Miettinen. A cell-type specific ganglioside of glomerular podocytes in rat kidney: an O-acetylated GD3. Kidney Int. 42: 624-631, 1992[Medline].

24.   Rodewald, R., and M. J. Karnovsky. Porous substructure of the glomerular slit diaphragm in the rat and mouse. J. Cell Biol. 60: 423-433, 1974[Abstract/Free Full Text].

25.   Saito, A., S. Pietromonaco, A. K. C. Loo, and M. G. Farquhar. Complete cloning and sequencing of rat gp330/"megalin," a distinctive member of the low density lipoprotein receptor gene family. Proc. Natl. Acad. Sci. USA 91: 9725-9729, 1994[Abstract/Free Full Text].

26.   Salant, D. J., and A. V. Cybulsky. Experimental glomerulonephritis. Methods Enzymol. 162: 421-461, 1988[Medline].

27.   Schnabel, E., J. M. Anderson, and M. G. Farquhar. The tight junction protein ZO-1 is concentrated along slit diaphragms of the glomerular epithelium. J. Cell Biol. 111: 255-1263, 1990.

28.   Schnabel, E., G. Dekan, A. Miettinen, and M. G. Farquhar. Biogenesis of podocalyxin: the major glomerular sialoglycoprotein in the newborn rat kidney. Eur. J. Cell Biol. 48: 313-326, 1989[Medline].

29.   Slot, J. W., and H. J. Geuze. A new method for preparing gold probes for multiple labeling cytochemistry. Eur. J. Cell Biol. 38: 87-93, 1985[Medline].

30.   Stevenson, B. R., J. D. Siliciano, M. S. Mooseker, and D. A. Goodenough. Identification of ZO-1: a high molecular weight polypeptide associated with the tight junction (zonula occludens) in a variety of epithelia. J. Cell Biol. 103: 755-765, 1986[Abstract].

31.   Tissari, J., H. Holthofer, and A. Miettinen. Novel 13 A antigen is an integral protein of the basolateral membrane of rat glomerular podocytes. Lab. Invest. 71: 519-527, 1994[Medline].

32.   Willott, E., M. S. Balda, M. Heintzelman, B. Jameson, and J. M. Anderson. Localization and differential expression of two isoforms of the tight junction protein ZO-1. Am. J. Physiol. 262 (Cell Physiol. 31): C1119-C1124, 1992[Abstract/Free Full Text].

33.   Zong, Y., T. Saito, T. Minasa, N. Sawada, K. Enomoto, and M. Mori. Monoclonal antibody 7H6 reacts with a novel tight junction-associated protein distinct from ZO-1, cingulin and ZO-2. J. Cell Biol. 120: 477-483, 1993[Abstract].


AJP Renal Physiol 273(6):F984-F993
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