The anti-neutrophil cytoplasm antibody (ANCA)-associated vasculitides (AAV) are a group of life-threatening multi-system diseases characterized by necrotising inflammation of small blood vessels and ...crescentic glomerulonephritis. ANCA are thought to play a direct pathogenic role. Previous studies have shown that spleen tyrosine kinase (SYK) is phosphorylated during ANCA-induced neutrophil activation in vitro. However, the role of SYK in vivo is unknown. Here, we studied its role in the pathogenesis of experimental autoimmune vasculitis, a pre-clinical model of myeloperoxidase-ANCA-induced pauci-immune systemic vasculitis in the Wistar Kyoto rat. Up-regulation of SYK expression in inflamed renal and pulmonary tissue during early autoimmune vasculitis was confirmed by immunohistochemical and transcript analysis. R406, the active metabolite of fostamatinib, a small molecule kinase inhibitor with high selectivity for SYK, inhibited ANCA-induced pro-inflammatory responses in rat leucocytes in vitro. In an in vivo study, treatment with fostamatinib for 14 days after disease onset resulted in rapid resolution of urinary abnormalities, significantly improved renal and pulmonary pathology, and preserved renal function. Short-term exposure to fostamatinib did not significantly affect circulating myeloperoxidase-ANCA levels, suggesting inhibition of ANCA-induced inflammatory mechanisms in vivo. Finally, SYK expression was demonstrated within inflammatory glomerular lesions in ANCA-associated glomerulonephritis in patients, particularly within CD68+ve monocytes/macrophages. Thus, our data indicate that SYK inhibition warrants clinical investigation in the treatment of AAV.
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Antibody mediated rejection is a major cause of renal allograft loss. Circulating preformed donor specific antibodies (DSA) can result as a consequence of blood transfusion, pregnancy or prior ...transplantation. Current treatment strategies are limited due to partial or transient efficacy, adverse side-effects or patient unsuitability. Previous in vivo studies exploring autoimmune diseases have shown that spleen tyrosine kinase (SYK) signalling is involved in the development of pathogenic autoantibody. The role of SYK in allogenic antibody production is unknown, and we investigated this in a rodent model of sensitization, established by the transfusion of F344 whole blood into LEW rats. Two-week treatment of sensitized rats with selective SYK inhibitor fostamatinib strongly blocked circulating DSA production without affecting overall total immunoglobulin levels, and inhibition was sustained up to 5 weeks post-completion of the treatment regimen. Fostamatinib treatment did not affect mature B cell subset or plasma cell levels, which remained similar between non-treated controls, vehicle treated and fostamatinib treated animals. Our data indicate fostamatinib may provide an alternative therapeutic option for patients who are at risk of sensitization following blood transfusion while awaiting renal transplant.
Anti-neutrophil cytoplasm antibody (ANCA) associated vasculitis with renal involvement requires treatment with potentially toxic drugs to reduce morbidity and mortality, and there is a major ...challenge to determine clinical and histological features predictive of renal prognosis. The aim of our study was to evaluate the use of the 2010 international histological classification for ANCA-associated glomerulonephritis (AAGN) as a predictor of renal outcome when used in conjunction with other prognostic factors.
One hundred and four patients with AAGN treated at our centre were included: 23 were classified as focal, 26 as crescentic, 48 as mixed and 7 as sclerotic. Renal outcomes were based on estimated glomerular filtration rate (eGFR) at 1 and 5 years, and on renal survival.
By univariate analysis, patients in the focal class had the best renal outcome, those in the sclerotic class the worst outcome, and those in the mixed and crescentic classes had intermediate renal survival. There was no significant difference in outcome between the mixed and crescentic classes. In multivariate models, histological class did not improve model fit or associate with renal outcome after adjusting for established prognostic factors. Lower percentage of normal glomeruli, greater degree of tubular atrophy (TA), MPO-ANCA positivity, increasing age and lower starting eGFR, all correlated with poorer renal outcomes.
We conclude that, in our cohort of patients, the international histological classification is predictive of renal outcome in AAGN, but did not appear to be additionally informative over other established prognostic factors in multivariate analysis. However, it may be of value to combine the current histological classification with other established parameters, such as TA and percentage normal glomeruli.
More reliable biomarkers using near-patient technologies are needed to improve early diagnosis and intervention for patients with renal disease. Infrared (IR) vibrational ...spectroscopy/microspectroscopy is an established analytical method that was first used in biomedical research over 20 years ago. With the advances in instrumentation, computational and mathematical techniques, this technology has now been applied to a variety of diseases; however, applications in nephrology are just beginning to emerge. In the present study, we used attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy to analyze urine samples collected from rodent models of inflammatory glomerulonephritis (GN) as well as from patients with crescentic GN, with the aim of identifying potential renal biomarkers; several characteristic mid-IR spectral markers were identified in urine samples. Specifically, a 1545 cm
band increased in intensity with the progression and severity of GN in rats, mice and humans. Furthermore, its intensity declined significantly in response to corticosteroid treatment in nephritic rats. In conclusion, our results suggest that specific urinary FTIR biomarkers may provide a rapid, sensitive and novel non-invasive means of diagnosing inflammatory forms of GN, and for real-time monitoring of progress, and response to treatment.
Abstract Background The anti-neutrophil cytoplasm antibody (ANCA)-associated vasculitides (AAV) are a group of life-threatening multisystemic diseases characterised by necrotising inflammation of ...small blood vessels and crescentic glomerulonephritis. Existing treatments are not always effective and are often complicated by substantial toxicity. Previous studies have shown that spleen tyrosine kinase (SYK) is phosphorylated during ANCA-induced neutrophil activation, and thus it represents a potential therapeutic target. We aimed to investigate the role of SYK in the pathogenesis of an in-vivo model of AAV, and to establish whether SYK is expressed in human disease. Methods We studied the effect of fostamatinib, a small molecule inhibitor selective for SYK, in experimental autoimmune vasculitis (EAV), which is a rodent model of AAV that is induced by immunisation of Wistar Kyoto rats with the ANCA target antigen, myeloperoxidase. Animals (eight per group) with established disease, as confirmed by onset of haematuria and proteinuria 4 weeks after immunisation, were treated with vehicle, fostamatinib 20 mg/kg, or fostamatinib 30 mg/kg by twice daily oral gavage from week 4 to 6 and then assessed for disease severity at the end of week 6. We also used immunohistochemical techniques to analyse SYK expression and activation in human renal biopsy specimens from patients with ANCA-associated glomerulonephritis. Findings In EAV, at the end of week 6, there was a dose-dependent reduction in proteinuria (median 2·88 mg/day IQR 1·80–6·37 vs 0·13 0·00–0·78 vs 0·23 0·00–0·59 in vehicle, fostamatinib 20 mg/kg, and fostamatinib 30 mg/kg groups, respectively; p=0·0004), haematuria (3 arbitrary units AU 2–3 vs 0 0–0·38 vs 0 0–0·038, p=0·0018), glomerular histological abnormalities (12% 3–29 vs 5 2–9 vs 0 0–6, p=0·0262), glomerular macrophage infiltration (1·77 1·28–2·58 AU vs 0·25 0·20–0·41 vs 0·20 0·06-0·41, p=0·0004), lung haemorrhage severity (3 AU 2·0–3·8 vs 1 0·3–1·8 vs 0 0·1–0·4, p<0·0001), and haemosiderin deposition in lung tissue (3·06 AU 0·93–4·14 vs 0·48 0·17–0·70 vs 0·19 0·06–0·42, p=0·0087). Immunohistochemical analysis confirmed SYK expression in human ANCA-associated glomerulonephritis. Staining for phospho-SYK demonstrated SYK activation that localised to diseased lesions (segmental necrosis, areas of extracapillary proliferation). Glomerular SYK expression correlated with histological class of disease (crescentic 3·0% 2·1–3·8, mixed 1·4% 0·92–2·4, focal 1·1% 0·7–1·8, sclerotic 0·3% 0·1–0·5; p=0·01). Interpretation Our results suggest that, in a preclinical model, SYK inhibition with fostamatinib is an effective treatment for crescentic glomerulonephritis and lung haemorrhage, the life-threatening manifestations of AAV, even after onset of disease. SYK is activated in human ANCA-associated glomerulonephritis, and expression correlates with disease severity, suggesting that SYK contributes to the pathogenesis of human disease. These observations support the potential investigation of targeting SYK in clinical studies of AAV. Funding UK Medical Research Council.
Co-presentation with both ANCA and anti-GBM antibodies is thought to be relatively rare. Current studies of such ‘double-positive’ cases report small numbers and variable outcomes. To study this ...further we retrospectively analyzed clinical features and long-term outcomes of a large cohort of 568 contemporary patients with ANCA-associated vasculitis, 41 patients with anti-GBM disease, and 37 double-positive patients with ANCA and anti-GBM disease from four European centers. Double-positive patients shared characteristics of ANCA-associated vasculitis (AAV), such as older age distribution and longer symptom duration before diagnosis, and features of anti-GBM disease, such as severe renal disease and high frequency of lung hemorrhage at presentation. Despite having more evidence of chronic injury on renal biopsy compared to patients with anti-GBM disease, double-positive patients had a greater tendency to recover from being dialysis-dependent after treatment and had intermediate long-term renal survival compared to the single-positive patients. However, overall patient survival was similar in all three groups. Predictors of poor patient survival included advanced age, severe renal failure, and lung hemorrhage at presentation. No single-positive anti-GBM patients experienced disease relapse, whereas approximately half of surviving patients with AAV and double-positive patients had recurrent disease during a median follow-up of 4.8 years. Thus, double-positive patients have a truly hybrid disease phenotype, requiring aggressive early treatment for anti-GBM disease, and careful long-term follow-up and consideration for maintenance immunosuppression for AAV. Since double-positivity appears common, further work is required to define the underlying mechanisms of this association and define optimum treatment strategies.
Maintenance therapy for antineutrophil cytoplasmic antibody-associated vasculitis (AAV) should reduce rates of relapse with minimal toxicity. The MAINRITSAN trial—the first randomized controlled ...trial to compare the efficacies of rituximab and azathioprine in AAV remission maintenance— has demonstrated a superior outcome using rituximab. These data have important implications for the management of AAV.
ABSTRACT
Background
Current guidelines advise that rituximab or cyclophosphamide should be used for the treatment of organ-threatening disease in anti-neutrophil cytoplasm antibody (ANCA)-associated ...vasculitis (AAV), although few studies have examined the efficacy and safety of these agents in combination.
Methods
We conducted a single-centre cohort study of 66 patients treated with a combination of oral corticosteroids, rituximab and low-dose pulsed intravenous cyclophosphamide followed by a maintenance regimen of azathioprine and tapered steroid for the treatment of biopsy-proven renal involvement in AAV. Patients were followed for a median of 56 months. Case–control analysis with 198 propensity-matched cases from European Vasculitis Study Group (EUVAS) trials compared long-term differences in relapse-free, renal and patient survival.
Results
At entry, the median Birmingham Vasculitis Activity Score (BVAS) was 19 and estimated glomerular filtration rate was 25 mL/min. Cumulative doses of rituximab, cyclophosphamide and corticosteroids were 2, 3 and 4.2 g, respectively, at 6 months. A total of 94% of patients achieved disease remission by 6 months (BVAS < 0) and patient and renal survival were 84 and 95%, respectively, at 5 years. A total of 84% achieved ANCA-negative status and 57% remained B cell deplete at 2 years, which was associated with low rates of major relapse (15% at 5 years). The serious infection rate during long-term follow-up was 1.24 per 10 patient-years. Treatment with this regimen was associated with a reduced risk of death {hazard ratio HR 0.29 95% confidence interval (CI) 0.125–0.675, P = 0.004}, progression to end-stage renal disease (ESRD) HR 0.20 (95% CI 0.06–0.65), P = 0.007 and relapse HR 0.49 (95% CI 0.25–0.97), P = 0.04 compared with propensity-matched patients enrolled in EUVAS trials.
Conclusions
This regimen is potentially superior to current standards of care, and controlled studies are warranted to establish the utility of combination drug approaches in the treatment of AAV.
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