Neutrophil exocytosis induces podocyte cytoskeletal reorganization and proteinuria in experimental glomerulonephritis.

Acute glomerulonephritis is characterized by rapid glomerular neutrophil recruitment, proteinuria, and glomerular hypercellularity. The current study tested the hypothesis that the release of neutrophil granule contents plays a role in both the loss of filtration barrier leading to proteinuria and the increase in glomerular cells. Inhibition of neutrophil exocytosis with a peptide inhibitor prevented proteinuria and attenuated podocyte and endothelial cell injury but had no effect on glomerular hypercellularity in an experimental acute glomerulonephritis model in mice. Cultivation of podocytes with neutrophil granule contents disrupted cytoskeletal organization, an in vitro model for podocyte effacement and loss of filtration barrier. Activated, cultured podocytes released cytokines that stimulated neutrophil chemotaxis, primed respiratory burst activity, and stimulated neutrophil exocytosis. We conclude that crosstalk between podocytes and neutrophils contributes to disruption of the glomerular filtration barrier in acute glomerulonephritis. Neutrophil granule products induce podocyte injury but do not participate in the proliferative response of intrinsic glomerular cells.

Matrix metalloproteinase-12 deficiency attenuates experimental crescentic anti-glomerular basement membrane glomerulonephritis.

AIM: Matrix metalloproteinase-12 (MMP-12; macrophage elastase) is an enzyme that can cleave various extracellular matrix proteins and is required for macrophage infiltration and pulmonary fibrosis in experimental emphysema. We have shown previously that MMP-12 is highly up-regulated in experimental anti-glomerular basement membrane (GBM) disease. The aim of this study was to determine whether MMP-12 is required for glomerular macrophage infiltration and crescent formation in anti-GBM glomerulonephritis. METHODS: Accelerated anti-GBM disease was induced in groups of MMP-12 gene deficient mice (MMP-12-/-) and wild-type C57BL/6J controls, which were killed 12 days after injection of anti-GBM serum. RESULTS: Wild-type and MMP-12-/- mice developed glomerular damage and glomerular tuft adhesions to Bowman's capsule. Both groups developed severe proteinuria. Wild-type mice also developed significant loss of renal function and crescents in 22% of glomeruli, which were associated with macrophage infiltration and Bowman's capsule rupture. In contrast, MMP-12-/- mice were partially protected from renal function decline, crescent formation and Bowman's capsule rupture. This was associated with reduced macrophage infiltration in both glomeruli and the interstitium, and with reduced expression of CCL2, TNF-α and iNOS mRNA in MMP-12-/- kidneys. In addition, KIM-1 mRNA levels were reduced in MMP-12-/- mice indicating less tubular damage. CONCLUSION: These data demonstrate that endogenous MMP-12 facilitates macrophage accumulation and activation in anti-GBM glomerulonephritis which is required for glomerular crescent formation, Bowman's capsule rupture, tubular damage and renal function decline.

Spleen tyrosine kinase inhibition attenuates autoantibody production and reverses experimental autoimmune GN.

Spleen tyrosine kinase (SYK) has an important role in immunoreceptor signaling, and SYK inhibition has accordingly attenuated immune-mediated injury in several in vivo models. However, the effect of SYK inhibition on autoantibody production remains unclear, and SYK inhibition has not been studied in an autoimmune model of renal disease. We, therefore, studied the effect of SYK inhibition in experimental autoimmune GN, a rodent model of antiglomerular basement membrane disease. We show glomerular SYK expression and activation by immunohistochemistry in both experimental and clinical disease, and we show that treatment with fostamatinib, a small molecule kinase inhibitor selective for SYK, completely prevents the induction of experimental autoimmune GN. In established experimental disease, introduction of fostamatinib treatment led to cessation of autoantibody production, reversal of renal injury, preservation of biochemical renal function, and complete protection from lung hemorrhage. B cell ELISpot and flow cytometric analysis suggest that short-term fostamatinib treatment inhibits the generation and activity of antigen-specific B cells without affecting overall B-cell survival. Additionally, fostamatinib inhibited proinflammatory cytokine production by nephritic glomeruli ex vivo and cultured bone marrow-derived macrophages in vitro, suggesting additional therapeutic effects independent of effects on autoantibody production that are likely related to inhibited Fc receptor signaling within macrophages in diseased glomeruli. Given these encouraging results in an in vivo model that is highly applicable to human disease, we believe clinical studies targeting SYK in GN are now warranted.

Galectin-9 ameliorates anti-GBM glomerulonephritis by inhibiting Th1 and Th17 immune responses in mice.

Antiglomerular basement membrane glomerulonephritis (anti-GBM GN) is a Th1- and Th17-predominant autoimmune disease. Galectin-9 (Gal-9), identified as the ligand of Tim-3, functions in diverse biological processes and leads to the apoptosis of CD4(+)Tim-3(+) T cells. It is still unclear how Gal-9 regulates the functions of Th1 and Th17 cells and prevents renal injury in anti-GBM GN. In this study, Gal-9 was administered to anti-GBM GN mice for 7 days. We found that Gal-9 retarded the increase of Scr, ameliorated renal tubular injury, and reduced the formation of crescents. The infiltration of Th1 and Th17 cells into the spleen and kidneys significantly decreased in Gal-9-treated nephritic mice. The reduced infiltration of Th1 and Th17 cells might be associated with the downregulation of CCL-20, CXCL-9, and CXCL-10 mRNAs in the kidney. In parallel, the blood levels of IFN-γ and IL-17A declined in Gal-9-treated nephritic mice at days 21 and 28. In addition, an enhanced Th2 cell-mediated immune response was observed in the kidneys of nephritic mice after a 7-day injection of Gal-9. In conclusion, the protective role of Gal-9 in anti-GBM GN is associated with the inhibition of Th1 and Th17 cell-mediated immune responses and enhanced Th2 immunity in the kidney.

Modulation of heparan sulfate in the glomerular endothelial glycocalyx decreases leukocyte influx during experimental glomerulonephritis.

The glomerular endothelial glycocalyx is postulated to be an important modulator of permeability and inflammation. The glycocalyx consists of complex polysaccharides, the main functional constituent of which, heparan sulfate (HS), is synthesized and modified by multiple enzymes. The N-deacetylase-N-sulfotransferase (Ndst) enzymes initiate and dictate the modification process. Here we evaluated the effects of modulation of HS in the endothelial glycocalyx on albuminuria and glomerular leukocyte influx using mice deficient in endothelial and leukocyte Ndst1 (TEKCre+/Ndst1flox/flox). In these mice, glomerular expression of a specific HS domain was significantly decreased, whereas the expression of other HS domains was normal. In the endothelial glycocalyx, this specific HS structure was not associated with albuminuria or with changes in renal function. However, glomerular leukocyte influx was significantly reduced during antiglomerular basement membrane nephritis, which was associated with less glomerular injury and better renal function. In vitro decreased adhesion of wild-type and Ndst1-deficient granulocytes to Ndst1-silenced glomerular endothelial cells was found, accompanied by a decreased binding of chemokines and L-selectin. Thus, modulation of HS in the glomerular endothelial glycocalyx significantly reduced the inflammatory response in antiglomerular basement membrane nephritis.

Lutheran/basal cell adhesion molecule accelerates progression of crescentic glomerulonephritis in mice.

Migration of circulating leukocytes from the vasculature into the surrounding tissue is an important component of the inflammatory response. Among the cell surface molecules identified as contributing to leukocyte extravasation is VCAM-1, expressed on activated vascular endothelium, which participates in all stages of leukocyte-endothelial interaction by binding to leukocyte surface expressed integrin VLA-4. However, not all VLA-4-mediated events can be linked to VCAM-1. A novel interaction between VLA-4 and endothelial Lutheran (Lu) blood group antigens and basal cell adhesion molecule (BCAM) proteins has been recently shown, suggesting that Lu/BCAM may have a role in leukocyte recruitments in inflamed tissues. Here, we assessed the participation of Lu/BCAM in the immunopathogenesis of crescentic glomerulonephritis. High expression of Lu/BCAM in glomeruli of mice with rapidly progressive glomerulonephritis suggests a potential role for the local expression of Lu/BCAM in nephritogenic recruitment of leukocytes. Genetic deficiency of Lu/BCAM attenuated glomerular accumulation of T cells and macrophages, crescent formation, and proteinuria, correlating with reduced fibrin and platelet deposition in glomeruli. Furthermore, we found a pro-adhesive interaction between human monocyte α4ß1 integrin and Lu/BCAM proteins. Thus, Lu/BCAM may have a critical role in facilitating the accumulation of monocytes and macrophages, thereby exacerbating renal injury.

Ribosomal protein S19 is a novel therapeutic agent in inflammatory kidney disease.

RPS19 (ribosomal protein S19), a component of the 40S small ribosomal subunit, has recently been identified to bind the pro-inflammatory cytokine macrophage MIF (migration inhibitory factor). In vitro experiments identify RPS19 as the first endogenous MIF inhibitor by blocking the binding of MIF to its receptor CD74 and MIF functions on monocyte adherence to endothelial cells. In the present study, we sought to establish whether recombinant RPS19 can exert anti-inflammatory effects in a mouse model of anti-GBM (glomerular basement membrane) GN (glomerulonephritis) in which MIF is known to play an important role. Accelerated anti-GBM GN was induced in C57BL/6J mice by immunization with sheep IgG followed 5 days later by administration of sheep anti-mouse GBM serum. Groups of eight mice were treated once daily by intraperitoneal injection with 6 mg of RPS19/kg of body weight or an irrelevant control protein (human secretoglobin 2A1), or received no treatment, from day 0 until being killed on day 10. Mice that received control or no treatment developed severe crescentic anti-GBM disease on day 10 with increased serum creatinine, declined creatinine clearance and increased proteinuria. These changes were associated with up-regulation of MIF and its receptor CD74 activation of ERK (extracellular-signal-regulated kinase) and NF-κB (nuclear factor κB) signalling, prominent macrophage and T-cell infiltration, as well as up-regulation of Th1 [T-bet and IFNγ (interferon γ)] and Th17 [STAT3 (signal transducer and activator of transcription 3) and IL (interleukin)-17A] as well as IL-1ß and TNFα (tumour necrosis factor α). In contrast, RPS19 treatment largely prevented the development of glomerular crescents and glomerular necrosis, and prevented renal dysfunction and proteinuria (all P<0.001). Of note, RPS19 blocked up-regulation of MIF and CD74 and inactivated ERK and NF-κB signalling, thereby inhibiting macrophage and T-cell infiltration, Th1 and Th17 responses and up-regulation of pro-inflammatory cytokines (all P<0.01). These results demonstrate that RPS19 is a potent anti-inflammatory agent, which appears to work primarily by inhibiting MIF signalling.

The sphingosine-1-phosphate receptor agonist FTY720 prevents the development of anti-glomerular basement membrane glomerulonephritis.

The sphingosine-1-phosphate (S1P) agonist FTY720 prolongs the survival of organ allograft and attenuates autoimmune-mediated injury in experimental models. Most cases of glomerulonephritis (GN) in human appear to be immunologically initiated. In this study, we evaluated the potential therapeutic role of FTY720 in GN via a mouse anti-glomerular basement membrane (GBM) model. Mice were immunized with rabbit IgG in complete Freund's adjuvant (CFA) followed by an intravenous injection of a rabbit anti-mouse GBM serum. Disease and immune responses were assessed on day 14. Mice were treated with FTY720 (0.3 or 3 mg/kg) and prednisone (10 mg/kg) from days 0 to 14. The S1P modulator reduced proteinuria, serum creatinine, crescent formation and serum IgG level. The expressions of splenic S1P receptor and renal Th-1 cytokine were also inhibited at the transcription stage. Treatment with FTY720 increased splenocyte production of protective Th2 cytokine IL-4 and promoted the apoptosis of splenic CD4+ T cells in the animal models, which suggests that FTY720 played a protective role at the induction stage of GN by inhibiting mRNA expressions of splenic S1P receptor 1, S1P receptor 2, and S1P receptor 5.

Glomerulonephritis therapy: is there a role for green tea?

Rapidly progressive glomerulonephritis is the most aggressive form of glomerulonephritis with the worst prognosis. The current unspecific immunosuppressive therapy with corticosteroids and cytotoxic agents is often complicated by severe side effects. Peng and colleagues studied the therapeutic potential of the green tea component (-)-epigallocatechin-3-gallate (EGCG) in a murine model of immune-mediated glomerulonephritis. Their results indicate that EGCG treatment ameliorates renal inflammation, tissue damage, and loss of renal function and might therefore represent a novel therapeutic approach for human glomerulonephritis.

Endogenous foxp3(+) T-regulatory cells suppress anti-glomerular basement membrane nephritis.

Foxp3(+) T-regulatory cells (Tregs) may suppress pathogenic inflammation; however, although transferred Tregs lessen glomerulonephritis in mice, the role of endogenous foxp3(+) cells is not known. To study this, we characterized endogenous foxp3(+) cells in accelerated anti-glomerular basement membrane (GBM) nephritis by using foxp3(GFP) reporter mice to track their responses in early and established disease. Further, diphtheria toxin was used to ablate foxp3(+) Tregs in foxp3(DTR) mice after establishing an immune response. In this model, mice were immunized with sheep globulin in adjuvant, and sheep anti-mouse GBM globulin was injected after 4 days to initiate progressive histological and functional injury. Intrarenal leukocytic infiltrates were increased by day 3 but intrarenal foxp3(+) Tregs, present in interstitial and periglomerular areas, were only increased at day 7. Ablation of foxp3(+) Tregs after injection of anti-GBM globulin increased renal injury and systemic T-cell responses, including increased interferon-γ and interleukin-17A (IL-17A) production, but no change in antibody titers. Compared with foxp3(+) Tregs isolated from naive mice, those from immunized mice produced more IL-10 and more effectively regulated CD4(+)foxp3(-) responder T cells. Thus, endogenous foxp3(+) Tregs infiltrate the kidney in glomerulonephritis, and deleting foxp3(+) cells after the induction of immune responses upregulated T-cell reactions and enhanced disease. Hence, endogenous foxp3(+) cells have increased suppressive capacity after immune stimuli.

The green tea polyphenol (-)-epigallocatechin-3-gallate ameliorates experimental immune-mediated glomerulonephritis.

The unchecked overproduction of reactive oxygen and nitrogen species by inflammatory cells can cause tissue damage, intensify inflammation, promote apoptosis, and accelerate the progression of immune-mediated glomerulonephritis (GN). Here we tested whether the anti-inflammatory and antioxidant properties of the green tea polyphenol (-)-epigallocatechin-3-gallate (EGCG) favorably affect the development of immune-mediated GN. Pretreatment of 129/svJ mice with EGCG from 2 days before to 2 weeks after the induction of GN led to reduced proteinuria and serum creatinine, and marked improvement in renal histology when compared with vehicle-pretreated diseased mice. This pretreatment reduced oxidative stress, and normalized osteopontin, p65/nuclear factor-κB, inducible nitric oxide synthase, nitric oxide metabolites, p-Akt, phosphorylated extracellular signal-regulated kinases 1 and 2, p47phox, and myeloperoxidase, all of which were elevated in vehicle-pretreated diseased mice. Levels of glutathione peroxidase and peroxisome proliferator-activated receptor-γ (PPARγ), both reduced in the vehicle-pretreated diseased mice, were normalized. This renoprotective effect was reversed by concomitant administration of the PPARγ antagonist GW9662 throughout the EGCG pretreatment period. Importantly, mortality and renal dysfunction were significantly attenuated even when the polyphenol treatment was initiated 1 week after the onset of GN. Thus, EGCG reversed the progression of immune-mediated GN in mice by targeting redox and inflammatory pathways.

Differential effects of rapamycin in anti-GBM glomerulonephritis.

The immunosuppressive mammalian target of rapamycin inhibitor rapamycin is widely used in solid-organ transplantation, but the effect of rapamycin on kidney disease is controversial. This study evaluated the effect of rapamycin in the autologous phase of anti-glomerular basement membrane (anti-GBM) glomerulonephritis. Disease was induced by preimmunizing the animals with rabbit IgG 5 d before administration of rabbit anti-mouse GBM antiserum. When rapamycin was started on the day of immunization (group 1), mice were protected from glomerulonephritis, suggested by a dramatic decrease in albuminuria, influx of inflammatory cells, and Th1-cytokine expression in the kidneys. Activation of T cells and production of autologous mouse anti-rabbit IgG were also significantly reduced in rapamycin-treated animals. In contrast, when rapamycin was started 14 d after immunization (group 2), mice had a significant increase in albuminuria and renal infiltration of inflammatory cells compared with vehicle-treated animals, and there were no differences in T and B cell responses. A significant decrease in vascular endothelial growth factor-A and an increase in IL-6 were detected in kidneys of these rapamycin-treated mice. In conclusion, rapamycin has the potential to significantly reduce the B and T cell responses and thereby protect from glomerulonephritis when administered early in disease. Once disease is established, however, rapamycin seems to worsen glomerulonephritis by disturbing the endothelial cell/vascular endothelial growth factor system in the kidney.

Protective effect of ligustrazine on accelerated anti-glomerular basement membrane antibody nephritis in rats is based on its antioxidant properties.

Ligustrazine has a renoprotective effect against nephritis. In this study, we further characterized the renoprotective properties of ligustrazine in an experimental model using accelerated anti-glomerular basement membrane antibody (AGBM-Ab). Ligustrazine was given i.p. once daily at 50, 100 mg/kg for 15 days after singly giving i.v. of rabbit anti-rat glomerular basement membrane serum, and showed dose-dependent inhibition the elevation of urinary protein, serum creatinine and blood urea nitrogen as well as the development of glomerular histological changes. Ligustrazine (50 mg/kg) had no affect on glutathione (GSH) content, glutathione peroxidase and catalase activities, but decreased the malondialdehyde (MDA) content and increased superoxide dismutase (SOD) activity in nephritis induced by AGBM-Ab. Ligustrazine (100 mg/kg) significantly decreased MDA content while significantly increased GSH content and SOD, glutathione peroxidase, catalase activities of kidney tissues in the rats treated with AGBM-Ab alone. In conclusion, our results show that ligustrazine has protective activity against accelerated AGBM-Ab nephritis, and its renoprotective effect may be due to its antioxidant properties and inhibition reactive oxygen species (ROS).

Blocking VLA-4 prevents progression of experimental crescentic glomerulonephritis.

BACKGROUND/AIMS: Integrins are adhesion molecules of fundamental importance to the recruitment of leucocytes in inflammation. The alpha4beta1 integrin (VLA-4) is a leucocyte ligand for endothelial vascular cell adhesion molecule-1 (VCAM-1), fibronectin and osteopontin. We addressed the role of VLA-4 in mediating progressive renal injury in vivo using a blocking monoclonal antibody (mAb) in a rat model of crescentic glomerulonephritis. METHODS: WKY rats with nephrotoxic nephritis were given anti-VLA-4 or control mAb at 2.5 mg/kg by i.p. injection on alternate days. In separate experiments, antibodies were given from days 5-13, from days 13-21 or from days 14-28. RESULTS: Early treatment with anti-VLA-4 mAb from days 5-13 showed a significant effect on renal function, with a reduction in albuminuria (p < 0.01) and a higher creatinine clearance (p < 0.05). Delayed treatment from days 13-21 also showed a reduction in albuminuria (p < 0.05) and serum creatinine (p < 0.05). However, there was no significant effect on glomerular or interstitial scarring in these two experiments. In the late treatment study, in which anti-VLA-4 mAb was administered from days 14-28, serum creatinine was reduced (p < 0.05), creatinine clearance was improved (p < 0.05), and renal survival was significantly prolonged (p < 0.05). Interstitial scarring was significantly less in treated rats (p < 0.05). Glomerular macrophage and CD8+ cell counts were higher in anti-VLA-4 mAb treated rats (p < 0.05), possibly reflecting greater glomerular scarring in control animals. CONCLUSION: Leucocyte VLA-4 mediates pro-inflammatory and pro-fibrotic effects within the kidney, independent of any role in recruitment of leucocytes into the kidney. Blocking VLA-4 is a promising therapeutic approach in human glomerulonephritis.

Kallikrein transduced mesenchymal stem cells protect against anti-GBM disease and lupus nephritis by ameliorating inflammation and oxidative stress.

Previously we have shown that kallikreins (klks) play a renoprotective role in nephrotoxic serum induced nephritis. In this study, we have used mesenchymal stem cells (MSCs) as vehicles to deliver klks into the injured kidneys and have measured their therapeutic effect on experimental antibody induced nephritis and lupus nephritis. Human KLK-1 (hKLK1) gene was transduced into murine MSCs using a retroviral vector to generate a stable cell line, hKLK1-MSC, expressing high levels of hKLK1. 129/svj mice subjected to anti-GBM induced nephritis were transplanted with 10(6) hKLK1-MSCs and hKLK1 expression was confirmed in the kidneys. Compared with vector-MSCs injected mice, the hKLK1-MSCs treated mice showed significantly reduced proteinuria, blood urea nitrogen (BUN) and ameliorated renal pathology. Using the same strategy, we treated lupus-prone B6.Sle1.Sle3 bicongenic mice with hKLK1-MSCs and demonstrated that hKLK1-MSCs delivery also attenuated lupus nephritis. Mechanistically, hKLK1-MSCs reduced macrophage and T-lymphocyte infiltration into the kidney by suppressing the expression of inflammation cytokines. Moreover, hKLK1 transduced MSCs were more resistant to oxidative stress-induced apoptosis. These findings advance genetically modified MSCs as potential gene delivery tools for targeting therapeutic agents to the kidneys in order to modulate inflammation and oxidative stress in lupus nephritis.

The selective mineralocorticoid receptor antagonist eplerenone is protective in mild anti-GBM glomeru-lonephritis.

BACKGROUND: Growing evidence suggests that blockade of the aldosterone-receptor may preserve kidney function by anti-inflammatory effects independent of the blood pressure. We hypothesized that the selective aldosterone-receptor antagonist eplerenone has a profound anti-inflammatory effect in the autologous phase of anti-glomerular basement membrane (GBM) glomerulonephritis (GN). METHODS: Mice received ≈200mg/kg body wt/day eplerenone via supplemented chow diet or standard chow starting at the day of immunization with rabbit IgG. Three days later the anti-GBM antibody was injected and the experiments were stopped at day 7 and 14. RESULTS: Mice receiving eplerenone showed significantly decreased albuminuria and glomerular sclerosis at day 7 and 14 after induction of anti-GBM GN. Eplerenone treatment significantly inhibited the infiltration of CD4+, CD8+ T cells and macrophages into the kidneys. Circulating levels and glomerular deposition of autologous IgG were comparable in both groups. At day 7 the pro-inflammatory cytokines MCP-1 and IL-6 were found to be significantly decreased in regional draining lymph nodes of eplerenone-treated mice, whereas the anti-inflammatory cytokine IL-10 was significantly upregulated. In line, splenocytes from eplerenone-treated nephritic mice produced significantly increased IL-10. CONCLUSION: Aldosterone-receptor blockade by eplerenone effectively attenuated proteinuria, kidney damage and the inflammatory response in anti-GBM GN by significantly decreasing pro-inflammatory cytokines in the regional draining lymph nodes of the kidney. Our results suggest that this selective aldosterone receptor antagonist is a possible additional tool in the treatment of GN.

Nasal administration of recombinant rat alpha3(IV)NC1 prevents the development of experimental autoimmune glomerulonephritis in the WKY rat.

Experimental autoimmune glomerulonephritis (EAG), an animal model of Goodpasture's disease, can be induced in Wistar Kyoto (WKY) rats by immunization with either collagenase-solubilized rat glomerular basement membrane (GBM) or the recombinant NC1 domain of the alpha3 chain of type IV collagen [alpha3(IV)NC1]. EAG is characterized by circulating and deposited anti-glomerular basement membrane antibodies, focal necrotizing glomerulonephritis with crescent formation, and glomerular infiltration by T cells and macrophages. Previous studies have demonstrated that oral administration of collagenase-solubilized GBM to WKY rats prevented the development of EAG. Nasal administration of specific autoantigens has been reported to be more effective than oral administration in other models of autoimmune disease. The main aim of this study was to investigate further the concept of mucosal tolerance in EAG by examining the effect of nasal administration of recombinant rat alpha3(IV)NC1. Groups of WKY rats with EAG, induced by immunization with recombinant rat alpha3(IV)NC1, were given alpha3(IV)NC1 nasally on 3 consecutive days before immunization, at total cumulative doses of 25, 100, or 250 microg per rat. A dose-dependent effect was observed on the development of EAG. A dose of 25 microg had no effect on disease; 100 microg resulted in a moderate reduction in the severity of nephritis; and 250 microg led to a marked reduction in circulating and deposited antibodies, albuminuria, severity of glomerular abnormalities, and numbers of glomerular CD8+ T cells and macrophages. In addition, there was a reduction in the proliferative response of splenocytes from rats in the high dose group (250 microg) to alpha3(IV)NC1 in vitro. The results from this study clearly demonstrate for the first time that mucosal tolerance in EAG can be induced by nasal administration of recombinant rat alpha3(IV)NC1 and that this approach is effective in the prevention of crescentic glomerulonephritis. Further work using new antigen-specific treatment strategies may provide a novel approach to the treatment of patients with anti-glomerular basement membrane disease.

LF15-0195 prevents the induction and inhibits the progression of rat anti-GBM disease.

BACKGROUND: LF15-0195 is a novel immunosuppressant that is currently in phase II clinical trials for the treatment of vasculitis. This study examined whether LF15-0195 could suppress the induction and progression of rat anti-glomerular basement membrane (anti-GBM) glomerulonephritis. METHODS: Rapidly progressive glomerulonephritis was induced in primed rats by the administration of anti-GBM serum. In the first experiment, LF15-0195 was given daily by subcutaneous injection (days 0 to 14) to treat the induction of anti-GBM disease analyzed at day 14. In a second experiment, rats received LF15-0195 as an intervention treatment from days 7 to 28 (continuous therapy) or days 7 to 12 (pulse therapy) to treat the progression of disease assessed at day 28. RESULTS: Continuous LF15-0195 treatment during the induction of anti-GBM disease (experiment 1) prevented proteinuria and loss of renal function, and markedly reduced histological kidney lesions and renal fibrosis. LF15-0195 also reduced kidney leukocyte infiltrate, urine excretion of interleukin-1beta (IL-1beta) and transforming growth factor-beta (TGF-beta), and the serum antibody response, but not kidney deposition of Ig and C3. When LF15-0195 treatment was initiated at day 7, both continuous and pulse therapy partially inhibited disease progression by suppressing the loss of renal function, interstitial macrophage and T-cell accumulation, tubular cell proliferation, and renal fibrosis. CONCLUSION: LF15-0195 prevents the induction and suppresses the progression of rat anti-GBM disease through multiple mechanisms of action, suggesting that this drug may have significant therapeutic potential in human glomerulonephritis. The similar efficacy of continuous and pulse intervention treatment in this model indicates that short-term LF15-0195 treatment may achieve optimal benefit without prolonged bone marrow suppression.

Anti-CD8 monoclonal antibody therapy is effective in the prevention and treatment of experimental autoimmune glomerulonephritis.

Experimental autoimmune glomerulonephritis (EAG), which is an animal model of Goodpasture's disease, can be induced in Wistar Kyoto rats by a single injection of rat glomerular basement membrane (GBM) in adjuvant. EAG is characterized by circulating and deposited anti-GBM antibodies, focal necrotizing glomerulonephritis with crescent formation, and glomerular infiltration by T cells and macrophages. Our hypothesis was that T cell-mediated immunity, in addition to humoral immunity, was necessary for the development of crescentic nephritis in this model. To investigate the role of CD8+ T cells in the pathogenesis of EAG, the in vivo effects of an anti-CD8 monoclonal antibody (OX8) were examined, with administration starting at the time of immunization (prevention) or 2 wk after immunization, when glomerular abnormalities were first detected (treatment). When administered intraperitoneally at 5 mg/kg, three times per week, from week 0 to week 4 (prevention), OX8 completely inhibited the development of albuminuria, deposits of fibrin in the glomeruli, glomerular and interstitial abnormalities, the influx of CD8+ T cells and macrophages, and glomerular expression of granzyme B and inducible nitric oxide synthase. Circulating anti-GBM antibody levels were not reduced, but there was a reduction in the intensity of antibody deposition on the GBM. When administered at the same dose from week 2 to week 4 (treatment), OX8 greatly reduced the severity of EAG; in particular, the formation of crescents was prevented. These studies demonstrate that anti-CD8 monoclonal antibody therapy is effective in both the prevention and treatment of EAG. They confirm the importance of T cell-mediated immunity in the pathogenesis of this model of Goodpasture's disease. Similar therapeutic approaches may be worth investigating in human crescentic glomerulonephritis.

Oral administration of glomerular basement membrane prevents the development of experimental autoimmune glomerulonephritis in the WKY rat.

Experimental autoimmune glomerulonephritis (EAG), an animal model of Goodpasture's disease, can be induced in Wistar Kyoto (WKY) rats by a single injection of collagenase-solubilized rat glomerular basement membrane (GBM) in adjuvant. EAG is characterized by circulating and deposited anti-GBM antibodies, accompanied by focal necrotizing glomerulonephritis with crescent formation. The inhibitory effect of orally administered antigens has been reported in various animal models of autoimmunity but not in EAG in the rat. The effects of feeding rat GBM by gavage, at total doses of 0.5, 2.5, or 5 mg, before immunization were examined. A dose-dependent effect was observed on the development of EAG. A dose of 0.5 mg of GBM had no effect on disease, 2.5 mg resulted in a moderate reduction in the severity of nephritis but no change in anti-GBM antibody production, and 5 mg resulted in a marked reduction in circulating and deposited anti-GBM antibodies, albuminuria, deposits of fibrin in the glomeruli, severity of glomerular abnormalities, and numbers of infiltrating T cells and macrophages. Animals that were fed 5 mg of GBM showed a significant reduction in IgG2a but not IgG1, anti-GBM antibody levels, suggesting downregulation of Th1 responses. There was also a dose-dependent reduction in the proliferative responses of splenic T cells from treated animals to GBM antigen in vitro. These results clearly demonstrate that mucosal tolerance can be induced by oral administration of GBM antigen and that this approach is effective in preventing EAG.