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The Development of Immunopathologic Investigation of Kidney Disease
The Development of Immunopathologic Investigation of Kidney Disease Frank J. Dixon, MD, and Curtis B. Wilson, MD
T
HE IMMUNOPATHOLOGIC study of kidney disease, glomerulonephritis, and more recently expanded to tubulointerstitial nephritis, has closely followed the technological advances in biomedicine and been periodically punctuated by perceptive observations and amazingly accurate interpretation and postulation. The role of immunologic events in the pathogenesis of glomerulonephritis was recognized both in the experimental laboratory and in the clinic in the first few years of this century. In 1900, Lindemann induced albuminuria and uremia in rabbits by injection of guinea pig anti-rabbit nephrotoxic serum. 1 A number of immunologic studies in the next two decades indicated first that the responsible nephrotoxic antigen was in the renal cortex and later more specifically in the glomeruli. 2 In the following two decades, a number of the most eminent immunologists such as Masugi, Smadel, and Kay analyzed the pathologic and immunologic aspects of this disease, concluding that morphologically the renal changes were quite similar to some cases of human glomerulonephritis, that the disease occurred in distinct acute and chronic stages, and finally that the host's immune response to the heterologous nephrotoxic serum was the crucial element in the autologous chronic progressive phase of the disease. 3-5 These findings afforded us as complete an immunopathologic picture of this disease model as pre-World War II technology would permit. During this same 40-year period, a very different trail of immunopathologic investigation developed that also implicated immunologic events in glomerulonephritis. This line of study began not in the experimental laboratory, but in the clinic where von Pirquet made some unbelievably astute From the Department of Immunology, Scripps Clinic and Research Foundation, La Jolla, CA Supported in part by National Institutes of Health Grants No. CA27489, AI07007, DK20043 , DK32353 , DK40251 , and AG04342. Address reprint requests to Frank J. Dixon, MD, Scripps Clinic and Research Foundation , 10666 N Torrey Pines Rd, La Jolla , CA 92037. © 1990 by the National Kidney Foundation , Inc. 0272-638619011606-0011 $3.0010
574
observations on patients suffering iatrogenic renal complications of serum therapy, on which he then based his prophetic explanation of the immunopathologic events transpiring in this disease that some 50 years later would be proven correct by newly developed technology.6 In essence, he saw that the various manifestations of serum sickness, including glomerulonephritis, developed at a time when, in the presence of circulating antigen, the host made an antibody response with resultant interaction in the circulation of antigen and antibody, resulting, in his terms, in a disease-producing "toxic body." Subsequently, meticulous laboratory studies of serum sickness, usually in the rabbit, defined in detail the immunopathologic developments in the course of this disease. Thus, prior to World War II, we also had a reasonable understanding of the sequence of events in serum sickness-type glomerulonephritis, although there was no clear-cut definition of the "toxic bodies" or precisely how the immunologic reaction occurring in the circulation produced the renal disease. During the period of World War II, two important technological developments occurred that would detennine the course of immunopathologic studies of glomerulonephritis. These were, first, the development of fluorescent antibody labeling by Coons and Kaplan,7 an advance completely unrelated except chronologically to military events; and second, the availability of isotope tracers for biological research, a happy byproduct of our military program in nuclear fission. The ability to put fluorescent labels on proteins, antigens, and antibodies of biological significance provided a means whereby these substances could be traced in vivo at the histologic and cytologic levels. We could now see where antibodies were made, where antigens and antibodies interacted along with components of complement, and finally what became of these immunologic reactants. Isotope labels, while not as precise at the cytologic or histologic levels, provided quantitative measures of the immunologic reactants being traced, and together with the histologic detail offered by immunofluorescence and electron microscopic tracers , afforded a powerful technology for in vivo immunologic study. This new technology provided the basis for the ex-
American Journal of Kidney Disease, Vol XVI, No 6 (December), 1990: pp 574-578
IMMUNOPATHOLOGY OF KIDNEY DISEASE
plosive development of immunopathologic investigation of renal disease during the subsequent three decades. There was another fortuitous development that provided new insights into clinical renal disease and this was the introduction and frequent use of percutaneous renal biopsies in the diagnosis and management of glomerulonephritis. Later, renal transplantation, often with pretransplant nephrectomy, provided additional tissue for study and, in some instances, created a situation to study the development of recurrent nephritogenic processes. With the application of new tracer techniques the few remaining mysteries about nephrotoxic or Masugi nephritis, or as it was more accurately renamed, antiglomerular basement membrane (antiGBM) antibody nephritis, were quickly revealed. Interestingly, much of the early observations employing radioiodinated anti-GBM antibodies came as a byproduct of Pressman's work on anticancer antibodies in which he used anti-GBM nephritis as a model. 8 The reversible fixation of nephrotoxic antibody to the GBM and other host antigens was defined in detail and with considerable precision. The host's antibody response to the heterologous antikidney antibodies was similarly measured and its reaction with the glomerular bound nephrotoxic antibodies recognized as the cause of the autologous phase of injury. Also, the phlogogenic contributions of complement, polymorphonuclear leukocytes, and monocytes were described and appropriately placed in the series of pathogenetic events. Considerable expansion of the number of inflammatory cell and intrinsic glomerular cell products that can contribute to the mediation of immunologically induced injury are now understood or under study; however, their discussion is beyond our space limitation. As a result of the availability of renal biopsies and occasional nephrectomy specimens obtained during renal transplantation, it was finally possible to completely define the pathogenetic mechanisms involved in the spontaneous anti-GBM antibodyinduced nephritis or Goodpasture syndrome in man. In the course of renal transplantation of such a patient a nephrectomy specimen became available providing the means to satisfy "Koch's postulates" in this particular type of nephritis. The antiGBM antibodies in this nephrectomy specimen were eluted and transferred to non-human primates where they promptly fixed in the glomeruli
575
and reproduced all manifestations of the nephritis seen in the patient from whom they were taken. Thus, in the spontaneous human version of nephrotoxic nephritis, it was clear that anti-GBM antibodies were indeed capable of producing the disease. Left unanswered then and to this date, is the question of what etiologic events and/or agents initiate the formation of anti-GBM antibody in these patients. However, better understanding of the reactive GBM antigen, currently thought to be a portion of the NCI domain of a type IV collagen a-chain, has led to identification of genetic variants in GBM antigenic structure and an association of defects with Alport's syndrome. The concept of a nephritogenic antibody reacting with a renal antigen has been expand~d since these seminal observations to include reactions with a number of glomerular and tubular cell-associated molecules. Antibody or immune cell reactions with these antigens provide models of selective cell damage or perturbation. For example, the rat glomerular mesangial cell can be selectively damaged and caused to proliferate with antibodies reactive with a Thy I. I-like antigen on its surface. The same quantitative and temporal factors of antibody fixation established in the initial anti-GBM antibody studies apply to the current crop of models. In addition, the observation of the host's antibody reaction to a foreign kidney-bound antigen in the autologous phase of the anti-GBM antibody model has been expanded to embrace a number of molecules (exogenous and endogenous) that can bind for physicochemical reasons to the renal tissue to serve as targets for nephritogenic antibody interaction. These molecules include lectins, charged molecules, and DNA. The application of the new tracer techniques to the study of serum sickness was equally productive. Since serum therapy in the post-war period was no longer in vogue in clinical medicine, there was little opportunity to study serum sickness as it occurred in man, but the similar disease produced in rabbits was extensively analyzed. Using radioiodinated bovine albumin as antigen in the rabbits, it was possible to show that the accelerated elimination of circulating antigen that occurred with the onset of the host's antibody response did indeed involve the formation of circulating antigen antibody complexes that reacted with complement and were deposited in various vascular structures including the glomeruli. Apparently, these im-
576
mune complexes were in fact the "toxic bodies" that von Pirquet had predicted in the course of his studies of serum sickness in man. 6 Not only were these complexes formed simultaneously with the onset of disease, they were also deposited specifically in the vascular lesions characteristic of serum sickness and appeared to be the pathogenetic agents of this disease. Because at the onset of disease antigen was present in excess in the circulation, only immune complexed antibody could reach the kidney until several days later when immune elimination had cleared circulating antigen. Free antibody was then able to continue to interact with previously deposited immune complex material. By a variety of manipulations of the serum sickness model, particularly that using small daily injections of foreign serum protein over a long period of time, it was possible to demonstrate that the clinical and morphologic form of glomerulonephritis produced was determined by the logistics of immune complex formation, ie, the amounts of antigen and antibody involved and the duration of time over which they were formed. Most pathogenic complexes were relatively small soluble complexes capable of remaining in the circulation, but still large enough to fix complement. Very large complexes formed in antibody excess were particulate and rapidly removed from the cirwere particulate and rapidly removed from the circulation by phagocytes and degraded. There is considerable local interchange of antigen and antibody within tissue immune complex deposits relative to the antigen to antibody ratio in the immediate environment. The interchange is modified by physicochemical properties ofthe antigen (or antibody), which may serve to retain it at the site, ie, charge interaction with the glomerular capillary wall. Secondary immune events such as the formation of anti-idiotypic antibodies or rheumatoid factors also modify the interchange. The transient glomerular lesion of acute serum sickness is largely infiltrative, with monocytes/ macrophages appearing to nearly occlude the capillary lumens. The daily injection model begins as mesangial proliferation/expansion and later subepithelial immune deposits lead to membraneous changes, with varying degrees of proliferation, including crescent formation when amounts of complexes are great. This pathogenetic mechanism was readily demonstrated in a variety of clinical glomeru-
DIXON AND WILSON
lonephritides associated with infectious or parasitic diseases where the nature of the antigens was known and their identification in circulating or glomerular deposited complexes could be recognized. Even in the nephritis that sometimes accompanies neoplasia, immune complexes appeared to play a role since complexes could be detected in the circulation and glomeruli and, in some cases, tumor antigens could be demonstrated in them. In iatrogenic complications of a variety of therapies, including penicillin administration, immune complexes containing the offending drug could be detected in the circulation and in the associated vascular lesions. However, it was disappointing that in the large number of non-anti-GBM nephritides where a specific antigen was not suspected, the presence of significant amounts of circulating immune complexes was usually not demonstrable and a search for specific antigens was unsuccessful. By the 1970s the pathogenetic mechanism of immune complex type, serum sickness nephritis, was clearly understood, but except for the instances mentioned above, its demonstration in terms of antigen identification in human nephritis remains infrequent. Considerable investigative effort has been expended on the study of systemic lupus erythematosus (SLE), which appears to have an immune complex pathogenesis. Autoantibody responses to a variety of autoantigens including DNA have been identified in this disease. Immune complexes of these autoreactants have been found in the circulation and have been detected in and eluted from the glomeruli of diseased kidneys. Many of the limitations inherent in studying disease in man have been surmounted by the development of a number of murine strains that regularly develop a SLE-like syndrome immunologically and pathologically virtually identical to that seen in man. The study of murine SLE has defined most of the pathogenetic events involved, but has been of little help in discovering the genetically based etiologic factors in this disease in either mouse or man. A third line of immunopathologic investigation of glomerulonephritis dealt with a variety of experimental situations in which animals were immunized with various kidney antigens and developed one or another kind of nephritis. Because the renal antigenic preparations in these experiments were complex and varied, as were the species of host subjects, the diseases themselves were variable and the pathogenetic mechanisms complicated.
IMMUNOPATHOLOGY OF KIDNEY DISEASE
The Steblay model of anti-GBM disease in sheep was one example and animals producing anti-heterologous GBM antibodies for experimental use could themselves develop anti-GBM antibody disease. 9 One of the most intensively studied of the kidney immunization nephritides was that originally described by Frick and later popularized by Heymann, which involved the injection of crude renal extracts incorporated in complete Freund adjuvant repeatedly over several months.tO,1I This model was attractive since the histopathology of the glomerular lesion resembled that of membranous nephritis in man. Since the renal extracts obviously contained numerous antigens, it took a good deal of sophisticated biochemical and physical chemical dissection to determine the antigen or antigens most responsible for the disease. It appears now that an antigen such as gp 330, present in large amounts in the tubular brush border and in smaller amounts in coated pits on glomerular epithelial cell membranes, is the most likely culprit. On the basis of perfusion studies in which circulating antigen can be essentially excluded, it appears clear that a large part of the disease-producing immune reaction in this model takes place between host antibody and the glomerular epithelial cell surface antigen , where a capping and shedding phenomenon results in the subepithelial immune deposit formation . Thus, the application of the new tracer technologies in the decades following World War II clearly defined two pathogenetic mechanisms capable of inducing glomerular disease. However, these mechanisms, at least in the pure forms demonstrated in the laboratory, could not account for all of clinical nephritis. Anti-GBM nephritis, the pathogenetic mechanism of which was clearly demonstrated, appears to be involved in a very small proportion , less than 5 %, of human glomerulonephritis. The immune complex pathogenetic mechanism can be invoked in a variety of nephritides secondary to obvious infection or drug exposure, but is less clear in the majority of spontaneous glomerulonephritis . Equally disappointing has been the failure to identify in man nephritogenic immune responses against non-GBM antigens chemically or anatomically similar to those best described in the kidney immunization nephritides . If, in fact , a significant number of human glomerulonephritides are the result of the patient's immune response to renal antigens, we have
577
not been able to identify these antigens, with the exception of the relatively small number of cases of anti-GBM nephritis. While a good bit can be learned in laboratory models about the phlogogenic mechanisms that operate secondary to the immunologic reactions involving the glomeruli and produce much of the injury, it may well be that it will take studies in man to determine which are the relevant renal antigens operative in this form of human autoimmunity. If this is true, it is unfortunate that the enthusiasm for renal biopsy has declined. It may be that the final immunologic answer to human glomerulonephritis will have to come from the limited amount of available, useful human tissue. It may take another technological or philosophical breakthrough to obtain the information necessary to satisfactorily explain the immunopathology, and more importantly the etiology, of human glomerulonephritis. The new advance may be upon us in the form of rapidly expanding molecular biologic techniques capable of identifying the precise nature of nephritogenic antigens and their reactive epitopes. In addition, these techniques can enhance understanding of autoimmune responses by identifying specific immune cell products and receptors such as their immunoglobulin genes and T-cell receptors . The contribution of a myriad of cell products in the form of cytokines and growth factors that can be studied in selective glomerular or tubular cell cultures and in vivo models can all combine to enhance our understanding of the mechanisms involved in initiation, mediation, and progression of immune renal injury. The use of in situ hybridization to localize the cell products and mRNA analysis, including the extremely sensitive polymerase chain reaction, as well as manipulation of critical mRNA production, may provide the next level of understanding of nephritogenic immune responses.
REFERENCES I. Lindemann W: Sur la mode d'action de certains poisons renaux. Ann Inst Pasteur 14:49-59, 1900 2. Krakower CA , Greenspon SA : Localization of the nephrotoxic antigen within the isolated renal glomerulus. Arch Pathol 51 :629-639, 1951 3, Masugi M: Uber die experimentelle glomerulonephritis durch das spezifische antinierenserum . Ein beitrag zur pathogenese der diffusen glomerulonephritis. Beitr Pathol 92:429466 , 1934 4. Smadel JE: Experimental nephritis in rats induced by in-
T
HE IMMUNOPATHOLOGIC study of kidney disease, glomerulonephritis, and more recently expanded to tubulointerstitial nephritis, has closely followed the technological advances in biomedicine and been periodically punctuated by perceptive observations and amazingly accurate interpretation and postulation. The role of immunologic events in the pathogenesis of glomerulonephritis was recognized both in the experimental laboratory and in the clinic in the first few years of this century. In 1900, Lindemann induced albuminuria and uremia in rabbits by injection of guinea pig anti-rabbit nephrotoxic serum. 1 A number of immunologic studies in the next two decades indicated first that the responsible nephrotoxic antigen was in the renal cortex and later more specifically in the glomeruli. 2 In the following two decades, a number of the most eminent immunologists such as Masugi, Smadel, and Kay analyzed the pathologic and immunologic aspects of this disease, concluding that morphologically the renal changes were quite similar to some cases of human glomerulonephritis, that the disease occurred in distinct acute and chronic stages, and finally that the host's immune response to the heterologous nephrotoxic serum was the crucial element in the autologous chronic progressive phase of the disease. 3-5 These findings afforded us as complete an immunopathologic picture of this disease model as pre-World War II technology would permit. During this same 40-year period, a very different trail of immunopathologic investigation developed that also implicated immunologic events in glomerulonephritis. This line of study began not in the experimental laboratory, but in the clinic where von Pirquet made some unbelievably astute From the Department of Immunology, Scripps Clinic and Research Foundation, La Jolla, CA Supported in part by National Institutes of Health Grants No. CA27489, AI07007, DK20043 , DK32353 , DK40251 , and AG04342. Address reprint requests to Frank J. Dixon, MD, Scripps Clinic and Research Foundation , 10666 N Torrey Pines Rd, La Jolla , CA 92037. © 1990 by the National Kidney Foundation , Inc. 0272-638619011606-0011 $3.0010
574
observations on patients suffering iatrogenic renal complications of serum therapy, on which he then based his prophetic explanation of the immunopathologic events transpiring in this disease that some 50 years later would be proven correct by newly developed technology.6 In essence, he saw that the various manifestations of serum sickness, including glomerulonephritis, developed at a time when, in the presence of circulating antigen, the host made an antibody response with resultant interaction in the circulation of antigen and antibody, resulting, in his terms, in a disease-producing "toxic body." Subsequently, meticulous laboratory studies of serum sickness, usually in the rabbit, defined in detail the immunopathologic developments in the course of this disease. Thus, prior to World War II, we also had a reasonable understanding of the sequence of events in serum sickness-type glomerulonephritis, although there was no clear-cut definition of the "toxic bodies" or precisely how the immunologic reaction occurring in the circulation produced the renal disease. During the period of World War II, two important technological developments occurred that would detennine the course of immunopathologic studies of glomerulonephritis. These were, first, the development of fluorescent antibody labeling by Coons and Kaplan,7 an advance completely unrelated except chronologically to military events; and second, the availability of isotope tracers for biological research, a happy byproduct of our military program in nuclear fission. The ability to put fluorescent labels on proteins, antigens, and antibodies of biological significance provided a means whereby these substances could be traced in vivo at the histologic and cytologic levels. We could now see where antibodies were made, where antigens and antibodies interacted along with components of complement, and finally what became of these immunologic reactants. Isotope labels, while not as precise at the cytologic or histologic levels, provided quantitative measures of the immunologic reactants being traced, and together with the histologic detail offered by immunofluorescence and electron microscopic tracers , afforded a powerful technology for in vivo immunologic study. This new technology provided the basis for the ex-
American Journal of Kidney Disease, Vol XVI, No 6 (December), 1990: pp 574-578
IMMUNOPATHOLOGY OF KIDNEY DISEASE
plosive development of immunopathologic investigation of renal disease during the subsequent three decades. There was another fortuitous development that provided new insights into clinical renal disease and this was the introduction and frequent use of percutaneous renal biopsies in the diagnosis and management of glomerulonephritis. Later, renal transplantation, often with pretransplant nephrectomy, provided additional tissue for study and, in some instances, created a situation to study the development of recurrent nephritogenic processes. With the application of new tracer techniques the few remaining mysteries about nephrotoxic or Masugi nephritis, or as it was more accurately renamed, antiglomerular basement membrane (antiGBM) antibody nephritis, were quickly revealed. Interestingly, much of the early observations employing radioiodinated anti-GBM antibodies came as a byproduct of Pressman's work on anticancer antibodies in which he used anti-GBM nephritis as a model. 8 The reversible fixation of nephrotoxic antibody to the GBM and other host antigens was defined in detail and with considerable precision. The host's antibody response to the heterologous antikidney antibodies was similarly measured and its reaction with the glomerular bound nephrotoxic antibodies recognized as the cause of the autologous phase of injury. Also, the phlogogenic contributions of complement, polymorphonuclear leukocytes, and monocytes were described and appropriately placed in the series of pathogenetic events. Considerable expansion of the number of inflammatory cell and intrinsic glomerular cell products that can contribute to the mediation of immunologically induced injury are now understood or under study; however, their discussion is beyond our space limitation. As a result of the availability of renal biopsies and occasional nephrectomy specimens obtained during renal transplantation, it was finally possible to completely define the pathogenetic mechanisms involved in the spontaneous anti-GBM antibodyinduced nephritis or Goodpasture syndrome in man. In the course of renal transplantation of such a patient a nephrectomy specimen became available providing the means to satisfy "Koch's postulates" in this particular type of nephritis. The antiGBM antibodies in this nephrectomy specimen were eluted and transferred to non-human primates where they promptly fixed in the glomeruli
575
and reproduced all manifestations of the nephritis seen in the patient from whom they were taken. Thus, in the spontaneous human version of nephrotoxic nephritis, it was clear that anti-GBM antibodies were indeed capable of producing the disease. Left unanswered then and to this date, is the question of what etiologic events and/or agents initiate the formation of anti-GBM antibody in these patients. However, better understanding of the reactive GBM antigen, currently thought to be a portion of the NCI domain of a type IV collagen a-chain, has led to identification of genetic variants in GBM antigenic structure and an association of defects with Alport's syndrome. The concept of a nephritogenic antibody reacting with a renal antigen has been expand~d since these seminal observations to include reactions with a number of glomerular and tubular cell-associated molecules. Antibody or immune cell reactions with these antigens provide models of selective cell damage or perturbation. For example, the rat glomerular mesangial cell can be selectively damaged and caused to proliferate with antibodies reactive with a Thy I. I-like antigen on its surface. The same quantitative and temporal factors of antibody fixation established in the initial anti-GBM antibody studies apply to the current crop of models. In addition, the observation of the host's antibody reaction to a foreign kidney-bound antigen in the autologous phase of the anti-GBM antibody model has been expanded to embrace a number of molecules (exogenous and endogenous) that can bind for physicochemical reasons to the renal tissue to serve as targets for nephritogenic antibody interaction. These molecules include lectins, charged molecules, and DNA. The application of the new tracer techniques to the study of serum sickness was equally productive. Since serum therapy in the post-war period was no longer in vogue in clinical medicine, there was little opportunity to study serum sickness as it occurred in man, but the similar disease produced in rabbits was extensively analyzed. Using radioiodinated bovine albumin as antigen in the rabbits, it was possible to show that the accelerated elimination of circulating antigen that occurred with the onset of the host's antibody response did indeed involve the formation of circulating antigen antibody complexes that reacted with complement and were deposited in various vascular structures including the glomeruli. Apparently, these im-
576
mune complexes were in fact the "toxic bodies" that von Pirquet had predicted in the course of his studies of serum sickness in man. 6 Not only were these complexes formed simultaneously with the onset of disease, they were also deposited specifically in the vascular lesions characteristic of serum sickness and appeared to be the pathogenetic agents of this disease. Because at the onset of disease antigen was present in excess in the circulation, only immune complexed antibody could reach the kidney until several days later when immune elimination had cleared circulating antigen. Free antibody was then able to continue to interact with previously deposited immune complex material. By a variety of manipulations of the serum sickness model, particularly that using small daily injections of foreign serum protein over a long period of time, it was possible to demonstrate that the clinical and morphologic form of glomerulonephritis produced was determined by the logistics of immune complex formation, ie, the amounts of antigen and antibody involved and the duration of time over which they were formed. Most pathogenic complexes were relatively small soluble complexes capable of remaining in the circulation, but still large enough to fix complement. Very large complexes formed in antibody excess were particulate and rapidly removed from the cirwere particulate and rapidly removed from the circulation by phagocytes and degraded. There is considerable local interchange of antigen and antibody within tissue immune complex deposits relative to the antigen to antibody ratio in the immediate environment. The interchange is modified by physicochemical properties ofthe antigen (or antibody), which may serve to retain it at the site, ie, charge interaction with the glomerular capillary wall. Secondary immune events such as the formation of anti-idiotypic antibodies or rheumatoid factors also modify the interchange. The transient glomerular lesion of acute serum sickness is largely infiltrative, with monocytes/ macrophages appearing to nearly occlude the capillary lumens. The daily injection model begins as mesangial proliferation/expansion and later subepithelial immune deposits lead to membraneous changes, with varying degrees of proliferation, including crescent formation when amounts of complexes are great. This pathogenetic mechanism was readily demonstrated in a variety of clinical glomeru-
DIXON AND WILSON
lonephritides associated with infectious or parasitic diseases where the nature of the antigens was known and their identification in circulating or glomerular deposited complexes could be recognized. Even in the nephritis that sometimes accompanies neoplasia, immune complexes appeared to play a role since complexes could be detected in the circulation and glomeruli and, in some cases, tumor antigens could be demonstrated in them. In iatrogenic complications of a variety of therapies, including penicillin administration, immune complexes containing the offending drug could be detected in the circulation and in the associated vascular lesions. However, it was disappointing that in the large number of non-anti-GBM nephritides where a specific antigen was not suspected, the presence of significant amounts of circulating immune complexes was usually not demonstrable and a search for specific antigens was unsuccessful. By the 1970s the pathogenetic mechanism of immune complex type, serum sickness nephritis, was clearly understood, but except for the instances mentioned above, its demonstration in terms of antigen identification in human nephritis remains infrequent. Considerable investigative effort has been expended on the study of systemic lupus erythematosus (SLE), which appears to have an immune complex pathogenesis. Autoantibody responses to a variety of autoantigens including DNA have been identified in this disease. Immune complexes of these autoreactants have been found in the circulation and have been detected in and eluted from the glomeruli of diseased kidneys. Many of the limitations inherent in studying disease in man have been surmounted by the development of a number of murine strains that regularly develop a SLE-like syndrome immunologically and pathologically virtually identical to that seen in man. The study of murine SLE has defined most of the pathogenetic events involved, but has been of little help in discovering the genetically based etiologic factors in this disease in either mouse or man. A third line of immunopathologic investigation of glomerulonephritis dealt with a variety of experimental situations in which animals were immunized with various kidney antigens and developed one or another kind of nephritis. Because the renal antigenic preparations in these experiments were complex and varied, as were the species of host subjects, the diseases themselves were variable and the pathogenetic mechanisms complicated.
IMMUNOPATHOLOGY OF KIDNEY DISEASE
The Steblay model of anti-GBM disease in sheep was one example and animals producing anti-heterologous GBM antibodies for experimental use could themselves develop anti-GBM antibody disease. 9 One of the most intensively studied of the kidney immunization nephritides was that originally described by Frick and later popularized by Heymann, which involved the injection of crude renal extracts incorporated in complete Freund adjuvant repeatedly over several months.tO,1I This model was attractive since the histopathology of the glomerular lesion resembled that of membranous nephritis in man. Since the renal extracts obviously contained numerous antigens, it took a good deal of sophisticated biochemical and physical chemical dissection to determine the antigen or antigens most responsible for the disease. It appears now that an antigen such as gp 330, present in large amounts in the tubular brush border and in smaller amounts in coated pits on glomerular epithelial cell membranes, is the most likely culprit. On the basis of perfusion studies in which circulating antigen can be essentially excluded, it appears clear that a large part of the disease-producing immune reaction in this model takes place between host antibody and the glomerular epithelial cell surface antigen , where a capping and shedding phenomenon results in the subepithelial immune deposit formation . Thus, the application of the new tracer technologies in the decades following World War II clearly defined two pathogenetic mechanisms capable of inducing glomerular disease. However, these mechanisms, at least in the pure forms demonstrated in the laboratory, could not account for all of clinical nephritis. Anti-GBM nephritis, the pathogenetic mechanism of which was clearly demonstrated, appears to be involved in a very small proportion , less than 5 %, of human glomerulonephritis. The immune complex pathogenetic mechanism can be invoked in a variety of nephritides secondary to obvious infection or drug exposure, but is less clear in the majority of spontaneous glomerulonephritis . Equally disappointing has been the failure to identify in man nephritogenic immune responses against non-GBM antigens chemically or anatomically similar to those best described in the kidney immunization nephritides . If, in fact , a significant number of human glomerulonephritides are the result of the patient's immune response to renal antigens, we have
577
not been able to identify these antigens, with the exception of the relatively small number of cases of anti-GBM nephritis. While a good bit can be learned in laboratory models about the phlogogenic mechanisms that operate secondary to the immunologic reactions involving the glomeruli and produce much of the injury, it may well be that it will take studies in man to determine which are the relevant renal antigens operative in this form of human autoimmunity. If this is true, it is unfortunate that the enthusiasm for renal biopsy has declined. It may be that the final immunologic answer to human glomerulonephritis will have to come from the limited amount of available, useful human tissue. It may take another technological or philosophical breakthrough to obtain the information necessary to satisfactorily explain the immunopathology, and more importantly the etiology, of human glomerulonephritis. The new advance may be upon us in the form of rapidly expanding molecular biologic techniques capable of identifying the precise nature of nephritogenic antigens and their reactive epitopes. In addition, these techniques can enhance understanding of autoimmune responses by identifying specific immune cell products and receptors such as their immunoglobulin genes and T-cell receptors . The contribution of a myriad of cell products in the form of cytokines and growth factors that can be studied in selective glomerular or tubular cell cultures and in vivo models can all combine to enhance our understanding of the mechanisms involved in initiation, mediation, and progression of immune renal injury. The use of in situ hybridization to localize the cell products and mRNA analysis, including the extremely sensitive polymerase chain reaction, as well as manipulation of critical mRNA production, may provide the next level of understanding of nephritogenic immune responses.
REFERENCES I. Lindemann W: Sur la mode d'action de certains poisons renaux. Ann Inst Pasteur 14:49-59, 1900 2. Krakower CA , Greenspon SA : Localization of the nephrotoxic antigen within the isolated renal glomerulus. Arch Pathol 51 :629-639, 1951 3, Masugi M: Uber die experimentelle glomerulonephritis durch das spezifische antinierenserum . Ein beitrag zur pathogenese der diffusen glomerulonephritis. Beitr Pathol 92:429466 , 1934 4. Smadel JE: Experimental nephritis in rats induced by in-