Role of a rat membrane inhibitor of complement in anti-basement membrane antibody-induced renal injury

In the kidneys of anti-glomerular basement membrane (anti-GBM) antibody disease, binding of antibodies to tubular basement membrane (TBM) is often observed. The present work was performed to explore the mechanisms of binding of anti-GBM antibodies to TBM in vivo with special reference to 5I2Ag, a rat membrane inhibitor of complement which regulates complement activation at C3 convertase level. To suppress functions of renal 5I2Ag, F(ab')2 fragment of 5I2 (a neutralizing mAb against 5I2Ag) was perfused in the left kidney and then blood circulation was restored. Mild proteinuria ( < 10 mg/16 hr) was observed during first several days. Five days later, there were tubulointerstitial injuries defined by tubular vimentin staining and leukocyte infiltration. Significant deposition of C3 was observed in the capillaries and in TBM. In rats intravenously injected with rabbit anti-rat GBM antibodies five minutes after kidney perfusion with 5I2, strong binding of rabbit IgG to TBM was observed at one and five days after injection. Although these rats showed mild proteinuria comparable to those perfused with 5I2 and those injected with normal rabbit serum, tubulointerstitial injury was significantly enhanced at Day 5. In contrast, rats perfused with irrelevant mAb and injected with anti-GBM antibodies did not show any significant binding of antibodies to TBM nor tubulointerstitial injury. Furthermore, rats which were made proteinuric by puromycin aminonucleoside and injected with anti-GBM antibodies did not show any significant binding of rabbit IgG to TBM. These results indicate that 5I2Ag, a rat membrane inhibitor of complement at the C3 convertase level, regulates vascular permeability in the living kidney, and that dysfunction or decreased expression of this molecule leads to increased accessibility of anti-GBM antibodies to TBM.     

Anti-vitronectin antibodies enhance anti-Thy-1-induced proteinuria in PVG/c, but not in Wistar rats

Injection of rats with mouse monoclonal IgG2a anti-Thy1.1 antibodies (ER4G) results in rapid development of proteinuria in Wistar rats, reaching average values of 160 mg/24 h on day 3 after antibody administration. In contrast, no overt proteinuria was observed in PVG/c+ rats (maximum, 40 mg/24 h on day 3). This study investigates whether differences in the inactivation of C5b-9 complexes in the glomerulus by complement inhibitors are responsible for the differences in proteinuria between the two rat strains. Regardless of the presence of proteinuria, an increased expression of Crry by mesangial cells (MC) was observed within 24 h after injection of ER4G in both Wistar and PVG/c+ rats. Double-label immunofluorescence using goat anti-mouse Ig antibodies demonstrated an expression of Crry exclusively on MC. Furthermore, Crry colocalized with C5b-9 complexes on MC, as detected by a monoclonal antibody against the rat C5b-9 neo-antigen. In PVG/c+ rats, C5b-9 complexes persisted in the mesangial area for at least 7 d and colocalized immediately (within 1 h) and homogeneously with vitronectin. However, in proteinuric Wistar rats, C5b-9 complexes disappeared from the glomerular mesangium within 6 d. In these rats, mesangial colocalization of C5b-9 with vitronectin could only occasionally be detected. Pretreatment of PVG/c+ rats with antibodies against vitronectin, followed by administration of ER4G, resulted in the immediate development of proteinuria (maximum, 119 mg/24 h on day 3; P < 0.05), whereas Wistar rats did not become more proteinuric. This study provides evidence that differences in susceptibility of PVG/c+ and Wistar rats to complement-mediated damage of the glomerulus may be related to the degree of inactivation of C5b-9 complexes by complement regulatory factors.     

Effect of a novel immunosuppressant, FK 506, on autoimmune glomerulonephritis in Brown Norway rats

Mercuric-chloride (HgCl2) induces a lymphoproliferative disorder and autoimmune glomerulonephritis in Brown Norway rats. The effects of a new immunosuppressant FK 506 on this model of glomerulonephritis were studied. Brown Norway rats were treated with HgCl2 according to a standard protocol (HgCl2 1 mg/kg s.c. 3 times/ week). Rats developed proteinuria at day 7, which reached a plateau level at day 14. On day 14, renal histology showed prominent mesangial cellular proliferation and the expansion of mesangial matrix. Electron microscopic study showed the effacement of visceral epithelial foot processes and the microvillous transformation of the visceral epithelium. Immunofluorescence study showed a strong linear staining for IgG and the adhesion molecule ICAM-1 in all glomeruli. Coadministration of FK 506 (1 mg/kg s.c. daily) prevented the appearance of proteinuria at day 14 (621.4 +/- 30.5 vs. 2.2 +/- 2.7 mg/day) and the morphological lesions. These findings suggest that FK 506 could be useful for the therapy of certain types of human glomerulonephritis.     

Suppressive effects of sairei-to on monoclonal antibody 1-22-3-induced glomerulonephritis: analysis of effective components

The effects of traditional Chinese medicine (Sairei-to) on experimental glomerulonephritis induced in rats by monoclonal antibody (mAb) 1-22-3 injection was examined. The level of proteinuria in the Sairei-to-treated group was significantly lower than that in the PBS treated group. This suppressive effect was caused by the major component of Sairei-to, Syo-saiko-to but not by another component, Gorel-san. The suppressive effect of Syo-saiko-to was identified in its components (Bupleuri radix, Pinelliae tuber and Zingiberis rhizoma), but not in the other combined components (Ginseng radix and Zizyphi fructus). Further study revealed that the suppressive effects of the combined components were mainly derived from Bupleuri radix. It was demonstrated that the actual active ingredient is probably Saikosaponin-d. Light microscopy revealed that Sairei-to and its effective components suppressed the proliferation of mesangial cells and mesangial matrix expansion. Semiquantitative morphological studies of glomerular lesions on the eighth day showed that Syo-saiko-to and its combined components (Bupleuri radix, Zingiberis rhizoma and Pinelliae tuber) suppressed mesangial matrix expansion significantly compared with phosphate-buffered saline control groups (matrix score: 28.0 +/- 19.1 vs 102.3 +/- 14.1; 30.9 +/- 30.1 vs 102.3 +/- 14.1, P < 0.005, respectively). It was concluded that Saikosaponin-d, as well as Bupleuri radix, Syo-saiko-to and Sairei-to can suppress proteinuria and morphological changes in the rat glomerulonephritis model induced by mAb 1-22-3.     

Cationic dextran induces mesangioproliferative nephritis in rats independent of glomerular IgA deposition

BACKGROUND: Dextran-induced mesangioproliferative glomerulonephritis in mice and, as recently reported, in rats is used as a model of IgA nephropathy. The pathogenetic role of the glomerular IgA deposits in this model, however, is unclear since IgG is often deposited in parallel. METHODS: Lewis rats were immunized with cationic DEAE-dextran. Following this, rats received 5 x/week i.v. injections of 3 mg DEAE-dextran each, from days 20 to 80 and were then followed until day 120. RESULTS: Rats developed transient proteinuria (range 63-152 mg/24 h) and haematuria on day 80. Renal biopsies obtained at days 60, 80, 100 and 120 showed mild to severe mesangioproliferative changes at days 80 and 100 which did not persist at day 120. Electron-microscopy revealed mesangial immune deposits, signs of endothelial activation and vacuoles in mesangial cells and podocytes. Compared to normal, age-matched controls, in the nephritic rats significant (P < 0.05) increases were noted for glomerular total cellularity, alpha-smooth-muscle actin expression (a marker of activated mesangial cells), monocyte/macrophage counts, and matrix proteins. Using three different antibodies, no evidence of glomerular IgA deposition was detected at any time point. In contrast, glomerular IgG, IgM, C3, and occasional small C5b-9 deposits were present in nephritic rats. Circulating IgG but not IgA anti-dextran antibodies could be demonstrated in nephritic rats. CONCLUSIONS: The data confirm that mesangioproliferative glomerulonephritis can be induced in rats by immunization and chronic challenge with cationic dextran. Our data also show that in rats glomerular IgG deposition rather than IgA, appears to play an important pathogenetic role in this mesangioproliferative glomerulonephritis model.     

Expression of types I, II, and III TGF-beta receptors in human glomerulonephritis

Protein and mRNA expression of transforming growth factor-beta (TGF-beta) receptor type I (TbetaRI), type II (TbetaRII), and type III (TbetaRIII) were studied in serial sections of kidney samples obtained from patients with glomerulonephritis. In minimal change disease, weak expression of TbetaRI and TbetaRII was observed mainly in glomerular endothelial cells, peritubular capillaries, and interstitial arteriolar endothelial cells, whereas TbetaRIII expression was found mainly in the interstitium. Expression of all three TGF-beta receptors (TbetaR) was increased remarkably in glomerular and Bowman's capsular cells comprising the tuft adhesions to Bowman's capsules in glomerulonephritis with increased matrix accumulation, including IgA nephropathy, lupus nephritis, focal and segmental glomerulosclerosis, myeloperoxidase-antineutrophil cytoplasmic antibody-associated crescentic glomerulonephritis, and membranoproliferative glomerulonephritis. Increased expression of the three TbetaR was also seen in glomerular epithelial cells in the vicinity of glomerulosclerotic lesions, in crescent cells, and in some tubules and infiltrative mononuclear cells found in the periglomerular and tubulointerstitial lesions with increased matrix deposition. In contrast, no remarkable TbetaRII expression was noted in mesangial proliferative lesions in IgA nephropathy, lupus nephritis, and membranoproliferative glomerulonephritis. These data suggest that distinctive modulation of TbetaR expression may be involved in the development of adhesive, sclerotic, and proliferative renal lesions in human glomerulonephritis.     

Polyamide microcapsules containing alginic acid: extractability of metal ions and surface characterization by XPS

The pathophysiological role of the Thy-1.1 molecule expressed on rat mesangial cells with regard to mesangial cell dysfunction and injury remains unknown. The mechanism of Thy-1.1-associated injury has now been investigated with two monoclonal antibodies, 1-22-3 and OX7, that recognize different epitopes of Thy-1.1. Mesangiolysis and mesangial cell proliferation were more marked in rats injected with 1-22-3 than in those treated with OX7. Immunostaining for rat complement component C3 and also C9 was similar in the kidneys of rats 1 h after injection of either antibody. Alpha smooth muscle actin was first detected 3 days after injection of 1-22-3 and peaked on day 5; type I collagen staining showed a mesangial pattern on days 5 and 10. The staining for alpha smooth muscle actin and type I collagen was less intense in OX7-treated rats than in the 1-22-3-injected rats. The amounts of mRNAs encoding collagen types I and III peaked 5 days after injection of 1-22-3 and 10 days after injection of OX7. Rats injected with 1-22-3 developed proteinuria that was already marked on day 1 and peaked at 150 mg/day on day 3, whereas OX7 induced a low grade of proteinuria with large interindividual variability on day 3. Immunostaining for rat C3 in the normal rat kidneys, incubated in vitro with 1-22-3 or OX7 followed by incubation with normal rat fresh serum as a complement source, as well as the levels of serum complement activity, CH50, 30 min after injection of 1-22-3 or OX7 were similar, suggesting that the difference in the nephritogenicity of these two antibodies is not attributable to a difference in their complement-fixing activities, but rather may result from the difference in epitope specificities. The epitope recognized by 1-22-3 thus appears to be important in the initiation and progression of antibody-induced nephritis.     

Effect of pancreas transplantation on the prevention of nephropathy in alloxan-induced diabetic rats

We studied the effects of pancreas transplantation on kidney lesions of rats with alloxan-induced diabetes. Ninety inbred male Lewis rats were randomly assigned to 3 experimental groups: group NC included 30 non-diabetic control rats, group DC included 30 alloxan-induced diabetic control rats, and group PT included 30 alloxan-induced diabetic rats that received pancreas transplants from normal donor Lewis rats. Each group was further divided into 3 subgroups of 10 rats which were sacrificed at 1, 3, and 6 months of follow-up, respectively. Clinical and laboratory parameters during these periods were documented. The kidneys of 5 rats in each subgroup were studied and 50 glomeruli and tubules from each kidney were analyzed by light microscopy by two different investigators in a double-blind study. There was progressive glomerular basement membrane thickening (GBMT), mesangial enlargement (ME), and Bowman's capsule thickening (BCT) in kidneys of rats in the 3 experimental groups during follow-up. These alterations were significantly higher in DC rats (GBMT: 1.99 +/- 0.31; ME: 2.00 +/- 0.33; BCT: 1.88 +/- 0.27) when compared to NC (GBMT: 1.54 +/- 0.30; ME: 1.56 +/- 0.47; BCT: 1.36 +/- 0.35) and PT rats (GBMT: 1.49 +/- 0.29; ME: 1.57 +/- 0.36; BCT: 1.35 +/- 0.28) at 6 months (P < 0.01). The extent of GBMT, ME, and BCT observed in DC rats at 1 and 3 months was not significantly different from NC and PT rats. The amount of kidney lesions in PT rats was similar to that of NC rats and lower than those of DC rats at 6 months (P < 0.01). In addition, Armanni-Ebstein lesions of the tubules (AE) and tubular lumen protein (PRO) observed in DC rats were not present in NC or PT rats. We conclude that pancreas transplantation in alloxan-induced diabetic rats prevents the development of kidney lesions beginning at 6 months after transplantation.     

Clinicopathological significance of intratubular giant macrophages in progressive glomerulonephritis

Very large macrophages, which we have termed "giant macrophages" (G-M phi), have been found in renal tubules, some containing cytoplasmic vacuoles. To elucidate their pathophysiological roles, we examined renal biopsy tissues from various primary glomerulonephritis (GN) and tubulointerstitial nephritis (TIN) using immunohistochemistry with monoclonal antibodies against M phi and other cell surface markers. Giant macrophages were absent or rare in TIN, minimal change nephrotic syndrome, and minor glomerular abnormalities, but G-M phi was plentiful in progressive glomerulonephrides such as IgA nephropathy with crescents, membranoproliferative GN, focal segmental glomerulosclerosis, and especially in crescentic GN. These G-M phi were usually seen in the lumen of renal tubules, but occasionally were found in the Bowman's spaces and glomerular tufts, and similar cells were also found in urine. Moreover, they frequently made contact with tubular epithelial cells expressing intercellular adhesion molecule-1, and the tubular epithelial cells in such lesions often had degenerative changes. Giant M phi may damage tubular epithelial cells from the luminal side. Phenotypically, G-M phi showed activated (CD71+) and mature (25F9+) characteristics along with features of M phi (CD68+), and the cytoplasm contained a great deal of lipids. The numbers of G-M phi in renal tissues closely correlated with the degree of hematuria (rho = 0.5, P < 0.001), serum creatinine value (r = 0.63, P < 0.001) in GN patients (N = 96) and with proteinuria in IgA nephropathy patients (r = 0.89, P < 0.001, N = 27). These data suggest that G-M phi are M phi that were activated and matured in certain active inflammatory sites, which flowed into tubules and then into urine. Thus, the existence of G-M phi in biopsy tissue or urine reflect the activity of GN and may have a predictive value for the progression of GN.     

Perfusion-weighted MRI using gadobutrol as a contrast agent in a rat stroke model

BACKGROUND: A number of studies have demonstrated a pathological role for interleukin-1 (IL-1) in experimental models of glomerulonephritis, but the cellular pattern of renal IL-1 production remains poorly characterized. The aim of this study, therefore, was to identify the cell types expressing IL-1 in normal and diseased rat kidney. METHODS: Renal IL-1 beta expression was examined in normal rats and during a 21-day time course of rat accelerated anti-GBM glomerulonephritis by northern blotting, in situ hybridization and double immunohistochemistry. RESULTS: Interleukin-1 beta mRNA expression was readily detectable in normal rat kidney by northern blot analysis and in situ hybridization. Immunohistochemistry staining demonstrated constitutive IL-1 beta expression by glomerular endothelial cells and cortical tubular epithelial cells. There was a marked increase in whole kidney IL-1 beta mRNA in rat anti-GBM glomerulonephritis. Glomerular IL-1 beta immunostaining was upregulated, being expressed by podocytes, mesangial cells and infiltrating macrophages, and was particularly prominent within glomerular crescents. Double staining with the ED1 antibody showed IL-1 beta expression in up to 13% of glomerular macrophages, whereas 48% of macrophages within crescents stained for IL-1 beta. However, the most marked increase in IL-1 beta expression was seen in cortical tubular epithelial cells, particularly in areas of tubular damage. In situ hybridization confirmed that tubular IL-1 beta staining was due to local cytokine synthesis rather than protein absorption. CONCLUSIONS: This study has identified constitutive IL-1 beta expression by glomerular endothelium and tubular epithelial cells in normal rat kidney. In addition, the marked upregulation of IL-1 beta expression by intrinsic glomerular cells and tubules in rat anti-GBM disease suggests an important role for these cells in IL-1 dependent crescent formation and tubulointerstitial injury.     

Prevalence and causes of proteinuria in 20-year-old Finnish men

The prevalence and causes of proteinuria were studied in a cohort of 36147 men aged 20 (born in 1956). Proteinuria was found in 139 men (0.4%) at the initial screening or examination. Further investigations reduced the number of proteinuria cases to 72 (0.2%). Persistent proteinuria was demonstrated in 46 men (0.13% of the series) and orthostatic proteinuria in 26 (0.07%). Urography revealed anomalies in 18 of 104 cases. Elevated blood pressure and reduced glomerular filtration rate were observed in a few men, mainly from the group with persistent proteinuria. Renal biopsy was performed in 61 cases--38 with persistent proteinuria, 12 with orthostatic proteinuria and 11 without proteinuria at the time of examination. Light microscopy gave normal findings or showed only slight mesangial or focal glomerulonephritis in the great majority of cases. Membranous, mesangiocapillary or chronic proliferative glomerulonephritis was present in one-fourth of the men with persistent proteinuria. This was the only group with such lesions.     

Contractile cells of the kidney in primary glomerular disorders: an immunohistochemical study using an anti-alpha-smooth muscle actin monoclonal antibody

Mesangial cells of the renal glomerulus are thought to have contractile properties, resembling those of smooth muscle cells. Since actin synthesis in mesangial cells is increased in selected animal models of glomerulonephritis, we evaluated the expression of alpha-smooth muscle actin (ASMA), the principal actin isoform found in smooth muscle cells, in biopsy specimens from patients with primary glomerular disorders and in control tissues. Normal glomeruli and glomeruli in acute tubulointerstitial disorders showed few or no ASMA-positive cells in the glomeruli. In contrast, ASMA expression in mesangial cells was increased in minimal change disease, focal segmental glomerulosclerosis, mesangial proliferative glomerulonephritis, membranous glomerulonephritis, and immunoglobulin A nephropathy. In membranoproliferative glomerulonephritis and cryoglobulinemic glomerulonephritis both mesangial and capillary loop ASMA-positive cells were observed with a segmental distribution. In addition, ASMA-positive interstitial cells were seen in many biopsy specimens and often were increased in number in biopsy specimens showing early interstitial fibrosis and tubular atrophy. We conclude that ASMA synthesis in mesangial cells is upregulated in a variety of glomerular disorders, frequently associated with increased cell proliferation and mesangial matrix production. This phenotypic change may be an indicator of mesangial cell activation after injury and may have important pathophysiologic consequences.     

Sex differences in energy intake and expenditure

Four patients with rheumatoid arthritis developed heavy proteinuria after five to 12 months of treatment with D-penicillamine. Light microscopy of renal biopsy samples showed minimal glomerular capillary wall thickening and mesangial matrix increase, or no departure from normal. Electron microscopy, however, revealed subepithelial electron-dense deposits, fusion of epithelial cell foot processes, and evidence of mesangial cell hyperactivity. Immunofluorescence microscopy demonstrated granular capillary wall deposits of IgG and C3. The findings were similar to those in early membranous glomerulonephritis, differences being observed however in the results of staining for the early-acting complement components C1q and C4. It is tentatively concluded that complement was activated by the classical pathway.     

Mechanisms involved in the pathogenesis of tubulointerstitial fibrosis in 5/6-nephrectomized rats

The 5/6 nephrectomy model is used to study pathogenetic mechanisms underlying chronic renal failure. We previously demonstrated that increased mesangial cell proliferation and glomerular PDGF B-chain expression precede glomerulosclerosis in this model. In the present study we have assessed the concomitant changes in the cortical tubulointerstitium. A wave of tubular and interstitial cell proliferation (as determined by immunostaining for PCNA) occurred at week 1 after 5/6 nephrectomy. This wave preceded the peak glomerular cell proliferation by one week. Tubulointerstitial cell proliferation decreased thereafter and reached control values by week 10. In situ hybridization and immunostaining for PDGF B-chain and beta-receptor in sham-operated controls showed labeling of distal tubules and collecting ducts, while no signal was present in the interstitium. PDGF B-chain mRNA and protein expression was markedly increased in tubules at weeks 2 and 4 after 5/6 nephrectomy and in the interstitium (particularly in areas of inflammatory infiltrates) at weeks 2 to 10. Similar changes occurred with PDGF receptor beta-subunit immunostaining. Interstitial expression of desmin and alpha-smooth muscle actin (markers of myofibroblasts) progressively increased after week 1. Interstitial influx of monocytes/macrophages with focal accentuation started at week 2. Counts of lymphocytes, neutrophils and platelets showed only minor changes. In parallel to the monocyte/macrophage influx, progressive interstitial accumulation of collagens I and IV, laminin, and fibronectin occurred. All of these changes were correlated with the increase in serum creatinine, proteinuria and an index of tubulointerstitial damage. We conclude that tubulointerstitial changes after 5/6 nephrectomy show similarities with those observed in the glomeruli. Tubular and interstitial overexpression of PDGF B-chain and its receptor may play a role in mediating fibroblast migration and/or proliferation in areas of tubulointerstitial injury.     

Acute nephrotoxic serum nephritis in complement knockout mice: relative roles of the classical and alternate pathways in neutrophil recruitment and proteinuria

BACKGROUND: The importance of complement in the pathophysiology of renal disease is still being appreciated. To further address the role of this mediator system, we evaluated the influence of absolute deficiency of C3 and C4 on acute nephrotoxic serum nephritis (NSN). METHODS: Selective 'knockout' of C3 and C4 was routinely confirmed in null mice by ELISA. NSN was induced by intravenous injection of a sheep anti-rat nephrotoxic serum that cross-reacts with murine glomerular antigens. Deposition of heterologous immunoglobulin in wild-type glomeruli was associated with rapid complement deposition and neutrophil infiltration, and followed by the development of proteinuria. RESULTS: Neutrophil infiltration was markedly inhibited in C3-deficient mice indicating a role for complement in PMN recruitment. In contrast, C3 deficiency afforded only partial protection against proteinuria. NSN was studied further in C4 null mice to probe the relative roles of the classical and alternate pathway in disease pathophysiology. C3 and C4 deficiency were associated with equivalent inhibition of PMN recruitment and proteinuria. CONCLUSIONS: In aggregate, the data support a major role for complement in PMN recruitment in this model and point to complement-independent mechanisms of proteinuria in antibody-mediated glomerulonephritis. These 'knockout' mice should prove valuable for defining the complement-activated mediator systems that regulate leukocyte recruitment and tissue injury in renal diseases.     

De novo glomerular osteopontin expression in rat crescentic glomerulonephritis

Osteopontin (OPN) is a secreted acidic glycoprotein that has potent monocyte chemoattractant and adhesive properties. Up-regulation of tubular OPN expression is thought to promote interstitial macrophage infiltration in experimental nephritis; however, the role of OPN in glomerular lesions, particularly crescent formation, is unknown. The present study used Northern blotting, in situ hybridization and immunohistochemistry to examine OPN expression in a rat model of accelerated anti-GBM glomerulonephritis. Osteopontin mRNA and protein is expressed by some parietal epithelial cells, thick ascending limbs of Henle and medullary tubules and collecting ducts in normal rat kidney. De novo OPN mRNA and protein expression was evident in glomerular visceral and parietal epithelial cells in anti-GBM glomerulonephritis. Glomerular OPN expression preceded and correlated with macrophage infiltration in the development of hypercellularity, focal and segmental lesions and, notably, crescent formation. There was marked up-regulation of OPN expression by tubular epithelial cells that also preceded and correlated with interstitial macrophage (r = 0.93, P < 0.001) and T-cell infiltration (r = 0.85, P < 0.001). Both glomerular and tubular OPN expression correlated significantly with proteinuria (P < 0.001) and a reduction in creatinine clearance (P < 0.01). In addition, double immunohistochemistry showed co-expression of osteopontin and one of its ligands, CD44, in intrinsic renal cells. CD44 and OPN expression by parietal epithelial cells was evident in crescent formation, while virtually all OPN-positive tubules expressed CD44. Infiltrating macrophages and T-cells were CD44-positive, but only a small proportion of T-cells and few macrophages showed OPN expression. Interestingly, strong OPN mRNA and protein expression was seen in macrophage multinucleated giant cells. In summary, this study suggests that OPN promotes macrophage and T-cell infiltration in the development of renal lesions in rat anti-GBM glomerulonephritis, including glomerular crescent and multinucleated giant cell formation.     

Antiproteinuric effect of angiotensin-converting enzyme inhibition and C5b-9 urinary excretion in membranous glomerulonephritis

BACKGROUND: Angiotensin-converting enzyme (ACE) inhibitors have an antiproteinuric effect in membranous glomerulonephritis (MGN). However, no studies have investigated whether this antiproteinuric effect is influenced by urinary C5b-9 excretion, a marker of immunological activity in this disease. METHODS: Eleven patients with biopsy-proven MGN were treated with captopril for 8 weeks. The evolution of several clinical and biochemical parameters, including 24-h urinary protein excretion was evaluated every 4 weeks. Urinary C5b-9 excretion was measured at the onset and at the end of captopril treatment. RESULTS: Patients with MGN had significantly higher C5b-9 excretions than a group of 14 healthy controls (89 +/- 23 vs 3.7 +/- 1.4 ng/mg UCr; P < 0.001). A significant correlation was found between urinary C5b-9 and the magnitude of proteinuria, both at the onset and at the end of treatment. After 8 weeks of captopril treatment, proteinuria had decreased from 8 +/- 1.8 to 5.2 +/- 1.3 g/day (P < 0.05). Four weeks after captopril discontinuation, proteinuria rose to 7.3 +/- 1.7 g/day (P < 0.05). A marked variability in the antiproteinuric response was observed, ranging from 0 to 85% with respect to baseline values. No correlation between decrease in proteinuria and baseline urinary C5b-9 levels was observed. Several patients with elevated urinary C5b-9 levels had captopril-induced decrease in proteinuria. CONCLUSIONS: ACE inhibition induces an antiproteinuric effect in patients with MGN. The urinary C5b-9 excretion does not predict the magnitude of this response.     

Complement inhibition with an anti-C5 monoclonal antibody prevents acute cardiac tissue injury in an ex vivo model of pig-to-human xenotransplantation

Prevention of hyperacute xenograft rejection in the pig-to-primate combination has been accomplished by removal of natural antibodies, complement depletion with cobra venom factor, or prevention of C3 activation with the soluble complement inhibitor sCR1. Although these strategies effectively prevent hyperacute rejection, they do not address the relative contribution of early (C3a, C3b) versus late (C5a, C5b-9) activated complement components to xenogeneic organ damage. To better understand the role of the terminal complement components (C5a, C5b-9) in hyperacute rejection, an anti-human C5 mAb was developed and tested in an ex vivo model of cardiac xenograft rejection. In vitro studies demonstrated that the anti-C5 mAb effectively blocked C5 cleavage in a dose-dependent manner that resulted in complete inhibition of both C5a and C5b-9 generation. Addition of anti-C5 mAb to human blood used to perfuse a porcine heart prolonged normal sinus cardiac rhythm from a mean time of 25.2 min in hearts perfused with unmodified blood to 79,296, or > 360 min when anti-C5 mAb was added to the blood at 50 micrograms/ml, 100 micrograms/ml, or 200 micrograms/ml, respectively. In these experiments, activation of the classical complement pathway was completely inhibited. Hearts perfused with blood containing the highest concentration of anti-C5 mAb had no histologic evidence of hyperacute rejection and no deposition of C5b-9. These experiments suggest that the activated terminal complement components C5a and C5b-9, but not C3a or C3b, play a major role in tissue damage in this porcine-to-human model of hyperacute rejection. They also suggest that targeted inhibition of terminal complement activation by anti-C5 mAbs may be useful in clinical xenotransplantation.     

Immunohistochemical localization of C3d fragment of complement and S-protein (vitronectin) in normal and diseased human kidneys: association with the C5b-9 complex and vitronectin receptor

The localization of C3d, a fragment produced by C3 activation and S-protein (vitronectin), a regulatory factor of C5b-9, was studied immunohistochemically in normal human kidney and renal biopsies from patients with several types of glomerulonephritis. Immunofluorescent staining of the normal kidneys showed that C3d was present along the glomerular basement membrane (GBM), tubular basement membrane (TBM) and arterioles, and that S-protein was present in the GBM, mesangium, TBM, and arterioles. Immunoelectron microscopy of isolated basement membranes showed that C3d was localized exclusively on the epithelial side of the GBM, and that S-protein was present along both the epithelial and endothelial sides. In nephritic tissues, glomerular staining of C3d, C5b-9, and S-protein was increased when compared with that in normal tissues. S-protein, frequently co-localized with C3d and C5b-9 neoantigen, was intensely positive in the immune deposits of glomerular capillaries and the mesangial area, overlapping the background staining of GBM and mesangial matrix. S-protein and its receptor were occasionally co-localized in the glomeruli. These findings indicate that C3d and S-protein are normally present in the glomeruli. Co-staining of C3d, C5b-9 neoantigen, and S-protein within the immune deposits of nephritic kidneys suggests in situ binding of S-protein to locally-formed C5b-9 complex, or merely co-distribution of S-protein with the complex, rather than trapping of large molecular SC5b-9 complex from the circulation.