Glomerulonephritis In Wistar-Kyoto Rats Research Articles

Treatment with a cyclin‐dependent kinase inhibitor, seliciclib, is effective in reducing glomerular macrophage numbers and the severity of established experimental glomerulonephritis

The cyclin-dependent kinase inhibitor, seliciclib (R-roscovitine, CYC202), has anti-proliferative activity through its inhibition of cyclin-dependent kinase 2. We hypothesized that treatment with seliciclib would reduce glomerular macrophage numbers and glomerular crescent formation in experimental crescentic glomerulonephritis even when treatment is started after onset of disease. Nephrotoxic nephritis (NTN) was induced in Wistar Kyoto rats. In experiment 1, seliciclib (150 mg/kg per day) was given by oral gavage from 1 h before induction of NTN and continued to day 14. In experiment 2, treatment was started on day 4 of NTN and continued to day 14 in order to examine the effect of seliciclib in established glomerulonephritis. In experiment 1, seliciclib reduced proteinuria (119.5 ± 13.9 vs 191.4 ± 18.8 mg/day, P < 0.01), serum creatinine (54.0 ± 3.0 vs 81.0 ± 2.5 µmol/L, P < 0.005) and glomerular crescent score (23.9 ± 2.1 vs 44.6 ± 2.2, P < 0.005) in comparison with controls. In experiment 2, seliciclib ameliorated established glomerulonephritis, with reduction in proteinuria (58 ± 16 vs 165 ± 13 mg/day, P < 0.005), serum creatinine (39 ± 3 vs 62 ± 5 µmol/L, P < 0.05), glomerular macrophage numbers (6.8 ± 2.5 vs 18.5 ± 1.2 ED1+ cells per glomerular cross section, P < 0.05), glomerular cell proliferation (1.2 ± 0.37 vs 4.2 ± 0.80 bromodeoxyuridine (BrdU)+ cells per glomerular section, P < 0.05) and crescent score (10.8 ± 1.6 vs 43.9 ± 1.4, P < 0.05), in comparison with the controls. Seliciclib is effective in both prevention and treatment of established crescentic glomerulonephritis in Wistar Kyoto rats, in association with a reduction in the number of glomerular macrophages. We suggest that seliciclib, or other cyclin-dependent kinase inhibitors, may represent a novel therapeutic approach for patients with proliferative glomerulonephritis.

Genetic Mapping Found Major QTLs for Antibody-Induced Glomerulonephritis in WKY Rats

Genetic bases of glomerulonephritis, a major cause of kidney dysfunction in humans and one of the most characteristic complications of autoimmune disorders such as Goodpasture syndrome, are complex. The Wistar-Kyoto (WKY) rat strain is well characterized for its susceptibility to autoantibodies against glomerular basement membrane (GBM), however the molecular mechanisms underlining the phenotype are largely unknown. Here we performed a whole genome scan using a backcross (BC) F(1) (WKY x DA) x WKY population, for which the DA rat is a nonsusceptible control strain. We found two significant QTLs on chromosomes 1 and 12, which were involved in elevated levels of proteinuria and kidney weight index, respectively. The relevance of these QTLs with the genetic factors involved in autoimmunity and renal disease is discussed.

Anti-perforin antibody treatment ameliorates experimental crescentic glomerulonephritis in WKY rats

The depletion of CD8+ cells has been shown to prevent the initiation and progression of antiglomerular basement membrane (GBM) crescentic glomerulonephritis (GN) in Wistar-Kyoto (WKY) rats. In this study, we asked whether CD8+ cells produce their effects by perforin/granzyme-mediated or by Fas ligand (FasL)-mediated pathways. The glomerular mRNA expression of perforin and granzyme B corresponded with the number of CD8+ cells, whereas that of granzyme A, Fas, and FasL did not. The enhanced mRNA level of perforin and granzyme B was not evident in CD8+-depleted rats. The number of apoptotic cells in the glomeruli was significantly increased at day 3. Perforin mRNA was found in cells infiltrating the glomerulus by in situ hybridization and by using dual-staining immunohistochemistry perforin protein was found in glomerular CD8+ cells. We found that perforin was readily visualized at the inner surface of the glomerular capillaries by immunoelectron microscopy. Based on these results, we treated animals with a perforin antibody in vivo and found that it significantly reduced the amount of proteinuria, frequency of crescentic glomeruli, and the number of glomerular monocytes and macrophages, although the number of glomerular CD8+ cells was not changed. Our results suggest that CD8+ cells play a role in glomerular injury as effector cells in part through a perforin/granzyme-mediated pathway in the anti-GBM WKY rat model of crescentic GN.

Histamine ameliorates anti-glomerular basement membrane antibody-induced glomerulonephritis in rats

Anti-glomerular basement membrane (anti-GBM)-induced glomerulonephritis involves T-helper type 1 (Th1) responses leading to rapid crescent formation. As many inflammatory and immune responses in general are affected by histamine, we examined the effects of histaminergic ligands on immune renal injury in the rat. Female Wistar-Kyoto rats were injected intraperitoneally with an antibody against the GBMs. Histaminergic ligands were then injected twice daily for 5 days after which renal function was assessed by proteinuria. Treatment with histamine led to significant dose-dependent reductions in proteinuria compared to the control antibody-injected group and markedly decreased the number of crescentic glomeruli and macrophage infiltration of the glomeruli. Furthermore, histamine significantly decreased the plasma concentration of interleukin-12, a Th1-type cytokine compared to the antibody-injected control animals. Dimaprit, an H(2)/H(4) agonist, mimicked the effects of histamine on proteinuria and crescent formation. Clozapine, an H(4) agonist, tended to mimic the effects of histamine, whereas an H(1), mepyramine, or an H(2) antagonist, ranitidine, did not reverse the protective effect of histamine. We suggest that histamine may alleviate renal injury in anti-GBM glomerulonephritis by suppressing the immune response.

Inhibition of CXCL16 Attenuates Inflammatory and Progressive Phases of Anti-Glomerular Basement Membrane Antibody-Associated Glomerulonephritis

Chemokines recruit and activate leukocytes during inflammation. CXCL16 is a recently discovered chemokine that is expressed as a transmembrane protein that is cleaved to form the active, soluble chemokine. We analyzed the role of CXCL16 in the development of inflammation and in the progression of the anti-glomerular basement membrane (GBM) antibody-induced experimental glomerulonephritis in Wistar-Kyoto rats. CXCL16 was expressed in glomerular endothelial cells and mediated adhesion of macrophages expressing CXCL16 and its cognate receptor, CXCR6. Glomerular infiltrates displayed a strong migratory response to soluble CXCL16. Soluble CXCL16 and its receptor CXCR6 were induced in nephritic glomeruli throughout the disease, and CXCL16 expression correlated with the up-regulation of ADAM10, suggesting that this disintegrin and metalloproteinase mediates the chemokine activity of CXCL16. Blocking CXCL16 in the acute inflammatory phase or progressive phase of established glomerulonephritis significantly attenuated monocyte/macrophage infiltration and glomerular injury; proteinuria also improved. We conclude that CXCL16/CXCR6 plays a critical role in stimulating leukocyte influx, which causes glomerular damage during anti-GBM glomerulonephritis. Blocking CXCL16 actions limits the progression of anti-GBM glomerulonephritis even when the disease is established.

Copy number polymorphism in Fcgr3 predisposes to glomerulonephritis in rats and humans

Identification of the genes underlying complex phenotypes and the definition of the evolutionary forces that have shaped eukaryotic genomes are among the current challenges in molecular genetics. Variation in gene copy number is increasingly recognized as a source of inter-individual differences in genome sequence and has been proposed as a driving force for genome evolution and phenotypic variation. Here we show that copy number variation of the orthologous rat and human Fcgr3 genes is a determinant of susceptibility to immunologically mediated glomerulonephritis. Positional cloning identified loss of the newly described, rat-specific Fcgr3 paralogue, Fcgr3-related sequence (Fcgr3-rs), as a determinant of macrophage overactivity and glomerulonephritis in Wistar Kyoto rats. In humans, low copy number of FCGR3B, an orthologue of rat Fcgr3, was associated with glomerulonephritis in the autoimmune disease systemic lupus erythematosus. The finding that gene copy number polymorphism predisposes to immunologically mediated renal disease in two mammalian species provides direct evidence for the importance of genome plasticity in the evolution of genetically complex phenotypes, including susceptibility to common human disease.

Activation of Glomerular Basement Membrane–Specific B Cells in the Renal Draining Lymph Node after T Cell–Mediated Glomerular Injury

Linear binding of IgG to the glomerular basement membrane (GBM) is the hallmark of anti-GBM glomerulonephritis (GN). However, the precise mechanism by which diverse autoantibodies to GBM are induced in GN has not been determined. It was demonstrated previously that a single T cell epitope pCol(28-40) derived from collagen IV alpha3 chain not only induced severe GN in Wistar Kyoto rats but also triggered a diversified anti-GBM antibody response through "B cell epitope spreading." In this study, an expansion of T and B cells in the renal draining lymph node (RDLN) of diseased animals after glomerular injury was observed. RDLN was demonstrated to be the location of GBM-specific B cell activation. First, B cells from RDLN of pCol(28-40)-immunized rats produced in vitro anti-GBM antibodies and antinuclear antibodies. Second, B cells specific to the peptidic B cell epitope in pCol(28-40) were absent among expanding B cells in RDLN. Those findings provided a unique opportunity to track activation of diverse GBM-specific B cells in RDLN. Expression of B lymphocyte-induced maturation protein-1, which is involved in differentiation of plasma cells, in B cells of RDLN was detected and further elevated only after T cell-mediated prominent glomerular injury (day 19). This was supported by the fact that anti-GBM antibodies became detectable only after day 20. Those results suggest that T cell-mediated glomerular injury may trigger de novo internal immunization of autoantigens released from damaged GBM, which further leads to activation of a group of GBM-specific B cells in RDLN.

Characterization of the T-cell epitope that causes anti-GBM glomerulonephritis

We have demonstrated that a single T-cell epitope pCol(28-40) (SQTTANPSCPEGT) alone, which is derived from NC1 domain of alpha3 chain of type IV collagen (Col4alpha3 NC1), can induce severe glomerulonephritis in Wistar Kyoto rats. This study further characterized this T-cell epitope. A series of synthetic peptides derived from pCol (28-40) were tested in vivo and in vitro for their T-cell epitope activity and nephritogenicity. Major histocompatability complex (MHC) class II molecules in Wistar Kyoto rats were cloned, and MHC restriction of pCol(28-40) was determined. The T-cell epitope pCol(28-40) was restricted by rat MHC class II RT.1Bl. Ten amino acid residues (29 to 38) were mapped to be the minimum core of the T-cell epitope, which was capable of inducing the T-cell response and severe glomerulonephritis. Only three residues were identified as absolutely critical for the T-cell epitope: position 31 (T) was an anchor residue to the class II molecule, and positions 33 (N) and 34 (P) contributed to the specificity of the T-cell epitope. Thus, only substitution at those positions completely abrogated nephritogenicity of the T-cell epitope. Interestingly, pCol (28-40) also bound to human MHC class II human MHC class II molecule HLA-DRB*1501, which has been linked to human anti-glomerular basement membrane (GBM) disease, suggesting that human homologue of pCol(28-40) could be a potential human T-cell epitope. Our study demonstrated that only few residues in the nephritogenic T-cell epitope pCol(28-40) were critical. Our finding also revealed that pCol(28-40) is a potential nephritogenic T-cell epitope in Goodpasture's syndrome.

T-cell epitope of α3 chain of type IV collagen induces severe glomerulonephritis

Anti-glomerular basement membrane (GBM) glomerulonephritis is among the earliest recognized human autoimmune diseases. However, the etiology of anti-GBM glomerulonephritis remains unclear. We have previously shown that CD4+ T cells, specific to alpha3 NC1 of type IV collagen (Col4alpha3NC1), were able to induce anti-GBM glomerulonephritis in Wistar-Kyoto (WKY) rats. In the present study, we continued to map the nephritogenic T cell epitope in Col4alpha3NC1. Synthetic peptides, which covered Col4alpha3NC1, were used as immunogens to induce glomerulonephritis in WKY rats. T-cell and B-cell responses to the peptides in the animals were analyzed. One potent nephritogenic T-cell epitope, pCol(28-40) (SQTTANPSCPEGT), was identified. A single immunization with pCol(28-40) induced extremely severe glomerulonephritis in all 23 rats. Renal pathology revealed nearly 100% of glomeruli with crescentic lesions or tuft necrosis in 21 animals. pCol(28-40) elicited a T-cell response to the peptide; T cells isolated from rats immunized with recombinant Col4alpha3NC1 reacted with pCol(28-40). pCol(28-40) elicited a peptide specific antibody response, which did not react with polypeptide Col4alpha3NC1 or native GBM. An 11-mer peptide, pCol(a30-40) (Ac-TTANPSCPEGT), was further mapped to be the core of the T-cell epitope in pCol(28-40). As expected, immunization with pCol(a30-40) induced severe glomerulonephritis in 10 out of 19 rats. Our study not only demonstrated that a single T-cell epitope of Col4alpha3NC1 is sufficient to induce severe glomerulonephritis, but also provides a unique model for studying T-cell-mediated mechanisms in anti-GBM glomerulonephritis pathogenesis.

Protective effect of a novel and selective inhibitor of inducible nitric oxide synthase on experimental crescentic glomerulonephritis in WKY rats.

Nitric oxide (NO) plays important roles in a variety of pathophysiological processes. It has been reported that inducible NO synthase (iNOS) is upregulated in the glomeruli of patients with glomerulonephritis, although there has been no direct evidence that NO generated by iNOS contributes to the progression of glomerulonephritis. ONO-1714, a novel cyclic amidine analog, is a selective inhibitor of iNOS. To elucidate the role of iNOS in the pathogenesis of experimental crescentic glomerulonephritis, we examined the effect of ONO-1714 given to rats with nephrotoxic serum (NTS) nephritis. We induced NTS nephritis in Wistar-Kyoto (WKY) rats. These rats were given ONO-1714 or physiological saline intraperitoneally for 14 days using an osmotic pump after intraperitoneal injection with NTS. Glomerular expression of iNOS and urinary excretion of NO metabolites (nitrite/nitrate) were increased in rats after injection of NTS. As compared with the control group, ONO-1714 significantly reduced proteinuria, crescent formation, glomerular infiltration of macrophages and urinary excretion of nitrite/nitrate. The present results suggest that NO radicals generated by iNOS contribute to the progression of experimental crescentic glomerulonephritis in WKY rats. The selective iNOS inhibitor ONO-1714 may be beneficial for the treatment of crescentic glomerulonephritis.

Type IV Phosphodiesterase Inhibitor Is Effective in Prevention and Treatment of Experimental Crescentic Glomerulonephritis

Background: Tumour necrosis factor alpha (TNF-α) has an important role in acute glomerular inflammation. Rolipram, a type IV phosphodiesterase inhibitor, has multiple anti-inflammatory effects including inhibition of TNF-α synthesis. Methods: We investigated the effects of rolipram in prevention and delayed treatment of crescentic glomerulonephritis in Wistar Kyoto rats. Glomerulonephritis was induced by injection of nephrotoxic serum. Results: In the preventive study, rolipram (6.25 mg/kg i.p. twice daily) was started 2.5 h before injection of nephrotoxic serum. Rolipram reduced the expression of TNF-α in glomeruli and renal tubules and abrogated glomerular injury on day 4 (99.7% reduction in albuminuria and 96.4% reduction in fibrin deposition). In the delayed-treatment experiment, rolipram was started 4 days after injection of nephrotoxic serum. Rolipram reduced renal excretion of TNF-α by 63% on day 7. TNF-α was not detected in the sera of treated or control rats. Delayed treatment was effective in crescentic glomerulonephritis, as shown by reduction in albuminuria by 38.1%, fibrin deposition by 60.8%, and crescent formation by 67% on day 7. Conclusions: Rolipram is effective both in prevention and treatment of experimental crescentic glomerulonephritis. This was associated with a reduction of renal production of TNF-α.

Effect of CTLA‐4 chimeric protein on rat autoimmune anti‐glomerular basement membrane glomerulonephritis

The interaction of the T cell receptor with the antigen/major histocompatibility class II complex is insufficient to induce optimal T cell activation. Co-stimulatory signals, including those provided by CD28/CTLA-4 on T cells and B7 molecules (B7-1, -2, and -3) on antigen-presenting cells, are also required. CD28-B7 interactions can be blocked by a soluble human CTLA-4 chimeric protein (CTLA4Ig). We tested the effect of administration of CTLA4Ig on experimental anti-glomerular basement membrane (GBM) autoimmune glomerulonephritis in Wistar-Kyoto rats induced by immunization with bovine GBM. The disease is characterized by development of antibody to the alpha 3 chain of type IV collagen (Goodpasture's antigen), deposition of rat IgG in GBM, infiltration of the kidney by T cells and macrophages, severe crescent formation and renal failure leading to death in 5-6 weeks. Animals injected with human CTLA4Ig from day 0 to day 14 or to day 35 had reduced disease severity. Beneficial effects were observed even when injections were begun after the onset of glomerulonephritis on day 14. However, the rats developed antibody to the human CTLA4Ig, associated with reduction in levels of circulating CTLA4Ig. The results provide evidence for CD28/CTLA-4 signaling in rat autoimmune glomerulonephritis, and suggest that more effective inhibition of B7-dependent T cell activation, such as might be achieved with homologous CTLA4Ig, could be useful in the treatment of autoimmune diseases.

Antibodies to intercellular adhesion molecule 1/lymphocyte function-associated antigen 1 prevent crescent formation in rat autoimmune glomerulonephritis.

In patients with glomerulonephritis widespread crescents are associated with a poor prognosis. Crescent formation appears to depend on the migration of mononuclear cells into Bowman's space, and therefore the interaction between leukocytes and glomerular endothelium may be a critical event in the genesis of crescents. We performed the present study to determine the effects of mouse monoclonal antibodies to the adhesion molecules intercellular adhesion molecule 1 (ICAM-1) and lymphocyte function-associated antigen 1 (LFA-1) in a model of crescentic glomerulonephritis in Wistar-Kyoto rats, induced by immunization with bovine glomerular basement membrane (GBM). By 10-14 d after immunization, the rats had developed circulating anti-GBM antibodies, reactive with the alpha 3 chain of type IV collagen (the Goodpasture antigen), accompanied by proteinuria, accumulation of rat immunoglobulin (Ig)G in the GBM, increased expression of ICAM-1 by glomerular endothelial cells, infiltration of glomerular tufts with LFA-1+ T cells and monocyte/macrophages, and early crescents. At 5 wk all rats had diffuse fibrocellular crescents, glomerular sclerosis, and tubulointerstitial damage. All rats developed severe renal insufficiency and died by 5 or 6 wk. The administration of monoclonal antibodies to rat ICAM-1 and LFA-1 markedly decreased the severity of the renal disease. In a group of rats injected three times a week with the monoclonal antibodies, from 2 d before immunization with GBM to day 14, glomerular abnormalities and proteinuria were virtually absent at day 14; even at 5 wk glomerular disease was quite mild, with only slight crescent formation and with only a mild decrease in renal function. When treatment was continued until 5 wk, the beneficial effects were even more marked, with virtual absence of crescents and with preservation of normal renal function. In a group of rats in which treatment was initiated on day 14, shortly after the appearance of glomerular abnormalities, progression of the disease was appreciably retarded, and the decrease in renal function was inhibited. The kidneys of rats treated from days -2 to 14 with antibodies to ICAM-1 and LFA-1 showed bright linear staining for rat IgG along the GBM, which did not differ in intensity from that seen in untreated rats. Furthermore, the titers of anti-GBM antibodies at 2 wk in treated rats were not lower than that seen in most of the untreated rats. There was, however, moderate reduction of anti-GBM antibodies at 5 wk in the treated rats.(ABSTRACT TRUNCATED AT 400 WORDS)