Anti-glomerular Basement Membrane Antibody-induced Glomerulonephritis Research Articles

A novel soluble complement receptor 1 fragment with enhanced therapeutic potential

Human complement receptor 1 (HuCR1) is a pivotal regulator of complement activity, acting on all three complement pathways as a membrane-bound receptor of C3b/C4b, C3/C5 convertase decay accelerator, and cofactor for factor I-mediated cleavage of C3b and C4b. In this study, we sought to identify a minimal soluble fragment of HuCR1, which retains the complement regulatory activity of the wildtype protein. To this end, we generated recombinant, soluble, and truncated versions of HuCR1 and compared their ability to inhibit complement activation in vitro using multiple assays. A soluble form of HuCR1, truncated at amino acid 1392 and designated CSL040, was found to be a more potent inhibitor than all other truncation variants tested. CSL040 retained its affinity to both C3b and C4b as well as its cleavage and decay acceleration activity and was found to be stable under a range of buffer conditions. Pharmacokinetic studies in mice demonstrated that the level of sialylation is a major determinant of CSL040 clearance in vivo. CSL040 also showed an improved pharmacokinetic profile compared with the full extracellular domain of HuCR1. The in vivo effects of CSL040 on acute complement-mediated kidney damage were tested in an attenuated passive antiglomerular basement membrane antibody-induced glomerulonephritis model. In this model, CSL040 at 20 and 60 mg/kg significantly attenuated kidney damage at 24 h, with significant reductions in cellular infiltrates and urine albumin, consistent with protection from kidney damage. CSL040 thus represents a potential therapeutic candidate for the treatment of complement-mediated disorders.

Glutathione S-transferase Mu 2-transduced mesenchymal stem cells ameliorated anti-glomerular basement membrane antibody-induced glomerulonephritis by inhibiting oxidation and inflammation

IntroductionOxidative stress is implicated in tissue inflammation, and plays an important role in the pathogenesis of immune-mediated nephritis. Using the anti-glomerular basement membrane antibody-induced glomerulonephritis (anti-GBM-GN) mouse model, we found that increased expression of glutathione S-transferase Mu 2 (GSTM2) was related to reduced renal damage caused by anti-GBM antibodies. Furthermore, mesenchymal stem cell (MSC)-based therapy has shed light on the treatment of immune-mediated kidney diseases. The aim of this study was to investigate if MSCs could be utilized as vehicles to deliver the GSTM2 gene product into the kidney and to evaluate its potential therapeutic effect on anti-GBM-GN.MethodsThe human GSTM2 gene (hGSTM2) was transduced into mouse bone marrow-derived MSCs via a lentivirus vector to create a stable cell line (hGSTM2-MSC). The cultured hGSTM2-MSCs were treated with 0.5mM H2O2, and apoptotic cells were measured by terminal dUTP nick-end labeling (TUNEL) assay. The 129/svj mice, which were challenged with anti-GBM antibodies, were injected with 106 hGSTM2-MSCs via the tail vein. Expression of hGSTM2 and inflammatory cytokines in the kidney was assayed by quantitative PCR and western blotting. Renal function of mice was evaluated by monitoring proteinuria and levels of blood urea nitrogen (BUN), and renal pathological changes were analyzed by histochemistry. Immunohistochemical analysis was performed to measure inflammatory cell infiltration and renal cell apoptosis.ResultsMSCs transduced with hGSTM2 exhibited similar growth and differentiation properties to MSCs. hGSTM2-MSCs persistently expressed hGSTM2 and resisted H2O2-induced apoptosis. Upon injection into 129/svj mice, hGSTM2-MSCs migrated to the kidney and expressed hGSTM2. The anti-GBM-GN mice treated with hGSTM2-MSCs exhibited reduced proteinuria and BUN (58% and 59% reduction, respectively) and ameliorated renal pathological damage, compared with control mice. Mice injected with hGSTM2-MSCs showed alleviated renal inflammatory cell infiltration and reduced expression of chemokine (C-C motif) ligand 2 (CCL2), interleukin (IL)-1β and IL-6 (53%, 46% and 52% reduction, respectively), compared with controls. Moreover, hGSTM2-MSCs increased expression of renal superoxide dismutase and catalase, which may associate with detoxifying reactive oxygen species to prevent oxidative renal damage.ConclusionsOur data suggest that the enhanced protective effect of GSTM2-transduced MSCs against anti-GBM-GN might be associated with inhibition of oxidative stress-induced renal cell apoptosis and inflammation, through over-expression of hGSTM2 in mouse kidneys.

Superoxide dismutase mimetic drug tempol aggravates anti-GBM antibody-induced glomerulonephritis in mice

Oxidative stress plays an important role in the pathogenesis of anti-glomerular basement membrane antibody-induced glomerulonephritis (anti-GBM-GN). Superoxide dismutase (SOD) is the first line of defense against oxidative stress by converting superoxide to hydrogen peroxide (H(2)O(2)). We investigated the effect of the SOD mimetic drug tempol on anti-GBM-GN in mice. 129/svJ mice were challenged with rabbit anti-mouse-GBM sera to induce GN and subsequently divided into tempol (200 mg.kg(-1).day(-1), orally) and vehicle-treated groups. Routine histology, SOD and catalase activities, malondialdehyde (MDA), H(2)O(2), and immunohistochemical staining for neutrophils, lymphocytes, macrophages, p65-NF-kappaB, and osteopontin were performed. Mice with anti-GBM-GN had significantly reduced renal SOD and catalase activities and increased H(2)O(2) and MDA levels. Unexpectedly, tempol administration exacerbated anti-GBM-GN as evidenced by intensification of proteinuria, the presence of severe crescentic GN with leukocyte influx, and accelerated mortality in the treated group. Tempol treatment raised SOD activity and H(2)O(2) level in urine, upregulated p65-NF-kappaB and osteopontin in the kidney, but had no effect on renal catalase activity. Thus tempol aggravates anti-GBM-GN by increasing production of H(2)O(2) which is a potent NF-kappaB activator and as such can intensify inflammation and renal injury. This supposition is supported by increases seen in p65-NF-kappaB, osteopontin, and leukocyte influx in the kidneys of the tempol-treated group.

Amelioration of Crescentic Glomerulonephritis by RhoA Kinase Inhibitor, Fasudil, through Podocyte Protection and Prevention of Leukocyte Migration

The small GTPase RhoA is activated by the angiotensin II (AngII) type 1 receptor (AT1R), which is part of the local renin-angiotensin system that is involved in podocyte injury preceding glomerular crescent formation. We demonstrated previously that inhibition of AT1R protects against crescentic glomerular injury in Fc receptor-deficient mice (gamma -/-) with anti-glomerular basement membrane antibody-induced glomerulonephritis (anti-GBM GN). Here, we hypothesized that the RhoA kinase inhibitor, fasudil, attenuates AT1R-dependent crescentic GN. We examined anti-GBM GN in gamma -/- mice with or without fasudil treatment, and further investigated the underlying mechanisms in cultured differentiated podocytes and leukocytes. Fasudil markedly attenuated crescentic GN with a significant decrease in proteinuria and hematuria, infiltration of T cells and monocytes/macrophages as well as their local proliferation, and preservation of podocyte-specific proteins, including WT-1 and nephrin, in glomeruli. In vitro studies showed that AngII induced the down-regulation of both nephrin and WT-1 expression in podocytes, which was reversed by fasudil in a dose-dependent manner. Additionally, fasudil blocked the AngII-induced migration of both macrophages and T cells. Furthermore, we also examined lipopolysaccharide-induced nephrotic syndrome in severe combined immunodeficiency disease mice and found that fasudil failed to block the development of proteinuria because of a B7-1-dependent podocyte injury. In conclusion, fasudil treatment prevents crescent formation and disease progression in anti-GBM GN by preventing AngII-induced podocyte injury and leukocyte migration.

Mast cells and inflammatory kidney disease

Inflammatory kidney disease involves a complex network of interactions between resident kidney and infiltrating hematopoietic cells. Mast cells (MCs) are constitutively found in kidneys in small numbers but increase considerably in various renal diseases. While this increase is usually interpreted as a sign of pathological involvement, recent data using MC-deficient animals show their ability to restore kidney homeostasis. In anti-glomerular basement membrane antibody-induced glomerulonephritis, MCs are protective by initiating repair and remodeling functions counteracting the devastating effects of glomerular injury. Protection may also include immunoregulatory capacities to limit autoreactive T-cell responses. MCs also control tubulointerstitial fibrosis by activating tissue remodeling and neutralizing fibrotic factors. Release of mediators by MCs during inflammation, however, could also promote unwanted responses that ultimately lead to destruction of kidney structure, as exemplified by data showing either protection or aggravation in related renal disease models. Similarly, while the action of proteases may initially be beneficial, the generation of fibrosis-promoting angiotensin II by chymase also shows the limits of adaptive responses to achieve homeostasis. Thus, it is likely the physiological context involving the interaction with other cells and inflammatory mediators that determines the final action of MCs in the development of kidney disease.

Blockade of VEGF accelerates proteinuria, via decrease in nephrin expression in rat crescentic glomerulonephritis

Vascular endothelial growth factor (VEGF) is a potent angiogenic factor that maintains the glomerular and peritubular capillary (PTC) network in the kidney. The soluble form of the VEGF receptor-1 (soluble fms-like tyrosine kinase 1 (sFlt-1)) is known to regulate VEGF activity by binding VEGF in the circulation. We hypothesized that VEGF may be beneficial for maintaining glomerular filtration barrier and vascular network in rats with progressive glomerulonephritis (GN). For blockade of VEGF activity in vivo, rats were transfected twice with plasmid DNA encoding the murine sFlt-1 gene into femoral muscle 3 days before and 2 weeks after the induction of antiglomerular basement membrane antibody-induced GN. Inhibition of VEGF with sFlt-1 resulted in massive urinary protein excretion, concomitantly with downregulated expression of nephrin in nephritic rats. Further, blockade of VEGF induced mild proteinuria in normal rats. Administration of sFlt-1 affected neither the infiltration of macrophages nor crescentic formation. In contrast, treatment of sFlt-1 accelerated the progression of glomerulosclerosis and interstitial fibrosis accompanied with renal dysfunction and PTC loss at day 56. VEGF may play a role in maintaining the podocyte function as well as renal vasculature, thereby protecting glomeruli and interstitium from progressive renal insults.

Immunohistochemical characterization of glomerular inflammatory cells and expression of adhesion molecules in anti-glomerular basement membrane (anti-GBM) glomerulonephritis induced in WKY rats with monoclonal anti-GBM antibodies of different subclasses

According to previous report, adhesion of CD8-positive cells and macrophages to glomerular endotherial cells through the lymphocyte function-associated antigen-1 (LFA-1)/intercellular adhesion molecule-1 (ICAM-1) pathway is crucial for the initiation and subsequent progression of anti-glomerular basement membrane (anti-GBM) antibody-induced glomerulonephritis (anti-GBM nephritis) in WKY rats. In the present study glomerular inflammatory cell infiltration and LFA-1/ICAM-1 expression were examined in anti-GBM nephritis induced in WKY rats with monoclonal anti-GBM antibodies of different subclasses: IgG1, IgG2a, and IgG2b. The IgG2a and IgG2b subclasses induced significant proteinuria from day 3 as compared with the IgG1 subclass. Glomerular infiltration of macrophages and CD8-positive cells after administration of IgG2a and IgG2b subclass antibodies was significantly elevated compared to that for the IgG1 subclass. The intensity of glomerular ICAM-1 immunostaining by the IgG2a and IgG2b subclass antibodies tended to be stronger than that by the IgG1 subclass. Glomerular LFA-1-positive cell infiltration by the IgG2a and IgG2b subclasses was significantly higher than that of the IgG1 subclass. These results demonstrate that monoclonal antibodies belonging to the IgG2a and IgG2b subclasses strongly induce glomerular infiltration of inflammatory cells and expression of adhesion molecules in rat anti-GBM nephritis.

Fibrin independent proinflammatory effects of tissuefactor in experimental crescentic glomerulonephritis

Tissue factor initiated glomerular fibrin deposition is an important mediator of injury in crescentic glomerulonephritis. Recent data have suggested noncoagulant roles for tissue factor in inflammation. To test the hypothesis that in addition to its effects in initiating coagulation, tissue factor has proinflammatory effects in glomerulonephritis, rabbits given crescentic anti-glomerular basement membrane (GBM) antibody-induced glomerulonephritis were defibrinogenated with ancrod. One group of defibrinogenated rabbits was also given anti-tissue factor antibodies. Comparisons were made between these groups, as well as a third group that was neither defibrinogenated with ancrod nor given anti-tissue factor antibodies. Defibrinogenation alone abolished glomerular fibrin deposition, reduced crescent formation, and limited renal impairment (ancrod-treated, serum creatinine 274 +/- 37 micromol/L; untreated 415 +/- 51 micromol/L; P < 0.01). Tissue factor inhibition in defibrinogenated rabbits resulted in further protection of renal function (creatinine 140 +/- 19 micromol/L, P < 0.01) and reduced proteinuria (0.4 +/- 0.2g/day, untreated 2.6 +/- 0.4 g/day, P <0.01), which was significantly increased by defibrinogenation alone (ancrod-treated, 5.6 +/- 1.2 g/day). Anti-tissue factor antibodies (but not defibrinogenation alone) attenuated glomerular T-cell and macrophage recruitment, and major histocompatibility complex (MHC) class II expression. These results demonstrate important proinflammatory effects of tissue factor in crescentic glomerulonephritis that are fibrin independent and provide in vivo evidence for tissue factor's proinflammatory effects on MHC class II expression and leukocyte accumulation.

High nephritogenicity of monoclonal antibodies belonging to IgG2a and IgG2b subclasses in rat anti-GBM nephritis

To examine a subclass-effect relationship and a dose-effect relationship of autoantibodies in the rat antiglomerular basement membrane (GBM) antibody-induced glomerulonephritis (anti-GBM nephritis) model, we injected homologous monoclonal antibodies against the NC1 domains of rat type IV collagen into inbred Wistar-Kyoto (WKY) rats. Eight different autoantibodies from each of the IgG1, IgG2a, and IgG2b subclasses were established and administered to groups of four WKY rats at a dose of 300 microg/rat. To examine the dose-effect relationship, we administered 0 to 300 microg/rat of autoantibodies from each subclass to rats. All IgG1 antibodies induced mild nephritis, whereas the IgG2a and IgG2b antibodies induced moderate to severe nephritis. Some IgG2a and IgG2b antibodies induced pulmonary hemorrhage as well. These antibodies were reactive with alpha3(IV)NC1, alpha4(IV)NC1, or alpha5(IV)NC1. The minimum dose of antibody required to induce nephritis was 30 microg/rat for IgG1, 3 microg/rat for IgG2a, and 1 microg/rat for IgG2b. At doses of 30 microg/rat or less, antibody deposition was generally restricted to the GBM. At doses of 100 microg/rat or greater, antibody deposition extended to both the GBM and tubular basement membrane (TBM). Pulmonary hemorrhage was observed only when a large amount of pulmonary hemorrhagic antibody was administered. The severity of nephritis was dependent on both subclass and dose of autoantibodies. It becomes clear that pulmonary hemorrhage in anti-GBM nephritis is induced by autoantibodies.

Distinct contribution of Fc receptors and angiotensin II-dependent pathways in anti-GBM glomerulonephritis

The contribution of antibody and/or immune-complex to the pathogenesis of immunologically-mediated glomerulonephritis is not fully understood, although it has been recently clarified that Fc receptors (FcRs) play critical roles in the inflammatory cascade. We therefore re-evaluated the classical model of glomerulonephritis, anti-glomerular basement membrane antibody-induced glomerulonephritis (Anti-GBM GN), from the standpoint of FcRs and also investigated the residual FcR-independent mechanisms. We adopted an Anti-GBM GN mouse model that has two strains deficient in the FcR gamma chain [gamma(-/-)] or Fc gammaRIIB [RII(-/-)], and analyzed functional (urinary protein, serum creatinine, BUN) and pathological changes of the glomeruli. For the analyses of FcR-independent mechanisms, several doses of nephrotoxic serum were applied, and then mice were treated either with cobra venom factor or an angiotensin II type 1 receptor antagonist in gamma(-/-) mice. In gamma(-/-) mice, renal injuries were dramatically attenuated with an absence of polymorphonuclear cell (PMN) influx, while RII(-/-) mice suffered accelerated glomerular injuries in spite of a normal PMN influx. In the absence of FcR-dependent effects in gamma(-/-) mice, the FcR-independent pathway lead to chronic renal damage characterized by mesangial proliferation and progressive expansion of mesangial area, with monocyte/macrophage accumulation and with the expression of alpha smooth muscle actin in the mesangial cells and interstitium. Those injuries in gamma(-/-) mice were not attenuated by the decomplementation, but completely abolished by using an angiotensin II type 1 receptor antagonist. Our results clearly demonstrate that FcRs play a pivotal role in Anti-GBM GN, especially in its acute phase. We further clarified the existence of FcR and complement-independent but antibody-dependent pathway. Furthermore, we found that those pathological changes were strongly related to the renin-angiotensin system.

Pathogenic significance of interleukin-6 in a patient with antiglomerular basement membrane antibody-induced glomerulonephritis with multinucleated giant cells

We report a patient with anti-glomerular basement membrane disease who developed renal failure associated with systemic manifestations, including acute-phase inflammatory reactions and plasmacytosis. Renal tissue obtained by an open surgical biopsy showed circumferential cellular crescents, multinucleated giant cells, and exudation of fibrin in all glomeruli. Immunofluorescence microscopy demonstrated deposition of immunoglobulin G, C3, and membrane attack complex along glomerular capillary walls. Multinucleated giant cells were suggested to be macrophage-monocyte lineage because they were CD68 positive. Bone marrow aspiration showed an increase of plasma cells. Immunostaining showed intensive expression of interleukin-6 (IL-6) in practically every part of the renal sites involving multinucleated cells, crescents, tubules, and infiltrating cells, suggesting that one of the sources of systemically elevated IL-6 was the kidney. Serum IL-6, anti-glomerular basement membrane antibody, and acute-phase proteins were markedly elevated, and returned dramatically to the normal level after corticosteroid therapy and plasmapheresis. We believe that IL-6 played an important role in the development of many symptoms in the present case.

Clinical efficiency of type-IV collagen in extracorporeal shock wave lithotripsy.

We previously reported a patient who developed anti-glomerular basement membrane antibody-induced glomerulonephritis (GBM-GN) after extracorporeal shock wave lithotripsy (ESWL). To determine whether ESWL causes anti-GBM antibody production thereby inducing GBM-GN, we measured serum levels of type-IV collagen (the most major component of GBM) before and after ESWL. Serum samples were obtained from 13 patients with renal stones and 15 with ureteral stones. The serum type-IV collagen level was determined by a radioimmunoassay method. No significant changes were observed in the type-IV collagen level after ESWL in each patient group. Type-IV collagen was abundant in the GBM but was not released into the blood after ESWL. Therefore, in patients with urinary tract stones, it is not likely that type-IV collagen, a hidden antigen in the renal glomeruli, is exposed or released into the blood after ESWL, resulting in autoantibody production and GBM-GN.

A role for P selectin in complement-independent neutrophil-mediated glomerular injury

Neutrophil recruitment and lung injury following complement activation have been demonstrated to be dependent on endothelial expression of P selectin. In anti-glomerular basement membrane antibody-induced glomerulonephritis (anti-GBM GN) in mice, acute glomerular injury results from complement-independent neutrophil accumulation. The signals for neutrophil recruitment in this model are unknown. Expression of P selectin on glomerular endothelium was demonstrated within 30 minutes of administration of anti-GBM antibody to C57/BL10 mice. This was associated with rapid accumulation of neutrophils in glomeruli which peaked at one hour (6.2 +/- 0.5 neutrophils per glomerular cross section [neut/gcs], normal 0.34 +/- 0.06 neut/gcs, P < 0.01) and significant proteinuria after 16 hours (3.6 +/- 0.5 mg/16 hr, control 0.62 +/- 0.13 mg/16 hr, P < 0.01). Complement depletion with cobra venom factor, which reduced serum C3 levels to less than 5% of normal, did not alter expression of P selectin, reduce glomerular neutrophil accumulation (6.7 +/- 0.8 neut/gcs) or proteinuria (3.7 +/- 0.5 mg/16 hr). Platelet depletion also failed to alter glomerular expression of P selectin, neutrophil accumulation or the development of proteinuria. Mice were treated with an affinity purified anti-human P selectin antibody, which cross reacted with mouse P selectin and blocked P selectin-dependent binding of thrombin-activated mouse platelets to HL60 cells and did not bind to mouse neutrophils. Treatment, one hour prior to the administration of anti-GBM antibody, markedly inhibited glomerular neutrophil accumulation (0.94 +/- 0.12 neut/gcs) and prevented proteinuria (1.0 +/- 0.2 mg/16 hr), and did not alter binding of anti-GBM globulin in the kidney. These data strongly suggest that the rapid up-regulation of P selectin expression in glomeruli following binding of anti-GBM antibody is an essential signal for neutrophil recruitment in this complement independent model of glomerular injury.

Cloning of Rat Interleukin-13 (IL-13) cDNA and Analysis of IL-13 Gene Expression in Experimental Glomerulonephritis

Using a reverse transcriptase-polymerase chain reaction method, a cDNA for the complete coding region and part of the 5′-untranslated region of rat IL-13 was cloned from rat renal cortex RNA following the induction of anti-glomerular basement membrane antibody-induced glomerulonephritis. The coding region of IL-13 cDNA displays 74% and 87% sequence identity with the coding regions of human and mouse IL-13 cDNA, respectively. The deduced amino acid sequence of rat IL-13 reveals 63% and 79% homology with the human and mouse proteins, respectively. Using the rat IL-13 cDNA as a molecular probe, increased production of IL-13 mRNA in renal and splenic tissue is demonstrated in the first 48 hours of antibody-induced glomerulonephritis in the rat. The data suggest a role for IL-13 in the inflammatory response in vivo.

Isologous monoclonal antibodies can induce anti-GBM glomerulonephritis in rats.

Injection of isologous monoclonal antibodies (SR2, SR3) caused anti-glomerular basement membrane antibody-induced glomerulonephritis (anti-GBM nephritis) in WKY/NCrj rats. The antibodies were obtained from hybridoma cells derived from fusion of the spleen of a nephritic WKY/NCrj rat injected with rat solubilized renal basement membranes with adjuvant, and mouse SP2-myeloma cells. They belonged to the rat IgG2a subclass and bound to rat kidney in a linear pattern along the glomerular and tubular basement membranes. Histological changes in glomeruli were detected at day 1 after the injection; proteinuria with haematuria appeared on day 2; and proteinuria became severe and reached a plateau by day 5. These results demonstrate that anti-GBM nephritis can even be induced by an isologous monoclonal antibody and that the rat IgG2a subclass is at least nephritogenic. The experimental model of anti-GBM nephritis with isologous monoclonal antibodies makes it possible and easier to analyse further the mechanism of anti-GBM nephritis.

Properties of bovine nephritogenic antigen that induces anti-GBM nephritis in rats and its similarity to the Goodpasture antigen

The nephritogenic antigen that induces antiglomerular basement membrane antibody-induced glomerulonephritis (anti-GBM nephritis) in rats was isolated from collagenase-solubilized bovine renal basement membranes. Purification was achieved using antibody-coupled affinity columns which were originally used for the purification of trypsin-solubilized nephritogenic antigen (Sado et al. 1984a). The nephritogenic antigen was a heteropolymer composed of P2 (Mr 28 kDa) and P3 (Mr 30 kDa) polypeptides as monomers and their dimers in sodium-dodecyl-sulfate (SDS) polyacrylamide gel electrophoresis. The P3 polypeptide was considered to be the nephritogenic epitope, since a fraction composed of the P2 polypeptide alone was not nephritogenic. The properties of the nephritogenic epitope were the same as those of the Goodpasture epitope (M2*), which is a noncollagenous domain of the alpha 3 chain of type IV collagen (Butkowski et al. 1985; Saus et al. 1988), indicating that the nephritogenic antigen is the same as the Goodpasture antigen.

Transfer of anti-glomerular basement membrane antibody-induced glomerulonephritis in inbred rats with isologous antibodies from the urine of nephritic rats.

Anti-glomerular basement membrane antibody-induced glomerulonephritis (anti-GBM nephritis) was transferred from nephritic rats to normal recipient rats with isologous antibodies obtained from the urine of the nephritic rats. The original actively-immunized anti-GBM nephritis was induced in inbred WKY/NCrj rats by injecting the nephritogenic antigen from bovine renal basement membranes. Excreted urinary antibodies were collected, purified, and then injected into recipient rats of the same strain. Haematuria and proteinuria appeared on day 2 and day 3, respectively; both became heavier and reached a plateau by day 5. Endocapillary hypercellularity of mononuclear cells in glomeruli was the first histological change, which was observed from day 2, and later extracapillary changes such as fibrin deposition, capsular adhesion, and crescent formation were observed. These histological changes were the same as those seen in the actively-immunized nephritis. The results demonstrate that anti-GBM nephritis is clearly induced by autoantibodies. This new passively-immunized model of anti-GBM nephritis makes it possible and easier to analyse further the mechanism of anti-GBM nephritis because only isologous antibodies are used. This study also indicates that the urine of the nephritic rat is a good source of autoantibodies.

Glomerular macrophages express augmented procoagulant activity in experimental fibrin-related glomerulonephritis in rabbits.

Glomerular fibrin deposition and augmentation of procoagulant activity (PCA) are dependent on glomerular macrophage infiltration in anti-glomerular basement membrane antibody-induced glomerulonephritis (anti-GBM GN) in rabbits. Expression of PCA on the surface of glomerular macrophages and/or augmentation of intrinsic glomerular cell PCA by macrophage cytokines (such as IL 1) are potential mechanisms by which macrophages may augment glomerular PCA. Macrophages were isolated from glomeruli of rabbits developing anti-GBM GN to measure their PCA expression. These macrophages were characterized by morphological and functional criteria. Glomerular macrophages expressed markedly augmented PCA (2.8 +/- 0.7 mU/10(3) cells) compared with blood monocytes (0.05 +/- 0.02 mU/10(3) cells) and alveolar macrophages (0.09 +/- 0.02 mU/10(3) cells) from the same rabbits. Glomerular macrophage PCA was functionally identical to the PCA of whole glomeruli, and was consistent with that of tissue factor. Supernatants from nephritic glomeruli contained IL 1 bioactivity and augmented endothelial cell PCA in vitro. However, these supernatants and purified IL 1 failed to augment the PCA of normal and macrophage-depleted nephritic glomeruli. These studies demonstrate that, in this model of anti-GBM GN, glomerular macrophages contribute directly to the augmented glomerular PCA by their expression of surface membrane PCA, and have the potential to indirectly augment glomerular PCA by their production of cytokines capable of enhancing endothelial cell PCA.

The influence of concomitant experimental hypertension and glomerulonephritis

Utilizing the two kidney-one clip Goldblatt model (CH), we have examined the influence of hypertension upon the course of antiglomerular basement membrane antibody-induced glomerulonephritis (GN) over a period of 10 to 14 weeks after induction of CH and GN, utilizing evaluations in the awake rat by renal micropuncture of the unclipped kidney and morphologic analysis (control [C], GN, CH, and GN+CH). Metabolic studies revealed that GN and GN+CH rats developed proteinuria, elevations in serum creatinine and blood pressure (P less than 0.05). GN+CH resulted in significant reductions in two kidney glomerular filtration rate (GFR) below values in C, and GFR in the unclipped kidney was markedly reduced when compared to CH. The glomerular capillary hydrostatic pressure and the pressure gradient( (delta P) were elevated approximately 4 to 5 mm Hg in GN and CH rats and 6 mm Hg above GN and CH values in the GN+CH rats. The glomerular ultrafiltration coefficient (LpA) was reduced to approximately 40% of C in both GN and GN+CH in spite of further increases in delta P. Surface nephron filtration rate was similar in all groups, suggesting glomerular dysfunction was primarily in nephrons below the surface in GN+CH. Morphologic evaluation revealed that GN+CH exhibited a combination of diffuse and focally sclerotic lesions characteristic of GN and CH, respectively. Documented further increases in PG did not worsen glomerular morphology, but kidney GFR did decrease. Hypertension induced deterioration of glomerular function in nephrons below the surface was not associated with marked morphologic deterioration, but a combination of the characteristics of GN and CH.

Lymphokine (MIF) production by glomerular T-lymphocytes in experimental glomerulonephritis

Glomerular T-lymphocyte infiltration has recently been demonstrated to precede glomerular macrophage influx in a pre-immunized model of anti-glomerular basement-membrane antibody-induced glomerulonephritis (antiGBM-GN). In the current study, the functional role of these glomerular T-lymphocytes in directing macrophage localization was sought by measuring their production of macrophage migration inhibition factor (MIF). MIF activity in supernatants from cultured isolated glomeruli was measured in a conventional capillary tube bioassay. Glomerular T-lymphocytes (OX19 positive cells) were maximal (1.95 +/- 0.19 cells/glomerular cross section, c/gcs) 24 hours after injection of antiGBM antibody into sensitized animals. Seventy-two hours after antibody injection, T-lymphocyte numbers were reduced (1.02 +/- 0.14 c/gcs) while macrophage accumulation was maximal (at 24 hrs 4.2 +/- 1.3 macrophages/glomerulus (m/g), at 72 hrs 19.8 +/- 3.7 m/g). MIF activity was only detected in supernatants from T-lymphocyte infiltrated glomeruli (12 hrs 40.81 +/- 4.32% migration inhibition, 24 hrs 45.11 +/- 4.11% migration inhibition, 48 hrs 38.24 +/- 3.53% migration inhibition, 72 hrs 20.86 +/- 3.85% migration inhibition, all P less than 0.05). Control glomeruli from normal animals, pre-immunized animals given normal sheep globulin, pre-immunized animals given anti-GBM antibody and Cyclosporin A, and non-pre-immunized animals given antiGBM antibody did not contain glomerular T-lymphocytes, and their supernatants contained no MIF activity. This data indicates that the glomerular T-lymphocytes in pre-immunized antiGBM-GN are sensitized cells which release MIF and thus may direct glomerular macrophage localization in this model of antibody-induced glomerulonephritis.