Mediators Of Glomerular Injury Research Articles

Pathogenic Role of Effector Cells and Immunoglobulins in Cationic Bovine Serum Albumin-Induced Membranous Nephropathy

Membranous nephropathy (MN) is an autoimmune-mediated glomerulonephritis. The roles of effector cells and immunoglobulins (Igs) in the mediation of glomerular injury in MN have not been fully elucidated. MN was induced by cationic bovine serum albumin (cBSA), and passive disease was induced by transferring effector cells or serum into severe combined immunodeficient (SCID) mice. MN could not be induced in SCID mice. Transfer of serum from MN mice, but not from normal control mice, to SCID mice induced granular immune complex deposits and pathologic proteinuria. Increased immunofluorescent staining for complement, oxidative stress, terminal deoxynucleotidyl transferase-mediated nick end-labeling assay-positive cells, and augmented phospho-NF-κB staining were evident in the kidneys of MN serum recipients. However, no histological or clinical manifestations were exhibited by SCID mice that received an adoptive transfer of splenocytes. Adaptive immunity was essential for the development of MN. Specific Igs and their subsequent response contribute to the development of renal injury in cBSA-induced MN.

C3 and C4 gene expression and interferon-gamma-mediated regulation in human glomerular mesangial cells.

The glomerular mesangial cell (GMC) plays a key role in the maintenance of glomerular structure and function and in the mediation of glomerular injury. To explore the potential of this cell to produce complement and react to local inflammatory signals, we studied the synthesis and regulation of the third and fourth components of complement in cultured human GMC. Using metabolic labelling and immunoprecipitation, we found that C3 and C4 polypeptide chains were synthesized and secreted by GMC. Interferon-gamma (IFN-gamma) led to an increase in C4 protein synthesis, but not C3 synthesis. There was a corresponding increase in C4 mRNA in IFN-gamma-activated cells, but no increase in C3 mRNA, as determined by semi-quantitative polymerase chain reaction (PCR) estimation. These results demonstrate that human GMC can synthesize C3 and C4 proteins, and that regulation of expression of the C4 gene is mediated by IFN-gamma. We hypothesize that GMC production of complement could influence the clearance of immune aggregates by the kidney and the mediation of glomerular injury.

Prostaglandin E1 suppresses macrophage infiltration and ameliorates injury in an experimental model of macrophage-dependent glomerulonephritis

Prostaglandin E1 (PGE1) suppresses macrophage infiltration and ameliorates injury in an experimental model of macrophage dependent glomerulonephritis. Macrophages are mediators of glomerular injury in models of proliferative glomerulonephritis. We have recently shown that macrophages in glomerulonephritis have low prostaglandin E2 (PGE) generation, and other evidence implicates eicosanoids as regulators of macrophage activation. Here we have studied in rats the effect of 15(s)-15-methyl PGE1 (M-PGE1) on accelerated nephrotoxic nephritis, a model of acute macrophage-dependent glomerular injury. M-PGE1 ameliorated proteinuria (day 4; 61 +/- 13 mg/24 h, n = 9; vehicle treated, 164 +/- 17 mg/24 h, n = 11; P less than 0.002) and glomerular hypercellularity; quantification of infiltrating macrophages by isolating glomerular cells showed reduction in the numbers of macrophages (44 +/- 9/glomerulus; vehicle treated, 119 +/- 15/glomerulus; P less than 0.02) with inhibition of Ia antigen expression on infiltrating macrophages (8 +/- 5%; vehicle treated 25 +/- 4% P less than 0.05). Glomerular binding of nephrotoxic globulin and levels of autologous antibodies were not affected by M-PGE1. Thus the mechanism of suppression involves inhibition of macrophage accumulation and activation. M-PGE1 administered to normal rats did not affect numbers of resident leucocytes (12.6 +/- 1.5/glomerulus; vehicle treated, 13.2 +/- 1.3/glomerulus) or alter Ia antigen expression (4.1 +/- 0.2 Ia + cells/glomerulus; vehicle treated, 3.9 +/- 0.6/glomerulus). This study suggests a therapeutic role for PGE1 in this type of glomerulonephritis, and has implications for the pathophysiology of macrophage-mediated inflammation.

Interaction of native and oxidized lipoprotein(a) with human mesangial cells and matrix

The trapping of apolipoprotein(a) and apolipoprotein B-100 in glomeruli of patients with the nephrotic syndrome seems to be linked to a less favorable course of renal disease. To evaluate the potential role of lipoprotein(a) as a mediator of glomerular injury, we measured uptake of native lipoprotein(a) [Lp(a)] and oxidatively modified Lp(a) by cultured human mesangial cells and matrix and studied the effects of Lp(a) on mesangial cell DNA-synthesis and cellular proliferation. Uptake of Lp(a) by mesangial cells occurred at a significantly lower affinity (KD 16 micrograms/ml vs. 39 micrograms/ml) and a lower maximum degradative capacity (6.7-fold) than for LDL. Specificity of receptor mediated uptake was 50% for Lp(a) compared to 84% for LDL. Oxidative modification of both Lp(a) and LDL was accompanied by a significant decrease in uptake and degradative capacities. Due to the limited uptake, native and oxidatively modified Lp(a) had only marginal effects on intracellular cholesterol metabolism, which was measured as inhibition of sterol synthesis and stimulation of cholesterol esterification. However, binding of Lp(a), oxidized Lp(a) and oxidized LDL to extracellular mesangial matrix was enhanced compared to LDL. Furthermore, incubation of mesangial cells with Lp(a) and oxLp(a) in concentrations of 2.5 micrograms/ml and higher resulted in a decrease of DNA synthesis. Regardless of the oxidative status, a maximal suppression of DNA synthesis was observed at 20 micrograms/ml Lp(a). Native Lp(a) also blunted the stimulatory effects of PDGF on mesangial cell DNA-synthesis. Lp(a) did not alter basal TGF-beta transcription in human mesangial cells. The avid interaction of Lp(a) and modified lipoproteins with mesangial matrix provides a concept for the enhanced entrapment of these lipoproteins in the diseased glomerulum. Native Lp(a) is a poor ligand for the LDL receptor; oxidation of Lp(a) even lowers the affinity towards this receptor. Further studies must be carried out to clarify the pathophysiological significance of Lp(a) trapping in the mesangial matrix.

Human chronic kidney allograft rejection is accompanied by increased intraglomerular cathepsin B and L activity.

The major reason for late graft losses is chronic rejection. Recently, a large number of studies have indicated that proteolytic enzymes play an important role as mediators of glomerular injury. The cysteine proteinases cathepsins B and L degrade structural matrix proteins such as type I collagen and laminin. We investigated intraglomerular protease activities in 12 patients after kidney graftectomy because of end-stage renal disease following chronic rejection. A group of 12 patients undergoing nephrectomy because of cancer served as controls using only non-involved parts of the kidney. The activities of cathepsins B and L in homogenates of isolated glomeruli were measured fluorometrically methylcoumarylamide substrates and related to DNA content. In rejected kidney allografts we observed significantly enhanced intraglomerular cathepsin B activity and cathepsin B + L activity.

Elevated Levels of Tumor Necrosis Factor-α in the Nephrotic Syndrome in Humans

To investigate the possible role of cytokines in the mediation of glomerular injury in the nephrotic syndrome, the levels of interleukin (IL)-1 beta, IL-2, interferon (IFN)-alpha, IFN-gamma, and tumor necrosis factor-alpha (TNF-alpha) were measured in patients with primary nephrotic syndrome. These patients had minimal change nephropathy (MCN), focal and segmental glomerulosclerosis (FSGS), or membranous nephropathy (MN) on biopsy. Cytokine levels were assessed by immunoradiometric assays, and specimens consisted of plasma, urine, and the culture supernate of mitogen-stimulated peripheral blood mononuclear cells (PBMC). Only TNF-alpha was found to be significantly elevated, in the plasma and urine of patients with FSGS and MN, above that found in healthy control subjects and patients with MCN. The elevation of TNF-alpha could not be shown to correlate with the length or severity of the nephrotic syndrome or with loss of body mass. IL-1 beta, IL-2, IFN-alpha, and IFN-gamma levels were not elevated. In culture, mitogen-stimulated PBMC from all three groups of nephrotic subjects released an excess of TNF-alpha compared with controls, a response not consistently observed for the other cytokines measured. The findings of this survey of cytokine levels in nephrotic patients support the possibility that TNF-alpha may play a pathogenic role in the induction or maintenance of glomerular barrier dysfunction in humans.

Immunopathological aspects of immunoglobulin A nephropathy and other mesangial proliferative glomerulonephritides.

Immunoglobulin A nephropathy (IgAN) is an immune complex (IC) glomerulonephritis (GN) that represents one of the most common forms of primary glomerular disease. Proliferation of mesangial cells and the increase of mesangial matrix are histological hallmarks of mesangioproliferative GN. Increased serum levels of IgA, polymeric IgA, IgA rheumatoid factor, IgA-IC, and spontaneous or pokeweed mitogen-induced production of IgA by peripheral blood mononuclear cells are major humoral immune alterations reported in IgAN. Recently, we focused on the role of cytokines and growth factors in the mediation of glomerular injury. Platelet-derived growth factor, transforming growth factor beta, interleukin (IL)-1 and IL-6 are expressed by and act on mesangial cells. Increased expression of platelet-derived growth factor was found in both an active model of IgAN and in renal biopsies of patients with proliferative GN. A strict correlation between increased expression of B-chain mRNA and mesangial proliferation was found. Cytokines such as IL-1, interferon gamma, and IL-6, released by infiltrating mononuclear cells or produced locally by mesangial cells, affect the glomerular response to IgA-IC. In a passive murine experimental model of IgAN, IL-1 and interferon gamma increased mesangial hypercellularity, whereas IL-6 was highly pathogenic when associated to IL-1. In conclusion, classical immunological mechanisms in mesangial GN could interact with other pathways involving cytokines and growth factors in the progression of glomerular injury.

The role of T cells in the mediation of glomerular injury in Heymann's nephritis in the rat.

Heymann's nephritis (HN), a rat model of the membranous glomerulonephritis in man, is thought to be mediated by auto-Ig with subsequent activation of C. Whether T cell mechanisms are involved in the mediation of HN, apart from CD4+ cells providing help for auto-Ig production, was examined by treatment with mAb specific for T cell subsets for 6 weeks after immunization to induce HN. Anti-CD4 mAb therapy totally prevented proteinuria, in that at 6, 8, and 12 week treated rats had less than 15 mg/day of protein compared to controls that all had greater than 260 mg/day. Ig and C deposition in the glomerulus was significantly less and auto-Ig titers in serum were partially suppressed by anti-CD4 therapy. Anti-CD8 mAb therapy markedly reduced proteinuria at all time points, for example at 6 weeks there was 51 +/- 40 mg/day compared to 183 +/- 120 mg/day (P = 0.0003), but had no effect on auto-Ig titers or on Ig and C deposition in the glomerulus. A non-specific effect of high dose mouse mAb therapy was excluded by the findings that a mAb that did not bind to rat cells had no effect on the induction of HN and that serum C was not depleted in any of the mAb treated animals. A role for T effector mechanisms was further supported by the finding that therapy with mAb to T cell receptor alpha/beta chain or with cyclosporine also markedly delayed the onset of proteinuria. Examination of renal biopsies showed a T cell infiltrate in glomeruli and the interstitium of the untreated HN controls that was not present in MRC Ox35 or MRC Ox8 treated groups. This infiltrate included CD4+ and CD8+ T cells and macrophages. These results suggest induction of proteinuria in HN was totally dependent upon CD4+ T cells, and that CD4+ and CD8+ cells may have a direct role in the mediation of glomerular dysfunction in HN.

Proteolytic enzymes as mediators of glomerular injury

Despite significant advances in our understanding of the pathogenesis of glomerular injury, the specific mechanisms responsible for the pathological changes in glomerular structure and function which occur in glomerular disease remain poorly understood. Several diverse lines of evidence, including studies with purified proteinases, studies with freshly isolated glomeruli and intact cells, and cells in culture, as well as recent in vivo studies indicate that proteolytic enzymes play important roles as mediators of glomerular injury. The concept of proteolytic enzymes as mediators of glomerular injury is not new. The ability of polymorphonuclear leukocyte (PMN)-derived proteinases to degrade glomerular basement membrane (GBM) in vitro coupled with the PMN-dependence of certain types of glomerulonephritis led Cochrane and coworkers in the mid-sixties to postulate that neutrophil-derived proteinases cause glomerular injury by proteolysis of the GBM [1–3]. Since then, a large number of studies have provided support for this hypothesis. In addition, recent studies documenting the presence of GBM-degrading proteinases in glomeruli, plasma, and a variety of other circulating cells indicate that GBM damage can also be mediated by proteinases originating from these sources as well. Thus proteinase-mediated GBM damage may be an important mechanism of glomerular injury in leukocyte-independent as well as leukocyte-dependent glomerular diseases. Furthermore, recent studies suggest that proteolytic enzymes may contribute to glomerular injury by mechanisms independent of GBM damage. Such mechanisms include: proteolysis of non-GBM proteins; proteinase-mediated alterations in the production of key glomerular metabolites; and proteinase-induced effects on cellular proliferation. In the present review, we attempt to bring together a large body of diverse information derived from in vitro and in vivo studies, which supports a pathogenic role for proteolytic enzymes in glomerular injury.

Role of eicosanoids as mediators of glomerular injury in immune-mediated nephropathies.

Role of eicosanoids as mediators of glomerular injury in immune-mediated nephropathies.

Leukotrienes and Reactive Oxygen Species as Mediators of Glomerular Injury

Leukotrienes and Reactive Oxygen Species as Mediators of Glomerular Injury

Immune mechanisms and molecular mediators of glomerular injury in experimental nephritis. Summary of current results and continuing studies.

Despite a growing body of evidence on the importance of macrophage accumulation in renal glomeruli to the subsequent injury in experimental immune nephritis, exact mechanisms of the monocyte/macrophage recruitment and phenomena leading to tissue damage are not completely clarified. Our experiments indicate that neutral proteinases released in situ by activated macrophages may, at least in part, be responsible for the development of autoimmune nephrotoxic glomerulonephritis in rats. Accordingly, apart from the well-established features of glomerular hypercellularity and basement membrane damage, a new mechanism of direct cytolysis by macrophage-derived proteinases is postulated to be of potential relevance to the nephritic glomerular injury.

Platelet cationic proteins are present in glomeruli of lupus nephritis patients

Synthetic polycations have been shown to bind and neutralize glomerular polyanions (GPA), thereby increasing the permeability of the glomerular capillary wall (GCW). In the present study it is demonstrated that human platelet-derived cationic proteins (HuPlt CP), which are able to increase cutaneous vascular permeability, bind in vitro to the GCW following incubation of normal human kidney sections with purified HuPlt CP or with washed human platelets stimulated with thrombin, immune complexes (IC) and platelet-activating factor (PAF), or stimulated with a suspension of washed human platelets and polymorphonuclear leukocytes in the presence of phagocytable substrate. The antiserum used in immunofluorescence test to detect the binding of HuPlt CP was specific for two different molecular types of HuPlt CP, both with an isoelectric point (pI) of 10.5. Glomerular deposits of HuPlt CP were also detectable by immunofluorescence microscopy in renal glomeruli present in tissue obtained by biopsy from patients with systemic lupus erythematosus (SLE), a disease in which platelets have been implicated as mediator of glomerular injury. These data indicate that when activated platelets release HuPlt CP in vivo, these proteins bind to glomerular structures. The binding of HuPlt CP to GCW appears to be ionic in nature since heparin, a polyanion, prevents this binding in vitro. In addition, heparin, as well as a high molarity buffer, removed deposits of HuPlt CP bound in vitro to normal GCW or bound in vivo to glomeruli of patients with SLE. The binding of HuPlt CP to GCW is associated with loss of colloidal iron staining, a qualitative technique that demonstrates primarily epithelial cell surface anionic sialoglycoproteins. In experiments of in vitro binding of purified HuPlt CP to section of normal kidney treatment with heparin completely restores the normal pattern of colloidal iron staining suggesting ionic neutralization of GPA. In contrast, heparin is only partially effective in restoring colloidal iron staining in normal kidney sections treated with platelets directly stimulated with IC or PAF or in kidney sections of patients with SLE. These observations indicate that under these conditions the ionic interaction of HuPlt CP with GCW is only partially responsible for the loss of colloidal iron staining. The results of the present study suggest that biologically active polycationic mediators released from stimulated platelets localize in GCW and participate in the induction of glomerular injury.

Cellular immune mechanisms in human glomerulonephritis: the role of mononuclear leucocytes.

Our understanding of the pathologic processes leading to injury in human glomerulonephritis remains limited. Several mediation pathways have been suggested from observations of human biopsies and studies of experimental glomerulonephritis in animals, although the pathogenic mechanism of injury in many situations is obscure. However cells of the monocyte/macrophages series can be identified within glomeruli in glomerulonephritis, especially in more aggressive forms of the proliferative disease in humans. Experimental studies have confirmed the capacity of these cells to induce injury; thus, the role of monocytes as a mediator of glomerular injury is now established. Indirect evidence suggests that T cell-dependent mechanisms may also be involved in glomerulonephritis, but more data are needed to define the extent to which sensitised mononuclear leucocytes participate in the pathogenesis of human glomerulonephritis.

Abrogation of macrophage-dependent injury in experimental glomerulonephritis in the rabbit. Use of an antimacrophage serum.

Macrophages were shown by the use of glomerular cell culture and morphologic techniques to be present in large numbers within the glomeruli of rabbits with acute serum sickness (AcSS) and in a passive model of the autologous phase of antiglomerular basement membrane (GBM) antibody-induced glomerulonephritis (PAGBMN). To determine the part played by these cells in the glomerular injury, animals were treated with a sheep anti-rabbit macrophage serum (AMS) or normal sheep serum (NSS). NSS administration had no effect on the development of either model of glomerulonephritis. The use of AMS reduced the number of circulating monocytes and prevented the accumulation of macrophages within glomeruli in both models (AcSS/NSS, mean 126/glomerulus, range 40-251; AcSS/AMS, mean 8, range 1-44; PAGBMN/NSS, mean 52, range 27-69; PAGBMN/AMS, mean 5, range 2-7). The AMS-treated rabbits had only minor histologic lesion and profound reduction in proteinuria (AcSS/NSS, mean 516 mg/24 h, range 200-991; AcSS/AMS, mean 41, range 3-161; PAGBMN/NSS, mean 335, range 55-975; PAGBMN/AMS, mean 10, range 2-24). Similar studies in the heterologous phase of glomerular injury induced by the same anti-GBM antibody revealed no effect of the AMS on this polymorphonuclear leukocyte-related phase of injury, demonstrating the selectivity of the antisera. Complement depletion, with cobra venom factor, did not affect the development of glomerulonephritis nor the accumulation of macrophages in either model. Inhibition of macrophage accumulation can largely prevent these forms of experimental glomerulonephritis, thereby implicating macrophages as mediators of glomerular injury and consequent proteinuria.

Renal Vein Thrombosis

To the Editor.— Nephrotic patients with membranous glomerulonephritis have a very high incidence of renal vein thrombosis, as high as 40%. 1 The patient reported by Luft et al (234: 1158,1975) had this type of renal disease, and he also had another important predisposing factor for the development thrombosis—his extreme obesity. Obese patients have a deficient fibrinolytic mechanism 2,3 that correlates with the degree of overweight. 4 Obesity has recently been described in association with large amounts of fibrin in the glomeruli, mesangial IgM, C3, and a form of nephrotic syndrome that is reversible with weight reduction. 5 It is conceivable that fibrin deposition in the glomeruli could limit the phagocytic action of the mesangial cells reducing their efficiency in the removal of immunocomplexes and other mediators of glomerular injury. I share the view that nephrotic patients with massive obesity, as well as those with membranous glomerulonephritis, should be carefully