Intraglomerular platelet aggregation and experimental glomerulonephritis

Oxygen free radical production inhibits ADPase-mediated antithrombotic action. Different forms of experimental glomerulonephritis (GN) are characterized by early glomerular influx of inflammatory cells and thrombus formation. The causal relationship of these inflammatory events is obscure. Previous studies have shown that glomerular ADPase in the rat kidney may function as a potent antithrombotic principle, whereas this enzyme is highly sensitive for oxygen free radicals. To study whether O2- producing inflammatory cells are able to induce intraglomerular thrombosis via impairment of ADPase, we investigated influx of inflammatory cells in relation to glomerular ADPase activity and platelet aggregation in three models of GN. In two of these models (anti-Thy1 and anti-GBM GN) influx of neutrophils and thrombus formation occurs, whereas in anti-FX1A nephritis this aspect of the inflammatory phase is not present. The results show a relationship between influx of oxygen free radical-producing cells, reduction of glomerular ADPase activity and increased platelet aggregation. Moreover, it is shown that impairment of glomerular ADPase and increased platelet aggregation in anti-Thy1 and anti-GBM GN could be reduced by treatment with superoxide dismutase and catalase. The demonstration that activated neutrophils perfused ex vivo in the rat kidney can directly affect glomerular ADPase and antithrombotic potential in an O2- dependent manner, further supports the proposed sequence of events; oxygen free radicals produced by activated neutrophils reduce glomerular ADPase activity, leading to facilitation of thrombus formation.     

The detection of monocytes in human glomerulonephritis

Renal biopsy specimens from 343 patients with primary or secondary glomerulonephritis (GN) were examined for monocytes by the non-specific esterase reaction. Large numbers of monocytes per glomerulus (M/G) were found in essential cryoglobulinemia GN (29 pts, M/G 30.6 +/- 22.4), in acute post-infectious GN (27 pts, M/G 9.1 +/- 8.3), in rapidly progressive crescentic GN (20 pts, M/G 5.6 +/- 2.7), in systemic lupus GN (61 pts, M/G 5.0 +/- 5.6), and in IgA-GN associated with chronic liver disease (5 pts, M/G 6.4 +/- 5.9) or Sch?nlein-Henoch purpura (15 pts, M/G 3.3 +/- 6.4). Clinico-histological correlation showed that monocyte infiltration was correlated with the extent of proteinuria (all groups), with the presence of endoluminal "thrombi" (cryoglobulinemia GN), of polymorphonuclear leukocyte infiltration (post-infectious GN), of cellular crescents (crescentic GN), of "active" lesions (lupus GN), and with the extension of lesions to the peripheral capillary walls (IgA-associated GN). The M/G index was negligible in renal amyloidosis (21 pts), in idiopathic membranoproliferative GN (10 pts), in idiopathic IgA mesangial GN (63 pts), in membranous GN (40 pts), in focal glomerulosclerosis (29 pts), in minimal change nephropathy (18 pts), and in diabetic glomerulosclerosis (5 pts). The results confirm the participation of cells of the monocyte-macrophage series in the genesis of proliferative lesions, both intracapillary and extracapillary, in immune-mediated human GN and suggest their direct involvement in glomerular injury.     

Intraglomerular T cells and monocytes in nephritis: study with monoclonal antibodies

Intraglomerular T cells, monocytes, total leucocytes and other mononuclear subsets were sought in renal biopsies from patients with glomerulonephritis, using monoclonal antibodies and immunoperoxidase techniques. Twenty-four biopsies with no significant glomerular proliferation on optical microscopy, thirty-two with only endocapillary hypercellularity, and twenty-one with extra capillary crescentic glomerular disease were studied. Few intra-glomerular leucocytes were seen in the non-proliferative group. In contrast, when compared with this group, biopsies with glomerular hypercellularity and particularly those with crescents showed increased numbers of intra-glomerular total leucocytes and monocytes/macrophages, as well as an excess of T lymphocytes and T cytotoxic/suppressor cells; T helper/inducer lymphocytes were significantly increased only in the crescentic group. Only small numbers of B lymphocytes and NK cells were found in all groups. The numbers of total glomerular T-cells and monocytes per glomerular cross section were highly correlated in the crescentic group. Only idiopathic IgA nephropathy failed to show a significant increase in the numbers of intra-glomerular leucocytes, in comparison with the non-proliferative group, Henoch-Sch?nlein purpura biopsies in contrast had an excess of both monocytes and T cell subsets. The finding of T lymphocytes as well as monocytes in glomeruli of proliferative nephritis suggests that cellular immune mechanisms may play a role in their pathogenesis, especially when crescents are present.     

Intraglomerular localization of platelet related antigens, platelet factor 4 and beta-thromboglobulin in glomerulonephritis

We examined renal biopsies from 121 patients with various forms of glomerulonephritis, using antisera against platelet membrane antigens, platelet factor 4, beta-thromboglobulin and fibrinogen using the indirect immunofluorescent technique. Eight biopsies were also studied by electron microscopy for recognizable platelets. Thirty-six of sixty-four (56%) biopsies from patients with severe forms of glomerulonephritis showed some intraglomerular platelet antigen, extensive fluorescence in seventeen (25%). Of forty-six patient with milder, usually non-progressive forms of nephritis, twenty (44%) were positive, but only four (9%) showed extensive fluorescence. Platelets were identified by electron microscopy within the capillary lumina equally in those with positive and negative results for platelet-related antigens. There was no correlation between the presence of fibrin and platelet-related material in the glomeruli, nor between the presence or extent of intraglomerular platelet antigen and simultaneous measurements of intraplatelet serotonin in circulating platelets. However, there was a strong correlation between presence, extent and distribution of platelet-related antigen and platelet factor 4 fluorescence. These studies provide further evidence for the involvement of platelets in some forms of glomerular disease.     

Intraglomerular coagulation and fibrinolysis in human primary glomerular diseases]

It is a well known fact that intraglomerular coagulation plays an important role in the development of human primary glomerular diseases. However, the precise mechanism of intraglomerular coagulation, and intraglomerular coagulability and/or fibrinolytic activity remains obscure. The present study was aimed to elucidate the role of the intraglomerular coagulation and fibrinolysis in human primary glomerular diseases. Subjects enrolled in this study were 27 patients with minimal change nephrotic syndrome (MCNS), 14 patients with focal glomerular sclerosis (FGS), 36 patients with membranous nephropathy (MN), 161 patients with mesangial proliferative glomerulonephritis (mesPGN), 9 patients with membranoproliferative glomerulonephritis (MPGN), and 40 healthy volunteers as controls. Normal human renal cortex as controls of isolated intraglomerular plasminogen activator activity (PAA) was obtained at the time of nephrectomy from the normal pole of kidneys removed because of an opposite pole tumor. Urinary urokinase (UK), fibrinopeptide A (FPA) and fibrinopeptide B beta 15-42 (B beta 15-42) antigens were measured by RIA. Urinary tissue plasminogen activator (t-PA) antigen was measured by ELISA. Urinary fibrin/fibrinogen degradation products (FDP) were measured by latex agglutination method. Moreover, PAA was measured by 125I-fibrin films. The following results were obtained: 1) In primary glomerular diseases, levels of urinary UK and t-PA were significantly lower than those in healthy volunteers, 2) Urinary UK and t-PA showed gradual decrease along with the development of mesangial proliferation, 3) Urinary UK and t-PA were significantly correlated with both the urinary FPA and B beta 15-42, 4) In mesPGN and FGS, PAA was significantly lower than that in normal controls, 5) PAA was significantly correlated with urinary UK, t-PA, FPA and B beta 15-42, 6) Urinary UK and t-PA in the patients with urinary FDP were significantly lower than those in patients without urinary FDP, 7) Urinary UK, t-PA and PAA were significantly lower in patients with intraglomerular fibrin deposition than those in patients without fibrin depositions. These findings suggest that the decrease of urinary UK and t-PA levels and the diminution of isolated intraglomerular plasminogen activator activity contribute to the progression of primary glomerular diseases.     

Intraglomerular proteinase activity in adriamycin-induced nephropathy.

Adriamycin (ADR)-induced nephropathy is characterized by focal and segmental glomerulosclerosis and is supposed to be an ideal model of chronic progressive renal disease. The aim of our study was to investigate whether there might be an altered activity of glomerular proteinases in ADR nephropathy, thereby aggravating glomerular protein accumulation as an important initiating hallmark of glomerulosclerosis. In fact, we could demonstrate significantly enhanced levels of intraglomerular protein and DNA content in the experimental animals at week 7, 12 and 22 after administration of ADR. When relating intraglomerular proteinase activity, which was measured in ultrasonically destroyed isolated glomeruli, obtained by differential sieving techniques, to the intraglomerular protein and DNA content, this enzyme activity was significantly reduced throughout the observation period. Based on these data, we suggest that this relatively decreased proteinase activity in glomeruli exposed to ADR might play a pathogenetic role in the development of glomerular hypertrophy, an important harbinger of glomerulosclerosis.     

Activity of circulating monocytes in patients with chronic glomerulonephritis

Monocytes of 95 patients with chronic glomerulonephritis (ch.g.) testedin vitro demonstrated characteristics of activation in proliferative, and of functional suppression in mesangiocapillary glomerulopathy. Fc and C₃ receptor function studied by rosette assay and metabolic potential measured by the NBT reduction test constituted result patterns. Receptor tests were supplemented with their counterparts after monocyte triggering with heat-inactivated sera and in case of NBT assay — stimulation with zymosan. Membranous, minimal change, mesangial and focal glomerulonephritis monocytes presented less specific configurations of data than those of proliferative and mesangiocapillary, with a uniform increase of trypsin-resistant Fc receptor activity. There was no appreciable correlation between the presence of circulating immune complexes (c.i.c.) in patient sera and parameters tested. The mesangiocapillary suppression pattern suggests mononuclear phagocyte defect in this glomerulopathy.     

Intraglomerular embolization by tubular cells

Intraglomerular embolization by tubular epithelial cell is a rare artefact found in needle biopsy specimens. The authors have observed this phenomenon in five out of 300 renal biopsy cases. Tubular epithelial cells, were present in the glomerular capillaries (in two cases) or in the capillaries and within the glomerular urinary space (in three cases).     

Glomerular deposition of mannose-binding lectin in human glomerulonephritis.

BACKGROUND: Mannose-binding lectin (MBL), a member of the collectin family, binds to various oligosaccharides and activates the classical pathway of complement independent from C1q. At present it is unknown whether this so-called lectin pathway of complement activation plays a role in the pathogenesis of human glomerulonephritis. METHODS: Direct immunofluorescence of 84 renal biopsies using an MBL-specific monoclonal antibody and antibodies directed against IgG, IgA, IgM, C1q, C3, and terminal complement complex (TCC) was performed. Serum MBL levels of 50 patients were determined by enzyme-linked immunosorbent assay. RESULTS: MBL was detected in the glomeruli of patients with lupus nephropathy (15 of 16), membranous nephropathy (10/15), membranoproliferative glomerulonephritis type I (5/6) and anti-GBM nephritis (2/4). MBL deposition paralleled that of immunoglobulins, C1q, C3, and TCC but was less intense as compared to C1q. Focal segmental deposits of MBL were present in focal segmental glomerulosclerosis (4/6), IgA nephropathy (3/11), amyloidosis AL (1/4), and advanced renal fibrosis (2/2). Here MBL staining was identical to IgM and C3 and considered an unspecific entrapment of MBL in sclerotic lesions in these cases. No significant difference in MBL serum levels was observed between normal controls and patients with lupus nephritis, membranous nephropathy, membranoproliferative glomerulonephritis, focal segmental sclerosis, minimal change disease or IgA nephropathy. In patients suffering from membranous nephropathy with (n=10) or without (n=5) glomerular MBL deposits serum creatinine, C3, C4, serum protein, and proteinuria were not statistically different. CONCLUSION: MBL is present in the glomeruli of patients with glomerulonephritis involving deposition of IgG and activation of the classical pathway of complement. We propose that MBL binds to agalactosyl oligosaccharides of IgG that terminate in N-acetylglucosamine. The extent to which the lectin pathway of complement contributes to overall complement activation in the glomeruli remains unknown, but is likely to be marginal.     

Intraglomerular lipid deposition in routine biopsies

Studies have suggested that lipids participate in the pathogenesis of chronic progressive glomerulosclerosis. To examine the frequency of intraglomerular lipid deposition in routine biopsies, renal biopsy material from 631 consecutive patients with glomerular lesions was studied by light, electron, and immunofluorescence microscopy. Fifty-three patients (8.4%) revealed ultrastructurally detectable lipid deposits in nonsclerotic glomeruli. Seven had minimal lesion, 13 focal-segmental glomerulonephritis, two membranous nephropathy, nine membranoproliferative glomerulosclerosis, 11 IgA nephropathy, and 11 other diseases. Ultrastructurally, the lesion affected small segments of glomeruli, with clusters of lipids which exhibited a heterogenous pattern. A subendothelial accumulation of lipids was observed in 60%, storage of lipids on mesangial matrix in 68%, and intramembranous deposition in 21%. Intracellular accumulation of fat was noted in 23%. Indirect immunofluorescence testing for apolipoprotein B (apo B) and oil red O (ORO) staining were performed in 94 cases. Positive results for apo B and ORO staining in nonsclerotic glomeruli were obtained in 24 patients (26%) and 16 cases (17%), respectively. The distribution of lipids seen in these patients was either in diffuse or focal segmental patterns. The clinical data of 40 patients with glomerular lipid deposition were compared with 80 controls from the residual group of patients matched for age, sex, and disease. Serum creatinine and cholesterol levels, and daily excretion of urinary protein were not significantly different between the two groups. These results suggest that an abnormal accumulation of lipids, mainly of apo B containing lipoprotein, in nonsclerotic glomeruli from routine biopsies is not as rare as previously thought. In addition, factors other than hypercholesterolemia seem to be operative for the glomerular lipid deposition observed in our patients.     

Intraglomerular distribution of fibronectin in primary glomerular diseases

The intraglomerular distribution of fibronectin was examined in 152 renal biopsy specimens with the direct immunofluorescence technique. Fibronectin was detected only in the mesangial area in normal glomeruli, while in proliferative glomerulonephritis, it extended from the mesangium to the pericapillary regions proportionately with the proliferative changes. In membranoproliferative glomerulonephritis, fibronectin globally extended in the glomeruli. Also in membranous nephropathy, fibronectin was observed in the capillary walls despite an absence of proliferation. Electron microscopic examinations revealed that the glomeruli with the pericapillary distribution of fibronectin were mostly found in stage II or III, as determined by WHO criteria. The double immunofluorescence technique demonstrated that in membranous nephropathy fibronectin was on the inner side of the glomerular basement membrane, and that in membranoproliferative glomerulonephritis fibronectin extended concomitantly with the expansion of mesangial matrix. These findings suggest that intraglomerular fibronectin is localized only in the mesangial area in normal glomeruli, and that the pericapillary distribution of fibronectin occurs not only by mesangial proliferation but also by additional unknown mechanisms.     

Antigens in human monocytes. III. Use of monocytes in typing for HLA-D related (DR) antigens.

Monocyte preparations were obtained from the peripheral blood by adherence to plastic dishes. The number of monocytes obtained was significantly greater than the number of B cells recovered from similar samples. Monocytes could be frozen and thawed with relatively little loss in cell numbers or viability. Cytotoxicity tests were performed most reliably at 20 degrees C. Under these conditions, autoantibodies were rare, and normal sera were consistently negative. Serologic reactions obtained with monocytes correlated well with the known specificities of the monocyte panel. Typing results in both normal and diseased populations were similar to results obtained with B lymphocytes. Moreover monocyte cytotoxicity tests were more convenient and easier to read than similar tests performed with B cells.     

Renal expression of matrix metalloproteinases in human ANCA-associated glomerulonephritis.

BACKGROUND: Expression of matrix metalloproteinases (MMPs) by infiltrating and intrinsic renal cells is increased in inflammatory conditions, and may correlate with disease activity of glomerulonephritis. We analysed renal expression of MMPs, tissue inhibitor of metalloproteinase-1 (TIMP-1) and markers of neutrophil and monocyte infiltration in renal biopsies of patients with active anti-neutrophil cytoplasmic antibody (ANCA)-associated glomerulonephritis. METHODS: Immunohistochemical expression of MMP-2, -3, -9, TIMP-1, the neutrophil- and monocyte-derived MMP activators cathepsin G, neutrophil elastase and myeloperoxidase (MPO), and the monocyte marker CD14 was determined in renal biopsies of active proteinase 3 (PR3)-ANCA (n = 7) and MPO-ANCA (n = 6) associated glomerulonephritis, and in normal renal tissue (n = 4). Double labelling experiments of MMPs and TIMP-1 were performed with MPO and CD68, labelling neutrophils and macrophages. RESULTS: MMP-2-, MMP-3-, MMP-9- and TIMP-1-positive cells were detected in ANCA-associated glomerulonephritis in glomeruli with active inflammation (cellular crescents or fibrinoid necrosis), only occasionally in normal appearing glomeruli, and not in sclerotic glomeruli and positive cells were found in the tubulo-interstitium. MMPs and TIMP-1 were expressed predominantly by MPO-and CD68-positive cells. In normal renal tissue, no expression was detected, with the exception of weak mesangial staining for MMP-2. In ANCA-associated glomerulonephritis, glomerular MMP-2, -9 and TIMP-1 correlated with glomerular cathepsin G expression, while the number of MMP-9-expressing cells per glomerulus correlated with the percentage of crescentic glomeruli. Tubulo-interstitial expression of MMPs correlated with all markers of neutrophil and monocyte infiltration, and interstitial MMP-9 and TIMP-1 expression correlated with renal function at the time of renal biopsy. CONCLUSIONS: Expression of glomerular and interstitial MMP-2, -3, -9 and TIMP-1 is increased in active ANCA-associated glomerulonephritis and correlates with inflammatory activity.     

Monocyte chemoattractant protein-1 and osteopontin differentially regulate monocytes recruitment in experimental glomerulonephritis

BACKGROUND: This study evaluated the mechanisms of monocyte/macrophage (M/M) infiltration in a rat model of anti-glomerular basement membrane glomerulonephritis (GN). We focused on chemokines and osteopontin, which are known regulators of M/M recruitment. METHODS: Using immunohistology, in situ hybridization, and Northern blotting, the expression levels of chemokines and osteopontin were evaluated in isolated glomeruli and tubules 4, 10, and 20 days after the induction of GN. In vivo blocking experiments were performed by application of neutralizing antibodies against osteopontin and monocyte chemoattractant protein-1 (MCP-1). RESULTS: In nephritic animals, high glomerular MCP-1 and RANTES (regulated upon activation normal T cell expressed and secreted) expression levels were observed on days 4 and 10. The tubular expression of MCP-1, however, was only slightly enhanced. In contrast, tubular osteopontin production was maximally stimulated (day 10) and paralleled with peaks of albuminuria and tubulointerstitial M/M infiltration. Application of an anti-osteopontin antibody ameliorated tubulointerstitial and glomerular M/M recruitment, whereas treatment with an anti-MCP-1 antibody selectively reduced glomerular M/M recruitment. However, tubulointerstitial M/M infiltration remained unchanged. CONCLUSION: These studies show that chemokines and osteopontin are differentially expressed in glomeruli and tubules in this model of GN. Chemokines play a primary role in the glomeruli, whereas osteopontin has a predominant role in tubulointerstitial M/M recruitment. The roles of chemokines and osteopontin may thus be dependent on the renal compartment and on the disease model.     

Osteopontin expression in human crescentic glomerulonephritis

Osteopontin expression in human crescentic glomerulonephritis. BACKGROUND: Osteopontin is a molecule with diverse biological functions, including cell adhesion, migration, and signaling. The expression of osteopontin has been demonstrated in a number of models of renal injury in association with accumulations of monocyte/macrophages, including recent reports of osteopontin expression in glomerular crescents in a rat model of anti-glomerular basement membrane glomerulonephritis. METHODS: Glomerular expression of osteopontin in biopsies of human crescentic glomerulonephritis (N = 25), IgA nephropathy with crescents (N = 2), and diffuse proliferative lupus glomerulonephropathy with crescents (N = 1) was studied by immunohistochemistry, in situ hybridization, and combined immunohistochemistry/in situ hybridization. Additionally, antibodies to cell-specific phenotypic markers were used to identify cellular components of the glomerular crescent, which express osteopontin protein and mRNA. RESULTS: All of the crescents present in the biopsies studied contained a significant number of cells that expressed osteopontin protein and mRNA, demonstrated by immunohistochemistry and in situ hybridization, respectively. Using replicate tissue sections and combined immunohistochemistry/in situ hybridization, we showed that the majority of the strongly osteopontin-positive cells are monocyte/macrophages. In addition to the very strong and cell-associated localization, a weaker and more diffuse pattern of osteopontin protein and mRNA expression could be seen in a number of crescents. None of the osteopontin mRNA-expressing cells could be identified as parietal epithelial cells, CD3-positive T cells, or alpha-smooth muscle actin-positive myofibroblasts. Interstitial monocyte/macrophages did not express osteopontin, except when located in a periglomerular inflammatory infiltrate. CONCLUSIONS: Macrophages present in the human glomerular crescent express osteopontin protein and mRNA at a high level. This expression supports a role for osteopontin in the formation and progression of the crescentic lesion via chemotactic and signaling properties of the molecule.     

Collagen distribution in human membranous glomerulonephritis

In membranous glomerulonephritis (MGN), thickening of the glomerular basement membrane (GBM) is partly due to the accumulation of basement membrane material between and around immune deposits located on the epithelial aspect of the GBM. We investigated the distribution of type IV collagen chains (1/2, 3, 4, 5, 6) and of types I, III, V, and VI collagen in the glomeruli from 16 patients, by indirect immunofluorescence in 13 and the high-resolution immunogold technique in 6. No changes were detected in stage I MGN. The spiky projections of the GBM in stage II MGN and the basement membrane layers encircling immune deposits in stage III contained the 3, 4, and 5 chains of type IV collagen. In contrast, the 1/2 chains of type IV, as well as type VI collagen accumulated in the subendothelial aspect of the GBM. No significant staining for types I, III, and V collagens or for the 6 chain of type IV collagen was detected. The results show that, as in the normal glomeruli, the different chains of type IV collagen are not co-distributed in the glomerular extracellular matrix in MGN. They also indicate that type IV collagen chains and type VI collagen play an important role in the thickening of the GBM in human MGN.     

Interleukin-1 production by monocytes from patients with glomerulonephritis after stimulation in vitro with soluble immune complexes.

Monocytes from 30 patients with glomerulonephritis (GN) were stimulated with soluble immune complexes (IC). In order to neutralize the effect of prostaglandins, some cultures were incubated in the presence of indomethacin. Interleukin-1 (IL-1) activity was assessed by the comitogenic activity of the crude monocyte supernatants on phytohemagglutinin (PHA)-stimulated murine thymocytes. Our results demonstrate that, upon stimulation with the soluble IC monocytes from GN patients possess an enhanced capacity to produce IL-1, and the levels of IL-1 correlate with disease activity only in one case of acute poststreptococcal GN (AGN), not in all other patients. This enhanced production of IL-1 may contribute to the disordered immunoregulation in GN.     

Intercellular adhesion molecule-1 expression in human kidneys with glomerulonephritis.

Intercellular adhesion molecule-1 (ICAM-1) plays an important role in immune responses. Expression of this molecule was examined on cryostat sections of 15 normal human and 112 glomerulonephritic kidneys using a specific monoclonal antibody and the indirect immunoperoxidase staining technique. The expression of ICAM-1 on renal structures was compared to that of HLA-DQ, -DR, -DR/DP and -DP antigens. On normal kidneys ICAM-1 was observed on some Bowman's capsular cells and on single glomerular cells which probably represent endothelial and mesangial cells. ICAM-1 was present on peritubular capillaries and on vascular endothelium of large vessels as well as on fibroblasts, whereas no expression of ICAM-1 on proximal tubular epithelial cells (PTECs) was detected. In normal renal tissues the distribution of ICAM-1 was similar to that of HLA-DQ and -DP antigens. In kidneys with different forms of glomerulonephritis especially in association with interstitial inflammation, an abnormal expression of ICAM-1 on PTECs was frequently correlated with aberrant expression of HLA-DQ and -DP antigens. These results further support the hypothesis that PTECs may participate in cell-mediated immune reactions in glomerulonephritis.