The Immunologic Basis of Human Renal Disease

Symposium on Pediatric Nephrology

The Immunologic Basis of Human Renal Disease Edmund J. Lewis, MD.,* and William G. Couser, MD.**

The pathogenesis of glomerulonephritis in man has long been suspected to be the result of immune mechanisms. Schick observed that the nephritis associated with scarlet fever or with tonsillitis did not appear during the acute stages of infection, when streptococci or streptococcal products would be expected to be present in greatest abundance. He suggested that the lapse of time between symptomatic infection and the onset of nephritis might represent the period required for the development of hypersensitivity of the host to remaining organisms. 20o Von Pirquet first called attention to the similarity between the latent period seen before the development of poststreptococcal glomerulonephritis and the lag period between the injection of horse serum and the development of serum sickness. He implied that glomerulonephritis was the result of the interaction of microorganisms with antibodies which were produced during the latent period. 239 ,240 This analogy was supported by the recognition of diffuse glomerulonephritis similar to that seen in postscarlatina nephritis as part of the serum sickness syndrome in man. 191 During the last 50 years considerable evidence has been put forth to show that glomerulonephritis can be produced in animals by immune mechanisms. Immunopathologic studies have provided means for the interpretation of the lesions seen in human renal disease in the light of our detailed understanding of experimentally induced glomerulonephritis. I t is the purpose of this review to consider the present state of knowledge regarding the role of immune mechanisms in the pathogenesis of renal damage in diseases of man (Table 1).

MECHANISMS CAUSING NEPHRITIS Antigen-Antibody Complexes and Experimental Serum Sickness In 1913 Longcope 139 reported the presence of glomerular abnormalities and proteinuria in rabbits and dogs who had received repeated ':'Assistant Professor of Medicine, Harvard Medical School; Chief, Renal Division, Thorndike Memorial Laboratory, Harvard Medical Unit, Boston City Hospital :'Research Fellow, Harvard Medical School and Thorndike Memorial Laboratory Supported by grants AI 08542 from the National Institutes of Health, U. S. Public Health Service, and RR-76 from the General Clinical Research Centers Program, National Institutes of Health

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Renal Diseases Associated with Immune Pathogenetic Mechanisms

Glomerulonephritis due to the deposition of antigen-antibody complexes on the glomerular basement membrane Serum sickness nephritis Systemic lupus erythematosus Poststreptococcal glomerulonephritis Rapidly progressive and chronic glomerulonephritis Chronic hypocomplementemic glomerulonephritis Cryoglobulinemia Henoch-Sch6nlein syndrome (anaphylactoid purpura) Glomerulitis associated with some infections (bacterial endocarditis, malaria, syphilis) Glomerulitis associated with viral diseases (mumps, vaccinia, varicella, adenoviruses, ECHO 9, Coxsackie B3, LCM) Glomerulonephritis associated with anti-glomerular basement membrane antibodies Goodpasture's syndrome Rapidly progressive and chronic glomerulonephritis Renal tubular acidosis associated with hyperglobulinemia and immune disorders

injections of small amounts of foreign protein such as horse serum or egg albumin. While the evidence that has accumulated suggests that Longcope most likely produced glomerulonephritis in these animals, his published photomicrographs failed to support this contention. Thirty years later, Rich and Gregoryl95 observed glomerulonephritis with morphological similarity to human disease in rabbits who were sensitized to horse serum protein. Hawn and JanewayS5 and Germuth 69 extended these observations to show that the glomerular changes in serum sickness appeared during the period when antigen was rapidly being removed from the circulation and before the appearance of free antibody. It was assumed that the lesions were related to the development of an immune reaction involving antigen-antibody interaction in the circulation, much as had been implied by von Pirquet 239 in 1911. Benacerraf et al. IS and McCluskey et al. l4l • 143 provided further evidence that the glomerular lesion was induced by antigen-antibody complexes, by infusing preformed complexes into animals in order to produce glomerulitis similar to that of serum sickness. Dixon et a1. 49 demonstrated that the localization of antigen and gamma globulin in the glomerulus coincided with the phase of immune elimination of the antigen from the serum, further indicating that the lesion is the result of antigen-antibody complexes. It is now clear that several conditions must be satisfied in order to induce experimental serum sickness nephritis (Table 2). McLean et aU 46 observed that rabbits given daily injections of horse serum for prolonged periods of time developed chronic glomerular lesions in a large proportion of instances. It has been shown that the amount of antibody which the animal forms in response to the foreign antigen is critical to the development of nephritis. 4s Dixon et al. 4S extended previous studies, utilizing the chronic administration of foreign antigen, and concluded that the development of chronic glomerulonephritis in these animals was determined by the nature of the antibody response. Using

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Events Leading to the Development of Immune Complex-Mediated Glomerulonephritis

1. Quantitative antibody response which leads to the formation of soluble antigen-

antibody complexes formed in antigen excess.'" 2. N onprecipitating antibody formed in response to the foreign antigen.'" 3. Localization of immune complexes within the glomerular capillary wall, under the influence of vasoactive amines and hydrodynamic factors. 37 • "8. 40. "2 4. Entrapment of immune deposits" and phagocytosis of deposited material by mesangial cells. 12 • 50. 71,225 5. Fixation of complement components by immune complexes."!' HI. " " 6. Production of leukochemotactic factor(s).""-245 7. Basement membrane damage produced by the release of leukocyte proteolytic enzymes.:m, 104

bovine serum albumin or bovine gamma globulin as the antigen to produce nephritis in rabbits, these workers found that the antibody response to daily antigen administration varied among animals: (1) Some animals produced large amounts of antibody. These animals tended to develop the lesion of acute serum sickness, which resolved despite continued antigen administration. There was no tendency to have persistent circulating soluble complexes in these animals because an excess of antibody was present in the circulation and the condition of antigen excess, which accounts for soluble complex formation, did not occur. (2) Animals which did not form antibody to the antigen did not develop glomerulonephritis. These animals cleared injected antigen from the circulation slowly, without forming antigen-antibody complexes. (3) Those animals which formed relatively little antibody in response to a relatively large amount of circulating antigen formed soluble antigen-antibody complexes in antigen excess. These animals developed chronic glomerulonephritis morphologically similar to the lesion seen in human disease. In addition to the quantitative aspects of the antibody response, the qualitative characteristics of the antibody formed may play an important role in the ultimate deposition of the immune complex along the glomerular basement membrane. Pincus et al,187 have shown that nonprecipitating antibodies to bovine serum albumin are more likely to be associated with the development of experimental chronic glomerulonephritis induced by the chronic administration of foreign protein to rabbits, than are antibodies that induce immune precipitation. Notwithstanding the importance of the quality and quantity of the antibody response in the development of immune-complex nephritis, other factors are required in order to promote the localization of the complexes in the vessel walls (Table 2). Thus Cochrane 37 . 38 observed that complexes tend to localize in vessels that have been pretreated with histamine, indicating that a factor (or factors) which increases vascular permeability permits the deposition of complexes. In addition, rabbits treated with agents which prevent the release or action of serotonin or histamine, such as antiplatelet antiserum, antihistamine (chlorprophenpyridamine maleate), or antiserotonin (methysergide maleate) are protected from the localization of immune complexes in the glomerulus and the development of glomerulitis. ll2 These authors also showed that

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hydrodynamic factors play a role in immune complex localization by demonstrating that areas of high turbulence had a predilection for the inflammatory lesions of serum sickness. The ability of immune complexes to deposit in vessel walls is also a function of the size of the complex. Soluble complexes with sedimentation rates greater than 19S tend to become trapped in vessel walls and are associated with the development of vascular and glomerular lesions. 40 These deposited complexes have been shown to contain antigen, antibody, and the C3 component of complement, using immunofluorescent microscopyYl, 142, 157, 242 Examination by electron microscopy characteristically reveals the deposition of electron-dense deposits, representing trapped complexes, along the epithelial side of the glomerular basement membrane (Figs. 1 and 2).7,58,59 The mechanisms whereby deposited immune complexes induce glomerular injury appear to relate in part to the ability of these complexes to fix complement. Immunofluorescence studies reveal the deposition of complement component C3 in the lesions of acute serum sickness. 111 Unlike the clear observations which have been made in nephrotoxic antibody-induced nephritis 81 (see p. 476), animals have not been chronically depleted of complement in order to determine the

Figure 1. Immunofluorescent photomicrograph from a patient with acute poststreptococcal glomerulonephritis showing the finely granular localization of IgG along the capillary basement membrane and in the mesangium. A similar pattern was noted when the biopsy was examined for the presence of the third component of complement (beta-lC/beta-lA globulin). An identical picture is seen in experimental acute serum sickness, where these deposits represent antigen-antibody complexes. The inset shows a high power magnification of a capillary loop, IgG deposits are noted along the basement membrane.

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Figure 2. Electron micrograph from a patient with acute poststreptococcal glomerulonephritis. The arrows point to typical subepithelial electron dense deposits seen in this disease as well as in experimental acute serum sickness. EP, epithelial cell; US, urinary; BM, basement membrane; E, endothelial cells; CAP, capillary. (Courtesy of Dr. R. Cotran.)

effect on the pathogenesis of the serum sickness lesion. The importance of complement in serum sickness is therefore based upon the known role of complement in other hypersensitivity reactions such as the Arthus phenomenon. 111,221,242 The activation of this latter system in turn causes elaboration of potent leukocyte chemotactic principles,28, 41, 221, 243-245 The resulting inflammatory exudate presumably results in damage to the glomerular basement membrane and will be discussed below (p, 476).

Anti-Glomerular Basement Membrane Antibody-Induced Nephritis Although immune mechanisms were seriously considered to be the basis for renal injury in nephritis from the early work of Longcope,139 the failure to provide convincing morphological evidence that the characteristic lesion of human glomerulonephritis could be experimentally reproduced cast some doubt upon this hypothesis, In 1934 MasugP55 found that glomerular damage similar to that of human disease could be produced by immune means, Masugi immunized rabbits and ducks with rat kidney tissue and produced glomerulonephritis in rats by injecting this rabbit or duck anti-rat kidney antiserum, These experiments proved that specific antibody directed against kidney tissue could be responsible for the induction of glomerulonephritis, Krakower and Greenspon 120 demonstrated that the heterologous antibodies capable of inducing this

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experimental model of glomerulonephritis were directed against antigens which resided in the glomerular basement membrane. The induction of glomerulonephritis using heterologous antibody to glomerular basement membrane obviously has no direct relationship to naturally occurring disease. However, Steblay 215. 216 was able to show that sheep could be induced to make autologous anti-glomerular basement membrane antibody if immunized with glomeruli from another species. The explanation for this phenomenon is based upon the similarities in the antigenic make-up of the glomerular basement membrane among several species. Thus, antibodies against the foreign glomeruli are capable of fixing to the host animal's own glomeruli and inducing nephritis (Fig. 3). Based on Steblay's observations, one can speculate that nephritis due to anti-glomerular basement membrane antibodies could occur naturally due to several possible mechanisms. 1. The exposure of the immune system to an endogenous "self-antigen" to which it is not ordinarily exposed and therefore not tolerant could result in the production of "auto-antibodies." It is possible that antigens residing in the glomerular basement membrane are not usually available to the immune system. Release of glomerular basement membrane antigens into the circulation could therefore lead to the formation of anti-glomerular basement membrane antibodies. It is of interest that the injection of normal rabbit urine into rabbits provokes nephritis owing to the production of anti-glomerular basement membrane antibodies. 129 The immunizing agents in this instance are soluble glycoproteins with antigenic characteristics of glomerular basement membrane and with molecular weights of 70,000 to 100,000. These glycoproteins are present in small amounts in normal urine. 2. A glomerular antigen which has been changed chemically by a biological agent, such as a virus, could appear foreign to the immune system and result in the development of anti-glomerular basement

Figure 3. Immunofluorescent photomicrograph taken from a guinea pig who developed experimental autoimmune glomerulonephritis, similar to that described in sheep by Steblay, after being immunized with human glomerular basement membrane antigen. The typical linear pattern of autologous gamma globulin deposition is seen along the glomerular basement membrane. This is typical of the deposition pattern of anti-glomerular basement membrane antibodies. 250)

ex

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membrane antibodies. There is no evidence that this phenomenon occurs naturally. 3. Antibodies to a foreign antigen which has an antigenic similarity to glomerular basement membrane, thus allowing immunologic cross reactivity, could lead to the development of anti-glomerular basement membrane antibodies. It is known that a cell wall antigen present in the streptococcus cross reacts with antibody made to glomerular basement membrane glycoprotein, and the opposite is also true. 150 . 152 Thus the streptococcus conceivably could induce nephritis in a manner similar to the heterologous glomeruli which Steblay used. Immunopathologic studies of poststreptococcal glomerulonephritis do not support this latter explanation as the pathogenesis of acute poststreptococcal glomerulonephritis (as discussed on page 486). The possibility that anti-glomerular basement membrane glomerulonephritis could result from streptococcal infection in rare instances remains extant. 4. Antibodies to a native nonrenal antigen which leads to the formation of antibody that cross reacts with glomerular basement membrane could also lead to anti-glomerular basement membrane mediated nephritis. Seegal and Loeb 206 showed that antiserum produced against placental tissue was capable of causing nephrotoxic nephritis. The antigenic similarity between several organs is emphasized by the fact that antiserum to placenta, aorta or lung can cause nephrotoxic nephritis in the same manner as anti-kidney antiserum. 15 . 20. 217. 223 The sequence of events which follows the fixation of nephrotoxic (anti-glomerular basement membrane) antibody to glomeruli has ·been noted (p. 476). The fixation of complement, leukochemotaxis, and subsequent glomerular basement membrane damage has been well described in nephrotoxic (Masugi) nephritis. BI The pattern of antibody deposition in anti-glomerular basement membrane mediated disease is quite distinctive and can be clearly differentiated immunochemically and morphologically from nephritis associated with antigen-antibody complexes, using either immunofluorescence Inicroscopy or electron microscopy. In this form of nephritis, the antibody deposits uniformly along the endothelial side of the basement membrane. Thus specific fluorescent staining for deposited antibody demonstrates a continuous, linear staining of the basement membrane (Fig. 4).B1. 156.17B Electron-dense deposits along the endothelial surface of the basement membrane are seen on electron Inicroscopy in animals who have nephrotoxic (Masugi) nephritis,6. Bin contrast to the electron-dense subepithelial humps in immune complex-induced disease.7. 5B. 59

MEDIATORS OF RENAL DAMAGE IN GLOMERULONEPHRITIS Although it is well established that mechanisms for glomerular damage analogous to those which occur in experimental models operate in human renal disease, histologic and immunopathologic studies have

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Figure 4. Fluorescent staining for IgG in a biopsy from a patient with rapidly progressive glomerulonephritis, who was shown to have circulating antiglomerular basement membrane antibodies. The typical linear pattern of deposition of these antibodies is present. An identical distribution of heterologous antibody to glomerular basement membrane occurs in experimental nephrotoxic nephritis. (250 x)

revealed that the pathogenesis of human renal diseases may be considerably more complex than that of experimental models. Thus, for example, although the pathogenesis of lupus glomerulonephritis appears almost certainly to be the result of antigen-antibody complexes which are composed of DNA and anti-DNA,4,1I7 there is a broad spectrum of histologic lesions and clinical courses which may be seen in lupus nephritis. 209 Baldwin et alY have described three typical patterns of glomerular lesion in lupus nephritis: (1) focal proliferative nephritis, (2) diffuse proliferative nephritis, and (3) membranous nephritis; all of these are associated with the glomerular deposition of IgG. Conversely, Koffler et al. 1l5 have reported finding immunoglobulin deposits in glomeruli without attendant clinical or histologic abnormalities in patients with systemic lupus erythematosus. The differences in morphologic lesions, clinical severity, and clinical course in patients with glomerular disease has stimulated investigation into possible differences in the pathogenetic mechanisms of renal damage operative in human disease.

Immunoglobulins I t is clear from the above evidence that glomerular inflammation can be induced by two separate mechanisms: (1) the deposition of antigenantibody complexes (in this instance the antibody is directed against an antigen which is non glomerular in nature), and (2) the fixation of specific antibody directed against antigens located in the glomerular basement membrane. The role of the magnitude of the antibody response in the predilection of an experimental animal to the development of chronic immunecomplex-induced glomerulonephritis has been discussed (page 468). It is clear that the formation of circulating soluble antigen-antibody complexes, formed in antigen excess, is required for the development of the chronic serum sickness lesion. 48 Complexes with a sedimentation con-

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stant greater than 19S appear to have the greatest tendency to localize in vascular walls. 40 Another important factor in the development of immune complexes which tend to deposit on the glomerular basement membrane is the quality of the antibody, nonprecipitating antibody being associated with the experimental chronic glomerular lesion. 187 There appear to be other factors involved in the determination of the nature of the glomerular lesion which occurs after immune complexes develop, as either proliferative or membranous glomerulonephritis may occur in association with immune deposits. 7o It is possible that the biological characteristics of the antibody which deposits because of either of the above mechanisms may playa role in determining the extent or type of glomerular damage which occurs. Thus the induction of nephrotoxic nephritis using avian antiserum, as originally reported by Masugi, is not associated with the immediate onset of a glomerular inflammatory response. 238 Although these antibodies localize on the glomerular basement membrane, they are unable to fix complement and therefore do not initiate leukocyte chemotaxis and inflammatory exudation. In contrast, nephrotoxic antiserum produced in rabbits is capable of fixing complement and initiating glomerular inflammation. 81 There is a known variability for different classes of antibodies to fix complement within a given species. It is known that two subgroups of gamma globulin exist in the guinea pig. 179 Each of the subgroups is capable of interacting with the complement system. Guinea pig gamma2 antibody is capable of activating the complement cascade in the conventionally accepted manner, with fixation of C1 and subsequent activation of C4, C2, and C3 through C9. Guinea pig gammal antibody activates the C3 to C9 portion of the complement system, not involving C1, C4, or C2. Thus, the two subgroups of gamma globulin may have different potentials for inducing inflammation, based upon their complement fixing properties. Similarly there are five classes of immunoglobulins in man: 64 IgM, IgG, IgA, IgD and IgE. Antibodies comprised of IgM and some IgG antibodies fix complement, while IgA, IgD, and IgE antibodies do not. In addition, four subgroups of gamma G globulin exist in man,l7l termed yG1, yG2, yG3, and yG4. Gamma G1 and yG3 fix complement well, yG2 fixes complement poorly and yG4 does not fix complement. l03 • 169 Thus the immunoglobulin or gamma G subgroup composition of antibody which is involved in either a deposited immune complex or in the makeup of anti-glomerular basement membrane antibody could determine the extent of complement fixation. Berger 20 has demonstrated that some patients with a focal nephritis have non-complement-fixing IgA deposited in the glomerulus and suggests that this accounts for the benignity of the lesion. It is known that the subgroups of IgG which deposit in a given case of glomerulonephritis vary from patient to patient. 131 It is therefore also possible that part of the explanation for the spectrum of glomerular lesions seen in human renal disease is dependent upon the biological characteristics of the gamma G globulin which comprises the antibody involved in the reaction.

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The Complement System The complement system of man comprises nine components whose interactions are required for immune hemolysis. These components (in order of their reaction in complement fixation) are termed CI, C4, C2, C3, C5, C6, C7, C8, and C9. The first component of complement, CI, is composed of three subunits, Clq, Clr, and CIs. The Clq subunit is that portion of the molecule which interacts with immunoglobulin molecules. The fixation of Clq to IgG or IgM results in the activation of CIs to CI esterase. CI esterase then acts on C4 and C2 to form a C4-2 complex which can enzymatically cleave the C3 molecule. At this stage of complement activation the immune adherence and immune phagocytosis reactions occur. In addition, the enzymatic cleavage of C3 liberates the fragment anaphylotoxin, which is a histamine liberator. 28 The involvement of C3 in the reaction is followed by the fixation of C5, C6, and C7, which are responsible for leukochemotaxis.243.244 Complement components C3 28 and C5 245 may also be individually responsible for leukocyte chemotaxis. The attraction of polymorphonuclear leukocytes and release of lysosomal enzymes into the local area of inflammation can cause glomerular damageY' 81 It is noteworthy that polymorphonuclear lysate or polymorphonuclear cytoplasmic granules are capable of causing proteolysis of glomerular basement membrane in vitro,39. 104 providing a mechanism for immunologically induced polymorphonuclear-mediated glomerular damage. The activation of terminal components C8 and C9 is required for immune hemolysis. The role of these latter components in damage to the glomerulus is unclear. The complement system appears clearly to be involved in the glomerular damage attendant to experimental nephrotoxic (Masugi) glomerulonephritisY' 238 Nephrotoxic antibody is capable of fixing complement in vivo 81 and in vitro. 31 Glomerular inflammation and damage are significantly diminished by either the diminution of the serum complement level or the depletion of polymorphonuclear leukocytes. 81 Thus, despite the fixation of specific antibody directed against glomerular basement membrane antigens, one may mitigate the nephritogenic potential of the reaction by interrupting the complementpolymorphonuclear leukocyte mediators of inflammation. It must be emphasized however that there is strong evidence that glomerular damage which is not complement mediated can be demonstrated in both the immune complex and anti-glomerular basement membrane (nephrotoxic) models. Nephrotoxic serum injected into complement depleted rats causes proteinuria and a mild glomerular lesion, as does avian nephrotoxic antiserum (e.g., duck anti-rat kidney antiserum) which does not fix complement. 81 Neutrophil depletion in serum sickness does not prevent glomerular lesions;111 however, the questionable adequacy of the prolonged neutropenia in the latter experiments makes the interpretation of these results difficult. U sing the techniques of immunofluorescence microscopy, C3 (/31C globulin) can be demonstrated to deposit in a lumpy fashion in immune complex-induced disease and in a linear fashion in anti-glomerular basement membrane induced nephritisY' 111 Analogous patterns of deposition have been found in human glomerulonephritis (Fig. 5),118,122,130

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Figure 5. Immunofluorescent staining for the fourth component of complement (beta1 E globulin) in the biopsy of a patient with systemic lupus erythematosus. The C4 is deposited in a granular pattern along the capillary wall and is also deposited in the mesangium. A similar pattern of deposition of complement components Clq and C3 was also found in the glomeruli in this patient. (x 250)

suggesting that the complement system is involved in human nephritis in much the same manner as has been shown in the experimental model. More recent investigations using antisera to other complement components, particularly C1q and C4, have shown that the complement components also may be found deposited on the glomerulus in human nephritis.4, 131 The involvement of the complement system in human glomerulonephritis has been suspected since Gunn's observation in 1914 of low serum complement levels in postscarlatina glomerulonephritis. 78 Depressed levels of serum complement have also been found in lupus glomerulonephritis and in other forms of acute, subacute, and chronic glomerulonephritis. 57, 61, 82. \25,202,236 The implications of these observations are discussed later in this review (see p. 488).

The Coagulation System A substantial amount of evidence has accumulated which implicates the coagulation system as a mediator of glomerular damage in both experimen tal and human glomerulonephritis. 144 . 234. 235 The mechanisms whereby hypersensitivity reactions lead to local thrombotic phenomena are not completely understood. Robbins and Stetson 196 have shown that immune complexes can augment the coagulation process in vitro. In addition to the more conventional forms of inflammatory tissue damage produced by antigen-antibody complexes, seen in acute serum sickness and the Arthus phenomenon, intravascular coagulation can be produced

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in vivo under the proper experimental conditions. Lee 127 was able to induce intravascular fibrin polymerization in vivo and produce renal cortical necrosis in animals given antigen-antibody complexes intravenously, provided that the animal had undergone reticuloendothelial blockade using thorium dioxide. McCluskey and his co-workers have shown that the character of the glomerular damage which occurs in nephrotoxic (Masugi) nephritis depends upon the interaction of the coagulation process as well as the deposition of specific antibody and complement in the glomerulus. These investigators demonstrated that the characteristic proliferative glomerulonephritis, with epithelial crescent formation, seen in this experimental model is intimately associated with the deposition of fibrin and fibrinogen derivatives. Immunofluorescence studies of the glomeruli of these animals showed accumulation of fibrin and fibrinogen between the epithelial cells of the crescents and within the intracapillary cells of the mesangium. In contrast heterologous gamma globulin is deposited only along. the basement membrane. 234 , 235 Treatment of the animals with the anticoagulant warfarin prior to the administration of heterologous nephrotoxic antibody resulted in the expected antibody deposition along the glomerular basement membrane and proteinuria; however, the characteristic proliferative component of the nephritis in this model appeared to be intimately related to local fibrin deposition within the glomerulus and could be prevented by pretreatment with the anticoagulant drug. The coagulation process may be involved in human disease in a manner analogous to experimental nephrotoxic nephritis. The first observation that the proteins of the coagulation system might be involved in human glomerulonephritis was made by Ellis,56 who demonstrated a substance with the tinctorial characteristics of fibrin to be present in the glomerular crescents. These observations were confirmed by immunofluorescent antibody studies of human renal biopsies which often show fibrinogen deposition in the mesangium, within capillaries and occasionally in glomerular crescents, a pattern identical to that of the experimental nephrotoxic model (Fig. 6). The deposition of fibrin or fibrinogen derivatives has been demonstrated in acute and subacute glomerulonephritis and lupus nephritis by Paronetto and Koffier 181 and McCluskey et al,144 Stiehm and Trygstad 222 have demonstrated the presence of split products of fibrinogen in the serum of children with poststreptococcal glomerulonephritis, lupus nephritis, hemolytic-uremic syndrome, and chronic nephritis, suggesting that local intravascular coagulation occurs in the glomerulus in these diseases. The significance of the coagulation process in human glomerular disease remains a subject for further study. Cellular Hypersensitivity The importance of humoral antibodies in the pathogenesis of experimental glomerulonephritis has been discussed. The broad spectra of clinical courses and histologic lesions seen in the human nephritides has raised the question of whether immune damage may be mediated by

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Figure 6. Specific immunofluorescent staining for fibrin is noted in an epithelial crescent in a glomerulus from a patient with rapidly progressive glomerulonephritis. Only traces of fibrin are noted within the glomerular tuft. (Courtesy of Dr. T. Cavallo.)

mechanisms other than those classically involved in the experimental acute serum sickness or nephrotoxic nephritis reactions. The role of cellular hypersensitivity in human renal disease is unknown. The possibility that sensitized lymphocytes may be involved in the mediation of renal damage in an animal model (autologous immune complex nephritis, Heymann) has been suggested by transfer experiments. 96 , 98 However, the lesion produced by these investigators may have been produced by the transfer of antibody producing cells, rather than sensitized lymphocytes. Other investigators have been unable to confirm the transfer of autologous immune complex nephritis (Heymann) (see p. 481) without the transfer of serum and immunizing antigen as well as lymphocytes. 73 The role of sensitized lymphocytes in human renal disease has become a subject of some controversyY' 197 It is not uncommon to find dense collections of lymphocytes and mononuclear cells in the renal cortex in the human nephritides, particularly rapidly progressive glomerulonephritis, Goodpasture's syndrome, and lupus nephritis. 21 . 44, 92 In addition, multinucleate giant cells of the Langhans variety similarly have been described in the glomeruli of affected kidneys.133 Although these histologic abnormalities may not represent a specific cellular immune reaction, their presence has raised this possibility. In vitro evidence for the existence of cellular hypersensitivity to glomerular basement membrane antigens using the macrophage migration inhibition assay has been shown in some patients with glomerulonephritis.197 Bendixen 19 had previously used an in vitro assay based upon the migration of peripheral leukocytes to determine evidence of sensitization to an antigenic preparation of fetal renal homogenate in some patients with glomerulonephritis. Rosenberg and David 198 have confirmed that the assay employed by Bendixen indeed measures delayed hypersensitivity. Zabriskie and his associates 255 have provided further interest in the role of sensitized cells by demonstrating that patients with

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progressive glomerulonephritis manifest in vitro cellular hypersensitivity to the particulate antigens of the streptococcus. These workers suggest that cellular hypersensitivity to the streptococcal antigen, or perhaps to a renal antigen (such as glomerular basement membrane) which is immunochemically related to streptococcal antigen, may be responsible for the continuing immunologically mediated damage in some patients with glomerulonephritis. Pirofsky et al. 188 have treated patients with Goodpasture's syndrome and other forms of nephritis with anti-human thymocyte antiserum, in order to selectively depress cellular hypersensitivity, and found that there was an improvement in the course of the disease in this group of patients. Unfortunately, therapeutic trials such as this have been uncontrolled. Similar suggestive evidence that the depression of cellular hypersensitivity ameliorates renal damage comes from the experience in renal transplantation, where the deposition of humoral antibodies to glomerular basement membrane has not necessarily resulted in the recurrence of severe glomerulonephritis. 134 , 148 Thus the role of specific cellular hypersensitivity in the mediation of renal damage in glomerulonephritis remains obscure. Similarly, the origin and role of the lymphocytes which are often noted to be present in large numbers in the renal cortex in patients with rapidly progressive glomerulonephritis and other forms of glomerulonephritis is unknown.

AGENTS CAPABLE OF INDUCING GLOMERULONEPHRITIS Viruses Investigators have explored the possibility that viral antigens are involved in the pathogenesis of glomerulonephritis through the formation of antigen-antibody complexes. Clinical glomerulonephritis with temporary impairment of kidney function has been reported in humans infected with mumps,102, 165 measles,203 vaccinia,94 Coxsackie B3 virus,29,233 varicella,253 adenoviruses,35,211 ECHO type 9 virus,254 infectious hepatitis,43 and acute lymphocytic choriomeningitis. 128 In addition, suggestion of viral etiology of human renal disease was made by Goodpasture,75 who described the association of proliferative glomerulonephritis with pulmonary hemorrhage in a patient who died of influenza. Duncan et al. 52 described "virus-like" particles in renal tissue of a patient with Goodpasture's syndrome; however, there is no convincing evidence that viruses are in fact involved in the pathogenesis of this disease (see p.496). In 1967 Baker and Hotchin lo reported that mice infected neonatally with lymphocytic choriomeningitis virus developed chronic glomerulonephritis with progressive decrease in kidney function. The glomerular lesion in these animals manifests basement membrane thickening and glomerular hypercellularity. Oldstone and Dixon 176 showed that newborn mice infected with lymphocytic choriomeningitis virus were not tolerant to this antigen and were, in fact, capable of making antibody to the virus. The glomerulonephritis which occurred in these animals was

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associated with the deposition of anti-lymphocytic choriomeningitis antibody in the glomerulus. The immunopathologic features of IgG and beta-1-C globulin deposition suggests immune complex-induced disease in these animals. Another animal model of virus-related glomerulonephritis was produced by Burch et al,'lo who inoculated suckling mice with ECHO virus and observed the development of acute glomerulonephritis 6 days after the inoculation. These authors were able to demonstrate viral antigen in the glomerulus in these mice, suggesting that a viral antigen-antibody reaction was responsible for the glomerulonephritis. Further evidence of the nature of the antibody depositing in these animals is required to determine whether antigen-antibody complexes are actually involved in the glomerular lesion seen by these investigators. A third observation correlating possible acquired viral infection with glomerular disease has been made by Lewis et al.,135 who demonstrated that hybrid mice with "allogeneic disease," induced by the injection of parental (BALB/c) strain spleen cells into the offspring FI hybrid (BALB/c X A/Jax), develop glomerulonephritis. Granular deposits of immunoglobulins, suggesting immune complex deposition, can be demonstrated along the glomerular basement membrane of these mice. The fact that mice with "allogeneic disease" are susceptible to viral infections has raised the possibility that the glomerular lesion is the result of antigen-antibody complexes involving viruses. The possibility that the nephritis is caused by other immune phenomena occurring in "allogeneic disease" and unrelated to viruses also remains a plausible explanation however. As noted above, human glomerulonephritis has been associated with viral infections in rare but well documented instances. In addition, the occurrence of glomerulonephritis in cases of viral hepatitis has been observed. 43 Gocke et al,74 have demonstrated that patients with polyarteritis nodosa with Australia antigenemia may exhibit evidence of vascular deposition of antigen-antibody complexes made up of Australia antigen and immunoglobulins. The viral nature of Australia antigen has been discussed by Blumberg. 27 Thus there is strong evidence for the role of immune complexes involving the Australia antigen in human disease. The possibility that other forms of vasculitis and glomerulonephritis may be caused by viral agents involved in immune reactions awaits further study. Autologous Antigens HEYMANN MODEL. In 1959 Heymann 97 described the induction of membranous glomerulonephritis in rats immunized by the intraperitoneal administration of autologous or homologous renal tissue in complete Freund's adjuvant. Immunofluorescent studies have shown the granular deposition of gamma globulin,175 and electron microscopic studies 59 similarly are compatible with immune complex induced nephritis. Edgington and Glassock 54 have shown that the antigen involved in the deposited complexes is derived from the apical brush border of normal proximal convoluted tubule cells. In addition, Edgington et al. 55 found that induction of this disease in rats using human tubular antigen

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resulted in the deposition of antigen-antibody complexes in which the antigen was autologous rather than human. Immunization with human antigen therefore stimulated the production of antibodies which crossreacted with autologous rat antigen. The formation of antigen-antibody complexes indicates that the "brush border antigen" was somehow available to the circulation and capable of combining with antibody. This latter observation has raised the possibility that a similar loss of tolerance to an auto-antigen could occur in man as the result of antibody formation to a foreign or altered-native antigen. At present there is no evidence that this pathogenetic mechanism is operative in human nephritis. THYROGLOBULIN. Weigle and High 246 have shown that rabbits immunized with heterologous thyroglobulin form antibody which crossreacts with autologous thyroglobulin. Immune complex-induced nephritis can occur in these animals if autologous thyroglobulin gains access to the circulation at a time when circulating antithyroglobulin antibodies are present. GLOMERULAR BASEMENT MEMBRANE ANTIGENS. In contrast to the nephritis produced by the autologous "brush border antigen" and thyroglobulin models, anti-glomerular basement membrane nephritis can be induced by the immunization of animals with homologous or heterologous glomerular basement membrane,215. 216 heterologous lung basement membrane,218 and autologous or homologous urinary antigens which are antigenically similar to glomerular basement membrane glycoprotein. 129 The pathogenetic mechanisms of this nephritis are discussed on page 471.

NATURALLY OCCURRING GLOMERULONEPHRITIS IN ANIMALS Glomerulonephritis in NZB and NZB/W Hybrid Mice In 1959 Bielschowsky et al. 26 described the occurrence of a spontaneous disease in New Zealand black mice (NZB), which was characterized by Coombs-positive hemolytic anemia. The F1 hybrid of NZB and NZW mice (NZB/W) was later found invariably to develop a severe membranous glomerulonephritis associated with the presence of the lupus erythematosus cell phenomenon. 87 Burnet and Holmes 32 showed that 98 per cent of female NZB/W mice died with glomerulonephritis before 1 year of age. These authors also noted the development of antinuclear antibodies and Coombs-positive hemolytic in these mice and suggested that the renal disease had an immunological basis analogous to that of systemic lupus erythematosus in man. Lambert and Dixon 124 have shown that the development of glomerulonephritis in these mice is closely related to the formation of antinuclear antibodies which appear to be antibodies directed against DNA. Analogous to the findings in human lupus nephritis, elution of immunoglobulins deposited in the glomeruli in these mice have revealed antibody to soluble nuclear antigens. 124 Seegal et al. 204 have demonstrated evidence of pyrimidine

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nucleosides in glomerular lesions. Therefore, as appears to be the case in systemic lupus erythematosus, DNA-anti-DNA complexes appear to be deposited in this disease. The similarity between the immunologic and the histologic abnormalities in this spontaneous renal disease in mice and systemic lupus erythematosus in man may provide important insight into the nature of the latter disease. New Zealand mice appear to have a genetic predisposition to produce antibody to DNA antigens. Cerottini et al. 34 showed that NZB/W mice invariably produce high titer antinuclear antibody when immunized with DNA coupled to methylated bovine serum albumin. Mice from the strains AJAX, DBA/2, AKR, and CBA produce much lower titers of antinuclear antibody when subjected to the same immunization, and do not develop glomerulonephritis. NZB mice are known to harbor viruses 53. 15B. 190 and also appear to be usually susceptible to immunization by viruses. Lambert and Dixon l23 have shown that newborn NZB/W mice infected with either polyoma virus or lymphocytic choriomeningitis virus develop antinuclear antibody by the age of 4 months, while !nice which remained uninfected rarely have antinuclear antibody at this time. Steinberg et al.219 have shown that immunization of NZB/W mice with polyinosic-polycytidylic acid (polyI-polyC) induces the formation of anti-RNA and anti-DNA. This abnormal immune responsiveness to viral stimuli can therefore lead to the development of immune-complex nephritis through the formation of immune complexes composed of DNA-anti-DNA. Evidence derived from New Zealand mice therefore indicates that genetic factors create an altered state of immunologic reactivity allowing the production of high titers of serum "autoantibodies," perhaps influenced by viral infection. Talal 226 has reviewed the evidence regarding the basis of the immunologic abnormalities found in New Zealand mice. Aleutian Mink Disease A naturally occurring disease in Aleutian mink is characterized by hypergammaglobulinemia, polyarteritis, plasma cell proliferation, and glomerulonephritis. B4. B6 The disease has been transmitted by cell-free filtrates BB, 90. 229 and it has been assumed to be of viral origin. Electrondense deposits have been described along the glomerular basement membrane,B9.110 and fluorescent antibody studies have demonstrated gamma globulin in the mesangium of the glomerulus. IB9 These findings are not characteristic of immune complex nephritis; however, they are compatible with that interpretation. Similar distributions of immunoglobulins can be seen in acute serum sickness. 62 . 71 The antibody nature of deposited immunoglobulins is unknown. 9o The possibility that immune complex-induced nephritis related to viral disease is the basis of the glomerular lesion in these animals make this disease relevant to the study of nephritis in man. The apparent transmission of Aleutian mink disease to man has been reported. 36 These latter authors do not describe a glomerular lesion in their patient.

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HUMAN RENAL DISEASES Poststreptococcal Glomerulonephritis The clinical relationship between infection with the streptococcus and urinary abnormalities dates back to the early eighteenth centuryY3. 247 Lohlein,138 in 1907, described the occurrence of acute diffuse proliferative glomerulonephritis in patients with a recent history of a streptococcalinfection. He concluded that the pathologic lesion in these patients was the result of direct toxic action on the kidney by the streptococcus. As noted above, von Pirquet239.240 described the similarity in the latent periods preceding the onset of serum sickness and postscarlatina nephritis and suggested that a similar mechanism could be operative. Dick 45 demonstrated that beta-hemolytic streptococci caused scarlatina, thus clearly establishing a relationship between the streptococcus, postscarlatina-phenomena, and acute glomerulonephritis. The exact manner whereby streptococci are capable of inducing glomerulonephritis has not as yet been determined. The role of immunologic mechanisms in the pathogenesis of poststreptococcal glomerulonephritis has had support on the basis of several lines of evidence: 1. The similarity in the latent period preceding the onset of nephritis to that occurring in serum sickness. There is no proof at present that an identical mechanism explains these two phenomena. 2. The appearance of a low serum complement level in postscarlatina glomerulonephritis. 78 The involvement of the complement system as a mediator in immune reactions would suggest that an immunopathogenetic mechanism accounts for complement depletion. As noted below, the metabolism of the serum complement components is complex, and hypocomplementemia cannot be considered as absolute proof of an immunologic mechanism. 3. The demonstration of immunoglobulins and the C3 component of complement on the glomerulus in poststreptococcal glomerulonephritis further suggests an immunologic mechanism for renal damage. The immunofluorescence and the electron microscopic lesions of acute poststreptococcal glomerulonephritis are virtually indistinguishable from the renal lesion seen in acute serum sickness (Figs. 1,2).5,66,118.149,205 This morphologic evidence provides strong arguments for the immune complex pathogenesis of poststreptococcal glomerulonephritis. Immunoglobulins deposited in the glomeruli in this disease have not yet been isolated or characterized regarding evidence for anti-streptococcal antibody activity. Numerous investigators have attempted to produce glomerulonephritis in experimental animals using live or killed streptococci or streptococcallysates. 1ol , 113, 128, 194 Kelley and Winn 108 produced renal lesions in mice by implanting diffusion chambers containing group A hemolytic streptococci into the peritoneal cavity. The chamber was capable of retaining the bacteria, but allowed soluble bacterial products to escape. Renal lesions were produced by type 12 nephritogenic streptococci, while streptococci of a non-nephritogenic strain did not produce lesions. The

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histology of these lesions was not similar to that of human acute glomerulonephritis, however. Kaplan 107 injected streptococcal M protein intravenously into mice and showed that the protein was localized in glomerular endothelial cells. Again, the renal lesion in these animals did not appear similar to that seen in human diseases. Lindberg and VostP:lH. 1:J7. 241 have developed a model which reproduces streptococcus-related nephritis in rats; however, the morphologic picture is not that seen in human glomerulonephritis. These investigators have indicated that the implantation of a Millipore diffusion chamber containing nephritogenic group A, type 12 streptococci is capable of inducing glomerulonephritis which appears to be due to an immunologic process. Immunoglobulins are deposited on the glomeruli in these rats and eluted IgG reacted with the M protein of type 12 streptococci, but not with other streptococcal antigens. 136 Thus, there is indication that this experimental model is mediated by virtue of the deposition on the kidney of specific antibody directed agaiIist a streptococcal antigen. The possibility that antibody is depositing as antigen-antibody complexes in these animals has been considered. However, the histologic abnormalities reported by Vosti et al. 241 are not similar to those found in human acute nephritis. Electron microscopic studies of the glomeruli from these animals have failed to demonstrate electron-dense deposits, despite the fact that gamma globulin is readily identified by immunofluorescence microscopy. The resemblance of this model to disease in man is therefore only partial. Becker and MurphylS have reported the induction of poststreptococcal glomerulonephritis in rabbits using injections of streptococci isolated from patients who had acute glomerulonephritis. These investigators present convincing detailed clinical and histopathologic evidence which supports their conclusion that this disease is similar to that in man. They report that the demonstration of immunoglobulin deposition by fluorescence microscopy was inconstant in these animals. Further detailed studies using fluorescence and electron microscopy are required in order to establish the identity of the immunopathologic lesions in the model described by Becker and Murphy and in human disease. Several possible pathogenic mechanisms have been postulated on the basis of experimental models in order to explain the occurrence of acute poststreptococcal glomerulonephritis in man. 1. The non-immune cytotoxic effects of streptococcal products has been considered an explanation for glomerular damage. Holm et al. 1oo have shown that injection of rabbits with an "autolysate" from type 12 streptococcus can cause the immediate induction of hypertension and proteinuria and hematuria within a few days of the injection, thus suggesting a non-immune mechanism for renal damage. The similarity between this lesion and that of acute poststreptococcal glomerulonephritis is not convincing. The possibility that the acute glomerulonephritis is associated with those strains of the streptococcus which produced DPNase has been considered;22,192 however, Holm et al.100 were not able to relate the production of DPNase with nephritogenicity of a particular streptococcal strain.

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In most cases, the typical glomerular lesion of acute glomerulonephritis has not been reproduced in animals given injections of streptococcal products. Further, the evidence supporting an immune reaction in the glomerulus would appear to outweigh that which supports the argument that the kidney lesions are the direct toxic effect of one or more streptococcal factors. 2. The antigenic relationship of streptococcal components to antigens residing in the glomerular basement membrane has been stressed by Markowitz and Lange. 152 These authors have shown that a common glycopeptide determinant is present in the cell membrane of nephritogenic streptococcus and the glomerular basement membrane. This supports the concept that poststreptococcal glomerulonephritis is the result of an immune reaction initiated by the response of the immune system to streptococcal products. However, taking this argument to its conclusion, one would expect that the deposition of immunoglobulins in the glomerulus would be uniform along the glomerular basement membrane, as if the immunoglobulins were anti-glomerular basement membrane antibodies. The observation that immunoglobulin deposition is present in a lumpy pattern, suggesting antigen-antibody complex~s,5. 66. 161. 205 is therefore not compatible with the above explanation in typical cases of poststreptococcal nephritis. Markowitz and Lange l49 have recently published further data regarding the induction of glomerulonephritis in monkeys using antistreptococcal antibodies. These authors have shown that there is a common glycopeptide residing in the protoplasmic membrane of nephrito genic streptococcus and human glomerular basement membrane. The injection of rabbit antiserum to streptococcal protoplasmic membrane induces glomerulonephritis associated with granular, discontinuous deposits of gamma globulin in Rhesus monkeys. This evidence suggests that there may be antigenic components of the glomerular basement membrane which are not uniformly distributed along the basement membrane and would account for noncontinuity of antibody deposition in glomerulonephritis. Evidence to support this hypothesis is preliminary at present. 3. The typical lesions seen in acute glomerulonephritis on electron microscopy, consisting of electron-dense deposits on the subepithelial side of the glomerular basement membrane, and the immunofluores-· cent finding of IgG and C3 protein in the deposits has provided strong evidence that the pathogenesis of acute glomerulonephritis is analogous to the experimental serum sickness nephritis model caused by the deposition of soluble antigen-antibody complexes. Andres et al. 5 have offered evidence that streptococcal antigen is also present in these deposits. Others have failed to demonstrate streptococcal antigens and have suggested that streptococcal antigen-antibody complexes may not be involved in this lesion. 144 The finding of circulating cryoglobulins in children with acute poststreptococcal glomerulonephritis has led to the possible explanation that the complex which deposits in the glomeruli consists of IgG and an antiglobulin (rheumatoid) factor which form a cryoglobulin. 13 In view of the known diffuse glomerulonephritis in some patients with

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circulating cryoglobulins,160 this explanation must be seriously entertained. 4. Treser et al.230 have shown that fluorescein-labeled IgG isolated from the serum of a patient with acute glomerulonephritis is capable of fixing to the glomerulus of biopsies taken from patients with acute glomerulonephritis. This antibody activity can be abolished by absorbing the serum with a preparation of streptococcal plasma membranes. These authors suggest that they are demonstrating streptococcal antigens which may be involved in deposited antigen-antibody complexes. A second possible explanation is that they are demonstrating free antigens which have deposited in the glomeruli independently and not as part of an immune complex. Miller 164 and Kaplan 107 have shown that streptococcal M protein, when injected into mice, will deposit in the glomerulus and persist. Thus, one may develop antibodies to a fixed or trapped streptococcal antigen that has deposited in the glomerulus. An immune reaction to an antigen might occur several days after its localization in the glomerulus. This explanation could account for the latent period seen in poststreptococcal glomerulonephritis. Although the great majority of the patients with acute poststreptococcal glomerulonephritis improve without residual damage, a small percentage will have progressive disease, leading to uremia. 56 Dixon et al. 48 and Germuth et al. 70 have shown that the natural history of chronic serum sickness glomerulonephritis from the acute stages through chronicity can be reproduced in the experimental animal. The repeated injection of a foreign antigen such as bovine serum albumin into rabbits is capable of inducing a "chronic serum sickness reaction" in those animals which have a quantitatively poor antibody response and therefore have circulating soluble antigen-antibody complexes which have been formed in antigen excess. 48 Evidence for the continuing participation of circulating complexes in patients with acute nephritis who have progressive disease is lacking at present. The explanation for the spectrum of natural histories seen in this disease is therefore difficult to understand on the basis of antigen-antibody reactions involving streptococcal antigens or other foreign antigens which are transiently present. The immune deposits which occur in experimentally induced chronic glomerulonephritis have been shown to diminish when the administration of antigen is stopped. 48 These observations have prompted several investigators to consider the possibility that immune mechanisms other than those described in the serum sickness model, particularly those relating to cellular hypersensitivity, may playa role in the progression of renal damage in nephritis. 255 Zabriskie et al.2 55 have shown that lymphocytes from patients with progressive glomerulonephritis manifested evidence in vitro of cellular hypersensitivity to the particulate antigens of group A streptococci using peripheral leukocyte migration and DNA synthesis assays. These authors speculate that continued damage in this form of renal disease could be the result of the activity of sensitized lymphocytes, as has been discussed on page 478. The interpretation made by these authors is similar to that of Rocklin et al. 197 who have demonstrated cellular hyper-

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sensitivity to glomerular basement membrane antigens in some forms of human glomerulonephritis and similarly suggest that cellular as well as humoral hypersensitivity may playa role in the renal damage in glomerulonephritis. It is clear from the foregoing evidence that although the glomerular damage in acute poststreptococcal glomerulonephritis appears to be immunologic in nature, the mechanisms for the development of this lesion in man remain obscure.

Chronic H ypocomplementemic Glomerulonephritis Chronic glomerulonephritis in children is sometimes accompanied by low serum hemolytic complement levels.76.250 The histologic lesion characteristically seen in these patients consists of a lobular appearance to the glomerular tufts with an increase in mesangial material and an apparent thickening of the glomerular capillary walls by eosinophilic material that is contiguous with the mesangial matrix. Examination of the glomeruli using the methenamine silver technique reveals the appearance of splitting of the basement membrane by nonargyrophilic material, giving the capillary wall a "tram track" appearance. Although the initial observations of cases with hypocomplementemic glomerulonephritis suggested that the serum complement levels reflected an active immune process,248. 250 it would appear that the probable explanation for the low serum complement levels is far more complicated. Alper and Rosen 3 have shown that the third component of complement (C3) is synthesized at a depressed rate in children with this disease. Pickering et al. 184 have described the presence of a complement inactivator in the serum of patients with hypocomplementemic glomerulonephritis which acts in the complement system at or after C3. The in vitro demonstration by Spitzer et al. m that serum from patients with hypocomplementemic glomerulonephritis contains a factor which can mediate an enzymatic non-immune destruction of C3 could also account for hypocomplementemia in this disease. Several authors have shown that C3 is selectively depressed relative to the earlier complement components Clq, C4 and C2 in these patients.u9. 132. 185 The activation of the third component of complement (C3) and subsequent activation of the later components can occur in vitro without the initial fixation of Clq or activation of C4 and C2 by an antigenantibody reaction. 72 Thus, endotoxin lipopolysaccharide and cobra venom factor are capable of depleting C3 without the apparent participation of antibody molecules or Clq in the reaction.72.170 The normal levels of Clq and C4 seen in patients with hypocomplementemic glomerulonephritis despite extremely low levels of C3 could conceivably occur' through an analogous non-immune mechanism. The nature of the factor which could be responsible for either the occurrence of glomerulonephritis in these children or the triggering of the complement system in this disease is still a matter of speculation. Immunofluorescence microscopy does reveal deposition of IgG and C3 protein in a limited number of biopsies that have been investigated (Fig. 7).95 In addition, small amounts of Clq and C2 have been noted to

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Figure 7. Immunofluorescent photomicrograph of a glomerulus from a patient with membranoproliferative glomerulonephritis accompanied by chronic hypocomplementemia. The biopsy was stained for the presence of C3 (beta-lC/beta-lA globulin). A similar distribution of IgG was seen in this patient. Immunofluorescent staining for complement components C 1 q and C4 were negative in this biopsy.

be deposited on the glomerulus of several patients, suggesting the possibility that complement fixation by an immune mechanism on the glomerulus occurs in a normal fashion despite the apparent selective abnormality of C3 in the serum. It is clear that the serum complement levels in hypocomplementemic glomerulonephritis do not correlate with the clinical course of the disease. West and McAdams 249 have described a spontaneous increase in serum C3 levels in these patients with no apparent improvement in their disease. Conversely, Northway et al.173 have shown that serum C3 protein levels can remain low in patients who have apparent clinical remission of their renal disease. West et al. 24S have reported that the serum C3levels can remain low after a patient with hypocomplementemic glomerulonephritis has sustained a bilateral nephrectomy in preparation for transplantation, indicating that the kidney is not essential for the breakdown or utilization of C3. Hypocomplementemic glomerulonephritis thus is a complex syndrome which may be associated with glomerular deposits of immunoglobulins and, therefore, may be the result of immune damage. However these patients also manifest abnormalities in the serum complement system, independent of the glomerular reaction, possibly due to other than the immune utilization of complement factors.

Systemic Lupus Erythematosus The glomerulonephritis which accompanies systemic lupus erythematosus is associated with a variety of immunologic abnormalities. The

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presence of diminished levels of total serum complement (and several complement components)57. 61, 82. 125, 132,202,236 and the evidence that inactivated C3 protein can be found in freshly drawn lupus serum l68 suggest the possibility that there is in vivo utilization of complement in immune reactions, Vaughn et al,236 were the first to observe that changes in the serum complement level vary with the degree of activity of the disease in these patients, Others have made similar observations,57, 61, 82, 125, 202 The localization of immunoglobulins and complement in the glomeruli in systemic lupus erythematosus are further evidence for the immune mediation of renal damage,122, 181 Using acid elution, Freedman and Markowitz 65 isolated the immunoglobulins deposited on the glomeruli from the kidneys of patients who had died with lupus nephritis, They showed that the eluted immunoglobulin had antibody activity to constituents of the cell nucleus. Koffler et al. 117 were able to elute antibodies which react mainly with DNA from the isolated glomeruli prepared from the kidneys of patients with lupus nephritis, using deoxyribonclease. In addition, these authors showed that DNA antigen was present in the glomeruli of two kidneys with lupus nephritis, using fluorescein-tagged anti-DNA antibody. Andres et al. 4 have reported the localization of fluorescein-conjugated antisera to the pyrimidine bases thymine and cytosine in glomeruli of patients with systemic lupus erythematosus. These results suggest the presence of denatured DNA in these lesions. It therefore appears established that immune complexes, at least, in part, DNA-anti-DNA, explain the pathogenesis of lupus nephritis. Several patterns of immunoglobulin deposition in the glomeruli of patients with systemic lupus erythematosus have been described. 115, 117, 181 As noted on page 473, there are wide variations in the pattern and extent of immunoglobulin deposition from patient to patient and there is a similarly broad clinicopathologic spectrum in this disease. Although there appears to be strong evidence that immune complexes mediate the glomerulonephritic process in systemic lupus erythematosus, knowledge of the factors governing the natural history of the renal lesion is incomplete. Present evidence indicates that the pathogenesis of lupus nephritis is immune in nature; however, the etiology of systemic lupus erythematosus remains obscure. Several observations have suggested that systemic lupus erythematosus may be a response to viral infection. (1) Elevated antibody titers to paramyxoviruses, measles, and parainfluenza type 1 virus have been described in patients with systemic lupus erythematosus.1 83 (2) Circulating antibodies to RNA have been described in systemic lupus erythematosus. 201 These antibodies do not react in vitro in precipitin reactions with single-stranded RNA, which represents most mammalian RNA. Positive reactions are found when the antigen is either synthetic polyinosinic-polycytidilic acid (polyI-polyC), polyadenylic-urydylic acid (polyA-polyU) or statalon viral double-stranded RNA. These results have been interpreted as suggesting that the antiRNA antibodies found in patients with systemic lupus erythematosus are formed primarily against double-stranded RNA, which is more char-

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acteristic of viral antigen than human autoantigen. (3) Tubular structures identified by electron microscopy in the endothelial cells of the glomerular capillary have been interpreted as "myxovirus" by some investigators (Fig. 8).79,174 These observations have led to the speculation that the structures represent double-stranded RNA virus that would be capable of inducing antinucleic acid antibodies. The viral nature of these structures remains the center of some controversy.186 (4) The pathologic and serologic similarities between human systemic lupus erythematosus and naturally occurring disease of New Zealand mice has already been commented upon (p. 482).

Chronic and Rapidly Progressive Glomerulonephritis Immunoglobulins and complement components have been demonstrated to be deposited on the glomeruli of patients with several forms of glomerulonephritis. 115 , 117, 118, 131, 144 In some cases, the immune deposit may appear strikingly similar to that which appears in the immune complex form of experimental glomerulonephritis or the glomerular basement membrane model (Figs. 2, 4, 9). The nature of the antigen which may be involved in deposited immune complexes in human glomerulonephritis is obscure in virtually all cases. Some patients with chronic or rapidly progressive glomerulonephritis have been shown to have circulating and deposited anti-glomerular basement membrane antibodies similar or identical to the antibody which has been found in patients with Goodpasture's syndrome (p. 496). It would appear from the studies of Lerner et al. laO that anti-glomerular basement membrane

Figure 8. Electron micrograph from the biopsy of a patient with systemic lupus erythematosus: numerous electron dense particles of uniform size are seen within an endothelial cell and have been postulated to represent myxoviruses. (Courtesy of Dr. R. Cotran.)

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Figure 9. Immunofluorescent staining for IgG in a patient with chronic membranous glomerulonephritis. The immunoglobulin deposit is noted to have a coarsely granular appearance, which follows the glomerular capillary wall and is believed to represent deposited antigen-antibody complexes. (Courtesy of Dr. M. Schwartz.)

antibodies are pathogenic in these patients and capable of mediating glomerular damage.

Lipoid Nephrosis The term lipoid nephrosis refers to the most frequent cause of nephrotic syndrome in children. This disease is associated with virtually normal glomerular histology. The characteristic lesion seen on electron microscopy is that of fusion of the foot processes of the epithelial cells. 212 Immunofluorescent studies are characteristically negative for immunoglobulins, complement, and fibrinY It is clear, therefore, that the usual immunopathologic mechanisms invoked to explain human glomerular pathology do not apply in this disease. Rarely, one finds a strong history of exposure to allergens as the apparent precipitating event in a child with the nephrotic syndrome. 252 With the exception of this clinical suggestion that hypersensitivity of the allergic type is related to the onset of the disease, there is only slender evidence that an immunologic reaction explains its occurrence. Lewis et al. 132 have reported low levels of Clq in children with this disease. The decreased serum level of this complement component could be the result of increased filtration and excretion in the urine. Another possibility is the existence of a primary abnormality in the metabolism of C 1 q. N gu and his associates 172 have measured immunoconglutinin levels in children with acute glomerulonephritis and nephrotic syndrome. Immunoconglutinin is a naturally occurring antibody directed against antigens present on C3 and C4 molecules which have partici-

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pated in an immune reaction. 121 Elevated immunoconglutinin levels were found in both acute glomerulonephritis and idiopathic nephrotic syndrome. The relationship of this observation to an immune pathogenesis of the renal lesion is speculative. Although there have been occasional reports of depressed total serum hemolytic complement levels in lipoid nephrosis, in general, CH50, C3 and C4levels have been normal in this disease. 57. 132 The evidence for an immunopathogenetic basis for this lesion is therefore virtually nonexistent at present.

Focal Nephritis Focal nephritis refers to the microscopic finding of pathology in some glomeruli while others are entirely normal. 91 The changes are often confined to one or two tufts of an involved glomerulus. Foci of endothelial and mesangial cell proliferation, usually adjacent to necrosis of a portion of a tuft, platelet aggregation, fibrin deposition, and capillary thromboses are common. 91 Minimal lesions may consist of platelet aggregates and fibrin visible only by electron microscopy, while severe lesions show intense epithelial cell proliferation with crescent formation. Most examples fall into a spectrum between these extremes. No characteristic basement membrane changes are recognized. Since first defined by Heptinstall and Joekes 93 in 1959, focal nephritis has been identified as the early lesion in glomerular diseases of diverse etiology, including lupus nephritis, Henoch-Schonlein purpura, benign recurrent hematuria, Goodpasture's syndrome, and lipoid nephrosis. 109 Prominent and sometimes persistent hematuria, a tendency to recur after a nonstreptococcal infection or other allergic insult, and a very slowly progressive course are common features of focal nephritis. Occasionally a rapidly progressive course with a diffuse lesion and uremia within weeks may evolve. 91 Whether the common clinicopathologic features reflect pathogenetic factors common to the several diseases characterized by focal nephritis is not known. Two examples of focal nephritis are discussed. HENOCH-SCHONLEIN PURPURA. Henoch-Schonlein or anaphylactoid purpura is preceded by a nonstreptococcal upper respiratory infection in more than half the cases. 2,67 Rare cases have been documented in association with food allergies,! drug reactions,237 and insect bites. 105 A diffuse vasculitis underlies lesions in the skin, intestines, and joints, and may affect interlobular and afferent arterioles in the kidney.91 The glomerular lesion, however, is that of focal nephritis 237 and occurs in about one third of cases, with figures of 6 to 62 per cent reported in different series, 199, 237 Progression from focal to diffuse involvement has not been documented in this disease, and the prognosis is correspondingly good, despite the tendency for hematuria to recur,2, 220, 237 In 1914 Osler 180 first suggested a role for hypersensitivity in this syndrome, In addition to the association with preceding infection, evidence assembled to support this thesis in the subsequent half century includes the following. Biopsy of active skin lesions discloses an acute vasculitis of small arteries with frequent capillary thrombosis. 237 This morphologic picture

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resembles the experimental Arthus reaction mediated by immune complexes. 68 However, serum complement levels during activity have invariably been normal,23.77 and immunofluorescent studies of involved vessels do not reveal deposition of globulins or complement. 163 Miescher162 described a hypersensitivity reaction to the subcutaneous injection of filtrates of several bacteria in patients with Henoch-Schonlein purpura that is not found in normal subjects. This reactivity has been transferred to normal individuals by preparations of leukocytes, but not by serum from patients with Henoch-Schonlein purpura. 162 The significance of these observations relative to the vasculitis is speculative. Immunofluorescence microscopy of the glomerular lesion reveals only minimal basement membrane involvement, but a characteristic mesangial distribution of granular deposits containing IgG, complement, fibrinogen, and IgA.2o.232 The deposits are accompanied by an acute inflammatory reaction in a similar distribution, involving only small adjacent segments of basement membrane. Electron micrographs show capillary thrombi and subendothelial and mesangial deposits, thought to correspond to those seen by fluorescence microscopy.232 The findings are consistent with an immune complex-mediated disease confined largely to the mesangium and accompanied by local involvement of platelets and the coagulation system. No data regarding possible antigenic components of an immune complex mediating this syndrome are available at present, nor is the role of the deposited IgA clear. The ability of mesangial cells to phagocytize immunoglobulin complexes may contribute to the mesangial localization of immunoglobulins. 225 Dreesman 50 and Bari 12 have reported that immune complexes formed at equivalence, which are heavier and less soluble than those formed in antigen excess, do not penetrate the basement membrane, and therefore deposit in the mesangium where they induce an inflammatory response. These observations could explain the pattern of immunoglobulin deposition in Henoch-Schonlein purpura and the Inild nature of the resulting nephritis. SiInilar mesangial deposits without significant renal damage have also been found in lupus.ll5 It is also possible that immune mechanisms analogous to serum sickness are not involved in the pathogenesis of Henoch-Schonlein purpura. Infusion of adenosine diphosphate alone produces platelet aggregation and focal glomerular lesions similar to those seen in HenochSchonlein purpura. 106 Viruses also may be capable of inducing local platelet aggregation without participating in an immune reaction. 60 While the mesangial deposits of immunoglobulin and complement suggest a role for immune mechanisms in the development of the vascular and glomerular lesions of Henoch-Schonlein purpura, such a role has not been convincingly demonstrated. The etiology and pathogenesis of this disease at present are unknown. BENIGN RECURRENT HEMATURIA. Patients with benign recurrent hematuria exhibit the common clinical and pathological features of focal nephritis with manifestations limited to the kidney. Bates 14 documented the nonstreptococcal nature of the upper respiratory infection which

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frequently precedes hematuria. Complement levels are usually normal,77. 224 and the course of the disease is benign, despite the often persistent hematuria and frequent recurrences. 9. 177 Several authors have reported focal nephritis in about 50 per cent of patients with benign recurrent hematuria undergoing biopsy, while normal histology is seen in the remainder.9, 14, 20 Most electron microscopic studies show no characteristic findings except a variation in thickness of basement membrane,83 with deposits occasionally seen on either surface. 21o In 1968 Berger reported fluorescence microscopy in some 300 renal biopsies, among which were 55 that had distinct intercapillary deposits of IgA, IgG, complement and fibrinogen in all glomerulPo Many were classified as focal nephritis pathologically, while others were normal or unclassified. Electron micrographs of these specimens confirmed a subendothelial deposit between the basement membrane and mesangial cells. When reviewed, the clinical history in each of these patients was that of benign recurrent hematuria. Evidence that immune mechanisms playa role in the disease is strengthened by Berger's description of one patient who progressed to renal failure with this lesion, received a renal homograft, and developed hematuria in the graft, which on biopsy contained the same intercapillary deposits of IgA. These changes were not found in 40 other renal allografts studied. 20 Berger's findings await confirmation, but the patients described appear to represent cases of benign recurrent hematuria. The similarity of Henoch-Schonlein purpura and benign recurrent hematuria suggests that similar etiologic considerations may apply to both.

Cryoglobulinemia Cryoglobulins are found in association with several renal diseases, including lupus nephritis,83 poststreptococcal and rapidly progressive glomerulonephritis,t3 the nephritis in NZB mice,99 and an animal model of serum sickness nephritis. 145 These observations have stimulated investigation of their possible pathogenic role in renal disease. Franklin and Meltzer 160 described an additional group of patients with a mixed IgG-IgM cryoglobulin with rheumatoid factor activity and a syndrome of purpura, arthralgia, weakness, vasculitis, and low complement level. In three of these an acute rapidly progressive glomerulonephritis developed, with granular deposits of IgG and IgM on the glomerular basement membrane. The analogy with experimental acute serum sickness led to studies attempting to characterize the cryoglobulin as a pathogenic soluble immune complex. The IgG component of one mixed cryoglobulin from a patient without renal disease was found to contain no sialic acid and was immunologically bound to the IgM component, which had antibody activity specific for the altered IgG component. 256 Denatured DNA has also been found to be a component of some mixed cryoglobulins, to which it is tightly bound both in solution and in the precipitate. 13 Similar complexes have been observed in lupus. 63 These studies suggest that mixed cryoglobulins can represent soluble antigen-antibody complexes. Immunoglobulins eluted from glomeruli have not yet been shown to possess cryoglobulin activity, although rheumatoid factor activity in a

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glomerular IgM deposit has been described. 114 Mixed cryoglobulins with rheumatoid factor activity have also been reported in association with the nephritis observed in some viral diseases. '3 A high incidence of cryoglobulins, composed in part of IgG and containing C3, is found in acute poststreptococcal and rapidly progressive glomerulonephritis, where their presence may correlate with disease activity.13 The IgG portion of this complex also lacks a sialic acid component. 13 Isolated samples of these complexes have been reported to fix complement and to localize in glomeruli of animals, with subsequent development of hematuria and proteinuria. 13 The association of cryoglobulins with glomerulonephritis in man and the observations described have made the possible role of these complexes in the pathogenesis of human nephritis the subject of much recent interest. Demonstration that cryoglobulins represent immune complexes which playa role in the pathogenesis of glomerulonephritis awaits future investigations.

Goodpasture's Syndrome In 1967 Lerner et alYo showed that the linear deposits of IgG in glomeruli in patients with Goodpasture's syndrome had anti-glomerular basement membrane activity in vitro, and when injected into squirrel monkeys would reproduce the renal disease seen in man. Goodpasture's syndrome is the prototype of this mechanism of immunologically induced glomerular damage in man, and the typical linear deposit of immunoglobulin on glomerular basement membrane is considered one of its principal characteristics. 120 The documentation of anti-glomerular basement membrane nephritis in man constituted a major advance in the understanding of the role of humoral immunity in the pathogenesis of human renal disease. 46 However it did not adequately explain all aspects of Goodpasture's syndrome, since anti-glomerular basement membrane antibody from these patients injected into monkeys does not provoke the characteristic pulmonary hemorrhage seen in the patients. '2o This observation led to a search for additional mechanisms to account for the pulmonary lesion. At present there is no evidence to support the participation of a second mechanism in the pathogenesis of the pulmonary lesion of Goodpasture's syndrome. However, data have accumulated which suggest that both the renal and pulmonary lesions may be mediated by a similar or identical antibody. The presence of linear deposits of immunoglobulin and complement in a focal distribution in the alveolar septa has been shown by several investigators. 17. 116. 147. 151 Many studies document the antigenic similarity of alveolar and glomerular basement membrane. '59 The proper dose of heterologous anti-lung serum will fix to both alveolar and glomerular basement membrane and induce nephritis and pulmonary hemorrhage. so, 251 Absorption of anti-lung serum with lung antigen abolishes fixation of antibody to both organs. so Alveolar basement membrane antigen can induce an anti-glomerular basement membrane nephritis identical to that produced using glomerular basement membrane antigen. 218 The anti-glomerular basement membrane antibody

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eluted from a kidney in a patient with Goodpasture's syndrome fixes to lung and glomerular basement membrane in a linear pattern in vitro, but does not react with basement membranes in other organs or vessels. Immunoglobulin has now been eluted from both the lung and kidney of two patients with Goodpasture's syndrome, and several properties of each eluate have been compared. 1I6 , 147 Immunoglobulin from both sites had antibody activity to antigenic determinants that was very similar or identical and showed fixation in a linear pattern in vitro on both human and monkey lung and glomerular basement membrane. 116 In vivo antibody from both sites fixed quantitatively to glomerular basement membrane and no pulmonary localization was detectable. \47 Anti-glomerular basement membrane antibody from patients with rapidly progressive glomerulonephritis without pulmonary involvement showed a slightly different specificity and did not fix to alveolar basement membrane in vitro, although the renal lesion in these patients was indistinguishable from that of Goodpasture's syndrome. 147 These findings indicate the close similarity or identity of the antibody deposited in lung and kidney in Goodpasture's syndrome. At present this evidence favors a single mechanism mediating both lung and kidney lesions in Goodpasture's syndrome. It provides no explanation for the failure of the eluted antibody to transfer the pulmonary lesion, however. Consequently, both the mechanism of the pulmonary hemorrhage and the nature of the stimulus that initiates anti-glomerular basement membrane antibody production remain unclear.

Renal Tubular Disorders Renal tubular acidosis has been described in patients who have hypergammaglobulinemia in association with a variety of diseases of apparent immune origin. The degree of hypergammaglobulinemia does not appear to correlate with renal tubular acidosis.1 67 Sjogren's syndrome,207 lupoid hepatitis,t93 cryoglobulinemia,t40 purpura hyperglobulinemica,42 thyroiditis,153 and idiopathic hyperglobulinemia have all been associated with this syndrome. In addition, some patients with kidney transplants have manifested renal tubular acidosis. 24 . 154. 166 Patients with Sjogren's syndrome and renal tubular acidosis have been found to have higher serum immunoglobulin concentrations and more antibodies to autologous antigens than patients with this disease who did not manifest an acidifying defect. Biopsies from patients with Sjogren's syndrome may show interstitial infiltrations of plasma cells and lymphocytes. 208 . 231 Pasternack and Linder l82 have described the localization of IgG in renal tubular cells and interstitial infiltrates surrounding the tubules of patients with Sjogren's syndrome and renal tubular acidosis. In addition, these authors described circulating antibodies which fix to renal tubular cells in vitro, using the indirect fluorescent technique. Talal et al. 227 have studied patients with Sjogren's syndrome of renal tubular acidosis and described IgG in the cytoplasm of renal tubular cells of two patients. At present, the relationship between hypergammaglobulinemia and renal tubular acidosis appears well documented. The role of antibody

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deposition or cellular hypersensitivity in the impairment of the ability of the distal tubule to maintain a normal hydrogen ion gradient is unknown. Data such as that noted above suggest that an immune reaction may be operative; however the non-immune effects of high serum gamma globulin levels or pathologic lesions unrelated to serum globulins, such as the arteritis that has been described in Sjogren's syndrome, may also playa role in this abnormality.

CONCLUDING REMARKS Evidence supporting the role of immunologic mechanisms in the pathogenesis of human renal diseases has been presented. Some insight into possible analogies between experimentally induced glomerulonephritis and human disease has been gained through morphologic and immunochemical studies. Nevertheless, although examples of both immune complex-induced as well as anti-glomerular basement membrane antibody induced glomerulonephritis can now be cited in man, the etiologic agents responsible for the development of these immunologic abnormalities remain obscure. With the exception of lupus erythematosus, bacterial endocarditis, some viral diseases, malaria, and syphilis, there is little indication regarding the nature of the antigen involved in immune complex formation in the nephritides of man. The present state of knowledge regarding the pathways determining the nature and extent of renal damage in human glomerulonephritis similarly is incompletely understood. Further definition of the etiologic agents and pathogenetic mechanisms involved in human disease is required in order to attain the goals of primary prevention and rational treatment in these disorders. ACKNOWLEDGMENTS

The authors wish to thank Miss Anne Wallace for her excellent technical support and Miss Maureen McDonough for secretarial assistance. We are particularly indebted to our colleagues, Drs. Ramzi Cotran and Tito Cavallo, who have collaborated in the immunopathologic studies with us and who provided illustrations for this review.

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