Effect of L-carnitine administration on erythrocyte survival in haemodialysis patients.
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Anemia↗
Biomedical subjects
Charles D Pusey
Publications and source records attributed to Charles D Pusey.
Copy number polymorphism in Fcgr3 predisposes to glomerulonephritis in rats and humans.
Identification of the genes underlying complex phenotypes and the definition of the evolutionary forces that have shaped eukaryotic genomes are among the current challenges in molecular genetics. Variation in gene copy number is increasingly recognized as a source of inter-individual differences in genome sequence and has been proposed as a driving force for genome evolution and phenotypic variation. Here we show that copy number variation of the orthologous rat and human Fcgr3 genes is a determinant of susceptibility to immunologically mediated glomerulonephritis. Positional cloning identified loss of the newly described, rat-specific Fcgr3 paralogue, Fcgr3-related sequence (Fcgr3-rs), as a determinant of macrophage overactivity and glomerulonephritis in Wistar Kyoto rats. In humans, low copy number of FCGR3B, an orthologue of rat Fcgr3, was associated with glomerulonephritis in the autoimmune disease systemic lupus erythematosus. The finding that gene copy number polymorphism predisposes to immunologically mediated renal disease in two mammalian species provides direct evidence for the importance of genome plasticity in the evolution of genetically complex phenotypes, including susceptibility to common human disease.
Animals↗
Therapeutic effect of anti-TNF-alpha antibodies in an experimental model of anti-neutrophil cytoplasm antibody-associated systemic vasculitis.
The therapeutic options for anti-neutrophil cytoplasm antibody (ANCA)-associated systemic vasculitis (AASV) remain limited and hampered by adverse effects. One potential novel therapeutic avenue involves inhibition of TNF-alpha, with encouraging uncontrolled data in humans with one agent (infliximab) but disappointing controlled data from another (etanercept). For investigating the potential role of TNF-alpha as a therapeutic target in AASV, the effect of an anti-rat TNF-alpha mAb (CNTO 1081) in a rat model of AASV was investigated. For testing the effect of TNF-alpha blockade in this model, starting on day 28 after immunization (a point when glomerulonephritis is established), animals were randomized to treatment with CNTO 1081 or control mouse IgG. Treatment with CNTO 1081 significantly reduced albuminuria (mean 1.1 +/- 0.3 mg/24 h CNTO 1081 versus 8.0 +/- 1.9 controls; P < 0.05) and crescent formation (0% CNTO 1081 versus 60% controls; P < 0.05). Lung hemorrhage was also reduced (CNTO 1081: median score 0, range 0 to 2; controls: 2, range 1 to 3; P < 0.05). When analyzed by intravital microscopy, there was a 43% inhibition of leukocyte transmigration in mesenteric venules in response to topical CXCL1 (a neutrophil chemoattractant) in the CNTO 1081 group compared with controls (P < 0.001). Anti-myeloperoxidase antibody titers were similar in both groups throughout the study. In conclusion, these findings indicate that TNF-alpha plays an important role in the pathogenesis of experimental autoimmune vasculitis and suggest that blockade of this cytokine with an mAb is effective in treating established vasculitis. The therapeutic action of anti-TNF-alpha reagents may be mediated, in part, by suppression of the enhanced leukocyte-endothelial interactions in this disorder.
Animals↗
Antineutrophil cytoplasm antibodies directed against myeloperoxidase augment leukocyte-microvascular interactions in vivo.
Systemic small vessel vasculitis is associated with antineutrophil cytoplasm antibodies (ANCAs). While there is mounting in vitro evidence to suggest that ANCAs are capable of enhancing leukocyte-endothelial interactions, no in vivo evidence for this has been provided. In this study a novel rat model of ANCA-associated experimental autoimmune vasculitis (EAV), induced by immunization with human myeloperoxidase (MPO), was used to analyze directly the potential effect of ANCAs on leukocyte-venular wall interactions in vivo as observed by intravital microscopy. These rats developed anti-MPO antibodies directed against rat leukocytes, showed pathologic evidence of small vessel vasculitis, and had enhanced leukocyte adhesion and transmigration in response to the chemokine Groalpha (CXCL1 [CXC ligand 1]). Passive transfer of immunoglobulin from rats with EAV to naive rats conferred enhanced adhesion and transmigration responses in the recipients. Furthermore, rats with EAV and recipients of ANCA-positive immunoglobulin developed extensive microvascular injury, as manifested by mesenteric hemorrhage, in response to CXCL1. This study provides the first direct in vivo evidence for the ability of ANCAs to enhance leukocyte-endothelial interactions and cause microvascular hemorrhage, thereby providing a mechanism by which ANCAs could exert pathogenic effects in systemic vasculitis.
Animals↗
Nasal administration of recombinant rat alpha3(IV)NC1 prevents the development of experimental autoimmune glomerulonephritis in the WKY rat.
Experimental autoimmune glomerulonephritis (EAG), an animal model of Goodpasture's disease, can be induced in Wistar Kyoto (WKY) rats by immunization with either collagenase-solubilized rat glomerular basement membrane (GBM) or the recombinant NC1 domain of the alpha3 chain of type IV collagen [alpha3(IV)NC1]. EAG is characterized by circulating and deposited anti-glomerular basement membrane antibodies, focal necrotizing glomerulonephritis with crescent formation, and glomerular infiltration by T cells and macrophages. Previous studies have demonstrated that oral administration of collagenase-solubilized GBM to WKY rats prevented the development of EAG. Nasal administration of specific autoantigens has been reported to be more effective than oral administration in other models of autoimmune disease. The main aim of this study was to investigate further the concept of mucosal tolerance in EAG by examining the effect of nasal administration of recombinant rat alpha3(IV)NC1. Groups of WKY rats with EAG, induced by immunization with recombinant rat alpha3(IV)NC1, were given alpha3(IV)NC1 nasally on 3 consecutive days before immunization, at total cumulative doses of 25, 100, or 250 microg per rat. A dose-dependent effect was observed on the development of EAG. A dose of 25 microg had no effect on disease; 100 microg resulted in a moderate reduction in the severity of nephritis; and 250 microg led to a marked reduction in circulating and deposited antibodies, albuminuria, severity of glomerular abnormalities, and numbers of glomerular CD8+ T cells and macrophages. In addition, there was a reduction in the proliferative response of splenocytes from rats in the high dose group (250 microg) to alpha3(IV)NC1 in vitro. The results from this study clearly demonstrate for the first time that mucosal tolerance in EAG can be induced by nasal administration of recombinant rat alpha3(IV)NC1 and that this approach is effective in the prevention of crescentic glomerulonephritis. Further work using new antigen-specific treatment strategies may provide a novel approach to the treatment of patients with anti-glomerular basement membrane disease.
Administration, Intranasal↗
Glomerulonephritis due to antineutrophil cytoplasm antibody-associated vasculitis: an update on approaches to management.
A patient with antineutrophil cytoplasm antibody-associated vasculitis frequently has multisystem disease and might present to a range of medical specialities. The manifestations that result in the greatest burden of morbidity and mortality are renal and pulmonary disease. In this review we will focus on rapidly progressive glomerulonephritis due to vasculitis, with specific reference to recent advances in our understanding of pathogenesis. The current standard of care for managing renal vasculitis, be it mild, moderate or severe, has largely been established in clinical trials of prednisolone, cyclophosphamide, methotrexate, azathioprine and plasma exchange. In addition to these, newer therapies such as mycophenolate mofetil and infliximab are being used more frequently. We will review the role of each of these approaches to management of small vessel vasculitis in 2005.
Adolescent↗
Antibody blockade of TNF-alpha reduces inflammation and scarring in experimental crescentic glomerulonephritis.
BACKGROUND: Tumor necrosis factor-alpha (TNF-alpha) is a proinflammatory cytokine produced by macrophages, and by renal mesangial and tubular epithelial cells. It stimulates the release of interleukin (IL)-1beta, monocyte chemoattractant protein-1 (MCP-1), and transforming growth factor-beta (TGF-beta). Blockade of TNF-alpha is currently used clinically in several autoimmune inflammatory diseases. We hypothesised that blocking TNF-alpha with a monoclonal antibody would prevent inflammation and renal fibrosis in crescentic glomerulonephritis. METHODS: Nephrotoxic nephritis was induced in Wistar Kyoto (WKY) rats by intravenous injection of rabbit antirat glomerular basement membrane (GBM) nephrotoxic serum (NTS). Anti-TNF-alpha monoclonal antibody or saline was given intraperitoneally three times per week in four protocols: experiment 1, days 0 to 7; experiment 2, days 0 to 14 and days 4 to 14; experiment 3, days 4 to 28; and experiment 4, days 14 to 28. RESULTS: In experiment 1, rats treated from disease induction had less glomerular fibrinoid necrosis and fewer glomerular macrophages at day 7. In experiment 2, rats treated from day 0 or day 4 showed improved renal function, as judged by serum creatinine, with a significant reduction in crescents. In experiment 3, anti-TNF-alpha treatment significantly reduced urine protein to creatinine ratio and urinary MCP-1 levels. Serum creatinine was preserved at both day 14 and day 28. Tubulointerstitial inflammation, glomerular and tubulointerstitial scarring, and markers of fibrosis [alpha-smooth muscle actin (alpha-SMA) and type IV collagen] were significantly less in treated rats at day 28. In experiment 4, serum creatinine was higher and tubulointerstitial scarring was less in delayed-treated animals. CONCLUSION: Neutralization of endogenous TNF-alpha reduces glomerular inflammation, crescent formation, and tubulointerstitial scarring, with preservation of renal function, in experimental crescentic glomerulonephritis. TNF-alpha blockade is effective even when introduced at the time of maximum glomerular inflammation.
Animals↗
Anti-proteinase 3 antibody binding to neutrophils as demonstrated by confocal microscopy.
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Antibodies, Antineutrophil Cytoplasmic↗
Urinary monocyte chemoattractant protein-1 (MCP-1) is a marker of active renal vasculitis.
BACKGROUND: Macrophage infiltration and cytokine production are important in the pathogenesis of crescentic glomerulonephritis in anti-neutrophil cytoplasm antibody (ANCA)-associated vasculitis. The aim of this study was to investigate whether urinary levels of chemokines, monocyte chemoattractant protein-1 (MCP-1) and fractalkine, were useful tools for non-invasive assessment of renal vasculitis. METHODS: In a prospective study, concentrations of chemokines were measured in urine and serum samples using specific enzyme-linked immunosorbent assays, and related to the patients' clinical status. Renal expression of MCP-1 was studied by immunohistochemical staining of renal biopsies. RESULTS: Urinary levels of MCP-1 were significantly higher in patients with active (P<0.01) or persistent (P<0.05) renal vasculitis, in comparison with healthy volunteers, control patients, patients with inactive vasculitis and patients with extra-renal disease only. There were no differences in serum concentrations of MCP-1 between these groups. Reduction in urinary MCP-1 levels following treatment preceded the improvement of renal function by a median of 2 weeks. In one patient, rising urinary levels of MCP-1, despite immunosuppressive therapy, was associated with progression to severe renal failure. There were no differences in urinary fractalkine levels between the different groups of patients and controls. Immunohistology of renal biopsies from patients with crescentic glomerulonephritis showed increased staining for MCP-1 in glomerular and interstitial cells. Urinary MCP-1 levels correlated with glomerular, but not tubulointerstitial, macrophage infiltration (P<0.05). CONCLUSIONS: This study shows that measurement of urinary MCP-1, but not fractalkine, is a useful non-invasive technique for the assessment of renal involvement and monitoring the response to therapy in ANCA-associated vasculitis.
Adult↗
Renal vasculitis--an update in 2004.
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Antibodies, Antineutrophil Cytoplasmic↗
Leptin-deficient mice are protected from accelerated nephrotoxic nephritis.
Leptin is an adipose tissue-derived hormone that signals nutritional status to the hypothalamus. Recent evidence indicates that leptin modifies proinflammatory immune responses and may provide a key link between nutritional deficiency and immune dysfunction. To study the influence of leptin deficiency on immune-mediated renal disease, susceptibility to accelerated nephrotoxic nephritis was examined in leptin-deficient C57BL/6-ob/ob mice and C57BL/6 wild-type controls. The model was induced with sheep anti-mouse glomerular basement membrane antibody injected to mice preimmunized against sheep IgG, and mice were sacrificed 8 days after induction of disease. The leptin-deficient ob/ob mice were strongly protected from glomerular crescent formation, macrophage infiltration, glomerular thrombosis, and albuminuria in this model. Our findings suggest that leptin is required for the induction and maintenance of immune-mediated glomerulonephritis, and that blockade of the leptin axis might provide an attractive therapeutic possibility in human autoimmune disease.
Animals↗
DNA adducts and p53 mutations in a patient with aristolochic acid-associated nephropathy.
BACKGROUND: Aristolochic acid-associated nephropathy (AAN) is a specific type of renal disease that predisposes patients to a high risk of urothelial carcinoma. The authors have analyzed DNA from a patient who had urothelial malignancy 6 years after presenting with AAN and later had a breast carcinoma that metastasized to the liver. METHODS AND RESULTS: DNA was isolated from the primary breast tumor, the liver tumor, and the original urothelial malignancy and assayed for aristolochic acid (AA)-DNA adducts and mutations in the p53 gene. The adduct detected was the adenosine adduct of aristolochic acid I 7-(deoxyadenosin-N6-yl)aristolactam I (dA-AAI). In DNA from the breast and liver tumors the authors showed the same missense mutation in codon 245 (GGC-->GAC; Gly-->Asp) of exon 7 of p53. In contrast, DNA extracted from the urothelial tumor showed an AAG to TAG mutation in codon 139 (Lys-->Stop) of exon 5. CONCLUSION: A to T transversions, as observed here, are the typical mutations observed in the H-ras gene of tumors induced when rodents are treated with AA and correspond with DNA adduct formation at adenosine residues. These data indicate the probable molecular mechanism whereby AA causes urothelial malignancy.
Aristolochia↗
Microscopic polyangiitis.
Microscopic polyangiitis is a systemic vasculitis affecting smal-l and medium-sized vessels and is characteristically associated with a focal and segmental necrotizing glomerulonephritis. It may present as a pulmonary-renal syndrome with rapidly progressive glomerulonephritis and alveolar hemorrhage, but the pattern of disease will vary according to the organ systems involved. Granulomatous disease of the upper or lower respiratory tract is not a feature, and its presence suggests the diagnosis of Wegener's granulomatosis. The etiology of the condition is unclear, but most patients have antineutrophil cytoplasm antibodies (ANCA) with specificity for either myeloperoxidase (MPO) or proteinase 3 (PR3), and there is increasing evidence that these may be pathogenic. Current treatment includes an induction phase using cyclophosphamide and steroids to attain remission, followed by a maintenance phase in which the levels of immunosuppression are gradually reduced. Azathioprine may be substituted for cyclophosphamide at 3 months. Adjunctive plasma exchange or intravenous methylprednisolone is used in the management of either or both severe renal disease and alveolar hemorrhage, and new evidence suggests that plasma exchange is more effective in recovery of renal function. Overall, 1-year survival in systemic vasculitis is around 85%, and up to 50% of patients relapse, although relapse is less common in those with MPO-ANCA. Newer therapies are being explored in an attempt to increase the efficacy and reduce the toxicity of treatment.
Journal Article↗
The changing profile of acute tubulointerstitial nephritis.
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Acute Disease↗
Retransplantation in Alport post-transplant anti-GBM disease.
BACKGROUND: Post transplant anti-glomerular basement membrane (GBM) disease affects up to 5% of patients with Alport's syndrome. Defects in the COL4A5 gene are responsible for most cases, and alpha 5(IV)NC1 is the usual target for alloantibodies. Gene deletions are more commonly associated with this complication than are point mutations. The disease is severe in renal allografts and nearly always results in graft loss. METHODS: Three cases of retransplantation in Alport's syndrome are described here in detail. All cases were started on immunosuppressive therapy early in the course of their disease and one patient (case 2) received pre-emptive anti-T-cell therapy (Campath IH). Anti-GBM antibodies in these cases were investigated by standard anti-GBM enzyme-linked immunosorbent assay (ELISA), by indirect immunofluorescence, and by Western blotting using collagenase-digested human GBM and recombinant type IV collagen NC1 domains made in insect cells. RESULTS: All cases showed early antibody and complement fixation to human GBM. Target alloantibodies were to alpha 5(IV)NC1 domain predominantly. Cases two and three gave negative results on standard ELISA for anti-GBM antibodies. Pathologic examination revealed crescentic glomerulonephritis, which was rapid in onset in case 1, blunted and less aggressive in case 3, and case 2 developed segmental necrosis without crescent formation. Neutrophilic infiltrates were an early feature in all 3 cases. All cases are compared with a review of all retransplanted cases in the literature. CONCLUSION: Alport anti-GBM disease is a severe disease in retransplanted patients. Anti-T-cell therapy seemed to modify the pathologic findings but did not prevent graft loss. Longer term plasma exchange and mycophenolate mofetil may attenuate the illness, but in these cases did not prevent graft loss. Western blotting detected alloantibodies to alpha 5(IV) NC1 domain and is more sensitive and specific for this disease than standard ELISAs.
Adolescent↗
Blockade of the CD154-CD40 costimulatory pathway prevents the development of experimental autoimmune glomerulonephritis.
BACKGROUND: Experimental autoimmune glomerulonephritis (EAG) was induced in Wistar-Kyoto (WKY) rats by immunization with rat glomerular basement membrane (GBM) in adjuvant. This model is characterized by anti-GBM antibody production, accompanied by focal necrotizing glomerulonephritis with crescent formation. There is also glomerular infiltration by T cells and macrophages. Our hypothesis was that blocking the interaction between CD154 (CD40L) on Th cells and CD40 on antigen-presenting cells should inhibit T-cell activation, and thus the development of EAG. METHODS: The in vivo effects of a hamster anti-rat monoclonal antibody to CD154 (AH.F5) were examined in EAG starting at day -1 prior to immunization, day +7 after immunization, or day +14 after immunization. RESULTS: When administered from day -1 at a dose of 10 mg/kg intraperitoneally three times per week for the duration of the study (4 weeks), AH.F5 resulted in a marked reduction in circulating anti-alpha3(IV)NC1 antibodies, deposits of IgG on the GBM, albuminuria, deposits of fibrin in the glomeruli, severity of glomerular abnormalities, and numbers of glomerular T cells and macrophages. When administered from day +7 at the same dose, AH.F5 resulted in a moderate reduction in the severity of disease, while administration from day +14 had no significant effect. CONCLUSION: These studies demonstrate for the first time that early blockade of the CD154-CD40 T-cell costimulatory pathway can prevent the development of crescentic nephritis, and that delayed treatment can reduce the severity of disease. This confirms the importance of T cell mediated immunity in the pathogenesis of EAG, and suggests that strategies targeting T-cell costimulation may provide a novel approach in the treatment of human glomerulonephritis.
Albuminuria↗
Clinical features and outcome of patients with both ANCA and anti-GBM antibodies.
BACKGROUND: Patients have been described who have both anti-neutrophil cytoplasm antibodies (ANCA) and anti-glomerular basement membrane (GBM) antibodies. We have attempted to define the true prevalence of such "double positive" patients, and describe in detail their clinical features and outcome. METHODS: We have reviewed all serologic assays performed between 1990 and 2000 in a single institution, and the case notes of patients having sera positive for both ANCA and anti-GBM antibodies. During this time 20,392 sera were initially tested for ANCA, and 4808 sera tested for anti-GBM antibodies. RESULTS: Five percent of all ANCA-positive serum samples were also positive for anti-GBM antibodies, and 32% of all anti-GBM positive samples had detectable ANCA. Of 27 patients with both antibodies, 82% had anti-myeloperoxidase specific P-ANCA. Pulmonary hemorrhage occurred in 44%. Renal biopsy showed extensive glomerular cellular crescents in most patients. Patient and renal survival rates were 52% and 26%, respectively, at one year. Sixty-eight percent of patients were dialysis-dependent at presentation, and none of these recovered renal function, despite immunosuppression with or without plasma exchange. CONCLUSION: Serologic evidence of double positivity for both ANCA and anti-GBM antibodies is common in patients with either antibody. In our study these patients have a poor prognosis when presenting with severe disease and initially behave more like anti-GBM disease than vasculitis. Recovery from severe renal failure is rare.
Anti-Glomerular Basement Membrane Disease↗
Glomerular T cells are of restricted clonality and express multiple CDR3 motifs across different Vbeta T-cell receptor families in experimental autoimmune glomerulonephritis.
Experimental autoimmune glomerulonephritis (EAG) is an animal model of Goodpasture's disease which can be induced in Wistar-Kyoto (WKY) rats by a single intramuscular injection of collagenase-digested rat glomerular basement membrane (GBM) in adjuvant. This model is characterised by anti-GBM antibody production, accompanied by focal necrotising glomerulonephritis with crescent formation and glomerular infiltration by T cells and macrophages. Previous work has shown that EAG is a T-cell-dependent disease. We proposed that intraglomerular T cells might be directly involved in pathogenesis and would be oligoclonal. In this study, EAG was induced by standard methods, the kidneys perfused with saline at week 2 and week 4, and the glomeruli separated by a sieving method. Glomerular RNA was extracted and reverse transcribed. RT-PCR showed overexpression of an average of two Vbeta families in each kidney analysed. However, no predominant single Vbeta family was overexpressed in any of the experimental animals. CDR3 spectratyping of Fam-labelled PCR products showed a marked restriction involving different Vbeta families. Sequencing demonstrated multiple CDR3 motifs, each expressed in association with different Vbeta gene segments. Our results show that glomerular T cells are of restricted clonality and suggest a role for antigen-specific effector T cells in the pathogenesis of EAG.
Animals↗