Experimental mesangioproliferative glomerulonephritis in rats induced by intravenous administration of anti-thymocyte serum

Focal glomerulonephritis was induced in rats, by a single intravenous injection of anti-Thy-1.1 antibody (ATS). One hour after the administration, the glomeruli of affected rats developed necrotic changes of the mesangial cells while after two hours, mesangiolytic changes appeared. From six days onwards, focal segmental mesangial proliferation which persisted until 30 days, occurred. This is thought to be the first report of experimental nephritis induced by pure anti-mesangial antibody.     

Distinctive roles of neutrophils and monocytes in anti-thy-1 nephritis

Anti-Thy-1.1 glomerulonephritis as an experimental model for mesangial proliferative glomerulonephritis was induced in Wistar rats by a single injection of monoclonal IgG2a-anti-Thy-1.1 antibody (ER4G). This transient model is complement-mediated and leads to mesangial-cell (MC) lysis followed by MC proliferation, glomerular microaneurysm formation, glomerular influx of polymorphonuclear leukocytes (PMNs) and macrophages, proteinuria, and hematuria. In this study we investigated the distinctive roles of infiltrating PMNs or monocytes/macrophages by treating rats with an antibody against rat integrin CD11b/CD18 (ED7) or by depletion of monocytes with multilamellar clodronate liposomes, respectively. ED7 administration resulted in reduction of the influx of PMNs in glomeruli during the first 6 days after induction of Thy-1.1 nephritis, whereas treatment with an isotype-matched irrelevant antibody (PEN9) or with phosphate-buffered saline had no effect on macrophage influx. Increased glomerular C3 and C6 deposition on days 1 and 3 was seen in the ED7-treated rats but not seen in the control groups. In addition, the ED7-treated group showed an increased number of aneurysmatic glomeruli and more severe hematuria. Monocyte/macrophage depletion led to a significant reduction of mesangial matrix expansion, although mesangial proliferation, proteinuria, and hematuria remained unaltered. These results, together with the known effects of PMN-derived enzymes on C3 cleavage, suggest that a reduction in the influx of PMNs results in sparing of C3 and consequently of more complement activation in the glomerulus with increased complement-mediated damage. Our data indicate that infiltrating PMNs and monocytes/macrophages play distinctive roles during inflammation in this model of MC glomerulonephritis.     

Bovine serum albumin chronic serum sickness nephropathy in rats.

We administered bovine serum albumin (BSA) 1 or 3 mg i.v. into hyper-immune Sprague-Dawley rats weekly for up to 6 months. Animals with free circulating antibody 1 h after BSA developed mesangial deposits of IgG and C-3 without proteinuria. Rats without free antibody at 1 h developed either mesangial or mesangial and glomerular basement membrane (GBM) deposits. Rats given 3 mg BSA tended to have GBM deposits, proteinuria and undetectable antibody at 1 h. By electronmicroscopy, all rats had mesangial and subendothelial dense deposits, while those with GBM lesions had intramembranous and subepithelial deposits with foot process obliteration. Light microscopic evaluation of kidney tissue revealed only mild histological changes similar to those in age-matched control rats. The present studies demonstrate that prolonged i.v. administration of BSA into rats results in the development of a chronic non-inflammatory nephropathy. Despite certain parallels to chronic serum sickness nephropathy in rabbits, species differences appear to modify the nephropathy in rats.     

Glomerulonephritis induced by monoclonal anti-Thy 1.1 antibodies. A sequential histological and ultrastructural study in the rat

The present report describes the natural history of an experimental acute glomerulonephritis with massive proteinuria induced by a single intravenous injection of a (mouse) monoclonal anti-rat Thy 1.1 antibody into the rat. The disease is characterized by direct although transient binding of this monoclonal antibody to glomerular basement membrane and mesangium after injection as demonstrated by immunofluorescence microscopy. Immediate activation of complement occurs as shown by glomerular deposition of C3 and C9. Concomitant activation of the coagulation cascade is reflected by the presence of fibrinogen deposits in the affected glomeruli. One hour after injection mesangial alterations are prominent including condensation of mesangial cell chromatin, and lysis of mesangial cells as shown by light- and electron- microscopy, leading to the formation of aneurysms in the capillary tuft. Commencing on day 4 mesangial cell proliferation can be observed, accompanied by glomerular crescent formation at day 14, which decreases gradually 3 weeks after antibody administration, whereas mesangial hypercellularity can be observed up week 10 after intravenous injection of the antibody. The disease is clinically characterized by a massive transient proteinuria starting immediately after antibody injection, reaching mean values of 300 mg/24 hours at days 2 to 4, gradually decreasing to normal levels after 3 weeks. It is concluded that in this unique model of glomerulonephritis induced by a monoclonal antibody, recognition of Thy 1.1 epitopes as well as activation of complement including the C5-C9 membrane attack complex may play a major role in the pathogenesis of this experimental disease.     

Mesangial sclerotic change with persistent proteinuria in rats after two consecutive injections of monoclonal antibody 1-22-3.

Irreversible mesangial changes with persistent proteinuria were induced in rats given two consecutive injections 2 weeks apart of a MoAb 1-22-3 to rat mesangial cell. The characteristics of the resulting lesions were investigated and compared with those of the reversible change induced by a single injection. At 24 h after the second injection, mesangiolytic changes similar to those after a single injection were evident. The accumulation of macrophage-like cells in glomeruli observed at 1 week after the first injection was not evident during the experimental period after the second injection. Hypercellularity with the characteristics of intrinsic mesangial cell and increased mesangial matrix were already present 1 week after the second injection. And mesangial sclerotic change progressed up to 6 months. Deposition of collagen type I and type III and accumulation of collagen fibril at the ultrastructural level were evident in rats 6 months after the second injection. Proteinuria started immediately and continued for more than 6 months after the second injection. The mesangial sclerotic change with persistent proteinuria described here is considered to be a better model for investigating the mechanism of chronic progression of human mesangial proliferative glomerulonephritis.     

Mesangial immune deposits induced in rats by antibodies to fibronectin

Immune deposits in the glomerular mesangium were induced in rats by injection of rabbit antibodies to rat plasma fibronectin (FN). By direct immunofluorescence and electron microscopy, deposits of immunoglobulins were detected in the mesangium of all rats injected with anti-FN IgG but not of control rats injected with normal rabbit IgG. By light microscopy, kidneys obtained 20 days after the antibody injection appeared to be normal. No proteinuria was detected during the experiment. Tissue uptake studies combined with direct immunofluorescence examination suggest that the initial accumulation of rabbit immunoglobulins in the glomerular mesangium is probably due to direct local binding of anti-FN antibody rather than trapping of immune complexes formed in the circulation. Quantitation of the direct binding using an in vivo perfused kidney system indicate that only a small fraction of the injected antibodies (less than 1 microgram/kidney) could bind. These studies indicate that (1) mesangial immune deposits may be induced by injection of antibodies to a glycoprotein, fibronectin, which is a normal structural component of the mesangium; (2) the initial accumulation of immunoglobulins in the mesangium is probably related to an in situ binding; and (3) mesangial antigens might be involved, in certain cases, in autoimmune reactions.     

An acute model for IgA-mediated glomerular inflammation in rats induced by monoclonal polymeric rat IgA antibodies.

An acute model for IgA-mediated glomerular inflammation in rats was induced by the in situ deposition of IgA directly into the glomerular mesangium. F(ab')2 anti-Thy1 MoAb was used to anchor an antigen, DNP (2,4-dinitrophenol), in the glomeruli of rats. Subsequent infusion of rat polymeric (p-) or monomeric (m-) IgA MoAb with specificity for DNP resulted in mesangial deposition of IgA in both groups of rats. However, acute proteinuria was observed only in p-IgA-treated rats and not in PBS- or m-IgA-treated rats. Immunofluorescence analysis revealed deposition of C3 in an identical pattern to that of IgA in the glomeruli of p-IgA-treated rats. No mesangial deposits of C4 or C1q were seen in these animals. Rats receiving m-IgA or PBS displayed no detectable C3, C4 or C1q deposition. The amount of proteinuria in p-IgA-treated rats was related to the amount of deposited C3. The presence of intraglomerular monocytes was only observed 2 days after p-IgA injection. By light microscopy, aneurysm formation, mesangial hypercellularity and matrix expansion were seen only in p-IgA-treated rats. However, by 37 days post-injection complete resolution of the lesions was observed. No histological renal changes were observed in PBS- or m-IgA-treated rats. In conclusion, an acute form of IgA-mediated nephritis in rats was induced by p-IgA but not by m-IgA. This reproducible model provides a basis for further study into the mechanisms of IgA-mediated glomerular inflammation.     

Chronic serum sickness glomerulonephritis: Passive immunisation inhibits the removal of glomerular antigen and electron dense deposits

Summary Chronic serum sickness glomerulonephritis was induced in 20 Wistar rats, using radio-labelled, chemically cationised bovine serum albumin (BSA) as antigen. Four days after the last injection of antigen, when relocation of antigen within the rat had effectively ceased, the rats were given a single large intraperitoneal dose of either non-immune rat gamma globulin or anti-BSA rat gamma globulin. Ten days later the rats were killed. The rats which had received the anti-BSA globulin had significantly more antigen in renal cortex and in isolated glomeruli than the control group. They also had larger mesangial deposits as assessed by morphometry at electron microscope level; assessment of subepithelial deposits provided equivocal results. These findings provide direct confirmation that circulating antibody which is directed against an antigen which is trapped within deposits in the glomerulus will inhibit the removal of the antigen and deposits from the mesangium.     

Long-term observation of glomerulonephritis induced by multiple injections with anti-Thy-1 antibody in rats

Multiple Injections with a mouse monoclonal anti-rat Thy-1 antibody (five times, at weekly intervals) induced marked glomerular sclerotic lesions which are characterized by adhesion of glomerular capillaries to Bowman's capsule and persistent proteinuria in rats. Abnormal production of type I collagen and increased accumulation of type IV collagen and flbronectln were observed in these glomeruli. The glomerular expression of mRNA for these matrix components and transforming growth factor-pi (TGF-β1) were markedly increased at 4 days after the last injections with anti-Thy-1 antibody, but decreased to below the levels of control rats at 5 weeks. This may be down-regulation of mRNA In mesangial cells. The glomerular sclerotic lesions were not progressive but the process of glomerular healing seemed to be retarded. The proteinuria and the glomerular adhesion were irreversible.     

Influence of antibody avidity on glomerular immune complex localization.

Immune-complex-mediated glomerulonephritis (IC-GN) was induced with daily intraperitoneal injections of horse apoferritin (HAF) for varying intervals in Swiss albino and BALB/c mice. Anti-HAF antibody avidity in plasma pools from mice with predominantly mesangial immune deposits was compared with avidity in plasma pools from mice with predominantly capillary wall deposits. Plasma from Swiss mice having predominantly mesangial deposits after 7 or 14 days of HAF had higher avidities than plasma from Swiss mice with predominantly capillary wall deposits after 14 days or more than 28 days of HAF. Plasma from BALB/c mice with exclusively mesangial deposits after 7 days of HAF had higher avidity than plasma from BALB/c mice with predominantly capillary wall deposits after 14 or more days of HAF. Therefore, there was a correlation between glomerular site of immune deposition and avidity of circulating anti-HAF antibodies, with higher avidity antibodies associated with mesangial immune deposits and lower avidity antibodies with capillary wall deposits.     

Focal mesangial proliferative glomerulonephritis in the rat caused by Habu snake venom: the role of platelets.

Platelet aggregates are a prominent early feature in the glomerular lesions of focal mesangial proliferative glomerulonephritis induced by Habu venom. After venom injection the peripheral blood platelet count falls rapidly, and in vitro the venom causes platelet aggregation and release of 5-HT. The role of platelets in this model has been studied in rats depleted of platelets by antiplatelet serum administration. No effect on the early (24 h) glomerular damage was found. Mesangial proliferation which appears 2--3 days after glomerular injury was significantly inhibited in platelet-depleted rats. These results show that platelet activity does not initiate the mesangial injury in this model, but suggest that platelets or their products act as a stimulus to mesangial proliferation.     

New rat model induced by anti-glomerular basement membrane antibody shows severe glomerular adhesion in early stage and quickly progresses to end-stage renal failure

The aim of the present study was to introduce a new anti-glomerular basement membrane nephritis model in which plasma creatinine levels dramatically increased only 4 weeks after a single administration of rabbit antirat glomerular basement membrane antibody in Sprague–Dawley rats. According to renal morphology, glomerular lesions characterized by mesangial expansion and adhesion of the glomerular tuft to Bowman's capsule were observed in the early stage at day 7 after disease induction; adhesion was detected in approximately 90% of glomeruli 14 days after antibody injection. After 21 days the rats exhibited pronounced glomerulosclerosis/hyalinosis and severe tubulointerstitial lesions characterized by interstitial fibrosis. Urinary podocytes excreted in nephritis rats were studied and it was found that urinary podocyte loss might be closely related to progression of renal injury. Because this new model simply and reproducibly demonstrates development of end-stage renal disease, it will be beneficial for elucidating mechanisms by which chronic renal injury irreversibly progresses, as well as for developing therapeutic agents for chronic renal failure.     

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.     

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.     

Involvement of dipeptidyl peptidase IV in immune complex-mediated glomerulonephritis

Dipeptidyl peptidase IV (DPPIV) is widely expressed in many tissues; however, its precise biological function is poorly understood. One of its possible physiologic roles is an involvement in the immune system, which plays a pivotal role in the pathogenesis of glomerulonephritis. The present study focused on the involvement of DPPIV in immune complex-mediated glomerulonephritis. Experimental nephritis was induced by anti-Thy-1.1 monoclonal antibody E30 using Wistar or F344 rats. The application of a new monoclonal antibody against DPPIV, F16, completely suppressed E30-induced proteinuria and mesangial proliferation in Wistar rats, whereas these preventive effects of F16 were not observed in F344 rats, which spontaneously lack DPPIV protein. Treatment with F16 inhibited glomerular deposition of complement C3 and complement C4 after the binding of E30 to the mesangial cell surface. Because the preventive effect of F16 was attributable to suppression of the complement cascade, we examined its influences on complement-dependent mesangial cell lysis in vitro. We discovered that the complement cascade was markedly inactivated in F16-treated Wistar rat serum but not in F16-treated F344 rats. These results indicate that DPPIV may play a somewhat crucial role in regulating the complement cascade and that inhibition of DPPIV may serve as a new target for preventing complement-dependent tissue injury.     

Quantitative morphological analysis of glomerular changes of the rabbit kidney on treatment with insulin preparations of different purity

Summary Thirty rabbits were immunized with MC insulin and high-molecular impurities of commercial insulin preparations (a+b component) over 30, 60 and 90 days. The serum insulin antibody titer was determined in animals as the insulin binding capacity. Further, a quantitative morphological analysis of the various types of glomerular cells and of the mesangium was performed on the glomeruli as a blind study. Significant mesangial cell proliferation and an increase in mesangial matrix were found on treatment with the a+b component whereas the animals treated with MC insulin exhibited only a transient and slight mesangial activation after 30 days. There was a positive correlation between the magnitude of the insulin binding capacity and the mesangial activation. Hence, the glomerular changes which are observed after treatment with insulin which is not highly purified must be attributed to the high molecular weight contaminants. Heterologous pure insulin must be regarded as having virtually no immunological effect.This work was supported by the Deutsche Forschungsgemeinschaft (We 468/6)     

Experimental glomerulonephritis induced by immune complexes of monoclonal antibodies produced by immunoglobulin class-switch variants

Experimental glomerulonephritis was induced in mice by injecting performed antigen-antibody complexes composed of monoclonal anti-dansyl antibody of switch-variant origin and dansyl-conjugated bovine serum albumin. A comparison was made of the ability of two kinds of monoclonal antibodies, IgE and IgG2a, of the same variant origin to induce glomerulonephritis. Both types of immune complexes (IC), (IgE-IC and IgG2a-IC), given daily elicited exudative proliferative glomerulonephritis accompanied by proteinuria. Significant glomerular hypercellularity including invasion by polymorphonuclear leukocytes was observed as early as 2 days and was prominent at 14 days after the start of daily injections. Deposits of IgE-IC and IgG2a-IC plus the third complement component were observed mainly in mesangial areas early in the experiment (3 to 8 days) and additionally along peripheral loops at a later stage (9 to 14 days). By electron microscopic examination, immune deposits were detected in the mesangial areas as well as in the subepithelial aspect of the peripheral loops. These results reveal that the isotype of the antibody used to prepare IC does not influence the form or severity of glomerulonephritis.     

Mycophenolate mofetil and roscovitine decrease cyclin expression and increase p27(kip1) expression in anti Thy1 mesangial proliferative nephritis

The response of mesangial cells to a phlogistic challenge includes cell proliferation and mesangial matrix expansion. Cell proliferation is a highly regulated process which includes enhancing factors such as cyclins, cyclin dependent kinases, and inhibitory proteins, such as p27(kip1). The aim of the study was to evaluate the effects of Mycophenolate mofetil (MMF), and roscovitine (R), on the cell cycle regulatory system when administered in the florid phase of the experimental model of mesangial proliferative nephritis induced by the anti Thy-1 antigen monoclonal antibody. Three days after nephritis induction, different groups were given MMF and R. Rats treated with MMF or R showed a slight decrease in mesangial proliferation and matrix expansion. Samples of cortical tissue were tested by 'real time' RT-PCR in order to study gene expression of cyclins B, D1, D2, D3, E, and the cyclin inhibitor p27(kip1). Localization of mRNA was evaluated by in situ hybridization. Real time RT-PCR analysis showed a significant decrease in cyclins B, D1, D2, and D3 in rats treated with either MMF or R as compared to controls. Both MMF and R treatment induced a significant increase in p27(kip1) mRNA expression. In situ hybridization showed a mesangial-endothelial expression pattern in glomeruli. The number of labelled cells per glomerulus, the number of positive glomeruli in each examined slide as well as cyclin D2 and D3 signal intensity was significantly lower in rats treated with MMF or R as compared to controls, whereas MMF or R treatment up-regulated p27(kip1) mRNA expression. Immunohistochemical evaluation of p27(kip1) aimed to examine the influence of MMF or R on protein expression confirmed up-regulation.     

Progressive renal lesions induced by administration of monoclonal antibody 1-22-3 to unilaterally nephrectomized rats.

A new animal model of progressive glomerulosclerosis was developed by administering a single i.v., injection of MoAb 1-22-3 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 glomeruli (ca 4%). Cellular infiltration was also noted in some parts of the interstitium. Increased expression of transforming growth factor-beta (TGF-beta) was observed in the unilaterally nephrectomized rats treated with MoAb 1-22-3, 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 in considered to be a great advantage of this model for clarifying the mechanisms involved in the chronic progression of mesangial proliferative glomerulonephritis.     

Glomerular macrophage modulation affects mesangial expansion in the rat after renal ablation.

Recent studies have provided evidence for the involvement of macrophages (m phi) in various types of human and experimental glomerular disease. The aim of the present study was to examine the effect of m phi depletion on glomerular injury after 3/4 renal ablation in the rat. This "remnant kidney" model is a widely used experimental model of focal and segmental glomerulosclerosis. Sustained glomerular m phi depletion was induced in remnant kidney rats by a regimen of sublethal triple systemic X-irradiation with shielding of the kidney remnants. Groups of 8 X-irradiated and 8 non-irradiated rats were studied at 5, 9, and 13 weeks after renal ablation. X-irradiated rats showed severe peripheral blood leukopenia at 5 and 9 weeks which had normalized at 13 weeks. The number of remnant glomerular m phi (immunohistochemistry with the monoclonal antibody ED1) in X-irradiated rats at 5 weeks was significantly lower when compared to non-irradiated remnant kidney rats. A rebound effect occurred at 9 and 13 weeks with increased m phi in remnant glomeruli of X-irradiated rats. Light microscopic examination disclosed significantly lower semiquantitative scores for mesangial cellularity and mesangial matrix expansion in remnant glomeruli of X-irradiated rats at 5 weeks when compared to non-irradiated remnant kidney rats. Mesangial matrix expansion had increased in X-irradiated rats at 9 and 13 weeks after ablation coincident with elevated glomerular m phi at these intervals. Multiple linear regression analysis indicated a highly significant contribution of m phi to the best fitting regression model predicting mesangial matrix expansion (multiple r2 = 0.81). In conclusion, these data provide evidence for a contributory role of m phi in the evolution of glomerular injury in the rat after renal ablation.