C5b-9 increases albumin permeability of isolated glomeruli in vitro

C5b-9 increases albumin permeability of isolated glomeruli in vitro. Deposition of antibody and activation of the complement cascade are important in both naturally occurring glomerulonephritis and in experimental models including passive Heymann nephritis. We studied the effect of antibody and complement on albumin permeability of isolated glomeruli to determine the role of the terminal complement components (C5-C9) in mediating the proteinuria in nephritis. Isolated glomeruli were treated with anti-Fx1a (Heymann antibody) and then incubated them with pooled human serum, serum in which complement had been inactivated by heat, or serum deficient in C6 or C7. The albumin reflection coefficient (σalbumin) was calculated from the volumetric response of glomeruli to transcapillary oncotic gradients produced by albumin or high molecular weight neutral dextran (252 kD). Convectional permeability to albumin (Palbumin) was calculated as 1-σalbumin. Albumin permeability of control glomeruli was not different from 0. Albumin permeability was not altered by antibody alone but was increased to 0.65 ± 0.04 when antibody treated glomeruli were incubated for 10 minutes with pooled serum as a source of complement. Heat treatment of serum to inactivate complement prevented the increase in permeability. Incubation for 10 minutes with serum without antibody pretreatment caused a lesser increase in permeability of isolated glomeruli (0.18 ± 0.06). Serum deficient in either C6 or C7 did not cause an increase in albumin permeability of antibody pre-treated glomeruli, but incubation with a combination of these sera (now containing the complete cascade) increased permeability to the same extent as did pooled normal serum (0.58 ± 0.04). We conclude that activation of the terminal complement components is required for the increase in glomerular macromolecular permeability caused by anti-Fx1a and that terminal complement activation is sufficient to alter the permeability independent of complement hemodynamic events or contribution by circulating cells

Detection of terminal complement components in experimental immune glomerular injury

Detection of terminal complement components in experimental immune glomerular injury. Complement mediates glomerulonephritis by inflammatory cell-dependent and non-inflammatory cell-independent effects on glomerular permeability. The latter may involve terminal components of the complement system. We examined several models of immunologic renal injury in the rat by immunofluorescence (IF) for terminal complement components C5, C6, C7, and C8 in glomeruli using antisera to human C5-8, which cross-react with the analogous rat complement components. Rats with the heterologous and autologous phases of passive Heymann nephritis (PHN) had proteinuria and 1 to 2+ capillary wall deposits of heterologous or rat IgG, rat C3, and C5-8. Complement depletion with cobra venom factor (CVF) significantly decreased proteinuria in both models and prevented deposition of all complement components. Rats with active Heymann nephritis had similar deposits of rat IgG and C5-8. Rats with anti-GBM nephritis and aminonucleoside nephrosis had severe proteinuria which was not affected by CVF treatment and deposits of C5-8 were absent. The presence of terminal complement components in immune deposits in experimental glomerular disease correlates with a functional role for complement in mediating glomerular injury. These data support the hypothesis that the terminal complement pathway may be a major mediator of some types of immune glomerular injury.Détection des constituants terminaux du complément au cours de lésions immunes glomérulaires expérimentales. Le complément est le médiateur d'une glomérulonéphrite par des effets inflammatoires cellule-dépendants, et non inflammatoires cellule-indépendants sur la perméabilité glomérulaire. Ces derniers pourraient mettre en jeu les constituants terminaux du système complémentaire. Nous avons examiné plusieurs modèles de lésions rénales immunologiques chez le rat par la immunofluorescence (IF) en ce qui concerne les constituants complémentaires terminaux C5, C6, C7, et C8 dans les glomérules en utilisant des antisérums contre C5-8 humain qui croisent avec les constituants complémentaires analogues du rat. Des rats dans les phases hétérologue et autologue d'une néphrite passive de Heymann (PHN) avaient une proléinurie et des dépôts sur les parois capillaires 1 à 2 + d'IgG hétérologue ou de rat, de C3 et de C5-8 de rat. Une déplétion complémentaire avec du facteur de venin de cobra (CVF) a diminué significativement la protéinurie dans les deux modèles et a prévenu le dépôt de tous les constituants complémentaires. Des rats atteints d'une nephrite active de Heymann avaient des dépôts identiques d'IgG et de C5-8 de rat. Des rats atteints de néphrite anti-GBM et de néphrose aux aminoglucosides avaient une protéinurie sévère non affectée par le traitement au CVF et les dépôts de C5-8 étaient absents. La présence des constituants terminaux de complément dans les dépôts immuns lors des glomérulopathies expérimentales est corrélée avec un rôle fonctionnel du complément dans la médiation des lésions glomérulaires. Ces données sont en faveur de l'hypothèse que la voie terminale du complément peut être un médiateur majeur de certains types de lésions glomérulaires immunes

Heparin suppresses mesangial cell proliferation and matrix expansion in experimental mesangioproliferative glomerulonephritis

Heparin suppresses mesangial cell proliferation and matrix expansion in experimental mesangioproliferative glomerulonephritis. Proliferation and extracellular matrix (ECM) overproduction by glomerular mesangial cells characterizes many types of glomerulonephritis and often precedes the development of glomerulosclerosis. Heparin is a potent inhibitor of mesangial cell growth in vitro. We examined whether standard heparin can inhibit mesangial cell proliferation in vivo in the mesangioproliferative anti-Thy 1.1 nephritis. Untreated control rats were compared to rats infused with heparin either early (day -2 to 1) or late (day 2 to 5) after induction of anti-Thy 1.1 nephritis. The results show that heparin treatment significantly reduced mesangial cell proliferation regardless of when it was initiated. Heparin (either early or late treatment) also reduced mesangial basic fibroblast growth factor (bFGF) expression and platelet-derived growth factor (PDGF) receptor up-regulation as reflected by immunostaining, whereas PDGF B-chain expression was reduced only by late heparin treatment. Furthermore, heparin treatment markedly inhibited the mesangial matrix expansion for a variety of ECM proteins, including laminin, type I and IV collagen, fibronectin and entactin. Heparin did not affect the initial mesangiolysis, glomerular macrophage influx, deposition of anti-Thy 1.1 IgG or fibrinogen, or the glomerular platelet influx. These results suggest that heparin, via its antiproliferative rather than anticoagulant effect, can inhibit mesangial cell proliferation, overexpression of polypeptide growth factors, and ECM protein overproduction in vivo. The beneficial effect of heparin can be demonstrated even if treatment is initiated after the development of nephritis. By virtue of these properties, heparin may be an effective agent in the treatment of human mesangioproliferative disease and in the prevention of glomerulosclerosis

Thrombospondin 1 precedes and predicts the development of tubulointerstitial fibrosis in glomerular disease in the rat

Thrombospondin 1 precedes and predicts the development of tubulointerstitial fibrosis in glomerular disease in the rat. Tubulointerstitial fibrosis is one of the most important histologic features that predicts progression in kidney disease. Thrombospondin 1 is an extracellular matrix protein that can activate latent TGF-β, a cytokine implicated in the pathogenesis of tubulointerstitial fibrosis. We examined the expression of thrombospondin 1 in several animal models of glomerulonephritis (anti-Thy1 model, aminonucleoside nephrosis, passive Heymann nephritis) that are associated with tubulointerstitial disease. Thrombospondin 1 mRNA and protein were transiently increased in tubular cells, myofibroblasts and some macrophages in areas of tubulointerstitial injury. Thrombospondin 1 expression always preceded the development of tubulointerstitial fibrosis, and correlated quantitatively and spatially with the later development of interstitial fibrosis. Thrombospondin 1 expression predicted the severity of tubulointerstitial fibrosis better than the degree of macrophage or myofibroblast accumulation. Thrombospondin 1 expression was associated with increased expression and activation of TGF-β1 and decreased expression of LAP-TGF-β in areas of tubulointerstitial injury. We conclude that thrombospondin 1 is an early marker predicting the development of tubulointerstitial kidney disease. De novo expression of thrombospondin 1 is associated and colocalized with increased expression of TGF-β1 and decreased expression of LAP-TGF-β during the development of tubulointerstitial disease in vivo. These data are consistent with the possibility that thrombospondin 1 may be an endogenous activator of TGF-β

Altered glomerular permeability induced by F(ab′)2 and Fab′ antibodies to rat renal tubular epithelial antigen

Altered glomerular permeability induced by F(ab′)2 and Fab′ antibodies to rat renal tubular epithelial antigen. Rats injected with F(ab′)2 and Fab′ antibody fragments directed against an antigen in the rat proximal tubular epithelial brushborder (Fx1A) developed immediate proteinuria [F(ab′)2 43.2 ± 6.7, N = 6; Fab′ 9.5 ± 2.8, N = 5; normal 1.6 ± 0.9 mg/day, N = 20]), that subsided after 3 to 5 days' duration. This reaction is in contrast to one exhibited by rats given intact IgG anti-Fx1A; the rats that did not develop immediate proteinuria (2.2 ± 0.3 mg/day, N = 5), and the glomerular binding of125I-antibody fragments was significantly less than that of intact IgG [F(ab′)2 0.11 ± 0.01; Fab′ 0.03 ± 0.01; IgG 0.17 ± 0.01% administered equimolar dose] at 24 hr. No proteinuria resulted from equimolar doses of nonantibody F(ab′)2 and Fab′. Less than 8% of the proteinuria induced by antibody fragments represented injected material, and 30 to 38% was albumin. Immunofluorescence revealed faint and diffuse glomerular capillary wall deposits of F(ab′)2 and Fab′ and tubular brushborder staining. Subepithelial, electrondense deposits and focal, podocyte effacement were seen by electron microscopy in rats given the F(ab′)2 antibody. Light microscopy and colloidal iron-staining were normal. In our study antibody fragments appear to interact directly with components of the outer, glomerular capillary wall to alter permeability in the absence of recognized mediators such as complement and inflammatory cells.Modification de la perméabilité glomérulaire déterminée par anticorps chez des rats anti-épithélium tubulaire F(ab′)2 et Fab′. Les rats été injectés avec des fragments F(ab′)2 et Fab′ contra anticorps de la bordure en brosse de l'épithélium tubulaire proximal de rat (Fx1A) ont immédiatement une protéinurie [F(ab′)2 43,2 ± 6,7, N = 6; Fab′ 9,5 ± 2,8, N = 5; normaux 1,6 ± 0,9 mg/d, N = 20] qui persiste pendant 3 à 5 jours. Cela réaction est différent de ce qui est observé chez les rats qui reçoivent l'IgG anti-Fx1A intacte; les rats quelles n'ont pas de protéinurie immédiate (2,2 ± 0,3 mg/d, N = 5) quoiqu'à 24 heures la liaison glomérulaire de fragments125I de l'anticorps soit significativement plus faible que celle de I'IgG intacte [F(ab′)2 0,11 ± 0,01; Fab′ 0,03 ± 0,01; IgG 0,17 ± 0,01 en % de la dose équimolaire administrée]. Aucune protéinurie n'a été la conséquence de l'administration équimolaire de F(ab′)2 et Fab′ non-anticorps. Moins de 8% de la protéinurie déterminée par les fragments d'anticorps représentent du matériel injecté et 30 à 38% est de l'albumine. L'immunofluorescence a montré des dépôts faibles et diffus, sur les parois des capillaires giomérulaires, de F(ab′)2 et Fab′ et le marquage de la bordure en brosse. En électronique, des dépôts denses sous-épithéliaux et l'effacement local des podocytes ont été observés chez les rats qui avaient reçu l'anticorps F(ab′)2. La microscopie optique et la coloration par le fer colloïdal n'ont pas montré d'anomalies. Dans notre étude les fragments d'anticorps semblent avoir paroi externe du capillaire glomérulaire et avoir modifié la perméabilité en l'absence de médiateurs connus tels le complément ou les cellules inflammatoires

The cyclin kinase inhibitor p21CIP1/WAF1 limits glomerular epithelial cell proliferation in experimental glomerulonephritis

The cyclin kinase inhibitor p21CIP1/WAF1 limits glomerular epithelial cell proliferation in experimental glomerulonephritis.BackgroundDuring glomerulogenesis, visceral glomerular epithelial cells (VECs) exit the cell cycle and become terminally differentiated and quiescent. In contrast to other resident glomerular cells, VECs undergo little if any proliferation in response to injury. However, the mechanisms for this remain unclear. Cell proliferation is controlled by cell-cycle regulatory proteins where the cyclin-dependent kinase inhibitor p21Cip1,WAF1 (p21) inhibits cell proliferation and is required for differentiation of many nonrenal cell types.MethodsTo test the hypothesis that p21 is required to maintain a differentiated and quiescent VEC phenotype, experimental glomerulonephritis was induced in p21 knockout (-/-) and p21 wild-type (+/+) mice with antiglomerular antibody. DNA synthesis (proliferating cell nuclear antigen, bromodeoxyuridine staining), VEC proliferation (multilayers of cells in Bowman's space), matrix accumulation (periodic acid-Schiff, silver staining), apoptosis (TUNEL), and renal function (serum urea nitrogen) were studied on days 5 and 14 (N = 6 per time point). VECs were identified by location, morphology, ezrin staining, and electron microscopy. VEC differentiation was measured by staining for Wilms’ tumor-1 gene.ResultsKidneys from unmanipulated p21-/- mice were histologically normal and did not have increased DNA synthesis, suggesting that p21 was not required for the induction of VEC terminal differentiation. Proliferating cell nuclear antigen and bromodeoxyuridine staining was increased 4.3- and 3.3-fold, respectively, in p21-/- mice with glomerulonephritis (P < 0.0001 vs. p21+/+ mice). At each time point, VEC proliferation was also increased in nephritic p21-/- mice (P < 0.0001 vs. p21+/+ mice). VEC re-entry into the cell cycle was associated with the loss of Wilms’ tumor-1 gene staining. Nephritic p21-/- mice had increased extracellular matrix protein accumulation and apoptosis and decreased renal function (serum urea nitrogen) compared with p21+/+ mice (P < 0.001).ConclusionThese results show that the cyclin kinase inhibitor p21 is not required by VECs to attain a terminally differentiated VEC phenotype. However, the loss of p21, in disease states, is associated with VEC re-entry into the cell cycle and the development of a dedifferentiated proliferative phenotype

Role of intrinsic renal cells versus infiltrating cells in glomerular crescent formation

Role of intrinsic renal cells versus infiltrating cells in glomerular crescent formation.BackgroundStudies were undertaken to characterize the cellular composition that occurs in glomeruli and the tubulointerstitium of a passive model of complement-independent crescentic nephritis in mice.MethodsGlomerulonephritis was induced by the injection of antibody to whole rabbit glomeruli, and tissue was examined histologically at 7, 14 and 28days.ResultsMice developed proteinuria, glomerular crescents, and progressive glomerulosclerosis and tubulointerstitial fibrosis. The majority of the cells within the crescents appeared to be intrinsic ezrin-positive epithelial cells of visceral or parietal origin. Many of the ezrin positive cells were proliferating and expressing the PDGF receptor. Despite expression of the macrophage adhesive protein, osteopontin, the early crescents were devoid of infiltrating macrophages, T cells or myofibroblasts, which could be explained by the finding that the Bowman's capsule remained intact. Tubulointerstitial damage also occurred, and included tubular dilation and atrophy, periglomerular and patchy interstitial infiltration and interstitial fibrosis with increased interstitial deposition of type IV collagen and laminin. Interstitial infiltrating cells included macrophages, CD4+ T lymphocytes, CD8+ T lymphocytes, and activated myofibroblasts. Tubular osteopontin expression was increased in the areas of tubulointerstitial damage and was associated with interstitial macrophage infiltration.ConclusionsWe describe an experimental model of complement-independent murine crescentic nephritis associated with tubulointerstitial injury. Proliferating glomerular epithelial cells are the main cellular components of the crescents in this model