ANIMAL MODEL OF HUMAN DISEASE

Anti- Glomerular- Basement-

Membrane Glomerulonephritis Animal Model: Experimental Autoimmune Anti-Glomerular-Basement-Membrane Glomerulonephritis in the Sheep

Contributed by: Raymond W. Steblay, MD, New York State Kidney Disease Institute, Division of Laboratories and Research, New York State Department of Health, Albany, NY 12201. Biologic Features

Sheep injected biweekly with 150 mg (wet weight) of either heterologous glomerular basement membranes (GBM) or homologous GBM with complete Freund's adjuvant (CFA) develop progressive glomerulonephritis and antibodies to the injected antigenic determinants. 1-4 Some of these antibodies react with the sheep's own GBM (ie, as autoantibodies) and form a characteristic linear deposit of IgG along the GBM, as seen by direct immunofluorescence (IF) (Figure 1).23 In most cases complement (C3) is seen in a similar pattern.3 Autoantibodies against GBM can be eluted from the nephritic sheep kidney by citric acid buffer, pH 3.2. They react with human GBM and lung basement membranes (LBM), sheep GBM and LBM, basement membranes of human skin and sebaceous glands, sarcolemma of rat muscle, human placenta, and the GBM of a 4-pound fetal sheep and of a 16-week-old human fetus.3-5 Thus, these autoantibodies are neither species- nor organ-specific.5 Intravenous infusion of nephritic renal eluates 3-5 or nephritic sheepserum globulins 6 into 3-month-old lambs resulted in deposition of antiGBM antibodies in the characteristic linear pattern along the GBM, fixation of C3, and a mild glomerular injury consisting of a brief infiltration of polymorphonuclear cells into the glomeruli and a transient proteinuria. The crucial evidence for the pathogenicity of autoantibodies is the transfer of the identical disease by donor autoantibodies within the same species. Progressive glomerulonephritis was transferred first by cross-circulation 7 and then by serum from nephritic donor sheep to well

recipients.5'8

Because the GBM is exposed to blood through the endothelial fenestrae and because adult antigenic determinants are present in fetal GBM, we postulated that GBM antigens might be present in the blood in both fetal and adult life.1'5 This assumption was confirmed when traces of GBM-like Publication sponsored by the Registry of Comparative Pathology of the Armed Forces Institute of Pathology and supported by Public Health Service Grant RR-00301 from the Division of Research

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Figure 1-Cryostat sections of nephritic sheep kidney stained with fluorescent-conjugated rabto sheep IgG. Note

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bit antiserum the sharp, smooth, linear, cons J_{ tinuous deposit of IgG along the . peripheral GBM. There is slight widening and increased intensity of staining along the mesangial ) Faint staining is present GBM. the tubular basement along membrane of some proximal tumem buresbut not along Bowman's

capsule. (X340)

antigenic material were found in rat and human serum.9 They may be present in all animals. We propose therefore that sheep have a low-zone tolerance to autologous GBM; that is, T cells are tolerant to autologous GBM, but B cells are still responsive.10 This state of tolerance is precarious, and the T-cell deficit can be circumvented by immunizing the sheep with a cross-reacting (heterologous) GBM and CFA. In this case, the sheep forms autoantibodies that are reactive only with the antigenic determinants shared by the sheep's own GBM and the injected heterologous GBM.3'4 Although tolerance to autologous GBM in sheep was easily broken by injections of either heterologous or homologous GBM with CFA, it was not broken by injections of autologous GBM and CFA.5 " Even though the kidney shares antigens with other organs and Group A streptococci, injections of nonrenal tissues such as human placenta, heart, synovia, and tonsil and whole, killed, Group A streptococci, together with CFA, did not induce anti-GBM nephritis.'2 Injections of human GBM without CFA also did not.' By contrast, injections of human LBM and CFA did induce typical anti-GBM nephritis in sheep.'2 Autoantibodies against sheep GBM were eluted from nephritic sheep kidneys. Absorption of the eluates with sheep or human LBM or GBM removed all autoantibodies against sheep GBM, indicating that the only autoantibodies formed were against antigenic determinants shared by all four tissues.'2 The use of human LBM in this model might explain the presence of nephritis in Goodpasture's syndrome, a pulmonary-renal syndrome in which hemorrhagic lung lesions frequently precede the onset of glomerulonephritis.'3"14 However, none of the sheep injected with either human GBM or LBM with CFA developed gross lung hemorrhages, despite the presence of autoantibodies that reacted with sheep LBM.5"12 In nephritic sheep, the clinical course is usually fulminating, with proteinuria and azotemia beginning, on the average, 3 weeks and 2 weeks, respectively, before death. The enlarged kidneys are covered with pe-

techiae.' The glomeruli are in various stages of exudative, hemorrhagic, necrotizing, and extracapillary (crescent) proliferative changes (Figure 2).56 The characteristic linear staining by IF for IgG along the GBM is evidence of anti-GBM autoantibodies (Figure 1 ) 5,8 The diagnosis is confirmed by identifying autoantibodies to sheep GBM in the serum or renal eluates.3 5,8,12

Comparison With Human Disease

The kidney lesions are similar in sheep and man. The early and/or mild glomerular changes in man are usually focal and segmental, and there is sometimes, within one kidney, an evolution (heterogenicity) of glomerular lesions, from essentially normal to obliterated glomeruli.'4 The chief difference between the experimental disease and Goodpasture's syndrome is the absence of lung hemorrhage in the experimental animal."2 In man the diagnostic linear staining is only presumptive evidence of anti-GBM antibodies. Linear staining may also be found in diabetes, periarteritis, and systemic lupus erythematosus; and "linear accentuation" may be present in normal kidneys at autopsy.13 It is best to confirm the diagnosis by identifying anti-GBM antibodies in serum and renal eluates by IF or radioimmunoassay.13 The specificity of the autoantibodies in man is more restricted than in animals. About a third of the cases in man either do not have C3 or have only a granular pattern of deposit. 3

Figure 2-Kidney section from a nephritic sheep. The glomerular tuft is disrupted and compressed by the exuberant fibroepithelial proliferation (crescent), obliterates Bowman's space and fuses with the periglomerular interstitium, making which difficult a delimiting of the glomerulus. Polymorphonuclear cells are infiltrating the tuft and crescent. Some erythrocytes and protein exudate are in the crescent. There is marked interstitial infiltration by mononuclear cells and some polymorphonuclear cells and edema. degenerative changes and casts in the lumens of tubules are evident. (H&E,Tubular-epithelial X360)

Problems needing solution are the etiology of the autoimmune response, improvements in therapy, the role of anti-basement-membrane antibodies in lung hemorrhage, the mechanism of C3-independent pathways of glomerular injury, and the presence of linear deposits of antiGBM antibodies without glomerular disease. Usefulness of the Model

This was the first model in which autoantibodies were shown to induce disease. It led directly to identification of anti-GBM human nephritis and should be useful for studying problems relevant to human disease, such as those listed above, as well as factors in progressive disease, the transfer of disease by serum, and the implications for tolerance and autoimmunity theory. Although the sheep is the most susceptible species, a similar anti-GBM disease may be induced in goats and monkeys and, less consistently or severely, in rats and rabbits.5 Most adult sheep have normally a primarily mesangial, granular deposit of IgG and CS and hypercellular glomeruli, which one should not confuse with the experimental anti-GBM disease.5 15 References 1. Steblay RW: Glomerulonephritis induced in sheep by injections of heterologous glomerular basement membrane and Freund's complete adjuvant. J Exp Med 116:253-272, 1962 2. Steblay RW, Rudofsky U: Specificities of autoantibodies induced in sheep by injections of homologous glomerular basement membrane and Freund's adjuvant. Fourth International Congress on Nephrology, Stockholm, Sweden, June 22-27, 1969, p 354 3. Steblay RW, Rudofsky U: In vitro and in vivo properties of autoantibodies eluted from kidneys of sheep with autoimmune glomerulonephritis. Nature (Lond) 218:1269-1271, 1968 4. Rudofsky U, Steblay RW: In vitro and in vivo properties of autoantibodies (AAb) against sheep glomerular basement membrane. Fed Proc 27:621, 1968 5. Steblay RW: Studies on experimental autoimmune glomerulonephritis: Their relevance to human disease. First International Convocation on Immunology, Buffalo, New York, 1968. Edited by NP Rose and F Milgrom. Basel, Karger, 1969, pp 286-299 6. Lerner RA, Dixon FJ: Transfer of ovine experimental allergic glomerulonephritis (EAG) with serum. J Exp Med 124:431-442, 1966 7. Steblay RW, Rudofsky U: An experimental model relevant to recurrent autoimmune glomerulonephritis in transplanted human kidneys. Transplant Proc 1:609613, 1969 8. Steblay RW, Rudofsky U: Further evidence for autoantibodies as mediators of experimental autoimmune nephritis in sheep. J Lab Clin Med 68:1021, 1966 9. McPhaul JJ Jr, Dixon FJ: Immunoreactive basement membrane antigens in normal human urine and serum. J Exp Med 130:1395-1409, 1969 10. Weigle WO: Immunologic unresponsiveness, Adv Immunol 16:61-122, 1973 11. Steblay RW: Studies on autoimmune nephritis in sheep and rats: IV. The relative efficacy of heterologous, homologous and autologous glomerular basement membrane (GBM) in inducing nephritis. Fed Proc 26:531, 1967 12. Steblay RW, Rudolfsky U: Autoimmune glomerulonephritis induced in sheep by injection of human lung and Freund's adjuvant. Science 160:204-206, 1968 13. Wilson CB, Dixon FJ: Anti-glomerular basement membrane antibody-induced glomerulonephritis. Kidney Int 3:74-89, 1973 14. Benoit FL, Rulon DB, Theil, GB, Doolan, PD, Watten RH: Goodpasture's syndrome: A clinicopathologic entity. Am J Med 37:424-444, 1964 15. Lerner RA, Dixon FJ: Spontaneous glomerulonephritis in sheep. Lab Invest 15:1279-1289, 1966

Animal model of human disease: anti-glomerular-basement-membrane glomerulonephritis.

ANIMAL MODEL OF HUMAN DISEASE Anti- Glomerular- Basement- Membrane Glomerulonephritis Animal Model: Experimental Autoimmune Anti-Glomerular-Basement...
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