Towards robust animal models of ANCA-associated Vasculitis

ANCA-associated systemic vasculitis (AASV) affects 1000 people each year in the UK, approximately half of whom will die or develop permanent kidney failure as a direct result of the disease or its treatment. During the past 20 years, a large body of evidence has been gathered together which suggests that it is a specific antibody termed ANCA which is responsible for initiating damage to blood vessels in the kidney and other organs in this disease. Current treatments are usually effective but they frequently cause serious side-effects such as infection and many patients are left with significant permanent organ damage by the time the disease is brought under control. Furthermore, AASV frequently relapses which requires treatment to be intensified again, further increasing the risk of side-effects. Recently understanding of AASV has been advanced by the development of 2 models which recreate the disease in rats and mice. These have been significant developments because the disease process is a complex one and whereas studying human cells outside of the body (in the test tube) provides important information, it cannot fully represent what happens inside human organs in the same way that the rodent models do. This proposal will further develop and improve the two rodent models so that they better represent the human disease and recreate the conditions where relapses occur. In particular, the proposal will compare damage to the kidney as it occurs in the animals with damage seen in humans, both at the level of the whole tissue and at the levels of cells and genes. These are important experiments because they will help to verify how closely the models reflect human disease and identify any significant differences. From these studies it will be possible to test new therapies in the models with a realistic expectation that they be can transferred to patient treatment where they show promising results. The importance of minimizing the use of animals has been a central theme in the development of this proposal and by carefully designing experiments to address specific scientific questions, the number of animals used and the extent of any suffering will be restricted as far as possible. It is because the human disease that is being studied is a complex, severe and life-threatening condition for which current treatments have significant limitations, that it is necessary undertake some studies in rodents.

Over the past two decades a paradigm has been developed using experiments with human cells describing potential mechanisms of microvascular injury in ANCA-associated vasculitis (AASV). This disease causes renal failure and lung haemorrhage, and is fatal if untreated. Current treatments are blighted by an unacceptable burden of adverse effects. The main issues to be addressed in this condition are: 1. Development of novel targeted therapies through increased understanding of basic pathogenic mechanisms and 2. Investigation of remission and relapse with a view to assessment of biomarker signatures.

For the first time we are now in a position to begin testing this paradigm using two recently described animal models of AASV: Experimental Autoimmune Vasculitis (EAV, a rat model relying upon immunisation with the ANCA antigen myeloperoxidase, MPO) and Murine Experimental Vasculitis (MEV, a passive mouse model induced by passive transfer of anti-MPO antibodies). We aim to optimise these two models by adjusting antigen dose, administration route or adjuvant components with a view to generating a model representative of human AASV: crescents affecting 50% of glomeruli and reproducible lung haemorrhage. We then plan to further develop the EAV model to mimic relapsing AASV using inflammatory stimulators and/or repeated MPO immunisation. We envisage the EAV model serving as a test bed for novel therapies as it more closely mimics human autoimmune AASV than the passive MEV model. We plan to further develop the MEV model to permit analysis of pathogenic mechanisms of anti-MPO antibody microvascular injury, with focus on the pathophysiologically relevant tissues in the kidney and lung using intravital microscopy of these organs. The optimised MEV model would permit use of the genetically modified mice currently available for probing specific molecular mechanisms.

We then intend to validate the phenotype of tissue injury in EAV and MEV against that seen in human AASV. In addition to standard readouts of histology and renal biochemistry, we will dissect out affected glomeruli and compare them using Affymetrix GeneChip technology and micro-fluidic targeted PCR microarrays to compare gene expression across the three species. We also intend to investigate whether the ANCA effects on neutrophils observed in human systems also pertain in the EAV and MEV models. By developing these two optimised models of AASV we will be well placed to begin translating the findings of the large body of in vitro work towards the clinic for patient benefit.