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
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ABSTRACT |
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
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INTRODUCTION |
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.
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MATERIALS AND METHODS |
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).
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RESULTS |
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.
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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.
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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.
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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.
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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.
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DISCUSSION |
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.
 |
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