The Effect of Cyclosporin A on Disease Progression in Proliferative Immune Complex Glomerulonephritis

In rats with the proliferative immune complex glomerulonephritis of chronic serum sickness, kidney function deteriorates in three discrete and readily distinguishable stages: Mild, Moderate, and Severe. The mononuclear cell composition of glomerular inflammation is also different in each stage. The immunosuppressive drug, cyclosporin A, was administered to rats with chronic serum sickness in order to investigate the relationship between glomerular immunopathology and pathophysiology in proliferative immune complex nephritis. When introduced after the onset of proteinuria, daily treatment with cyclosporin A failed to prevent the progression from Moderate to Severe nephritis, which is characterized by the abnormal differentiation and local proliferation of glomerular macrophages, as well as grave deterioration in kidney function. In contrast, when cyclosporin A therapy started before the onset of proteinuria, the course of proliferative glomerulonephritis was altered significantly. Although the levels of proteinuria and macrophage accumulation that are characteristic of the Moderate stage of nephritis were not reduced, progression to Severe nephritis did not occur. The number of glomerular macrophages appeared to increase in two separate phases in this chronic serum sickness model of proliferative immune complex glomerulonephritis. The first phase, which coincided with the onset of proteinuria, did not require T cells and culminated only in moderate hypercellularity and proteinuria. The second increase in the number of glomerular macrophages, which was accompanied by the expression of abnormal macrophage phenotypes, was closely linked to the development of severe kidney insufficiency. The protective effect of cyclosporin A therapy was consistent with, although not conclusive proof for, the hypothesis that local T cell activation may contribute to the progression of proliferative immune complex glomerulonephritis. Since cyclosporin A can also directly influence the responses of macrophages and mesangial cells, the effect of the drug on the course of nephritis in this model might not be related to its immunosuppressive action.     

Experimental glomerulonephritis induced by human IgG in rats.

Experimental immune complex glomerulonephritis was induced with human IgG (HIgG) in two strains of rats, Wistar-King-Aptekman (WKA) and spontaneously hypertensive rats (SHR). The rats were pre-immunized s.c. with 1 mg of HIgG in Freund's complete adjuvant, and 8 weeks later were given a daily i.v. 2 mg of HIgG injections for 4 weeks. Renal tissue, obtained at weekly intervals after the beginning of i.v. injections, was examined by light, immunofluorescence and electron microscopic tests. SHR developed an endocapillary proliferative glomerulonephritis with heavy proteinuria after the administration of HIgG for 4 weeks. They had massive depositions of HIgG, rat IgG, and rat C3 both in the mesangial area and along the capillary wall. On the other hand, in WKA rats, the proliferative lesions were scarcely seen and the immune deposits were observed almost exclusively in the mesangium. Moreover, urinary protein excretion of these rats was within normal range. In comparison with bovine serum albumin (BSA) nephritis in SHR, HIgG-induced glomerular lesion was relatively mild. This difference seemed attributable to the nature of the HIgG, such as its molecular weight and immunogenicity.     

Therapeutic Approach by a Novel Designed Anti-Inflammatory Drug,M2000, in Experimental Immune Complex Glomerulonephritis

The therapeutic efficacy of novel designed nonsteroidal anti-inflammatory drug, M2000 (ß- D- mannuronic acid) on experimental immune complex glomerulonephritis was evaluated. Bovine serum albumin (BSA) nephritis was induced in rats by a subcutaneous immunization and daily intravenous administration of BSA. M2000 solution (30 mg/kg) was administered intraperitoneally at regular 48-hr intervals for 4 weeks. Onset of treatment was day 56. Urinary protein was measured weekly and serum anti-BSA antibody was assessed by ELISA method at different intervals. Animals were killed on day 84 and blood samples and kidney specimens were obtained. Serum (creatinine, blood urea nitrogen, cholesterol, and triglyceride) and urine (protein, urea, and creatinine) determinants were measured at the time of sacrifice. Kidney specimens were processed for light and immunofluorescent microscopic examination. The fibrosarcoma cell line was used for assaying tolerability and matrix metalloproteinase type 2 (MMP-2) activity. MMP-2 activity was assessed using zymography. Our data showed that M2000 therapy could significantly reduce the urinary protein excretion in treated rats versus non-treated controls. Anti-BSA antibody titer was lower in treated rats than in controls at the 12th experimental week. Polymorphonuclear neutrophil leukocytes infiltration and glomerular immune complex deposition were less intense in treated rats than in controls. Cytotoxicity analysis of M2000 showed a much higher tolerability compared with other tested drugs (diclofenac, piroxicam and dexamethasone). The inhibitory effect of M2000 in MMP-2 activity was significantly greater than that of dexsamethasone and of piroxicam at a concentration of 200 μg/ml. Moreover, the toxicological study revealed that M2000 had no influence on serum (BUN, creatinine, triglyceride and cholesterol) determinants, urinary protein excretion and glomerular histology in healthy group receiving drug.

Conclusions: These findings suggest that treatment with M2000 can reduce proteinuria, diminish antibody production, and suppress the progression of disease in a rat model of immune complex glomerulonephritis.     

The interaction of anti-glomerular basement membrane antibody deposition with immune elimination of bovine serum albumin in the rabbit.

We studied the interaction of two different forms of immune glomerular damage occurring simultaneously: anti-glomerular basement membrane (GBM) antibody fixation and immune elimination of bovine serum albumin (BSA). 125I-radiolabelled BSA anti-BSA immune complexes, formed in response to a single small intravenous dose (150 mg/kg) of 125I BSA, did not cause proteinuria in control animals within 15 days, despite evidence of immune elimination of the antigen. Similarly, a small dose of nephrotoxic globulin (NTG)(3.0 mg/kg) did not cause immediate proteinuria in controls. Test animals received the BSA injection followed by the NTG injection 5, 7 or 9 days later. In this way, antibody fixed to glomerular basement membrane antigens at various times after BSA anti-BSA complexes first appeared in the circulation. Animals were killed on day 15. Fifteen of the eighteen test animals developed moderate to severe clinical nephritis. The onset of the nephritis coincided with BSA elimination irrespective of when the NTG was given. Greatly increased amounts of nonlinear immunofluorescent deposits were demonstrated in the glomeruli of test animals. We concluded that there was a marked synergistic effect between two forms of immune glomerular damage (i.e. that mediated by anti-GBM antibody and immune complexes), which appeared to be due to the increased deposition of complex material in the presence of active fixation of anti-GBM antibody. The relevance of this finding to human glomerulonephritis is discussed.     

Bovine serum albumin (BSA) nephritis in rats. I. Experimental model.

Proliferative glomerulonephritis with proteinuria was induced in Wistar rats by bovine serum albumin (BSA). Rats were first immunized with 1 mg or 2.5 mg of BSA and complete Freund's adjuvant (CFA) and eight weeks later 1 mg of BSA were given intravenously six times a week for four weeks. Immunofluorescence revealed granular deposits of IgG, C3, and BSA in the mesangial area with or without deposition of the same components along the capillary wall. Evaluation of the circulating antibody disclosed an apparent correlation between the level of antibody and histological findings. Rats with an intermediate amount of antibody production developed mesangial widening with mesangial immune deposits and no proteinuria. Rats with a low response developed proliferative glomerulonephritis with immune deposits along the capillary wall as well as in the mesangial area and proteinuria.     

Urinary excretion of the C5b-9 membrane attack complex of complement is a marker of immune disease activity in autologous immune complex nephritis.

The urinary excretion of the C5b-9 membrane attack complex of complement correlates with glomerular deposition of antibody in the passive Heymann nephritis (PHN) model of membranous nephropathy (MN). To determine if this parameter can be correlated with antibody deposition in a model of MN induced by an autologous mechanism and thus more analogous to human MN, the relationship of urinary C5b-9 to ongoing glomerular immune complex formation late in autologous immune complex nephritis (AICN) was studied. Based on urinary C5b-9, the animals were divided into two groups at 12 weeks after induction of AICN, those with persistently high urinary C5b-9 excretion and those in whom urinary excretion of C5b-9 returned to undetectable levels. While all rats developed glomerular deposition of rat IgG and significant proteinuria, high C5b-9 excretors had greater proteinuria and prolonged positive staining for glomerular C3. When normal syngeneic kidneys were transplanted into rats (n = 3) from each group, only those with persistent C5b-9 excretion developed subepithelial immune deposits of rat IgG in the transplanted kidney. As in the PHN model of MN, proteinuria was dissociated widely from urinary C5b-9 excretion, glomerular C3 staining, and evidence of circulating antibody. Thus these findings demonstrate that urinary excretion of C5b-9 serves as an index of on-going immunologic disease activity in the AICN model of MN, while proteinuria does not.     

Bovine serum albumin (BSA) nephritis in rats. III. Antigen distribution in various organs.

We established an experimental model of immune complex glomerulonephritis (ICGN) in rats by daily i.v. administration of 2.0 mg of bovine serum albumin (BSA) for 4 weeks (Yamamoto et al., 1978). In the present study, antigen distribution among various organs was evaluated in the whole course of the experimental rat model using the paired radiolabel technique and the direct immunofluorescent method. In the late stage of chronic serum sickness (CSS), antigen distribution to the liver decreased and that to the glomeruli increased remarkedly whereas hepatic distribution of the antigen was the highest among the organs in the early stage of CSS. BSA distribution in the glomeruli correlated positively with the distribution in blood and there was an inverse relationship between glomerular distribution of BSA and hepatic distribution throughout the course of CSS. These observations may show direct evidence that events in the liver influence the development of ICGN.     

Suppressive effect of cyclosporin A on the induction of chronic serum sickness nephritis in the rat

The suppressive effect of cyclosporin A (CyA) on the development of antibody-induced glomerulonephritis was analyzed in rats with serum sickness nephritis. The induction of serum sickness was established by preimmunization and subsequent continuous administration of the same antigen starting 1 month later. When CyA was given in the preimmunization stage, neither induction of glomerulonephritis nor specific antibody response was observed. In contrast, when CyA was administered in the stage of continuous sensitization, mild glomerulonephritis as well as specific antibody response developed. This phenomenon implies that the significant inhibition of the occurrence of immune complex nephritis results from the suppression by CyA of its primary response to the sensitizing antigen.     

Induction of an Autologous Immune-complex Glomerulonephritis in the Rat by Intravenous Injection of Heterologous Anti-rat Kidney Tubular Antibody: I. Production of Chronic Progressive Immune-complex Glomerulonephritis

An autoimmune kidney disease morphologically and functionally similar to the Heymann autoimmune nephrosis can be produced in rats by the injection of BSA followed by heterologous anti-rat kidney tubular antibody. Animals injected with the anti-kidney tubular antibody only, developed a milder form of the typical renal lesion without proteinuria. Control animals injected with BSA or normal rabbit serum alone and BSA and normal rabbit serum together did not develop a progressive type of kidney disease.Gamma-globulin eluted from the developed lesions of the heterologous antibody induced glomerulonephritis was autologous IgG which reacted with the periluminal zone of the proximal tubules on normal rat kidney sections in a fluorescent antibody test. Gamma-globulin eluted from kidneys of homologous renal antigen induced autologous immune complex (AIC) nephritis reacted with normal rat kidney sections in a similar manner.It is suggested that heterologous anti-tubular antibody reaching the proximal convoluted tubules is reabsorbed and releases the nephritogenic antigen with subsequent formation of autoantibody to it. The continuous release of the nephritogenic antigen and the development of the chronic progressive autologous immune-complex glomerulonephritis is maintained by autoantibody produced as the result of the ongoing autoimmune processes.     

BOVINE SERUM ALBUMIN (BSA) NEPHRITIS IN RATS I. EXPERIMENTAL MODEL

Proliferative glomerulonephritis with proteinuria was induced in Wistar rats by bovine serum albumin (BSA). Rats were first immunized with 1 mg or 2.5 mg of BSA and complete Freund's adjuvant (GFA) and eight weeks later 1 mg of BSA were given intravenously six times a week for four weeks. Immunofluorescence revealed granular deposits of IgG, G3, and BSA in the mesangial area with or without deposition of the same components along the capillary wall. Evaluation of the circulating antibody disclosed an apparent correlation between the level of antibody and histological findings. Rats with an intermediate amount of antibody production developed mesangial widening with mesangial immune deposits and no proteinuria. Rats with a low response developed proliferative glomerulonephritis with immune deposits along the capilary wall as well as in the mesangial area and proteinuria.     

SUPPRESSIVE EFFECT OF CYCLOSPORIN A ON THE INDUCTION OF CHRONIC SERUM SICKNESS NEPHRITIS IN THE RAT

The suppressive effect of cyclosporin A (CyA) on the development of antibody-induced glomerulonephritis was analyzed in rats with serum sickness nephritis. The induction of serum sickness was established by preimmunization and subsequent continuous administration of the same antigen starting 1 month later. When CyA was given in the preimmunization stage, neither induction of glomerulonephritis nor specific antibody response was observed. In contrast, when CyA was administered in the stage of continuous sensitization, mild glomerulonephritis as well as specific antibody response developed. This phenomenon implles that the significant inhibition of the occurrence of immune complex nephritis results from the suppression by CyA of its primary response to the sensitizing antigen. ACTA PATHOL JPN 38: 11–19, 1988.     

Identification and quantitation of cells that process immune complexes in nephritic rats induced with bovine serum albumin

A light microscopic autoradiography was used to determine which cell types positively disposed of the daily circulating immune complexes produced in rats with experimental immune complex glomerulonephritis. Our results indicated that polymorphonuclear leucocytes (PMNs) remove large quantities of immune complexes in the livers, spleens, and lungs of these rats. Semiquantitative autoradiography indicated further that the function of mononuclear phagocyte system decreased late in the course of glomerulonephritis; whereas, in compensation, the role of PMNs appeared to increase. Our data uncovered a significant role for PMNs in the disposition of immune complexes during the induction of experimental immune complex nephritis.     

Therapeutic approach by a novel designed anti-inflammatory drug, M2000, in experimental immune complex glomerulonephritis

The therapeutic efficacy of novel designed nonsteroidal anti-inflammatory drug, M2000 (beta- D- mannuronic acid) on experimental immune complex glomerulonephritis was evaluated. Bovine serum albumin (BSA) nephritis was induced in rats by a subcutaneous immunization and daily intravenous administration of BSA. M2000 solution (30 mg/kg) was administered intraperitoneally at regular 48-hr intervals for 4 weeks. Onset of treatment was day 56. Urinary protein was measured weekly and serum anti-BSA antibody was assessed by ELISA method at different intervals. Animals were killed on day 84 and blood samples and kidney specimens were obtained. Serum (creatinine, blood urea nitrogen, cholesterol, and triglyceride) and urine (protein, urea, and creatinine) determinants were measured at the time of sacrifice. Kidney specimens were processed for light and immunofluorescent microscopic examination. The fibrosarcoma cell line was used for assaying tolerability and matrix metalloproteinase type 2 (MMP-2) activity. MMP-2 activity was assessed using zymography. Our data showed that M2000 therapy could significantly reduce the urinary protein excretion in treated rats versus non-treated controls. Anti-BSA antibody titer was lower in treated rats than in controls at the 12th experimental week. Polymorphonuclear neutrophil leukocytes infiltration and glomerular immune complex deposition were less intense in treated rats than in controls. Cytotoxicity analysis of M2000 showed a much higher tolerability compared with other tested drugs (diclofenac, piroxicam and dexamethasone). The inhibitory effect of M2000 in MMP-2 activity was significantly greater than that of dexsamethasone and of piroxicam at a concentration of 200 microg/ml. Moreover, the toxicological study revealed that M2000 had no influence on serum (BUN, creatinine, triglyceride and cholesterol) determinants, urinary protein excretion and glomerular histology in healthy group receiving drug. CONCLUSIONS: These findings suggest that treatment with M2000 can reduce proteinuria, diminish antibody production, and suppress the progression of disease in a rat model of immune complex glomerulonephritis.     

Participation of the interstitium in acute immune-complex nephritis: interstitial antigen accumulation,cellular infiltrate,and MHC class II expression

Bovine serum albumin (BSA) injected into the rabbits induces acute immune complex glomerulonephritis. Since albumin is filtered and reabsorbed in the tubules, we investigated whether tubulointerstitial cells participate in the pathogenesis of this experimental condition. For this purpose, we induced immune-complex nephritis in 45 rabbits with the injection of 125I-BSA and urinary BSA excretion, glomerular and tubulointerstitial BSA accumulation, lymphocyte infiltration, proliferative activity and MHC class II antigens were examined 2, 4-5 and 6-8 days after immunization. Proteinuria developed day 6-8. BSA was found in urine from day 2 (mean +/- SE; 1089 +/- 339 micro g/24 h) and peaked on day 4 after immunization (2249 +/- 1106). BSA content (cpm/g tissue) in tubulointerstitium (TI) and glomeruli were similar at day 2 (457 +/- 45 and 407 +/- 75, respectively), but afterward increased significantly in TI, reaching a peak level on day 5 (1026 +/- 406) while remained unchanged in glomeruli (388 +/- 95). At the same time, preceding the onset of proteinuria, maximal intensity of the lymphocyte infiltration, proliferative activity and MHC class II antigen expression in tubular cells, monocytes/macrophages and interstitial cells were observed. Our study shows that antigen is excreted in the urine and concentrated in TI in association with overexpression of MHC class II molecules and lymphocyte infiltration. These findings occur prior to the development of proteinuria and suggest that the tubulointerstitial cells play a critical role in the pathogenesis of this model.     

Treatment of murine immune complex glomerulonephritis with prostaglandin E2: dose--response of immune complex deposition, antibody synthesis, and glomerular damage

A model of immune complex glomerulonephritis (ICGN) produced in mice by the daily injection of apoferritin was employed to study the effect of treatment with various doses of prostaglandin E2 (PGE2) on glomerular damage, immune complex deposition, proteinuria, and serum anti-apoferritin antibody. Administration of PGE2, 200 micrograms twice daily, resulted in a significant decrease in glomerular damage and immune complex deposition, prevented the development of proteinuria, and significantly reduced serum levels of anti-apoferritin antibody. PGE2, 100 micrograms twice daily, resulted in a decrease in immune complex deposition as assessed by immunofluorescence microscopy, but this dosage did not significantly alter glomerular damage, proteinuria, or antibody levels. PGE2 dosages of 50 and 25 micrograms twice daily had no effect on any of these parameters. The protective effect of PGE2 on the development of ICGN occurred only at dosages that were associated with decreased anti-apoferritin antibody.     

Platelet involvement in the nephritis of acute serum sickness in rabbits: protection by dipyridamole and FUT-175.

In the acute serum sickness model in rabbits, we investigated platelet release of 5-HT, platelet surface immunoglobulins, and platelet aggregation in response to ADP, together with the effect of dipyridamole and the Clr antagonist FUT-175. The immune release of 5-HT from platelets occurred between 4 and 6 days after injection of bovine serum albumin (BSA), before immune elimination and proteinuria, but coincident with the appearance of immune complexed BSA in the circulation. Nevertheless, platelet turnovers were not detectably accelerated. Treatment with dipyridamole 50 mg/kg/24 h prevented the release of 5-HT and inhibited proteinuria, glomerular hypercellularity and immune complexes in the glomeruli. Using the Clr antagonist FUT-175, similar abrogation of the disease was obtained. We conclude that in the nephritis of acute serum sickness in rabbits, some of the immune release from platelets may be the result of immune complex binding to the platelet, perhaps through the receptor for C3b.     

Cyclosporin A inhibits acute serum sickness nephritis in rabbits.

The effect of the immunosuppressive agent cyclosporin A (Cy A) on the renal injury in acute serum sickness was examined in rabbits. Serum sickness was induced in 23 untreated NZW rabbits by a single intravenous injection of bovine serum albumin (BSA) 250 mg/kg with E. coli endotoxin (5 micrograms/kg): BSA was eliminated after 8.6 +/- 0.16 days (mean +/- s.e. (mean]; proteinuria occurred in 19 (84%) and glomerular proliferation in 20 (87%) rabbits. When Cy A (15 or 25 mg/kg) was given daily by intramuscular injection, starting either 2 days before or at the time of induction of acute serum sickness, proteinuria was profoundly reduced and glomerular proliferation was inhibited. Even when rabbits were first treated with Cy A (25 mg/kg) 5 days after the induction of disease proteinuria and glomerular proliferation were similarly inhibited. When the treated animals were compared with controls there were no differences in the following: time to elimination of BSA, amount or size of circulating immune complexes, fall in serum C3 at immune elimination, or deposition of immune reactants in the glomeruli. These results show that Cy A inhibits the renal injury of acute serum sickness and indicate that T cells may play a role in mediating the nephritis in this condition.     

Active in situ immune complex glomerulonephritis using the hapten-carrier system: role of epitope density in cationic antigens.

The role of epitope density in cationic antigen was investigated in an active model of in situ immune complex glomerulonephritis (ICGN) using the hapten-carrier system. Trinitrophenol (TNP) was conjugated with variable density to cationic human immunoglobulin (C-HIgG) to yield TNP6.2-C-HIgG (low-valency antigen) and TNP31.3-C-HIgG (high-valency antigen). In rats preimmunized with TNP17.3-bovine serum albumin (BSA), endocapillary proliferative GN with proteinuria developed in rats receiving high-valency antigen. In contrast, no significant abnormalities in renal histology or urinalysis were observed when a low-valency antigen was injected. These results indicate that glomerular injury produced by hapten-specific immune reaction is affected by the number of haptenic groups conjugated to the carrier molecule (epitope density) in active in situ ICGN.     

Rat bovine serum albumin (BSA) nephritis. VI. The influence of chemically altered antigen.

Bovine serum albumin (BSA) used to incite chronic serum sickness glomerulonephritis in rats was chemically modified to study the effect of antigenic alteration. The BSA was used in its native form (n-BSA) as well as anionic (a-BSA), cationic (c-BSA) or glycosylated (g-BSA) forms. Spontaneously hypertensive rats (SHR) preimmunized 8 weeks earlier received daily intravenous injections of the respective BSA preparations for the ensuing 3 weeks. Histological examination of their kidneys revealed that c-BSA given for 2 weeks induced a severe diffuse proliferative glomerulonephritis and profound proteinuria. Electron-dense deposits localized preferentially in the subepithelial spaces of renal glomeruli from these rats, but a few in the mesangia. Quite differently, rats receiving n-BSA or g-BSA developed a less severe form of glomerulonephritis even after 3 weeks of injections. Besides the massive mesangial deposits, the subepithelial deposits were conspicuous in the glomeruli from rats given g-BSA for 2 weeks, but deposition in glomerular capillaries was rare in rats given n-BSA for the same duration. In contrast, the administration of a-BSA resulted in minimal abnormalities visible by light microscopy and a few immune deposits in the mesangia even at the third week. The antibody response in rats given c-BSA or a-BSA was apparently different from n-BSA treated rats. The present study shows the important role of the antigen's electric charge in the pathogenesis of proliferative glomerulonephritis. The foregoing results also foster our proposal that the carbohydrate content of the antigen influences the development of this renal disease.