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By immunization with rabbit immunoglobulins and the injection of a subnephritogenic dose of rabbit nephrotoxic serum (NTS), accelerated-type nephrotoxic serum nephritis (NTN) was induced in heterozygous (rnu/+) rats but not in athymic nude (rnu/rnu) rats. By transferring rat antibody against rabbit immunoglobulins, marked proteinuria was induced also in nude rats (202.0 +/- 98.4 mg/day on day 3) as in rnu/+ rats (122.6 +/- 35.3 mg/day on day 3). No marked differences in histological findings could be found between both groups. The most marked increase in the number of intraglomerular infiltrating cells was observed in heterozygous rats indicating that the presence of thymus-derived cells leads to the accumulation of more cells in glomeruli. We conclude that humoral immunity alone is enough to accelerate the pathogenic mechanism which induces glomerular injury with heavy proteinuria in this model.

Immunoferritin conjugates consist of conjugates of different size. They can be separated in 4.5% polyacrylamide gel electrophoresis into at least 4 bands. The amount of the smallest 1:1 conjugate, in which one antibody molecule is linked to one ferritin molecule, is highest in coupling products with low overall yields of conjugated ferritin. Therefore relatively mild reaction conditions are recommended. With increasing size the antibody binding capacity of the conjugate is reduced.

We have experimentally induced the 'linear pattern' in immunofluorescence: the linear deposition of endogenous immunoglobulin (Ig) along the glomerular basement membrane (GBM) in rats injected with both protamine and nephrotoxic serum. This Ig was demonstrated to have no specific antibody activity against GBM or rabbit serum. Our findings could be of value in the analysis of the mechanism and pathological meaning of the 'linear pattern' of endogenous Ig in immunofluorescence.

A method, by which the more precise localization of immunofluorescent deposits in the glomerulus is easily demonstrated with the fluorescence microscopy, was described. By means of this method, the deposits even in the Bowman's space could be identified, as well as the deposits in the subepithelial, subendothlial and mesangial regions. From this result, it was suggested that this method should be widely used for the immunofluorescence studies on human renal biopsied sections, in order to elucidate the relationship between the localization of deposits and renal diseases.

We have developed hybridoma cell lines which secrete monoclonal antibodies to some rat renal antigens, namely the brush border of proximal tubular epithelium and the cytoplasm of tubular cells. The immunoglobulin class of the hybridoma was found to be IgG1. Specific antibody activity against either glomerular basement membrane (GBM) and tubular basement membrane (TBM) or Bowman's capsule and a part of TBM was observed, although these hybridoma cell lines have not yet been successfully established. In particular, the hybridoma secreting antibdodies to TBM did not remain stable during antibody production, and was lost during the culture and cloning procedures. These monoclonal antibodies should be of value in research on the pathogenesis of human glomerulonephritis. [ABSTRACT FROM AUTHOR]

In vivo studies in rats demonstrated that the binding of a highly cationic antigen (cationized human IgG, pI >9-5) to glomerular polyanion could be significantly reduced by prior application of a small polycation, protamine sulfate. The degree of inhibition was dose dependent and the highest dose used, 4 mg/100 g body weight, reduced antigen binding by approximately 70%. In further experiments the ability of protamine sulfate to enhance elimination of cationic antigen-antibody immune complexes from the glomerular capillary wall was examined. Daily treatment with protamine sulfate, starting after induction of nephritis, produced a significant but only moderate reduction in the persistence of the antigen, without having any effect on proteinuria. Proteinuria could only be prevented when protamine sulfate was given immediately before induction of nephritis. Proiamine sulfate had little influence on the course of established renal disease in the model employed. These results do not substantiate the concept of charge competition as a potentially useful therapeutic strategy. [ABSTRACT FROM AUTHOR]

Glycophosphatidylinositol (GPI-linked Thy-1 molecules, well known cell surface markers of murine T cells., are present on the glomerular mesangial cells of the rat kidney. The administration of anti-Thy-1.1 MoAbs 1-22-3 and OX-7 to rats induces severe and mild complement-dependent mesangial proliferutive glomerulonephritis, respectively. In order lo determine whether proteintyrosine kinase (PTK) activity is associated with Thy-1 molecules on rat mesangial cell surface, we performed an immune complex kinase assay, using anti-Thy-1 MoAbs 1-22-3 and OX-7, followed by reimmunoprecipitation with anti-phosphotyrosine, anti-fyn, anti-lck and anti-lyn antibodies, Physical association of PTK, p59fyn and p56/53lyn with Thy-1 molecules was demonstated in cultured rat mesangial cells. The activities of these kinases detected in MoAb 1-22-3 precipitates were higher than those in MoAb OX-7 precipitates. These results suggest that Thy-1 molecule transduces some signals also in rat mesangial cells. [ABSTRACT FROM AUTHOR]

Histone can mediate the binding of both free DNA and DNA complexed to anti-DNA antibody to the glomerular capillary wall. We tested whether preformed histone--DNA--anti-DNA immune complexes (IC) could bind to the glomerular capillary wall. The immune complex, generated with anti-DNA antibody derived from an SLE patient and excess of 125I-DNA followed by digestion with DNase, was mixed with histones. The complex containing 4 μg DNA was injected via the aorta into the left kidney of rats. At 15 min, 1.3% of the histone-DNA--anti-DNA antibody complex bound (measured as 125I-DNA), when histone was omitted less than 0.1% of the DNA anti-DNA antibody complex bound. By immunofluorescence human immunoglobulins and histones, representing the IC, could be observed in a capillary pattern; but no complement deposition was detected. Electron microscopy revealed discrete, electron dense deposits in a subendothelial, subepithelial and mesangial localization at 15 min. These results provide direct evidence that antibodies from serum of SLE patients can form soluble histone-DNA--anti-DNA immune complexes that bind to the glomerular capillary wall in vivo. [ABSTRACT FROM AUTHOR]

A new animal model of progressive glomerulosclerosis was developed by administering a single i.v injection of MoAb I-22-3 to unilaterally nephrectomized rats. Renal morphological analysis revealed that glomerular lesions characterized by mesangial cell proliferation and mesangial matrix expansion were induced in about 95% of the glomeruli. Approximately 20% of the glomeruli of the unilaterally nephrectomized rats showed sclerosis or segmental sclerosis by week 6 after MoAb injection and crescent formation was observed in some glomeruti (Ca 4%). Cellular infiltration was also noted in some parts of the interstitium. Increased expression of transforming growth factor-beta (TGF-β) was observed in the unilaterally nephrectomized rats treated with MoAb I-22-3. but we could not demonstrate pathological involvement of platelet-derived growth factor (PDGF), even though early-stage mesangial cell proliferation was observed. The mechanism of mesangial cell proliferation in this model remains to be elucidated. The relatively short period of time needed to induce the sclerotic changes is considered to be a great advantage of this model for clarifying the mechanisms involved in the chronic progression of mesangial proliferative glomerulonephritis. [ABSTRACT FROM AUTHOR]

A single i.v. injection of 3 mg of the F(ab')2 fragment of MoAb 5-1-6 into rats induced immediate proteinuria (128.1±80.7 mg/24 h on day 1) which lasted 1-2 days. In contrast, rats administered 10 mg of the corresponding Fab fragment did not develop abnormal proteinuria even though an equivalent dose of the intact MoAb 5-1-6 far exceeded the nephritogenic dose. The total kidney binding of 125-Fab fragment was 209.5 ± 34.3 μg/2 kidneys. This exceeded that obtained by injection of 3 mg MoAb 5-1-6 IgG1 (58.9 ± 12.5 μg/2 kidneys at 1 h) and was similar to that obtained following injection of 3 mg F(ab)2 fragment (235.3 ± l6.9 μg/2 kidneys). Immunofluorescence (IF) showed a linear pattern along the glomerular capillary wall at 1h after the administration of MoAb 5-1-6 IgG 1, F(ab )2 or Fab fragment. On day 5, fine to coarse granules were observed scattered in F(ab')2-injected rat glomeruli, whereas granules were densely localized in Fab-injected rat glomeruli. Complement-depleted rats injected with 3 mg of MoAb 5-1-6 IgG1 developed proteinuria with the same time course as non-depleted rats. This observation, together with the ability of F(ab')2 to induce proteinuria, indicates that proteinuria induced by MoAb 5-1-6 is complement-independent. This study suggests that MoAb 5-l-6-induced proteinuria is initiated by cross-linking of the epitopes by divalent MoAb 5-1-6 and is independent of complement activity. [ABSTRACT FROM AUTHOR]

A description is made of renal lesions in rats induced by heterologous (rabbit) nephrotoxic serum with or without subsequent host immune reactions against it and the effects of immune reactions on the course of classical nephrotoxic serum (Masugi) nephritis are discussed. The disease was induced by injecting congenitally athymic ACI nude rats (rnu/rnu) and their normal heterozygous littermates (rnu/+) with rabbit anti-rat glomerular basement membrane (GBM) antiserum. In the autologous phase, rat IgG and immunoglobulins were localized in a linear pattern along capillary walls only in nephritic heterozygous rats. In the indirect plaque-forming cell (PFC) assay against rabbit immunoglobulins in the autologous phase, significantly more PFC could he detected in nephritic heterozygous rats than in nephritic nude rats. The nude and heterozygous rats were essentially the same with respect to the amount of urinary protein, histological change and clinical course. At least in classical nephrotoxic serum nephritis in rats, host immune reactions against GBM bound heterologous nephrotoxic serum were concluded to have no effect on the course of the disease. [ABSTRACT FROM AUTHOR]

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-HlgG (low-valency antigen) and TNP13.3-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. [ABSTRACT FROM AUTHOR]

IgM and IgG class rheumatoid factor (RF) containing glomerular immune complex (IC) deposits were detected in renal biopsy material from 10 out of 123 patients with various forms of glomerulonephritis (GN) by an immunofluorescent assay using FITC conjugated aggregated human IgG FAHIgG). In lupus GN, six out of 28 cases (235%), showed such activity. Solubilization of glomerular IC deposits in frozen sections of lupus GN was observed after a 5 h incubation with native 7S IgG, The binding of FAH IgG to glomerular IC deposits and solubilization of deposits by excess HIgG were also confirmed in sections of two types of rat experimental GN in which HIgG acted as exogenous antigen; in situ IC formation type GN in cationized HIgG planted kidneys and chronic serum sickness type GN. These results suggest a possible antigen excess mechanism contributing to the solubilization of glomerular IC deposits via RF and IgG interactions in lupus GN. [ABSTRACT FROM AUTHOR]

Objective: Progression is a hallmark of chronic renal diseases and histologically characterized by fibrosis and inflammation of the tubulointerstitial compartment independent of the underlying disorder. To define the role of lymphocytes in this process, the novel lymphocytespecific inhibitor FTY720 was administered to rats with progressive renal fibrosis, i.e. anti-thy1-induced chronic glomerulosclerosis (cGS). In this model, the initial and short-term inflammatory glomerular injury progresses self-perpetuatedly over months towards tubulointerstitial fibrosis by not primarily immune-mediated mechanisms. Methods: cGS was induced by murine anti-thy1 antibody injection into uni-nephrectomized male Wistar rats. Treatment with FTY720 (0.3 mg/kg body weight/d) was started 7 days after disease induction. In week 20, the following parameters were determined: proteinuria, blood lymphocyte number, plasma markers of kidney function, both glomerular and tubulointerstitial histological matrix accumulation (matrix score 0-4) and protein expression of transforming growth factor (TGF)-beta1, fibronectin and plasminogen-activator-inhibitor(PAI)-1 as well as renal macrophage and lymphocyte infiltration. Results: Treatment with FTY720 markedly reduced blood lymphocyte counts (-90%, p < 0.001) and renal lymphocyte infiltration (-64%, p < 0.01). In comparison to the untreated cGS animals, the lymphocyte reduction achieved significantly limited disease progression in this model, as shown by lowered proteinuria (73 ± 19 vs 171 ± 35 mg/d, p < 0.05), tubulointerstitial matrix expansion (matrix score 0.6 ± 0.2 vs 1.9 ± 0.5, p < 0.05) and TGF-beta1 (136 ± 7 vs 187 ± 16 pg/mL, p < 0.05), fibronectin (1,139 ± 145 vs 1,837 ± 228 ng/mL, p < 0.05) and PAI-1 expression (54 ± 8 vs 64 ± 6 ng/mL), as well as by improved renal function (plasma creatinine 0.64 ± 0.01 vs 0.75 ± 0.05 mg/dL, plasma urea 51 ± 4 vs 73 ± 11 mg/dL, both p < 0.05). Glomerular matrix protein expression and accumulation was moderately reduced by FTY720. Glomerular macrophage infiltration was not, tubulointerstitial macrophage infiltration was moderately, but not significantly decreased by FTY720 treatment. Conclusions: Lymphocyte inhibition by FTY720 limits disease progression towards tubulointerstitial fibrosis and renal insufficiency in experimental anti-thy1-induced, chronic glomerulosclerosis, although this process is not primarily immune-mediated. The data suggest that lymphocytes actively participate in the progression of chronic kidney diseases, and that FTY720 may be a novel approach to slow the progressive course of human chronic renal diseases. [ABSTRACT FROM AUTHOR]