Jul 21, 1982 - Freund's adjuvant. So-called passive Heymann nephritis in the rat, on the other hand, can be initiated by a single i.v. injection of a heterologous.
Br. J. exp. Path. (1982) 63, 667
PASSIVE HEYMANN NEPHRITIS IN THE RAT PRODUCED BY A HETEROLOGOUS ANTIBODY TO A HETEROLOGOUS KIDNEY FRACTION 3 ANTIGEN A. Z. BARABAS, J. CORNISH AND R. LANNIGAN Fxromsl the Departmnentt of Pathology, Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada T2N 1N4 Received for
pllblication July 21, 1982
Summary.-Antibody to rabbit kidney Fraction 3 antigen was raised in guinea-pigs. In an indirect fluorescent antibody test the guinea-pig sera showed high levels of antibody to both rabbit and rat tubular nephritogenic antigen and a much lower level of antibody against their own kidney tubular antigen. I.v. injection of this guinea-pig anti-rabbit kidney Fraction 3 antibody into rats produced immune-complex glomerulonephritis, morphologically identical to passive Heymann nephritis. H EYMANN NEPHRITIS in the rat can be produced by repeated i.p. injections of homologous (Heymann et al., 1959) and heterologous (Heymann et al., 1.963; Hess, Ashworth and Ziff, 1965; Edgington, Glassock and Dixon, 1967; Nagi et al., 1971) kidney tubular antigens in complete Freund's adjuvant. So-called passive Heymann nephritis in the rat, on the other hand, can be initiated by a single i.v. injection of a heterologous antibody to rat kidney Fraction 3 (F3) antigen (Barabas, Nagi and Lannigan, 1970). These initial observations have been confirmed (Feenstra et al., 1975; Barabas and Lannigan, 1 974b; Leendert et al., 1977; Zanetti and Druet, 1980). The injected antibody to the tubular antigen localizes immediately to a preexisting glomerular fixed antigen and forms beaded immune complexes (Couser etal., 1978; VanDammeetal., 1978). In most of the experiments antibody to the rat tubular antigen is raised in the rabbit or the sheep (Barabas and Lannigan, 1 974a; Salant, Darby and Couser, 1980). We have shown that rabbits will produce, after a prolonged period of immunization, high levels of antibodies to the rat kidney tubular antigen, but not against their own kidneys. Nevertheless,
we have demonstrated immune-complex glomerulonephritis in these antibody(Barabas and producing animals Lannigan, 1981). Our investigationshowed that the antigen in the glomerulus was derived from the injected rat tubular preparation and the rabbit IgG was directed to the rat F3 antigen only and not against rabbit kidney components. During the course of the present investigation we have found that guinea-pigs injected with rabbit kidney F3 antigen, in complete Freund's adjuvant, produced high levels of antibodies to the rabbit kidney tubular antigen. At the same time, they also produced high levels of crossreacting antibodies against rat kidney tubular antigens and low levels of antibodies which reacted with guinea-pig kidney tubules. These observations prompted us to inject rats with guinea-pig anti-rabbit kidney F3 antibody to see whether such antibodies are capable of producing passive Heymann nephritis. MATERIALS AND METHODS
Preparation of rabbit kidney tubular fraction 3 (F3) antigen.-Adult New Zealand white rabbits were obtained. Fraction 3 antigen, a mitochondrial preparation, was made by a method
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TABLE I.-Antibody activity of guinea-pig anti-rabbit kidney F3 antibody against rabbit, rat and guinea-pig kidney sections in an indirect fluorescent antibody test On rabbit kidney On rat kidney Gpa serum with sections anti-rabbit kidney sections ~ , _ F3 antibody activity 1:1500 1:2000 1:3000 Serum dilutions of: 1:1500 1:2000 1:3000 GP 1 + ++ + +++ + +++ + ++ + + + + + +++ GP 2 + + ++ ++ + + + + ++ + + + + ++++ ++ ++ ++ GP 3 + + + + + +++ + + + + GP 4 + + + + ++++ ++ ++ +++ ++ a GP =guinea pig. Scoring signifies intensity of fluorescence against tubular brush-border regions.
On GP kidney sections 1:110 ++ ++ ++ ++
1:20 ++ ++ ++ +
1:40 ++ ++ ++ +
TABLE II.-Graded deposition of immune complexes along the glomerular capillaries staining for rat and guinea pig IgG at 7 and 40 days 40 days
7 days Rat No. 1 2 3 4 5 6 7
Rat IgG
Guinea pig IgG
+
+
-
+ + + + + +
8
9 10 11 12
Grading:
+
+ + + + +
Rat IgG
Guinea pig IgG
+
++
+ + ++ ++ +
+ ++
+ hardly visible small beaded deposits.
+ easily visible small beaded deposits. + + bright beaded deposits.
previously described (Barabas and Lannigan, ] 974a). Production of antibody to the rabbit kidney tubular F3 antigen in the guinea-pig.-Four adult guinea-pigs were immunized by multiple i.m. injections of 5 mg rabbit kidney fraction 3 antigen in complete Freund's adjuvant. The animals were injected every other week and were bled 6 days after the 5th injection of the antigen. Serum from each guinea-pig was serial diluted and tested by the indirect fluorescent antibody technique on normal rabbit, rat and guinea-pig kidney sections. Samples of guinea-pig serum were pooled, inactivated at 560 for 30 min and absorbed with normal rat red blood cells before
Immunofluorescence. Cryostat sections of rat kidneys were stained for guinea-pig and rat IgG, using fluorescein-labelled goat anti-guinea-pig IgG and rabbit anti-rat IgG. Suitable controls were included. Experimental groups of rats.-Twelve male Wistar rats weighing approximately 250 g were injected i.v. with 1 ml of guinea-pig anti-rabbit kidney F3 antibody. Animals were biopsied at 7 days and kidney samples were obtained at the end of the experiment on Day 40. RESULTS
Light microscopy Rat kidney sections obtained at Day 7 Light microscopy. Blocks of kidneys were fixed in 10% formol saline and embedded in and on Day 40 showed no abnormal paraffin. 4,um-thick sections were cut and changes. stained with haematoxylin and eosin and the methenamine silver stain. Electron microscopy. 1 mm3 blocks of renal Fluorescent microscopy cortex were fixed in buffered glutaraldehyde Indirect fluorescent antibody test.postfixed in osmium tetroxide and embedded in Results shown on Table I indicate that all Epon. Glomeruli were stained with uranyl acetate and lead citrate and examined on an the guinea-pigs produced high levsls of circulating antibodies to tubular brushAEI Corinth microscope.
injection.
HEYMAN NEPHRITIS CAUSED BY ANTIBODY
669
Direct fluorescent antibody test. Rat kidney samples, obtained on Day 7 and at the end of the experiment on Day 40, were stained for guinea-pig and rat IgG. Table II shows the results. It can be seen that guinea-pig IgG is present with beaded deposits along the glomerular capillary blood vessels on Day 7 and on Day 40 (Fig. 1). Rat IgG on the other hand is present in the early biopsy specimens at Day 7 in the glomeruli of a few rats only with a beaded fashion. By Day 40 all the rat kidneys stained for rat IgG. Electron microscopy Numerous small electron-dense deposits were found in the sub-epithelial areas of the glomerular basement membrane (Fig. 2). In relation to the larger deposits, epithelial foot processes were fused. The thickness of the glomerular basement membrane was not altered to any significant degree. DISCUSSION
Heymann nephritis can be induced in rats by injections of homologous tubular antigens (Heymann et al., 1959) as well as by injections of heterologous tubular antigens (Heymann et al., 1963; Hess et al., 1965; Edgington et al., 1967; Nagi et al., 1971). However, up until this time, passive Heymann nephritis has only been produced by injection of antibodies to rat renal tubular antigens (Barabas et al., 1970; Feenstra et al., 1975). In the present experiment we have produced a glomerular lesion in rats, w Beaded e's.*.~. identical to that observed in passive FIG. deposition of rat IgG can be Heymann nephritis, by the i.v. injection of observe(l aroun(l glomerular capillary )loo0( a heterologous antibody directed against a vessels at Day 40. x 350. FIG. 2. -Numerous elcctron-(lense deposits heterologous renal tubular F3 antigen. (arrows) ar e pr esent on the epitlielial sicle of We have shown that antibodies raised in thle glomerular basement membrane (GBM1). guinea-pigs to rabbit kidney F3 antigen Foot processes (Fp) ar-e ftuse(d in relation to (leposits. x 16,300. reacted with rabbit and rat and to a lesser extent with guinea - pig kidney tubular border antigens of rabbit and rat kidneys brush-border antigens in an indirect and a much lower level of circulating fluorescent antibody test. These results antibody against the same components of showed in an in vitro test that guineapigs produced a cross-reactive antibody gtuinea-pig kidneys. 1.
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A. Z. BARABAS, J. CORNISH AND R. LANNIG-AN
Experimental Glomerulonephritis in the Isolated capable of reacting with kidney tubular Perfused Rat Kidnev. J. din. Invest, 62, 1275. antigens of other sepcies. Therefore it is EDGINGTON, T. S., GLrssocx, R. J. & Dimo-, F. J. (1967) Autologous Immune Complex Pathogenesis evident that rat, rabbit and guinea-pig of Experimental Xllergic Glomerulonephritis. kidneys. and possiblv kidneys of other Science, 155, 1432. species of animals. share identical or FEENSTrRA, K., LEE, R. Y. D., GREBEN-, H. A., ARENDs, A. & HOEDEMAEKER, P. J. (1975) structurallv similar tubular nephritogenic Experimental Glomerulonephritis in the Rat antigens. In the rat glomerulus a fixed Indued by Antibodies Directed against Tubular antigen has been demonstrated (Couser et Antigens. The Natural History: A Histologic and Immunohistologic study at the Light Microscopic al., 1978; Van Damme et al., 1978) to and the l,trastructural Level. Lab. Invet., 32, which the injected antibody attaches. This 235. observation has not been made in other HESs, E. V., AsHWORTH, C. T. & ZIrFF, M. (1965) Xephrosis in the Rat Induced by Rat Kidney species. If local conditions could be altered Extracts. Ann. N.Y. Acad Sci., 124, 323. to allow antigen trapping in the glomer- HEYMAN-, W., H.cxisL, D. B., HARWOOD, S.. WiILsox-, S. G. F. & Hu---E, J. L. P. (1959) ular basement membrane, then it may be Production of Nephrotic Syndrome in Rats bv possible to induce immune-complex Freund's Adjuvants and Rat Kidney Suspensions. identical to glomerulonephritis, similar or Proc. Soc. exp. Biol. Med., 100, 660. W., KMETEc, E. P., WnisoX, S. G. F., passive Heymann nephritis of rats, in HEX-mAN-, HuTEm, J. L. P., HACKEL, D. B. & C'UPPAGE, F. other species. (1963) Experimental Autoimmune Renal Disease
REFERENCES BXRXBAS, A. Z.. NAGI. A. H. & LANNIGAN, R. (1970) Induction of Autologous Immune-Complex Nephritis in Rats by Heterologous Anti-Kidnev Mitochondrial Antiserum. IJt. U-rol. Nephrol.. 2, 303.
BARABBAS. A. Z. & LAN-NIGAN. R. (1974a) Induction of an Autologous Immune Complex Glomerulonephritis in the Rat byEIntravenous Injection of Heterologous Anti-Rat Kidney Tubular Antibody. I. Production of Chronic Progressive ImmuneComplex Glomerulonephritis. Br. J. erp. Path.. 55, 47. BARABAS. A. Z. & LAN-NQIGAN-. R. (1974b) Induction of an Autologous Immune C'omplex Glomerulonephritis in the Rat bv Intravenous Injection of Heterologous Anti-Rat Kidney Tubular Antibody. II. Early Glomerular Lesions. Br. J. exp. Path., 55, 282. BARABAS, A. Z. & LANNIG AN. R. (1981) ImmuneComplex Nephritis in the Rabbit Produced bv Injections of Rat Renal Tubular Fraction 3 Antigen: Br. J. exp. Path.. 62, 94. COUSER. W. G.. STEINMYLI.ER. D. R.. STILMANT. Mt. M., SALA-NT. D. J. & LOwENSTEIN. L. J. (1978)
in Rats. In Irnmunopathologv. P. Graber & P. A. MNiescher, editors. Basle: Schwabe. p. 240. LEENDERT, A. vAx- Es., Bi.K, A. P. R., SCHOENFELD. L. & GLAssocK, R. J. (197) Chronic Xephritis Induced by Antibodies Reacting with Glomerular Bound Immune Complexes. Kidney Int., 11, 106. -NAGI. A. H., BARABAs, A. Z., AEXAN_DER, F. & L.-iIGAN, R. (1971) Production of Autologous Immune Complex Glomerulonephritis in Rats by Injections of Heterologous Renal Tubular Antigen. Acta med. Aced. Sci. hung., 28, 249. SALANT, D. J.. DARBY, C. & CousER, W. G. (1980) Experimental Membranous Glomerulonephritis in Rats. Quantitative Studies of Glomerular Immune Deposit Formation in isolated Glomeruli and W%hole Animals. Clin. Invest., 66, 7 1. vA- D nim, B. J. C., FLEruREN, G. J., BAKKER, W. W., VERNIER, R. L. & HOEDEMAEKER, P. J. (1978) Experimental Glomerulonephritis in the Rat Induced by Antibodies Directed Against Tubular Antigens. V. Fixed Glomerular Antigens in the Pathogenesis of Heterologous Immune Complex Glomerulonephritis. Lab. Inrvest., 38, 502. ZANE-ET. M. & DRUET, P. (1980) Passive Heymanns' Nephritis as a 3Model of Immrune Glomerulonephritis Mediated by Antibodies to Immunoglobulins. Clin. exp. Immunol., 41, 189.
