Clin. exp. Immunol. (1991) 83, 143-148
ADOMS
000991049100027T
Formation of subepithelial dense deposits in rats induced by a monoclonal antibody against the glomerular cell surface antigen K. NISHIKAWA, A. FUKATSU, H. TAMAI, N. SUZUKI, Y. ITO, N. SAKAMOTO & S. MATSUO Third Department of Internal Medicine, Nagoya University, School of Medicine, Nagoya, Japan
(Accepted for publication 15 August 1990)
SUMMARY We developed a monoclonal antibody, H5H3, of IgGl subclass by hybridization technique using spleen cells of mice immunized with plasma membrane fraction of isolated rat glomeruli. H5H3 recognized main bands at about 220 kD by immuno-overlay technique and bound to the glomerulus as well as brush border of proximal tubules by indirect immunofluorescence (IF) microscopy on normal rat kidney frozen sections. By immunoelectron microscopy (IEM) it bound to the surface of mainly glomerular epithelial cell and weakly to the endothelial cell. After injection to Wistar rats it remained granularly in the glomerulus for more than 2 weeks seen by IF. When rats were preimmunized with murine IgG 4 days before the injection of H5H3, mouse IgG, rat IgG and C3 were strongly visible granularly in the glomerulus in 14 days by IF. Numerous dense deposits were formed at subepithelial area seen by transmission electron microscopy. Perfusion experiment of H5H3 into rat left kidney showed granular distribution of mouse IgG in 48 h, indicating that the reaction occurred in situ. H5H3 bound diffusely in fine granular pattern on the surface of cultured glomerular epithelial cells (GEC) studied by IF and IEM. Antigenic redistribution occurred on GEC after incubation of H5H3 at 37 C. These results suggested the required conditions to form subepithelial immune dense deposits, namely that H5H3 after reaction with antigen could stay for long time in the glomerulus; that H5H3 became an antigen in autologous phase to induce large immune complexes; and H5H3 could induce antigenic modulation. Keywords dense deposits monoclonal antibodies Heymann's nephritis INTRODUCTION Heymann's nephritis is a rat model of human membranous glomerulonephritis because it induces proteinuria and subepithelial immune deposits (Heymann et al., 1959). Antigens responsible for the nephritis have been investigated and characterized. Several antigens, including the most relevant GP330 (Kerjaschki & Farquhar, 1983), were reported to be localized on glomerular epithelial cells and on brush borders of the proximal tubules (Kamata et al., 1985; Chatelet et al., 1988a; Natori, Hayakawa & Shibata, 1987). Recent studies strongly suggested that immune deposits at subepithelial area of the glomerular basement membrane were formed in situ (Kerjaschki & Farquhar, 1983; Bhan et al., 1985; Kerjaschki, Miettinen & Farquhar, 1987). Recently, monoclonal antibodies were raised against these antigens and were used to study the pathogenesis of Heymann's nephritis (Ronco et al., 1984; Bhan et al., 1985; Allegri et al., 1986). Because of their monospecificity, mono-
clonal antibodies are useful to investigate the precise localization of the antigen at ultrastructural level or to study the mechanism to form immune deposits; however it is difficult to produce proteinuria or to form immune deposits visible by transmission electron microscopy (TEM) when monoclonal antibodies are injected into rats. In the present study we developed and characterized a monoclonal antibody that reacted with glomerular epithelial cells and produced electron dense immune deposits at a subepithelial area visible by TEM during the autologous phase. The mechanisms of the formation of subepithelial dense deposits in the model and the relevance to those in Heymann's nephritis are discussed.
MATERIALS AND METHODS Animals Female Wistar rats, weighing 150-200 g, and female BALB/c mice weighing about 20 g were purchased from Chubu Kagaku Shizai (Nagoya, Japan) and allowed free access to food and water.
Correspondence: Atsushi Fukatsu, MD, The Third Department of Internal Medicine, Nagoya University, School of Medicine, 65 Tsuruma-cho, Showa-ku, Nagoya, 466, Japan.
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Monoclonal antibody Antigen preparation. Kidneys were perfused through abdominal aorta with 10 mm Tris-buffered saline, pH 7 4, containing 0-2 mm benzylsulphonyl fluoride and 1 mm Nethylmaleimide (Sigma Chemical Co., St Louis, MO). Glomeruli were separated from minced renal cortices by sieving (Krakower & Greenspon, 1954). Purity of separated glomeruli was about 90% with the contamination of 10% tubules. Plasma membrane was separated from glomeruli by sonication using Sonifier Disruptor (Branson, Shelton, CT) and centrifuged at 1200 g for 15 min. The supernatant was then ultracentrifuged at 30 000g for 45 min and the resulting pellet was treated with 0-1 % Triton-X to solubilize the plasma membrane proteins. After centrifugation at 30000 g for 45 min the supernatant was dialysed against 0 01 M phosphate-buffered saline (PBS), pH 7 4, concentrated using Minicon (Amicon, Denver, CO) and used as the antigen (glomerular protein). Immunization. BALB/c mice were immunized three times with 2 mg of the antigen mixed with Freund's complete adjuvant (FCA) every 2 weeks. Hybridization. Spleen cells were separated 3 days after the last immunization and fused with murine myeloma cells NS- 1 using PEG (mol. wt 4000; Merck, Rahway, NJ). Positive hybridoma clones were screened by indirect immunofluorescence (IIF) microscopy on frozen normal kidney sections. One of the positive clones, H5H3, was harvested and implanted into the abdominal cavity of a BALB/c mouse to obtain large amount of monoclonal antibodies. Immunoglobulin fraction was purified by 50% ammonium sulphate precipitation. Subclass of H5H3 was determined by ELISA. A monoclonal antibody (9C2) of the same subclass as H5H3, which did not react with renal structure studied by IIF was used for control. Western blotting Glomerular protein was mixed in sample buffer (4 6% SDS, 20% glycerol, 0 1 % bromophenol blue) with or without 10% 2mercaptoethanol and heated in boiled water for a few minutes and applied for SDS-PAGE on 8-25% gradient gel using PhastSystem (Pharmacia Fine Chemicals, Uppsala, Sweden). Electroblotting was also done using PhastSystem at 60 V for 30 min on PVDF papers. After blocking with 0 5% bovine serum albumin, the paper was reacted with H5H3 or 9C2 (diluted 10 pg/ml) and then with horseradish-peroxidase (HRP) labelled goat anti-mouse IgG antibody (1:1000). Positive reaction was visualized by 0 05% DAB with 0 005% H202.
Immunohistochemistry Frozen sections (4 Mm) of kidney, intestine, lung, heart, liver and spleen from normal rats were cut by a cryostat and incubated first with properly diluted H5H3 or with control monoclonal antibodies of the same subclass for 30 min at room temperature and then with FITC-labelled rabbit anti-mouse IgG antibody (Cappel) for 30 min. Slides were covered with medium containing p-phenylenediamine (Platt & Michael, 1983) and observed with an Olympus BH-2 epifluorescence microscope. Immunoelectron microscopy (IEM) was performed as reported (Matsuo et al., 1987). Briefly, 7-pm frozen sections were cut from a normal kidney perfused with periodate-lysine paraformaldehyde fixative (PLP) (McLean & Nakane, 1974) and immersed further for 4 h in PLP and incubated with 0-05% sodium borohydride solution, diluted H5H3 or 9C2 in PBS, diluted
HPR-labelled goat anti-mouse IgG antibody, 2% glutaraldehyde serially. Sections were reacted with 0-02% DAB solution and then with the same solution containing 0 005% H202 to visualize the positive products and fixed with 2% OS04. Sections were dehydrated, embedded in Epon 812 and processed for electron microscopy. Intravenous injection of H5H3 Sixteen rats (group 1) were pre-immunized with 0 5 mg of mouse IgG purified by protein A column mixed with CFA (day -4). Four days later (day 0) rats were injected with 5 mg of H5H3 intravenously. Kidney tissues were obtained by biopsy or killing on days 1, 3, 7, 14, 21 and 28. A fragment of kidney tissue was fixed in 10% buffered formalin and processed for light microscopy. Ultra-thin sections were cut from another fragment of tissue fixed in 2% glutaraldehyde and were stained with lead and uranium for electron microscopy. They were observed with a JOEL 100CX electron microscope. Part of kidney tissue was snap-frozen in liquid nitrogen and used for IF microscopy. Ten rats (group 2) were treated as above with the injection of the same amount of 9C2 instead of H5H3. Eight rats (group 3) were treated as group 1, and four rats (group 4) as group 2 without pre-immunization. Light microscopy, direct IF for mouse IgG, rat IgG, and rat C3 using FITC-labelled reagents (Cappel) were performed as described.
Perfusion of H5H3 Perfusion of left kidney with H5H3 was done, as reported previously (Matsuo et al., 1989), in rats pre-immunized twice with normal mouse IgG 1 and 3 weeks previously. Briefly left renal artery and vein were exposed and were cannulated with polyethylene tubes. Then 10 mg of H5H3 in tyrode buffer was perfused using peristaltic pump (Pharmacia) for 15 min at room temperature while all venous flow was recovered. Artery and vein were sutured to reconstruct the circulation. Kidney tissue was obtained at 3 and 48 h after the perfusion and studied histologically and immunohistologically as described above. Protein excretion in urine Urinary protein was measured by quantitative sulphosalicylic
acid method. Detection of mouse IgG and rat antibodies to mouse IgG in sera Sera taken on days 0, 1, 3, 7, 14, 21, and 28 were checked for circulating mouse IgG and for autologous antibodies to mouse IgG by ELISA. Microtitre plates (Immulon, Dynatech, Chantilly, VA) were coated with goat anti-mouse IgG antibody (Cappel) or mouse IgG, 500 ng/well, and reacted with serially diluted sera followed by the incubation with HRP-labelled goat anti-mouse IgG antibody or HRP-labelled goat anti-rat IgG antibody (Cappel). ELISA was done as described previously (Fukatsu et al., 1987).
Glomerular cell culture Primary glomerular epithelial cells (GEC) were cultured from separated glomeruli of Wistar rat kidneys in 8-well slides (Miles Scientific, Naperville, IL) or in 6-well plates (Costar) as described previously (Fukatsu et al., 1987). Characterization of GEC was based on the following criteria: (i) morphologic characteristics; (ii) positive cytokeratin (Miles); (iii) short
Subepithelial dense deposits in rats
145
Table 1. Comparison of the tissue localization of H5H3 recognizing antigen (H5H3 Ag) and Heymann-related antigens
A B C D E F 200 .
Tissue
97 . 68* 43 . 25 m 18
Kidney Brush border of proximal tubules Glomerular epithelium Glomerular endothelium Liver Endothelium Hepatocyte Gut
a
Brush boder
H5H3 Ag
GP330
GP90
GPl08
+
+
+
+
+
+
+
+
+*
-
+
+
-
+
?
+
+t
+
+
+ ?
Spleen 41.
...4.I
-
'..F'.4,1
A
Endothelium
-
-
+
Heart Endothelium
+
-
+
+
-
+
+
Chatelet et al. (1988a, 1988b)
Chatelet et al. (1988a,) 1988b)
Natori et al. (1987)
., 4
4.
4
4,
C. Fig. 1. (a) Results of Western blots. Bands at 220 kD are reacted with H5H3 (lanes B, C); lanes A, B, C, reacted with H5H3; lanes D, E, F, reacted with 9C2; lanes A, D, SDS-solubilized spleen homogenate (reduced condition) was run as control; lanes B, C, E, F, glomerular protein was run (B, E, reduced; C, F, non-reduced condition). (b) H5H3 binds to glomerular capillary wall as well as brush border of proximal tubules in the normal kidney. Indirect immunofluorescence; magnification x 190. (c) By indirect immunoelectron microscopy H5H3 reacts mainly with the cell surface of glomerular epithelial cells and weakly with that of endothelial cells. Magnification x 4700.
peripheral localization of actin filament; and (iv) positive Heymann's antigen. Immunohistochemical study of GEC GEC in 8-well slides were fixed with cold ethanol for 5 min and incubated with H5H3 for 15 min at room temperature; then with FITC-labelled rabbit anti-mouse antibody for 15 min. GEC in other slides were first incubated with H5H3 at 37°C for 5 to 15 min and fixed with cold ethanol then treated with FITC-labelled antibody as above. For IEM, GEC in 6-well plates were first fixed with PLP and incubated with H5H3 for 30 min at room temperature and then with HRP-labelled goat anti-mouse IgG antibody for 15 min. Peroxidase reaction and processing for electron microscopy were done as previously described (Fukatsu et al., 1987). RESULTS Western blotting H5H3 recognized two bands at about 220 kD in reduced or nonreduced condition (Fig. la). 9C2 did not show positive results.
Immunohistochemistry on normal tissue On normal frozen kidney sections, H5H3 was localized in glomerular capillary wall (GCW) and in brush border of proximal tubules (Fig. lb). By IEM positive reaction products
Lung Endothelium Pneumocyte References
?-
*Weakly stained by immunoelectron microscopy; t restricted to biliary pole; t hard to recognize by indirect immunofluorescence. were found mainly on the surface of GEC and more weakly on endothelial cells (Fig. lc). Extra renal distribution seen by IIF was listed in Table 1. Immunohistochemistry using 9C2 was negative.
Histology and immunohistology of rat kidneys injected with HSH3 In rats of group 1, mouse IgG was seen granularly along GCW on day 1, increased its intensity and granularity on day 7, peaked on day 14 (Fig. 2a), and slightly weakened on day 28 by direct IF. Rat IgG was positive on day 7, increased on day 14 (Fig. 2b) and remained so until killing (on day 28). Rat C3 was found with rat IgG but more weakly and coarsely (Fig. 2c). Light microscopy showed normal renal histology. By TEM, discrete dense deposits were visible mainly in slit membranes on day 14 (Fig. 2d) and later. In rats of group 3, mouse IgG was visible in the same pattern as seen in rats of group 1, but decreased on day 21, and disappeared on day 28. Rat IgG and C3 was not detected at any time. Light microscopy was normal and electron dense deposit was not found by TEM. In rats of groups 2 and 4, mouse IgG, rat IgG or C3 was not seen throughout the experiments. LM and TEM were normal. No proteinuria was detected throughout the experiment in any group of rats. Rat anti-mouse IgG antibody was not detected on day 0, weakly on day 7 and steadily on day 14 and after in rats of
K. Nishikawa et al.
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Fig. 3. When H5H3 is perfused into left kidney of rats pre-immunized with mouse IgG, mouse IgG was found granularly along glomerular capillary wall in 48 h. Direct immunofluorescence; magnification x 400.
cytokeratin was found positive; (iii) actin filament was stained peripherally; and (iv) Heymann's antigen was stained positive using sheep anti-rat brush border vesicle antibody (Camussi et al., 1985). When GEC were reacted with H5H3, H5H3 bound to the cell surface and to intracellular organellae (Fig. 4a). When reacted at 370C for 15 min, H5H3 was found on the cell in
To
Ss,o ,W f At) tow v, ,. ~ ~ ~ t-XHa
N
_
e.,* Fig. 2. When H5H3 is injected into rats of group f, mouse IgG is localized granularly along glomerular capillary wall (a) with rat IgG (b) and C3 (c) on day 14. Direct immunofluorescence; magnification (a, b, c) x 400. (d) Electron dense deposits are visible at subepithelial area; magnification x 6000.
_.A.'
.4.
groups 1 and 2. In rats of groups 3 and 4 it was detected on day 7 and onward but titre was low at any time. Mouse IgG was found in serum up to day 7.
Histology and immunohistochemistry of rat kidneys perfused with H5H3 Mouse IgG was found in glomeruli diffusely at 3 h and granularly along GCW at 48 h (Fig. 3) after the perfusion of H5H3 with deposition of rat IgG and C3. Light microscopy showed normal histology. Electron dense deposits were not TEM. byby TEM. found found
Immunocytochemistry of GEC Thecells grownoutof glomerulihadcthenaturerofepithelialcells because: (i) they had cobble stone appearance in monolayer; (ii)
,:
,, Fig. 4. H5H3 binds to the surface and to cytosomal organellae off t indirect glomerular epithelial cells (GEC) fixed with ethanol are immunofluorescence; magnification x 200. When GEC (a);incubated with H5H3 at 370C for 15 min H5H3 is localized in patchy pattern (b); some cells show capping; indirect immunofluorescence; magnificationI x 200. By immunoelectron microscopy H5H3 binds to the surface off GEC diffusely in fine granular pattern (c); magnification x 5000.
147
Subepithelial dense deposits in rats patchy pattern. Some cells showed capping (Fig. 4b). By TEM H5H3 bound to the cell surface diffusely in fine granular pattern. Apparent staining of coated pits was not seen. 9C2 did not bind to GEC. DISCUSSION
H5H3 is a monoclonal antibody of IgGl subclass which recognizes the antigen of molecular weight of about 220 kD localized mainly on GEC surface. The antigen recognized by H5H3 is most likely different from well-characterized Heymann's antigen like GP330 because of the difference of molecular weight and of renal or extrarenal distribution, as listed in Table 1. Although molecular weight was different, renal and extrarenal distribution of H5H3 was similar to that of GP90 (Chatelet et al., 1986a, 1986b) Allegri et al. (1986) injected five monoclonal antibodies to GP330 into rats but could not find subepithelial dense deposits and explained that polyvalent antigen-antibody interaction on the cell surface was necessary to form dense immune deposits. Kerjaschki et al. (1987) described that in initial phase of passive Heymann's nephritis, immune complexes were formed on the surface of epithelial cells, mainly at coated pits and resulting immune complexes attached to the basement membrane firmly. Mendrick & Rennke (1986) reported that a monoclonal antibody could bind to the mesangial matrix antigen which is fixed in the structure and formed electron dense immune deposits. From these studies it is conceivable that the prerequisites for dense deposit formation are (i) fixation of antigen or immune complex to the extracellular structure; (ii) sufficient supply of antigens and antibodies; and (iii) antigen redistribution in the case of cell surface antigen (Camussi et al., 1985). Previously described monoclonal antibodies were, when injected, fixed in glomeruli and disappeared within a week (Ronco et al., 1984; Bhan et al., 1985; Allegri et al., 1986), while H5H3 stayed for more than 2 weeks in glomeruli even without pre-immunization. This fact suggests that the antigens or immune complexes attach to extracellular sites rather firmly. When autologous antibody appeared in the rats preimmunized with mouse IgG, H5H3 itself became the antigen and complexes, consisting of H5H3, presumably antigens, rat immunoglobulins, and complements might become large enough to be visible as dense deposits. We cannot exclude the possibility that circulating immune complexes of rat antibodies and remaining mouse IgG were deposited on day 7 and later, although autologous antibody was not detected when H5H3 was injected. Our perfusion experiment showed that granular deposits were formed in situ. H5H3 can induce antigenic redistribution on GEC like Heymann's antigen (Camussi et al., 1985). These characteristics may be the reason why the interaction of H5H3 with its antigen on the surface of GEC results in the formation of immune dense deposits. H5H3 did not induce proteinuria even when subepithelial dense deposits were formed. The precise cause of proteinuria is not fully understood. Orikasa et al. (1988) described a monoclonal antibody which produced proteinuria in rats when bound to epithelial cells without forming dense deposits. Mendrick & Rennke (1988a) reported that a monoclonal antibody which recognized the sialoglycoprotein of glomerular cells induced proteinuria without dense deposits. They also described (Mendrick & Rennke, 1988b) the epitope specificity to the same
molecule for proteinuria. In Heymann's nephritis, Salant et al. (1980) showed that complement was necessary for the induction of proteinuria which presumably acted on the surface of epithelial cells. H5H3 lacked the capability of complement binding when bound to antigens on plasma membrane although rat C3 was found in deposits with rat antibodies. From these studies it seems that certain conditions are required for proteinuria induction, which seem to be different from those required for the formation of dense deposits. We developed a monoclonal antibody that stayed in glomeruli for a long period and formed subepithelial immune dense deposits when reacted with autologous antibodies in the rat.
ACKNOWLEDGMENTS We thank Mrs Nobuko Kamei, Ms Naoko Kuno and Ms Minako Miyawaki for excellent technical assistance. The suggestive discussions with Dr H. Kawahara (Nagoya Kyoritsu Hospital) and with Drs C. Yamazaki and A. Ito (Masuko Institute) are appreciated. This work was partly supported by The Monbusho Scientific Research Grants (63480189 and 63044065) and by the research grants from Nagoya Kyoritsu Hospital and The Masuko Institute for Medical Research.
REFERENCES ALLEGRI, L., BRIANTI, E., CHATELET, E., MANARA, G.C., RONCO, P. & VERROUST, P. (1986) Polyvalent antigen-antibody interactions are required for the formation of electron dense immune deposits in passive Heymann's nephritis. Am. J. Pathol. 126, 1. BHAN, A.K., SCHNEEBERGER, E.E., BAIRD, L.G., COLLINS, A. B., KAMATA, K., BRADFORD, D., ERIKSON, M.E. & MCCLUSKEY, R.T. (1985) Studies with monoclonal antibodies against brush border antigens in Heymann nephritis. Lab. Invest. 53, 421. CAMUSSI, G., BRENTJENS, J. R., NOBLE, B., KERJASCHKI, D., MALAVASI, F., ROHOLT, O.A., FARQUHAR, M.G. & ANDRES, G. (1985) Antibodyinduced redistribution of Heymann antigen on the surface of cultured glomerular epithelial cells. Possible role in the pathogenesis of Heymann glomerulonephritis. J. Immunol. 135, 2409. CHATELET, F., BRIANTI, E., RONCO, P., ROLAND, J. & VERROUST, P. (1986a) Ultrastructural localization by monoclonal antibodies of brush border antigens expressed by glomeruli. I. Renal distribution. Am. J. Pathol. 122, 500. CHATELET, F., BRIANTI, E., RONCO, P., ROLAND, J. & VERROUST, P. (1986b) Ultrastructural localization by monoclonal antibodies of brush border antigens expressed by glomeruli. II. Extrarenal distribution. Am. J. Pathol. 122, 512. FUKATSU, A., BRENTJENS, J.R., KILLEN, P.D., KLEINMAN, H.K., MARTIN, G.R. & ANDRES, G.A. (1987) Studies on the formation of glomerular immune deposits in brown Norway rats injected with mercuric chloride. Clin. Immunol. Immunopathol. 45, 35. HEYMANN, W., HACKEL, D.B., HARWOOD, J., WILSON, S.G.F. & HUNTER, J.L.P. (1959) Production of the nephrotoxic syndrome in rats by Freund's adjuvant and rat kidney suspensions. Proc. Soc. Biol. Med. 100, 660. KAMATA, K., BAIRD, L.G., ERIKSON, M.E., COLLIN, A.B. & MCCLUSKEY, R.T. (1985) Characterization of antigens and antibody specificities involved in Heymann nephritis. J. Immunol. 135, 2400. KERJASCHKI, D. & FARQUHAR, M.G. (1983) Immunocytochemical localization of the Heymann nephritis antigen (GP330) in glomerular epithelial cells of normal Lewis rats. J. exp. Med. 157, 667. KERJASCHKI, D., MIETTINEN, A. & FARQUHAR, M.G. (1987) Initial events in the formation of immune deposits in passive Heymann nephritis. gp330-anti gp330 immune complex form in epithelial coated pits and rapidly become attached to the glomerular basement membrane. J. exp. Med. 166, 109.
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KRAKOWER, C.A. & GREENSPON, S.A. (1951) Localization of the nephrotoxic antigen within the isolated renal glomerulus. Arch. Pathol. 51, 629. MATSUO, S., FUKATSU, A., TAUB, M.L., CALDWELL, P.R.B., BRENTJENS, J.R. & ANDRES, G. (1987) Glomerulonephritis induced in the rabbit by antiendothelial antibodies. J. clin. Intest. 79, 1798. MATSUO, S., YOSHIDA, F., YUZAWA, Y., HARA, S., FUKATSU, A., WATANABE, Y. & SAKAMOTO, N. (1989) Experimental glomerulonephritis induced in rats by a lectin and its antibodies. Kidney, Int. 36, 1011. McLEAN, IW. & NAKANE, P.K. (1974) Periodate-lysine paraformaldehyde fixative. A new fixative for immunoelectron microscopy. J. Histochem. Cytochem. 22, 1077. MENDRICK, D.L. & RENNKE, H.G. (1986) Immune deposits formed in situ by a monoclonal antibody recognizing a new intrinsic rat mesangial matrix antigen. J. Immunol. 137, 1517. MENDRICK, D.L. & RENNKE, H.G. (1988a) I. Induction of proteinuria in the rat by a monoclonal antibody against SGP-1 15/107. Kidney Int. 33, 818.
MENDRICK, D.L. & RENNKE, H.G. (1988b) II. Epitope specific induction of proteinuria by a monoclonal antibodies. Kidney' Int. 33, 831. NATORI, Y., HAYAKAWA, I. & SHIBATA, S. (1987) The detection and characterization of renal brush border antigen (gplO8) in various rat tissues. Clin. exp. Immunol. 67, 135. ORIKASA, M., MATSUI, K., OITE, T. & SHIMIZU, F. (1988) Massive proteinuria induced in rats by a single intravenous injection of a monoclonal antibody. J. Immunol. 141, 807. PLATT, J.L. & MICHAEL, A.F. (1983) Retardation of fading and enhancement of intensity of immunofluorescence by p-phenylenediamine. J. Histochem. Cytochem. 31, 840. RONCO, P., ALLEGRI, L., MELCION, C., PIROTSKY, E., APPAY, M.-D., BARIETY, J., PONTILLON, F. & VERROUST, P. (1984) A monoclonal antibody to brush border and passive Heymann nephritis. Clin. e.xp. Imnmunol. 55, 319. SALANT, D.J., BELOK, S., MADAIO, M.P. & COUSER, W.G. (1980) A new role for complement in experimental membranous nephropathy in rats. J. clin. Invest. 66, 1339.
ADOMS
000991049100027T
Formation of subepithelial dense deposits in rats induced by a monoclonal antibody against the glomerular cell surface antigen K. NISHIKAWA, A. FUKATSU, H. TAMAI, N. SUZUKI, Y. ITO, N. SAKAMOTO & S. MATSUO Third Department of Internal Medicine, Nagoya University, School of Medicine, Nagoya, Japan
(Accepted for publication 15 August 1990)
SUMMARY We developed a monoclonal antibody, H5H3, of IgGl subclass by hybridization technique using spleen cells of mice immunized with plasma membrane fraction of isolated rat glomeruli. H5H3 recognized main bands at about 220 kD by immuno-overlay technique and bound to the glomerulus as well as brush border of proximal tubules by indirect immunofluorescence (IF) microscopy on normal rat kidney frozen sections. By immunoelectron microscopy (IEM) it bound to the surface of mainly glomerular epithelial cell and weakly to the endothelial cell. After injection to Wistar rats it remained granularly in the glomerulus for more than 2 weeks seen by IF. When rats were preimmunized with murine IgG 4 days before the injection of H5H3, mouse IgG, rat IgG and C3 were strongly visible granularly in the glomerulus in 14 days by IF. Numerous dense deposits were formed at subepithelial area seen by transmission electron microscopy. Perfusion experiment of H5H3 into rat left kidney showed granular distribution of mouse IgG in 48 h, indicating that the reaction occurred in situ. H5H3 bound diffusely in fine granular pattern on the surface of cultured glomerular epithelial cells (GEC) studied by IF and IEM. Antigenic redistribution occurred on GEC after incubation of H5H3 at 37 C. These results suggested the required conditions to form subepithelial immune dense deposits, namely that H5H3 after reaction with antigen could stay for long time in the glomerulus; that H5H3 became an antigen in autologous phase to induce large immune complexes; and H5H3 could induce antigenic modulation. Keywords dense deposits monoclonal antibodies Heymann's nephritis INTRODUCTION Heymann's nephritis is a rat model of human membranous glomerulonephritis because it induces proteinuria and subepithelial immune deposits (Heymann et al., 1959). Antigens responsible for the nephritis have been investigated and characterized. Several antigens, including the most relevant GP330 (Kerjaschki & Farquhar, 1983), were reported to be localized on glomerular epithelial cells and on brush borders of the proximal tubules (Kamata et al., 1985; Chatelet et al., 1988a; Natori, Hayakawa & Shibata, 1987). Recent studies strongly suggested that immune deposits at subepithelial area of the glomerular basement membrane were formed in situ (Kerjaschki & Farquhar, 1983; Bhan et al., 1985; Kerjaschki, Miettinen & Farquhar, 1987). Recently, monoclonal antibodies were raised against these antigens and were used to study the pathogenesis of Heymann's nephritis (Ronco et al., 1984; Bhan et al., 1985; Allegri et al., 1986). Because of their monospecificity, mono-
clonal antibodies are useful to investigate the precise localization of the antigen at ultrastructural level or to study the mechanism to form immune deposits; however it is difficult to produce proteinuria or to form immune deposits visible by transmission electron microscopy (TEM) when monoclonal antibodies are injected into rats. In the present study we developed and characterized a monoclonal antibody that reacted with glomerular epithelial cells and produced electron dense immune deposits at a subepithelial area visible by TEM during the autologous phase. The mechanisms of the formation of subepithelial dense deposits in the model and the relevance to those in Heymann's nephritis are discussed.
MATERIALS AND METHODS Animals Female Wistar rats, weighing 150-200 g, and female BALB/c mice weighing about 20 g were purchased from Chubu Kagaku Shizai (Nagoya, Japan) and allowed free access to food and water.
Correspondence: Atsushi Fukatsu, MD, The Third Department of Internal Medicine, Nagoya University, School of Medicine, 65 Tsuruma-cho, Showa-ku, Nagoya, 466, Japan.
143
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Monoclonal antibody Antigen preparation. Kidneys were perfused through abdominal aorta with 10 mm Tris-buffered saline, pH 7 4, containing 0-2 mm benzylsulphonyl fluoride and 1 mm Nethylmaleimide (Sigma Chemical Co., St Louis, MO). Glomeruli were separated from minced renal cortices by sieving (Krakower & Greenspon, 1954). Purity of separated glomeruli was about 90% with the contamination of 10% tubules. Plasma membrane was separated from glomeruli by sonication using Sonifier Disruptor (Branson, Shelton, CT) and centrifuged at 1200 g for 15 min. The supernatant was then ultracentrifuged at 30 000g for 45 min and the resulting pellet was treated with 0-1 % Triton-X to solubilize the plasma membrane proteins. After centrifugation at 30000 g for 45 min the supernatant was dialysed against 0 01 M phosphate-buffered saline (PBS), pH 7 4, concentrated using Minicon (Amicon, Denver, CO) and used as the antigen (glomerular protein). Immunization. BALB/c mice were immunized three times with 2 mg of the antigen mixed with Freund's complete adjuvant (FCA) every 2 weeks. Hybridization. Spleen cells were separated 3 days after the last immunization and fused with murine myeloma cells NS- 1 using PEG (mol. wt 4000; Merck, Rahway, NJ). Positive hybridoma clones were screened by indirect immunofluorescence (IIF) microscopy on frozen normal kidney sections. One of the positive clones, H5H3, was harvested and implanted into the abdominal cavity of a BALB/c mouse to obtain large amount of monoclonal antibodies. Immunoglobulin fraction was purified by 50% ammonium sulphate precipitation. Subclass of H5H3 was determined by ELISA. A monoclonal antibody (9C2) of the same subclass as H5H3, which did not react with renal structure studied by IIF was used for control. Western blotting Glomerular protein was mixed in sample buffer (4 6% SDS, 20% glycerol, 0 1 % bromophenol blue) with or without 10% 2mercaptoethanol and heated in boiled water for a few minutes and applied for SDS-PAGE on 8-25% gradient gel using PhastSystem (Pharmacia Fine Chemicals, Uppsala, Sweden). Electroblotting was also done using PhastSystem at 60 V for 30 min on PVDF papers. After blocking with 0 5% bovine serum albumin, the paper was reacted with H5H3 or 9C2 (diluted 10 pg/ml) and then with horseradish-peroxidase (HRP) labelled goat anti-mouse IgG antibody (1:1000). Positive reaction was visualized by 0 05% DAB with 0 005% H202.
Immunohistochemistry Frozen sections (4 Mm) of kidney, intestine, lung, heart, liver and spleen from normal rats were cut by a cryostat and incubated first with properly diluted H5H3 or with control monoclonal antibodies of the same subclass for 30 min at room temperature and then with FITC-labelled rabbit anti-mouse IgG antibody (Cappel) for 30 min. Slides were covered with medium containing p-phenylenediamine (Platt & Michael, 1983) and observed with an Olympus BH-2 epifluorescence microscope. Immunoelectron microscopy (IEM) was performed as reported (Matsuo et al., 1987). Briefly, 7-pm frozen sections were cut from a normal kidney perfused with periodate-lysine paraformaldehyde fixative (PLP) (McLean & Nakane, 1974) and immersed further for 4 h in PLP and incubated with 0-05% sodium borohydride solution, diluted H5H3 or 9C2 in PBS, diluted
HPR-labelled goat anti-mouse IgG antibody, 2% glutaraldehyde serially. Sections were reacted with 0-02% DAB solution and then with the same solution containing 0 005% H202 to visualize the positive products and fixed with 2% OS04. Sections were dehydrated, embedded in Epon 812 and processed for electron microscopy. Intravenous injection of H5H3 Sixteen rats (group 1) were pre-immunized with 0 5 mg of mouse IgG purified by protein A column mixed with CFA (day -4). Four days later (day 0) rats were injected with 5 mg of H5H3 intravenously. Kidney tissues were obtained by biopsy or killing on days 1, 3, 7, 14, 21 and 28. A fragment of kidney tissue was fixed in 10% buffered formalin and processed for light microscopy. Ultra-thin sections were cut from another fragment of tissue fixed in 2% glutaraldehyde and were stained with lead and uranium for electron microscopy. They were observed with a JOEL 100CX electron microscope. Part of kidney tissue was snap-frozen in liquid nitrogen and used for IF microscopy. Ten rats (group 2) were treated as above with the injection of the same amount of 9C2 instead of H5H3. Eight rats (group 3) were treated as group 1, and four rats (group 4) as group 2 without pre-immunization. Light microscopy, direct IF for mouse IgG, rat IgG, and rat C3 using FITC-labelled reagents (Cappel) were performed as described.
Perfusion of H5H3 Perfusion of left kidney with H5H3 was done, as reported previously (Matsuo et al., 1989), in rats pre-immunized twice with normal mouse IgG 1 and 3 weeks previously. Briefly left renal artery and vein were exposed and were cannulated with polyethylene tubes. Then 10 mg of H5H3 in tyrode buffer was perfused using peristaltic pump (Pharmacia) for 15 min at room temperature while all venous flow was recovered. Artery and vein were sutured to reconstruct the circulation. Kidney tissue was obtained at 3 and 48 h after the perfusion and studied histologically and immunohistologically as described above. Protein excretion in urine Urinary protein was measured by quantitative sulphosalicylic
acid method. Detection of mouse IgG and rat antibodies to mouse IgG in sera Sera taken on days 0, 1, 3, 7, 14, 21, and 28 were checked for circulating mouse IgG and for autologous antibodies to mouse IgG by ELISA. Microtitre plates (Immulon, Dynatech, Chantilly, VA) were coated with goat anti-mouse IgG antibody (Cappel) or mouse IgG, 500 ng/well, and reacted with serially diluted sera followed by the incubation with HRP-labelled goat anti-mouse IgG antibody or HRP-labelled goat anti-rat IgG antibody (Cappel). ELISA was done as described previously (Fukatsu et al., 1987).
Glomerular cell culture Primary glomerular epithelial cells (GEC) were cultured from separated glomeruli of Wistar rat kidneys in 8-well slides (Miles Scientific, Naperville, IL) or in 6-well plates (Costar) as described previously (Fukatsu et al., 1987). Characterization of GEC was based on the following criteria: (i) morphologic characteristics; (ii) positive cytokeratin (Miles); (iii) short
Subepithelial dense deposits in rats
145
Table 1. Comparison of the tissue localization of H5H3 recognizing antigen (H5H3 Ag) and Heymann-related antigens
A B C D E F 200 .
Tissue
97 . 68* 43 . 25 m 18
Kidney Brush border of proximal tubules Glomerular epithelium Glomerular endothelium Liver Endothelium Hepatocyte Gut
a
Brush boder
H5H3 Ag
GP330
GP90
GPl08
+
+
+
+
+
+
+
+
+*
-
+
+
-
+
?
+
+t
+
+
+ ?
Spleen 41.
...4.I
-
'..F'.4,1
A
Endothelium
-
-
+
Heart Endothelium
+
-
+
+
-
+
+
Chatelet et al. (1988a, 1988b)
Chatelet et al. (1988a,) 1988b)
Natori et al. (1987)
., 4
4.
4
4,
C. Fig. 1. (a) Results of Western blots. Bands at 220 kD are reacted with H5H3 (lanes B, C); lanes A, B, C, reacted with H5H3; lanes D, E, F, reacted with 9C2; lanes A, D, SDS-solubilized spleen homogenate (reduced condition) was run as control; lanes B, C, E, F, glomerular protein was run (B, E, reduced; C, F, non-reduced condition). (b) H5H3 binds to glomerular capillary wall as well as brush border of proximal tubules in the normal kidney. Indirect immunofluorescence; magnification x 190. (c) By indirect immunoelectron microscopy H5H3 reacts mainly with the cell surface of glomerular epithelial cells and weakly with that of endothelial cells. Magnification x 4700.
peripheral localization of actin filament; and (iv) positive Heymann's antigen. Immunohistochemical study of GEC GEC in 8-well slides were fixed with cold ethanol for 5 min and incubated with H5H3 for 15 min at room temperature; then with FITC-labelled rabbit anti-mouse antibody for 15 min. GEC in other slides were first incubated with H5H3 at 37°C for 5 to 15 min and fixed with cold ethanol then treated with FITC-labelled antibody as above. For IEM, GEC in 6-well plates were first fixed with PLP and incubated with H5H3 for 30 min at room temperature and then with HRP-labelled goat anti-mouse IgG antibody for 15 min. Peroxidase reaction and processing for electron microscopy were done as previously described (Fukatsu et al., 1987). RESULTS Western blotting H5H3 recognized two bands at about 220 kD in reduced or nonreduced condition (Fig. la). 9C2 did not show positive results.
Immunohistochemistry on normal tissue On normal frozen kidney sections, H5H3 was localized in glomerular capillary wall (GCW) and in brush border of proximal tubules (Fig. lb). By IEM positive reaction products
Lung Endothelium Pneumocyte References
?-
*Weakly stained by immunoelectron microscopy; t restricted to biliary pole; t hard to recognize by indirect immunofluorescence. were found mainly on the surface of GEC and more weakly on endothelial cells (Fig. lc). Extra renal distribution seen by IIF was listed in Table 1. Immunohistochemistry using 9C2 was negative.
Histology and immunohistology of rat kidneys injected with HSH3 In rats of group 1, mouse IgG was seen granularly along GCW on day 1, increased its intensity and granularity on day 7, peaked on day 14 (Fig. 2a), and slightly weakened on day 28 by direct IF. Rat IgG was positive on day 7, increased on day 14 (Fig. 2b) and remained so until killing (on day 28). Rat C3 was found with rat IgG but more weakly and coarsely (Fig. 2c). Light microscopy showed normal renal histology. By TEM, discrete dense deposits were visible mainly in slit membranes on day 14 (Fig. 2d) and later. In rats of group 3, mouse IgG was visible in the same pattern as seen in rats of group 1, but decreased on day 21, and disappeared on day 28. Rat IgG and C3 was not detected at any time. Light microscopy was normal and electron dense deposit was not found by TEM. In rats of groups 2 and 4, mouse IgG, rat IgG or C3 was not seen throughout the experiments. LM and TEM were normal. No proteinuria was detected throughout the experiment in any group of rats. Rat anti-mouse IgG antibody was not detected on day 0, weakly on day 7 and steadily on day 14 and after in rats of
K. Nishikawa et al.
146
Fig. 3. When H5H3 is perfused into left kidney of rats pre-immunized with mouse IgG, mouse IgG was found granularly along glomerular capillary wall in 48 h. Direct immunofluorescence; magnification x 400.
cytokeratin was found positive; (iii) actin filament was stained peripherally; and (iv) Heymann's antigen was stained positive using sheep anti-rat brush border vesicle antibody (Camussi et al., 1985). When GEC were reacted with H5H3, H5H3 bound to the cell surface and to intracellular organellae (Fig. 4a). When reacted at 370C for 15 min, H5H3 was found on the cell in
To
Ss,o ,W f At) tow v, ,. ~ ~ ~ t-XHa
N
_
e.,* Fig. 2. When H5H3 is injected into rats of group f, mouse IgG is localized granularly along glomerular capillary wall (a) with rat IgG (b) and C3 (c) on day 14. Direct immunofluorescence; magnification (a, b, c) x 400. (d) Electron dense deposits are visible at subepithelial area; magnification x 6000.
_.A.'
.4.
groups 1 and 2. In rats of groups 3 and 4 it was detected on day 7 and onward but titre was low at any time. Mouse IgG was found in serum up to day 7.
Histology and immunohistochemistry of rat kidneys perfused with H5H3 Mouse IgG was found in glomeruli diffusely at 3 h and granularly along GCW at 48 h (Fig. 3) after the perfusion of H5H3 with deposition of rat IgG and C3. Light microscopy showed normal histology. Electron dense deposits were not TEM. byby TEM. found found
Immunocytochemistry of GEC Thecells grownoutof glomerulihadcthenaturerofepithelialcells because: (i) they had cobble stone appearance in monolayer; (ii)
,:
,, Fig. 4. H5H3 binds to the surface and to cytosomal organellae off t indirect glomerular epithelial cells (GEC) fixed with ethanol are immunofluorescence; magnification x 200. When GEC (a);incubated with H5H3 at 370C for 15 min H5H3 is localized in patchy pattern (b); some cells show capping; indirect immunofluorescence; magnificationI x 200. By immunoelectron microscopy H5H3 binds to the surface off GEC diffusely in fine granular pattern (c); magnification x 5000.
147
Subepithelial dense deposits in rats patchy pattern. Some cells showed capping (Fig. 4b). By TEM H5H3 bound to the cell surface diffusely in fine granular pattern. Apparent staining of coated pits was not seen. 9C2 did not bind to GEC. DISCUSSION
H5H3 is a monoclonal antibody of IgGl subclass which recognizes the antigen of molecular weight of about 220 kD localized mainly on GEC surface. The antigen recognized by H5H3 is most likely different from well-characterized Heymann's antigen like GP330 because of the difference of molecular weight and of renal or extrarenal distribution, as listed in Table 1. Although molecular weight was different, renal and extrarenal distribution of H5H3 was similar to that of GP90 (Chatelet et al., 1986a, 1986b) Allegri et al. (1986) injected five monoclonal antibodies to GP330 into rats but could not find subepithelial dense deposits and explained that polyvalent antigen-antibody interaction on the cell surface was necessary to form dense immune deposits. Kerjaschki et al. (1987) described that in initial phase of passive Heymann's nephritis, immune complexes were formed on the surface of epithelial cells, mainly at coated pits and resulting immune complexes attached to the basement membrane firmly. Mendrick & Rennke (1986) reported that a monoclonal antibody could bind to the mesangial matrix antigen which is fixed in the structure and formed electron dense immune deposits. From these studies it is conceivable that the prerequisites for dense deposit formation are (i) fixation of antigen or immune complex to the extracellular structure; (ii) sufficient supply of antigens and antibodies; and (iii) antigen redistribution in the case of cell surface antigen (Camussi et al., 1985). Previously described monoclonal antibodies were, when injected, fixed in glomeruli and disappeared within a week (Ronco et al., 1984; Bhan et al., 1985; Allegri et al., 1986), while H5H3 stayed for more than 2 weeks in glomeruli even without pre-immunization. This fact suggests that the antigens or immune complexes attach to extracellular sites rather firmly. When autologous antibody appeared in the rats preimmunized with mouse IgG, H5H3 itself became the antigen and complexes, consisting of H5H3, presumably antigens, rat immunoglobulins, and complements might become large enough to be visible as dense deposits. We cannot exclude the possibility that circulating immune complexes of rat antibodies and remaining mouse IgG were deposited on day 7 and later, although autologous antibody was not detected when H5H3 was injected. Our perfusion experiment showed that granular deposits were formed in situ. H5H3 can induce antigenic redistribution on GEC like Heymann's antigen (Camussi et al., 1985). These characteristics may be the reason why the interaction of H5H3 with its antigen on the surface of GEC results in the formation of immune dense deposits. H5H3 did not induce proteinuria even when subepithelial dense deposits were formed. The precise cause of proteinuria is not fully understood. Orikasa et al. (1988) described a monoclonal antibody which produced proteinuria in rats when bound to epithelial cells without forming dense deposits. Mendrick & Rennke (1988a) reported that a monoclonal antibody which recognized the sialoglycoprotein of glomerular cells induced proteinuria without dense deposits. They also described (Mendrick & Rennke, 1988b) the epitope specificity to the same
molecule for proteinuria. In Heymann's nephritis, Salant et al. (1980) showed that complement was necessary for the induction of proteinuria which presumably acted on the surface of epithelial cells. H5H3 lacked the capability of complement binding when bound to antigens on plasma membrane although rat C3 was found in deposits with rat antibodies. From these studies it seems that certain conditions are required for proteinuria induction, which seem to be different from those required for the formation of dense deposits. We developed a monoclonal antibody that stayed in glomeruli for a long period and formed subepithelial immune dense deposits when reacted with autologous antibodies in the rat.
ACKNOWLEDGMENTS We thank Mrs Nobuko Kamei, Ms Naoko Kuno and Ms Minako Miyawaki for excellent technical assistance. The suggestive discussions with Dr H. Kawahara (Nagoya Kyoritsu Hospital) and with Drs C. Yamazaki and A. Ito (Masuko Institute) are appreciated. This work was partly supported by The Monbusho Scientific Research Grants (63480189 and 63044065) and by the research grants from Nagoya Kyoritsu Hospital and The Masuko Institute for Medical Research.
REFERENCES ALLEGRI, L., BRIANTI, E., CHATELET, E., MANARA, G.C., RONCO, P. & VERROUST, P. (1986) Polyvalent antigen-antibody interactions are required for the formation of electron dense immune deposits in passive Heymann's nephritis. Am. J. Pathol. 126, 1. BHAN, A.K., SCHNEEBERGER, E.E., BAIRD, L.G., COLLINS, A. B., KAMATA, K., BRADFORD, D., ERIKSON, M.E. & MCCLUSKEY, R.T. (1985) Studies with monoclonal antibodies against brush border antigens in Heymann nephritis. Lab. Invest. 53, 421. CAMUSSI, G., BRENTJENS, J. R., NOBLE, B., KERJASCHKI, D., MALAVASI, F., ROHOLT, O.A., FARQUHAR, M.G. & ANDRES, G. (1985) Antibodyinduced redistribution of Heymann antigen on the surface of cultured glomerular epithelial cells. Possible role in the pathogenesis of Heymann glomerulonephritis. J. Immunol. 135, 2409. CHATELET, F., BRIANTI, E., RONCO, P., ROLAND, J. & VERROUST, P. (1986a) Ultrastructural localization by monoclonal antibodies of brush border antigens expressed by glomeruli. I. Renal distribution. Am. J. Pathol. 122, 500. CHATELET, F., BRIANTI, E., RONCO, P., ROLAND, J. & VERROUST, P. (1986b) Ultrastructural localization by monoclonal antibodies of brush border antigens expressed by glomeruli. II. Extrarenal distribution. Am. J. Pathol. 122, 512. FUKATSU, A., BRENTJENS, J.R., KILLEN, P.D., KLEINMAN, H.K., MARTIN, G.R. & ANDRES, G.A. (1987) Studies on the formation of glomerular immune deposits in brown Norway rats injected with mercuric chloride. Clin. Immunol. Immunopathol. 45, 35. HEYMANN, W., HACKEL, D.B., HARWOOD, J., WILSON, S.G.F. & HUNTER, J.L.P. (1959) Production of the nephrotoxic syndrome in rats by Freund's adjuvant and rat kidney suspensions. Proc. Soc. Biol. Med. 100, 660. KAMATA, K., BAIRD, L.G., ERIKSON, M.E., COLLIN, A.B. & MCCLUSKEY, R.T. (1985) Characterization of antigens and antibody specificities involved in Heymann nephritis. J. Immunol. 135, 2400. KERJASCHKI, D. & FARQUHAR, M.G. (1983) Immunocytochemical localization of the Heymann nephritis antigen (GP330) in glomerular epithelial cells of normal Lewis rats. J. exp. Med. 157, 667. KERJASCHKI, D., MIETTINEN, A. & FARQUHAR, M.G. (1987) Initial events in the formation of immune deposits in passive Heymann nephritis. gp330-anti gp330 immune complex form in epithelial coated pits and rapidly become attached to the glomerular basement membrane. J. exp. Med. 166, 109.
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KRAKOWER, C.A. & GREENSPON, S.A. (1951) Localization of the nephrotoxic antigen within the isolated renal glomerulus. Arch. Pathol. 51, 629. MATSUO, S., FUKATSU, A., TAUB, M.L., CALDWELL, P.R.B., BRENTJENS, J.R. & ANDRES, G. (1987) Glomerulonephritis induced in the rabbit by antiendothelial antibodies. J. clin. Intest. 79, 1798. MATSUO, S., YOSHIDA, F., YUZAWA, Y., HARA, S., FUKATSU, A., WATANABE, Y. & SAKAMOTO, N. (1989) Experimental glomerulonephritis induced in rats by a lectin and its antibodies. Kidney, Int. 36, 1011. McLEAN, IW. & NAKANE, P.K. (1974) Periodate-lysine paraformaldehyde fixative. A new fixative for immunoelectron microscopy. J. Histochem. Cytochem. 22, 1077. MENDRICK, D.L. & RENNKE, H.G. (1986) Immune deposits formed in situ by a monoclonal antibody recognizing a new intrinsic rat mesangial matrix antigen. J. Immunol. 137, 1517. MENDRICK, D.L. & RENNKE, H.G. (1988a) I. Induction of proteinuria in the rat by a monoclonal antibody against SGP-1 15/107. Kidney Int. 33, 818.
MENDRICK, D.L. & RENNKE, H.G. (1988b) II. Epitope specific induction of proteinuria by a monoclonal antibodies. Kidney' Int. 33, 831. NATORI, Y., HAYAKAWA, I. & SHIBATA, S. (1987) The detection and characterization of renal brush border antigen (gplO8) in various rat tissues. Clin. exp. Immunol. 67, 135. ORIKASA, M., MATSUI, K., OITE, T. & SHIMIZU, F. (1988) Massive proteinuria induced in rats by a single intravenous injection of a monoclonal antibody. J. Immunol. 141, 807. PLATT, J.L. & MICHAEL, A.F. (1983) Retardation of fading and enhancement of intensity of immunofluorescence by p-phenylenediamine. J. Histochem. Cytochem. 31, 840. RONCO, P., ALLEGRI, L., MELCION, C., PIROTSKY, E., APPAY, M.-D., BARIETY, J., PONTILLON, F. & VERROUST, P. (1984) A monoclonal antibody to brush border and passive Heymann nephritis. Clin. e.xp. Imnmunol. 55, 319. SALANT, D.J., BELOK, S., MADAIO, M.P. & COUSER, W.G. (1980) A new role for complement in experimental membranous nephropathy in rats. J. clin. Invest. 66, 1339.
