Rat Glomerular Basement Membrane Research Articles

Targeting of oxidized Macrophage Migration Inhibitory Factor (oxMIF) with antibody ON104 attenuates the severity of glomerulonephritis.

The oxidized form of Macrophage Migration Inhibitory Factor (oxMIF) has been identified as the disease-related isoform of MIF, exerting pathological functions in inflamed tissue. In this study, we aimed to explore the in vivo effects of the neutralizing anti-oxMIF antibody ON104 in a rat model of crescentic glomerulonephritis (CGN), to better understand its disease modifying activities. WKY rats received a single intravenous injection of a rabbit nephrotoxic serum (NTS), targeting rat glomerular basement membrane to induce CGN. On day 4 and day 6, ON104 was given intraperitoneally (i.p.) and on day 8 urine, blood and kidney tissue were collected. ON104 substantially attenuated the severity of CGN demonstrated by reduced proteinuria, hematuria, as well as lower levels of kidney injury molecule (KIM)-1. ON104 treatment preserved the glomerular morphology and suppressed crescent formation, a hallmark of the disease. On the cellular level, oxMIF neutralization by ON104 strongly reduced the number of macrophages and neutrophils within the inflamed kidneys. In vitro, we identified human neutrophils, but not monocytes, as main producers of oxMIF among total peripheral cells. The present study demonstrates that oxMIF is a pertinent therapeutic target in a model of CGN which mechanistically resembles human immune mediated CGN. In this model, neutralization of oxMIF by ON104 leads to an improvement in both urinary abnormalities and histological pathological characteristics of the disease. ON104, thus has the potential to become a novel disease-modifying drug for the treatment of glomerulonephritis and other inflammatory kidney diseases.

Annexin A1 exerts renoprotective effects in experimental crescentic glomerulonephritis.

Non-resolving inflammation plays a critical role during the transition from renal injury towards end-stage renal disease. The glucocorticoid-inducible protein annexin A1 has been shown to function as key regulator in the resolution phase of inflammation, but its role in immune-mediated crescentic glomerulonephritis has not been studied so far. Methods: Acute crescentic glomerulonephritis was induced in annexin A1-deficient and wildtype mice using a sheep serum against rat glomerular basement membrane constituents. Animals were sacrificed at d5 and d10 after nephritis induction. Renal leukocyte abundance was studied by immunofluorescence and flow cytometry. Alterations in gene expression were determined by RNA-Seq and gene ontology analysis. Renal levels of eicosanoids and related lipid products were measured using lipid mass spectrometry. Results: Histological analysis revealed an increased number of sclerotic glomeruli and aggravated tubulointerstitial damage in the kidneys of annexin A1-deficient mice compared to the wildtype controls. Flow cytometry analysis confirmed an increased number of CD45+ leukocytes and neutrophil granulocytes in the absence of annexin A1. Lipid mass spectrometry showed elevated levels of prostaglandins PGE2 and PGD2 and reduced levels of antiinflammatory epoxydocosapentaenoic acid regioisomers. RNA-Seq with subsequent gene ontology analysis revealed induction of gene products related to leukocyte activation and chemotaxis as well as regulation of cytokine production and secretion. Conclusion: Intrinsic annexin A1 reduces proinflammatory signals and infiltration of neutrophil granulocytes and thereby protects the kidney during crescentic glomerulonephritis. The annexin A1 signaling cascade may therefore provide novel targets for the treatment of inflammatory kidney disease.

Effects of Diaceto-Dipropyl-Disulphide on Plasma Sialic Acid and Renal Tissue Thiol Levels in Alloxan Diabetic Rats.

Plasma sialic acid levels are elevated in Diabetes Mellitus (DM) patients with proteinuria. Renal damage is mainly caused by free radicals that are excessively generated in DM. Thiols play an important role in the cellular antioxidative defence mechanisms mainly through thiol-disulphide exchange reaction. Diallyl disulphide, a garlic oil principle component, is known for its anti-diabetic properties. Its structural analogue, Diaceto-Dipropyl Disulphide (DADPDS), is a less toxic and more palatable disulphide and possesses similar anti-diabetic actions. This study was undertaken to assess the usefulness of DADPDS in prevention of de-sialation of Glomerular Basement Membrane (GBM) in alloxan diabetic rats and to assess effect of DADPDS on renal tissue thiol levels. Rats were divided into Normal, Diabetic and DADPDS treated diabetic groups. Diabetes was induced by intraperitoneal injection (IP) of alloxan. DADPDS was fed by gastric intubation. Plasma Sialic acid was determined by Ehrlich's method and renal tissue thiol levels by Nitroprusside reaction method. This study showed a significant decrease (p<0.001) in plasma sialic acid, plasma glucose and renal tissue TBARS levels along with significant increase (p<0.001) in renal tissue thiol levels in DADPDS treated alloxan diabetic rats when compared to diabetic control rats. Hence it may be concluded that DADPDS helps in preventing de-sialation of GBM in alloxan diabetic rats and improves renal tissue antioxidant defence mechanisms, may be through thiol-disulphide exchange reaction and thereby exhibits a possible clinical use in prevention of renal complications like diabetic nephropathy.

Unchanged Distribution Density of Anionic Sites on the Glomerular Wall in Rats with Active Heymann Nephritis

In various kinds of glomerulonephritis, alteration of anionic charge on the glomerular basement membrane (GBM) and podocytes has been controversial for more than decade. To elucidate the relation between glomerular protein leakage and anionic sites on the glomerular wall, we examined the distribution of anionic sites on the GBM and podocytes of rats with active Heymann nephritis (AHN). Urinalysis for protein levels was conducted, and the kidneys were examined using electron microscopic cytochemistry for the assessment of anionic charge with two cationic probes. The anionic sites on podocytes were decreased in number in the AHN rats; however, the distributions of anionic sites on the GBM were similar in density to those seen in the control animals. From these results, we consider that the decrease in anionic charge density on podocytes might be attributable to protein leakage and that the charge barrier of the GBM is irrelevant to the protein leakage in AHN rats.

Unchanged Distribution Density of Anionic Sites on the Glomerular Wall in Rats with Streptozotocin-Induced Diabetic Nephropathy

As a cause of proteinuria in diabetic nephropathy, a decrease in anionic charge on the glomerular basement membrane (GBM) is considered to be related to protein leakage. However, the constancy of the anionic charge has been reported in several types of nephropathy. To elucidate the relation between glomerular protein leakage and anionic charge, we examined the distribution of anionic sites on the GBM and podocytes in diabetic rats induced by a single intravenous injection of 60 mg/kg of streptozotocin (STZ). Five months after the treatment with STZ, urinalysis for glucose and protein levels was conducted, and the kidneys were examined using electron microscopic cytochemistry for the assessment of anionic charge with two cationic probes. The distributions of anionic sites on the GBM demonstrated by two kinds of cationic markers in the diabetic rats were similar in density to those seen in the control animals. The distributions of anionic sites on the foot processes and cell membrane of podocytes were regular and also similar in density to that of the control group. From these results, we consider that the charge barrier of the GBM and podocytes is irrelevant to the protein leakage in diabetic rats.

Pentoxifylline Ameliorates Glomerular Basement Membrane Ultrastructural Changes Caused by Gentamicin Administration in Rats

Gentamicin is commonly used for the treatment of severe gram negative bacterial infections but inevitably cause renal failure during prolonged use. The aim of our study was to emphasize protective effects of pentoxifylline on glomerular basement membrane (GBM) alterations induced by gentamicin in rats. Experiments were done on 40 male Wistar rats divided in three experimental groups. GM-group was treated daily with gentamicin in dose of 100 mg/kg during 8 days. PTX-group was treated daily with pentoxifylline in dose of 45 mg/kg and the same dose of gentamicin as in GM-group during 8 days. The control group received 1 ml/day saline intraperitoneally. Morphometric parameter measured during the analysis was glomerular basement membrane thickness. In GM-group of animals glomeruli were enlarged and GMB was diffusely and unequally thickened with neutrophil cells infiltration. In proximal tubules epithelial cells, vacuolization of cytoplasm with coagulation-type necrosis were observed. In PTX-group of animals glomeruli were somewhat enlarged and GBM was thickened only in some segments. Coagulation-type necrosis was not found. Blood urea and serum creatinine concentration in GM-group were significantly elevated in comparison with PTX-group while potassium level was decreased. Our results suggest that PTX has protective effects on GBM and proximal tubules in GM-treated rats.

Glomerular subendothelial and subepithelial immune complexes, containing the same antigen, are removed at different rates

To examine the persistence of immune deposits in the subendothelial and subepithelial areas of the glomerular basement membrane in rats, immune deposits were formed by injection of radiolabelled, cationized human serum albumin (HSA) as antigen, followed by rabbit antibodies to HSA. The disappearance of the radiolabelled antigen from immune deposits in glomeruli was described by a curve consisting of two exponential components. By electron microscopy, subendothelial and subepithelial immune deposits were initially present in glomeruli. At later time-points, only subepithelial immune deposits were present. The fast component of disappearance, attributed to subendothelial deposits, had a half-life of 3.89 +/- 0.32 h. The slow component of disappearance from glomeruli, attributed to subepithelial deposits, had a half-life of 85.5 +/- 3.1 h. Since some of the injected, radiolabelled antigen was sequestered in other compartments of the body, the possibility was raised that antigen from these sites might be released and contribute to the persistence of deposits in glomeruli. This possibility, however, was excluded when transplantation of kidneys with immune deposits to untreated recipients revealed no difference in the amount of antigen persisting in nontransplanted and transplanted kidneys.

In Vivo Degradation of Heparan Sulfates in the Glomerular Basement Membrane Does Not Result in Proteinuria

Heparan sulfates (HS) are long, unbranched, negatively charged polysaccharides that are bound to core proteins. HS in the glomerular basement membrane (GBM) is reported to be important for charge-selective permeability. Aberrant GBM HS expression has been observed in several glomerular diseases, such as diabetic nephropathy and membranous glomerulopathy, and a decrease in HS generally is associated with proteinuria. This study, with the use of a controlled in vivo approach, evaluated whether degradation of HS in rat GBM resulted in acute proteinuria. Rats received two intravenous injections of either heparinase III to digest HS or neuraminidase to remove neuraminic acids (positive control). Urine samples were taken at various time points, and at the end of the experiment, kidneys were removed and analyzed. Injection with heparinase III resulted in a complete loss of glomerular HS as demonstrated by immunofluorescence staining using anti-HS antibodies and by electron microscopy using cupromeronic blue in a critical electrolyte concentration mode. In the urine, a strong increase in HS was found within 2 h after the first injection. Staining for agrin, the major HS proteoglycan core protein in the GBM, was unaltered. No urinary albumin or other proteins were detected at any time point, and no changes in glomerular morphology were noticed. Injection of rats with neuraminidase, however, resulted in a major increase of urinary albumin and was associated with an increase in urinary free neuraminic acid. An increased glomerular staining with Peanut agglutinin lectin, indicative of removal of neuraminic acid, was noted. In conclusion, removal of HS from the GBM does not result in acute albuminuria, whereas removal of neuraminic acid does.

A Nephritogenic Peptide Induces Intermolecular Epitope Spreading on Collagen IV in Experimental Autoimmune Glomerulonephritis

This group previously identified a peptide p13 of alpha3(IV)NC1 domain of type IV collagen that induces experimental autoimmune glomerulonephritis (EAG) in rats with generation of antibodies to sites on alpha3(IV)NC1 external to the peptide as a result of intramolecular epitope spreading. It was hypothesized that intermolecular epitope spreading to other collagen IV chains also was induced. Rats were immunized with nephritogenic peptide that was derived from the amino terminal part of rat alpha3(IV)NC1 domain, and serum and kidney eluate were examined for antibodies to both native and recombinant NC1 domains of collagen IV. Peptide induced EAG with proteinuria and decreased renal function and glomerular basement membrane IgG deposits. Sera from these rats were examined by ELISA, which revealed reactivity not only to immunizing peptide but also to human and rat alpha3(IV)NC1 and to human alpha4(IV)NC1 domains. Kidney eluate that was depleted of alpha3(IV)NC1 antibodies still reacted to alpha4(IV)NC1, and alpha3(IV)NC1 column-bound antibody reacted with alpha3(IV)NC1. There was minimal reactivity to other collagen chains. Eluate that was adsorbed to NC1 hexamer from rat glomerular basement membrane lost all reactivity to glomerular constituents, and the eluted antibodies reacted to alpha3(IV)NC1 and alpha4(IV)NC1 domains. These studies show that a T cell epitope of alpha3(IV)NC1 induces EAG, intramolecular epitope spreading along alpha3(IV)NC1, and intermolecular epitope spreading to alpha4(IV)NC1 domain with minimal or no reactivity to other collagen chains or glomerular constituents. This is the first demonstration in EAG of intermolecular epitope spreading and identification of the spread epitopes.

Strain susceptibility to active induction and passive transfer of experimental autoimmune glomerulonephritis in the rat

Previous studies have shown that different inbred rat strains vary in their susceptibility to experimental autoimmune glomerulonephritis (EAG). The Wistar Kyoto (WKY) rat is highly susceptible and develops crescentic glomerulonephritis, while the Lewis (LEW) rat is resistant. When immunized with collagenase-solubilized rat glomerular basement membrane (GBM), both strains produce circulating autoantibodies reactive with rat GBM by enzyme-linked immunosorbent assay, but only the WKY rat shows strong linear deposits of IgG on the GBM. We investigated the hypothesis that differences in the characteristics of the anti-GBM antibodies produced, or in the inflammatory response to antibody deposition, could account for susceptibility. We found that circulating anti-GBM antibodies from WKY rats immunized with GBM were present at a higher concentration than those from LEW rats. Antibodies from WKY rats also recognized the rat alpha3 chain of type IV collagen [alpha3(IV)NC1], whereas those from LEW rats did not. Antibody eluted from the kidneys of WKY rats with EAG induced by GBM showed a higher affinity for GBM and recombinant rat alpha3(IV)NC1 than circulating antibody. This eluted antibody bound strongly to normal kidney sections from both WKY and LEW rats. Passive transfer of eluted anti-GBM antibodies from WKY rats with EAG resulted in similar binding of IgG to the GBM of WKY and LEW rats at 24 h. However, only the WKY recipients went on to develop crescentic glomerulonephritis by 28 days. This study demonstrates that the characteristics of the anti-GBM antibodies induced in WKY rats contribute to their susceptibility to EAG. However, the passive transfer experiments reveal that factors related to the inflammatory response to antibody deposition are also important in determining susceptibility. A combination of these genetic influences could explain the variation in severity of human anti-GBM disease.

Expression and localization of MT1-MMP and furin in the glomerular wall of short- and long-term diabetic rats

Diabetic glomerulopathy has been linked to shifts in balance between the synthetic and degradative pathways of the glomerular basement membrane (GBM), a key player in the permselectivity properties of the glomerular wall. The goal of this study was to trace the expression and localization of membrane type-1 metalloprotease (MT1-MMP) and its activating enzyme furin, key proteins involved in basement membrane turnover, in short- and long-term diabetic rat renal tissues. Quantitative immunogold was carried out for MT1-MMP and furin and their expression was evaluated in renal tissues of young and old, control and diabetic rats. To corroborate immunocytochemical findings, Western blots were performed on glomerular lysates. Electron microscopy revealed that the overall expression of MT1-MMP and furin is reduced in plasma membranes of all glomerular cell types of old normoglycemic animals, a phenomenon that is exacerbated in long-term diabetic animals. This observation supports the prevailing theory that diabetes fosters acceleration in the aging process. Interestingly, while biochemical results confirmed a decrease in MT1-MMP expression, an increase in furin was observed. Immunocytochemical studies resolved this discrepancy by tracing the increased furin expression in endoplasmic reticulum and Golgi membranes of podocytes, indicating that furin is retained in the secretory pathway in a diabetic environment. Disturbances at the molecular level of the otherwise tightly regulated MT1-MMP/furin interactions found at the cell surface must account for a lack in extracellular matrix remodeling, increased deposition of GBM material, and loss of glomerular filtration integrity.

The number of podocyte and slit diaphragm is decreased in experimental diabetic nephropathy

To determine the number of podocyte, slit diaphragms, slit diaphragm extensions and GBM thickness in diabetic nephropathy. Sixty "Rattus Wistar"of both sexes weighing 200-300 g were divided in two experimental groups: normal group 10 animals, and alloxan diabetic rats--50 animals. Alloxan was administered in a single IV dose of 42 mg/kg body weight. Body weight, water and food intake, diuresis, and blood and urine glucose were determined in both groups before alloxan injection and two weeks, six and twelve months after alloxan injection. Proteinuria was measured at 12 months in both groups. After 12 months animals were sacrificed, and the right kidney processed for electron microscopy. Clear clinical and laboratory signs of severe diabetes were seen, in all alloxan-diabetic rats at all follow-up times. Glomerular basement membrane (GBM) thickening, podocyte number, and slit diaphragm number and extension were determined. GBM of all diabetic rats was significantly thicker (median=0.29 microm; semi-interquartile range=0.065 microm) than in the normal rats (0.23 microm; 0.035 microm). Diabetic rat podocyte number (8; 1), slit diaphragm number (4; 1), and slit diaphragm extension (0.021 microm; 0.00435 microm) were significantly lower than in normal rats (11; 1) and (7; 1.5), and (0.031 microm; 0.0058 microm). Diabetic rat proteinuria (0.060 mg/24 h; 0.037 mg/24 h) was higher than in normal rats (0.00185 mg/24 h; 0.00055 mg/24 h). Experimental diabetes is associated with significant (p<0.05) changes in podocyte foot process, slit number, slit diaphragm extension, and GBM thickness.

Regulatory effects of inducible nitric oxide synthase on cyclooxygenase-2 and heme oxygenase-1 expression in experimental glomerulonephritis

We explored whether inducible nitric oxide synthase (iNOS) driven nitric oxide (NO) production regulates expression of iNOS, endothelial NOS (eNOS), Cyclooxygenase-2 (COX-2), and Hemeoxygenase-1 (HO-1) proteins in a rat model of glomerulonephritis induced by antibody raised in rabbits against rat glomerular basement membrane (anti-GBM). Rats were injected either with non-immune serum (control), or anti-GBM serum. In a group of rats N6-(1-iminoethyl)-L-lysine (L-NIL) was administered prior to injection of anti-GBM serum to inhibit iNOS activity. Urinary nitrite plus nitrate (NOx) excretion was assessed to determine the extent of iNOS inhibition by L-NIL. Urinary albumin excretion was assessed to determine extent of proteinuria. Urinary PGE2 was assessed as a marker of COX activity. Glomeruli were harvested 24 h after injection of anti-GBM serum and ED1, COX-2, iNOS, eNOS and HO-1 expression was analysed by Western blot analysis. iNOS activity in glomeruli was effectively reduced in L-NIL-treated nephritic animals. In these animals, there was exacerbation of proteinuria and reduction in urinary PGE2 levels without changes in the extent of macrophage infiltration in glomeruli. In nephritic animals, there was an increase in glomerular protein levels of COX-2, HO-1 and iNOS, but not of eNOS. While L-NIL treatment reduced glomerular HO-1, levels of COX-2 and iNOS increased; but not that of eNOS. The observations indicate that in glomerulonephritis iNOS-driven NO production acts as a negative feedback regulator of iNOS itself, suppresses COX-2 levels, and maintains HO-1 levels.

Nasal Administration of Recombinant Rat α3(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.

Oral Adsorbent Prevents Reduction of Anionic Sites of the Glomerular Basement Membrane in Diabetic Nephropathy

Background: AST-120, an oral adsorbent, inhibits progression of diabetic nephropathy by reducing albuminuria. The purpose of the present study was to explore the mechanism underlying the protective effect of AST-120 against albuminuria. Methods: Twenty-four male Sprague-Dawley rats underwent intravenous injection of streptozotocin and subsequent uninephrectomy. Half of the rats were fed standard rat chow, and the other half were fed rat chow containing AST-120. All rats were killed at week 6 after a clearance study. Results: There were no significant differences in body weight, kidney weight, urinary volume, blood pressure, blood glucose or inulin clearance between the two groups at week 6. However, urinary albumin excretion at week 6 was significantly lower in the AST-120 group than in the control group (p < 0.05). Light microscopic observation showed that the planar area of glomeruli was significantly smaller in the AST-120 group than in the control group (p < 0.01), and the shortest diameter of proximal tubules was less in the AST-120 group than in the control group (p < 0.01). Electron microscopic observation showed a significantly greater number of anionic sites on the lamina rara externa of the glomerular basement membrane in the AST-120 group than in the control group (p < 0.01). Conclusion: We conclude that AST-120 reduces albuminuria by preventing the decrease in the number of anionic sites in the glomerular basement membrane in rats with diabetic nephropathy. In addition, AST-120 inhibits the appearance of glomerular and tubular hypertrophy.

Glomerular T Cells Are of Restricted Clonality and Express Multiple CDR3 Motifs across Different Vβ T-Cell Receptor Families in Experimental Autoimmune Glomerulonephritis

Experimental autoimmune glomerulonephritis (EAG) is an animal model of Goodpasture’s disease which can be induced in Wistar-Kyoto (WKY) rats by a single intramuscular injection of collagenase-digested rat glomerular basement membrane (GBM) in adjuvant. This model is characterised by anti-GBM antibody production, accompanied by focal necrotising glomerulonephritis with crescent formation and glomerular infiltration by T cells and macrophages. Previous work has shown that EAG is a T-cell-dependent disease. We proposed that intraglomerular T cells might be directly involved in pathogenesis and would be oligoclonal. In this study, EAG was induced by standard methods, the kidneys perfused with saline at week 2 and week 4, and the glomeruli separated by a sieving method. Glomerular RNA was extracted and reverse transcribed. RT-PCR showed overexpression of an average of two Vβ families in each kidney analysed. However, no predominant single Vβ family was overexpressed in any of the experimental animals. CDR3 spectratyping of Fam-labelled PCR products showed a marked restriction involving different Vβ families. Sequencing demonstrated multiple CDR3 motifs, each expressed in association with different Vβ gene segments. Our results show that glomerular T cells are of restricted clonality and suggest a role for antigen-specific effector T cells in the pathogenesis of EAG.

Blockade of the CD154-CD40 costimulatory pathway prevents the development of experimental autoimmune glomerulonephritis

Experimental autoimmune glomerulonephritis (EAG) was induced in Wistar-Kyoto (WKY) rats by immunization with rat glomerular basement membrane (GBM) in adjuvant. This model is characterized by anti-GBM antibody production, accompanied by focal necrotizing glomerulonephritis with crescent formation. There is also glomerular infiltration by T cells and macrophages. Our hypothesis was that blocking the interaction between CD154 (CD40L) on Th cells and CD40 on antigen-presenting cells should inhibit T-cell activation, and thus the development of EAG. The in vivo effects of a hamster anti-rat monoclonal antibody to CD154 (AH.F5) were examined in EAG starting at day -1 prior to immunization, day +7 after immunization, or day +14 after immunization. When administered from day -1 at a dose of 10 mg/kg intraperitoneally three times per week for the duration of the study (4 weeks), AH.F5 resulted in a marked reduction in circulating anti-alpha3(IV)NC1 antibodies, deposits of IgG on the GBM, albuminuria, deposits of fibrin in the glomeruli, severity of glomerular abnormalities, and numbers of glomerular T cells and macrophages. When administered from day +7 at the same dose, AH.F5 resulted in a moderate reduction in the severity of disease, while administration from day +14 had no significant effect. These studies demonstrate for the first time that early blockade of the CD154-CD40 T-cell costimulatory pathway can prevent the development of crescentic nephritis, and that delayed treatment can reduce the severity of disease. This confirms the importance of T cell mediated immunity in the pathogenesis of EAG, and suggests that strategies targeting T-cell costimulation may provide a novel approach in the treatment of human glomerulonephritis.

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.

Segregation of Experimental Autoimmune Glomerulonephritis as a Complex Genetic Trait and Exclusion of Col4a3 as a Candidate Gene

Experimental autoimmune glomerulonephritis (EAG), an animal model of Goodpasture’s disease, can be induced in Wistar-Kyoto (WKY) rats (RT1-l) by immunization with rat glomerular basement membrane (GBM) in adjuvant. The model in this rat strain is characterized by anti-GBM antibody production accompanied by focal necrotizing glomerulonephritis with crescent formation. The main autoantigen in humans and rats has been identified as the non-collagenous domain of the α3 chain of type IV collagen (α3(IV)NC1). By contrast, Lewis (LEW) rats with the same MHC background (RT1-l), immunized with the same antigen, develop similar levels of circulating anti-GBM antibodies, but no histological evidence of nephritis. In order to investigate the genetic basis of susceptibility to EAG, we examined the response of both F1 (WKY × LEW) and backcross (BC1; WKY × F1) rats to immunization with rat GBM. F1 animals were completely resistant to the development of EAG, while BC1 animals showed a range of responses from severe crescentic glomerulonephritis to no histological evidence of disease. The results indicate that EAG is inherited as a complex trait under the control of WKY genes unlinked to the MHC. cDNA sequence analysis of α3(IV)NC1 in the two parental strains was identical, indicating no predicted amino acid sequence variation in the α3(IV)NC1 domain between these strains. Radiation hybrid mapping, using two separate PCR amplicons from rat α3(IV)NC1, localized rat Col4a3 to a region of chromosome 9. Since Col4a3 (encoding the autoantigen) is a candidate for susceptibility to EAG, we screened the region of rat chromosome 9 where Col4a3 is localized, using polymorphic microsatellite markers in segregating BC1 progeny. No significant linkage was detected. These results exclude Col4a3 as a recessive susceptibility gene for EAG in the BC1 progeny.

Non-Neoplastic Kidney Diseases, Abstract 110–113, Symposium

Renal antigens in mercuric chloride induced, anti-GBM autoantibody glomerular disease. Two antibody probes were used to characterize the putative renal antigens of HgCl2-induced antiglomerular basement membrane renal disease in Brown Norway (BN) rat. The first probe was the linear immunofluorescence imparting, in vivo bound, nephritogenic antiglomerular-basement-membrane autoantibody (anti-GBM-Ab). The second probe was a rat monoclonal antibody to the B subunit of laminin that was obtained from fusion of spleen cells of HgCl2 injected BN rat. By enzyme-linked immunosorbent assay (ELISA) the anti-GBM-Ab reacted with laminin, type IV collagen, collagenase-resistant noncollagenous portion of glomerular basement membrane (GBM), saline soluble proteins of kidney cortex homogenate and fibronectin. Western blot analysis of laminin indicated that the reactive epitopes detected by both probes were on the B chain subunit but not the A subunit. In nonreduced collagenase-digested GBM the epitopes were present on 27 kD and 42 to 48 kD polypeptides. A similar pattern was seen on collagenase-digested human GBM. On rat and human GBM the patterns obtained with rat autoantibody and autoantibody from a patient with Goodpasture syndrome were similar, suggesting that some of the in vivo bound anti-GBM autoantibodies in HgCl2-induced disease in rat are directed against epitopes which are similar to the Goodpasture antigen of human. Reactive epitopes were also detected on saline soluble proteins of kidney cortex homogenate with the predominant antigen being a 31 kD polypeptide. In the saline soluble proteins the reactive polypeptides including the major 31 kD polypeptide did not originate from laminin, type IV collagen, or the collagenase-resistant noncollagenous part of GBM. The precise structural origin of soluble proteins was not defined. The epitopes in type IV collagen and fibronectin could not be further characterized for molecular mass by Western blot analysis.