OSAI

A rare disease which affects the kidney, membranous nephropathy is the second-most common cause of nephrotic syndrome. The OSAI project is using novel technological approaches to shed new light on the disease, research which could lead to the development of rapidly acting new therapies, as Principal Investigator Professor Pierre Ronco explains

A rare disease which affects structures inside the kidney, membranous nephropathy is caused by a thickening of the base membrane of the glomerulus, a filtering unit in the kidney. This thickening is caused by the accumulation of immune deposits, as Professor Pierre Ronco explains. “There are deposits of antibodies and of antigens. The consequence of that is the development of proteinuria – loss of proteins through the urine – and the decrease of the albumin serum in the blood,” he says. Based at the INSERM Institute and University Pierre et Marie Curie in Paris, Professor Ronco is the Principal Investigator of the OSAI project, an EC-backed initiative investigating the pathophysiology of membranous nephropathy, which he coordinates with his co-worker, Dr Hanna Debiec (Research Director at INSERM). “The disease can evolve in three ways; spontaneous remission, persistent proteinuria, or endstage renal failure,” he says. “It has been shown that this disease is auto-immune in nature – in most cases the antigen is localised in the glomerulus. The patient produces antibodies directed to this antigen, which become deposited in the glomerulus and this induces the disease.”

The project’s primary objective is to understand the triggering events that lead to the disease and the pre-disposing factors behind positive or negative outcomes. This builds from the current understanding of how the disease develops and progresses. “The first step is the presentation of the antigens to the immune system by the HLA class 2 molecules – for instance HLA-DQA1,” explains Professor Ronco. The immune system produces antibodies which circulate in blood and are then deposited in the glomerulus, where Professor Ronco says they react with the antigens. “This activates a complement cascade, leading to the formation of the membrane attack complex of complement. This induces injury to the podocyte, and to the capillary wall, which in turn induces proteinuria and can eventually lead to nephrotic syndrome,”

he outlines. “What is not known is the trigger event which induces the production of antibodies in patients with predisposing variants. Molecular mimicry with microbes or toxic agents could play an important role.”

This forms a key element of the project’s research agenda. Membranous nephropathy is a complex disease, and typically develops as the result of a sequence of events. “We are interested in each step of the disease. We are interested

in the first step, in the triggering event that stimulates the production of antibodies. We are also interested in the last step, the formation of the complex of complement, which attacks the podocyte membrane, and the development of

Figure 1. Mechanisms of immune mediated podocyte injury in MN and pathogenesis based therapeutic approach.

proteinuria,” says Professor Ronco. One major area of interest is the gene variants which could be involved in complement activation. “We think that there are maybe genetic variants in the complement cascade, which are responsible – to varying degrees – for activation of the complement, and which thus may regulate formation of the membrane attack complex of complement, resulting in varying severity of disease,” continues Professor Ronco. “We aim to find new drugs that could potentially block the formation of this complex of complement.”

The disease does not invariably progress to a point where treatment is required though, as while in some cases patients require dialysis, around a third of patients will go into spontaneous remission. “Most patients present with nephrotic syndrome and a third of them will undergo remission,” says Professor Ronco. Researchers are also interested in the mechanisms that induce spontaneous remission of nephrotic syndrome. “It could be the disappearance of the triggering event, or possibly another mechanism is involved,” says Professor Ronco. Membranous nephropathy is known to be the second most common cause of nephrotic syndrome, underlining the wider importance of research in this area. “There are different causes of nephrotic syndrome. Some are immunological in nature and others are metabolic, including diabetes,” explains Professor Ronco.

Researchers are using cutting-edge techniques to investigate the successive steps of membranous nephropathy. The first technique is called HLA peptidomics, a method which is being used to identify epitopes, the relevant immune response targets. “Our objective is to identify T-cell epitopes of the major antigen PLA2R, that are recognised by the T-lymphocytes,” outlines Professor Ronco. A T-cell epitope is the part of a complex formed with HLA class-2 molecules that is presented to the immune system, which leads to the production of antibodies. “These HLA class-2 molecules can circulate in blood in combination with the epitope, which is recognised and presented by this molecule. However, the amounts of circulating HLA epitope-complexes are very low. So we have embarked on a collaboration with our colleagues in Zurich (Tim Fugmann, Dario Neri), who are very experienced in peptidomics,” continues Professor Ronco. “They are trying to set up a very sensitive method to identify the epitopes combined to HLA class-2 molecules in the blood.”

This technique has never previously been used to study auto-immune disorders where antigens or epitopes are presented by HLA class 2 molecules. While the technique is relatively untested, Professor Ronco says the early signs are promising. “Our collaborators have reached the first step, where epitopes complexed with HLA class-2 molecules can be identified. We are now starting a second step, where we will try to identify these complexes in patients with membra漀 us nephropathy or other immunological glomerular diseases,” he outlines. This approach will help researchers to monitor the progression of the disease and predict patient outcomes. “We think that the patients with active disease will have circulating HLA epitope complexes in the blood, whereas those patients in remission will probably have a very low amount of these complexes, or no complexes at all,” says Professor Ronco.

The second key technique being used within the project is molecular modelling of the membrane attack complex of complement. This is a highly complex molecule, with multiple components, and once assembled it can damage key structures within the kidney. “When assembled, this complex induces injury to podocytes and increases the permeability of the capillary wall to proteins,” explains Professor Ronco. Project collaborator Bogdan Iorga at CNRS in Gif-sur-Yvette, a research campus close to Paris, has developed a dynamic model, encompassing each step of the assembly process. “With this model, we will be able to identify each of the possible intermediate steps of the membrane attack complex assembly. Then we can potentially propose new compounds for the treatment of membranous nephropathy,” continues Professor Ronco. “The objective will be to block the assembly of the membrane attack complex of complement, and consequently prevent the onset of proteinuria.”

The current methods of treating membranous nephropathy rely primarily on immuno-suppressive agents, such as corticosteroids, cyclosporine and cyclophosphamide. However, Professor Ronco says that alongside the toxicity of immuno-suppressive agents, there are two main problems with this method of treatment. “The first is that a maximum of only around 70 percent of patients will enter remission of proteinuria. The second is that the effect of the treatment is delayed,” he explains. The project aims to develop alternative new therapies that will work more rapidly. “We want to develop new drugs that can block complement assembly on the surface of the podocyte in the glomerulus, because we hope that this new treatment will be active very rapidly,” outlines Professor Ronco. “Also, for those patients who are resistant to immuno-suppressive therapy, we hope to at least limit the level of inflammation in the glomerulus.”

Figure 2. Schematic description of the new diagnostic methodology based on analysis of plasma soluble HLA peptidome.

Figure 3. The MAC, C5b-9 complex formation. C5b-6 initiates pore formation via the sequential recruitment of homologous proteins: C7, C8, and 12–18 copies of C9. The C5b-9 complex forms transmembrane pores in cell membranes. Red arrows: designed inhibitors that blocks assembly and membrane insertion of the terminal complement complex.

This research could also hold implications beyond membranous nephropathy, as the condition is considered to be a paradigm of organ-specific autoimmune disease. However, while these approaches could potentially be used to study autoimmune conditions affecting other organs, Professor Ronco says the project’s current focus is very much on membranous nephropathy. “We hope to deliver additional biomarkers, especially T-cell epitopes, as identified by the peptidomic approach, that are associated with the disease,” he says. Cases of membranous nephropathy can vary in severity, so Professor Ronco says it’s important to identify those patients who need treatment and those who don’t. “A major objective is to propose predictive biomarkers to the clinicians, in order to determine those patients who will need treatment with immuno-suppressive agents and complement antagonist drugs, and those who won’t,” he says.

Membranous nephropathy : a model for solving organ-specific auto-immunity (OSAI)

Membranous nephropathy (MN) is a paradigm of organ-specific autoimmune disease which affects the kidney glomerulus, resulting in the formation of immune deposits, complement-mediated proteinuria, and renal failure. My group has recently identified 2 causative antigens (neutral endopeptidase and bovine serum albumin) in children, and 2 predisposing genes HLA-DQA1 and PLA2R1 coding for another antigen in adult MN.

ERC-AG - ERC Advanced Grant

Project Coordinator, Pierre Marie Victor Ronco Institut National de la Santé et de la Recherche Médicale T: + 33 1 56016639 E: pierre.ronco@tnn.ap-hop-paris.fr W: http://cordis.europa.eu/project/ rcn/108575_en.html

Pierre Marie Victor Ronco

Pierre Ronco was appointed full Professor of Nephrology at UPMC in 1986. He became head of the Renal division in 1995 and director of the INSERM research unit in 1998. He was appointed senior member of the prestigious Institut Universitaire de France in 2008. He served as President of the Scientific Councils of Medical Faculty Saint-Antoine and Francophone Kidney Foundation, President of the Francophone Society of Nephrology, councillor of the International Society of Nephrology (ISN) and of the European Society of Nephrology ERA-EDTA and President of the 49th ERA-EDTA congress in Paris (May 2012, 8600 delegates). He is currently Vice-President of the Francophone Kidney Foundation, and member of the NIH supported network on rare kidney diseases (Neptune).