The role of neutrophil serine proteases in vasculitic glomerulonephritis and potential as therapeutic targets.

Lead Research Organisation: University of Birmingham
Department Name: Immunity and Infection

Abstract

Kidney disease and kidney failure may be caused by inflammation within the filtering units of the kidney, so called glomerulonephritis. In some forms of glomerulonephritis, white blood cells release proteins that can cause tissue damage if the normal control processes are not working properly. One protein that we are very interested in is called proteinase 3 (PR3). We believe that PR3 is not only released by white blood cells but that it activates the endothelial cells lining the tiny capillaries of the glomerular filters, units that cleanse the blood and form urine. Activated endothelial cells attract more white blood cells into the glomerular filters, so that inflammation and damage quickly escalate to involve the whole of the glomerular filter.
The project aims to understand the effects of PR3 on the specialised endothelial cells within the glomerular filters (GEnC) - these special cells may be particularly vulnerable to the activating and damaging effects of PR3. We would also like to understand how these GEnC interact with the circulating white blood cells, called neutrophils, which express PR3 on their surface and release PR3 within glomerular capillaries in patients with vasculitis. We will monitor the development of inflammation products and how these relate to biomarkers of cell damage. We will then be able to tell if drugs that inhibit the effects of PR3 can prevent cell damage. Finally, we will culture GEnC with another specialised kidney cell, glomerular epithelial cells (GEpiC) which are separated from GEnC by a membrane, and which together form the glomerular filter. We will examine whether PR3 and neutrophils that have escaped across GEnC, then draw GEpiC into the inflammation process. Again, we will test whether drugs that inhibit the effects of PR3 can stop GEpiC responding in this way. Some of the drugs that inhibit PR3 are already being used for treating other diseases, so if our studies suggest benefit, it should be possible to undertake clinical trials in patients with vasculitis.

Technical Summary

Vasculitic glomerulonephritis is the commonest cause of rapidly progressive glomerulonephritis leading to kidney failure. There is increasing evidence that neutrophil serine proteases such as proteinase 3 (PR3) and elastase, are linked pathogenically to development of the glomerulonephritis. Further, PR3 has some unique affiliations with development of vasculitis that make it of particular interest. PR3 is an antigenic target for the anti-neutrophil cytoplasm autoantibodies (ANCA) that develop in vasculitis and, through neutrophil activating abilities, PR3-ANCA have been linked to vascular damage. Our recent in vitro studies that model neutrophil-endothelial cell interactions under flow and static conditions, have suggested a key role for neutrophil serine proteases, rather than products of the respiratory burst, in endothelial cell (EnC) injury caused by ANCA-activated neutrophils.
To address the hypothesis that neutrophil serine proteases, particularly PR3, are the key mediators of vasculitic glomerulonephritis, we will examine the effects of PR3 and elastase on inflammatory responses by glomerular endothelial cells (GEnC), utilising our in vitro static and flow models of vasculitis. This will be the first time that specialised GEnC have been tested in vasculitis models. We will compare the effects of PR3 and elastase on cell survival, cytokine and growth factor responses, adhesion molecule expression and neutrophil adhesion, and permeability responses; mRNA microarrays for inflammatory mediators will be performed to test for diverse differences between PR3 and elastase. The PR3-activated GEnC will be incorporated into flow models with ANCA-activated neutrophils; readouts will comprise neutrophil adhesion/migration and endothelial biomarkers of injury. How these responses may be inhibited by targeting PR3 will be examined. To model the pathology of vasculitic glomerulonephritis, we will recapitulate the glomerular capillary wall by novel co-cultures involving GEnC and glomerular epithelial cells (GEpiC) since the inflammatory responses of GEnC and GEpiC may be modified in this environment; as before, PR3 will be targeted to determine whether this inhibits injury responses. Finally, to confirm relevance of our models to the in vivo situation we will relate our findings to mediator expression in renal biopsies from patients with vasculitic RPGN.
Defining injury mechanisms induced by neutrophil serine proteases and their potential for inhibition will permit transition from pre-clinical studies to clinical trial, thereby leading to better therapies to prevent development of renal failure in vasculitic glomerulonephritis.

Publications

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Kuravi SJ (2012) Neutrophil serine proteases mediate inflammatory cell recruitment by glomerular endothelium and progression towards dysfunction. in Nephrology, dialysis, transplantation : official publication of the European Dialysis and Transplant Association - European Renal Association

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Kuravi SJ (2014) Podocytes regulate neutrophil recruitment by glomerular endothelial cells via IL-6-mediated crosstalk. in Journal of immunology (Baltimore, Md. : 1950)

 
Description Influenced training of practitioners directly and via guidelines
Geographic Reach National 
Policy Influence Type Influenced training of practitioners or researchers
Impact Invited to speak at educational updates - nationally and internationally
 
Description PhD Studentship
Amount £28,750 (GBP)
Organisation Kidney Research UK 
Sector Charity/Non Profit
Country United Kingdom
Start 09/2007 
End 09/2010
 
Description Project grant/Stuart Strange Vasculitis Trust
Amount £102,510 (GBP)
Organisation Vasculitis UK 
Sector Charity/Non Profit
Country United Kingdom
Start 01/2007 
End 12/2010
 
Description Studentship
Amount £75,000 (GBP)
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 08/2010 
End 08/2014
 
Title Chimeric antibodies 
Description Chimeric antibodies to proteinase 3 of IgG1, IgG3 and IgG4 subclasses 
Type Of Material Antibody 
Year Produced 2009 
Provided To Others? Yes  
Impact Dissection of neutrophil Fc receptor engagement by different IgG subclass antibodies 
 
Title In vitro modeling of glomerular cell-cell interactions 
Description Co-culture of glomeraulr epithelail cells (podocytes) and glomeraulr endothelial cells to model the human glomerulus more closely 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Provided To Others? No  
Impact To explore glomerular cell cross-talk during infallamtory process 
 
Description Patient group 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Geographic Reach National
Primary Audience Patients, carers and/or patient groups
Results and Impact Gave evidence orally. Answered questions. Subsequently interviewed on Radio

Patient engagement with research. Funding. Application of new technologies enabled
Year(s) Of Engagement Activity 2008,2009,2015,2016,2017