Membranous nephropathy (MN) although a rare disease affecting 1/100,000 individuals each year, is the most common cause of abundant protein loss in the urine (nephrotic syndrome) in the Caucasian adult, ending to end-stage renal failure in 20 to 40% of patients. The disease can also occur in young children and recur in the grafted kidney compromising renal function. MN is very challenging to treat as 30 to 40% of patients will benefit from spontaneous remission in the first 12 months and immunosuppressive treatment may induce severe side effects. The most common form in the adult referred to as idiopathic MN (iMN) is considered a paradigm of autoimmune disease with the pathogenic antibodies being directed to antigens localized at the surface of epithelial cells of the glomerulus (the kidney filtrating structure). In the last ten years, substantial advances have been made in the understanding of the molecular bases of MN with the identification of several antigens (such as the phospholipase A2 receptor-1) and predisposing genes although they have brought more complexity with the description of several antigen-antibody systems and the finding that circulating anti-PLA2R1 antibodies are not always associated with PLA2R1 antigen deposits in glomeruli and with recurrence of MN in the graft. Further progress based on new insights into the disease pathophysiology and characterization of robust biomarkers should be achieved to determine which patient to treat, when to stop treatment, and how the patients should be monitored.

We have funded a European consortium for the study of membranous nephropathy which forms the core research teams involved in work package-3. The consortium constituted of British, Czech, Dutch and French teams has collected national cohorts of >1,000 patients with iMN and a large biorepository of serum and DNA from these patients.

Patients database and biobanking
Starting from the existing resources (clinical phenotype, sera, DNA, biopsies) accessible for most patients we will first unify datasets, produce a template for collaborative studies in and outside EURenOmics and standardize quality controlled sampling. We will expand our resources by enrolling new patients with idiopathic and secondary (SLE-, hepatitis B-, cancer- and drug-related) MN, with rare familial cases of MN, as well as pairs of donors/recipients with recurrent and de novo MN occurring in the graft). Particular emphasis will be put on a careful follow-up of patients. Completion of this task is a prerequisite to international collaborative studies, biomarker discovery and patenting, and pseudorandomized trials using propensity scoring.

Identification of pathogenic B-cell epitopes and gene variants responsible for disease initiation, progression and recurrence in the graft
Disease-specific epitopes and antigens will be identified using patients’ sera and Ig eluted from MN glomeruli, by immunoproteomics of glomerular protein extracts, reactivity studies with PLA2R1 domains, and holistic approaches. In PLA2R1-positive patients, we will sequentially analyse the target epitopes, the IgG subclasses and the antibody avidity in spontaneous or treatment-induced remission versus active disease. In PLA2R1- negative patients with primary MN (about 20 to 30%) or secondary MN, we will focus on the identification of new antigens. Studies will be focused on patients with a regular follow-up to investigate intra- and inter-molecular epitope spreading and relevance of anti-idiotype network to disease outcome.

Based on the results of our GWAS study, we hypothesize that sequence variations in PLA2R1 may control expression at the podocyte membrane and the pattern of antigen processing, anti-PLA2R1 avidity and quality of anti-idiotype network. We will search for PLA2R1 and HLA class II gene variants by exon (coding region) sequencing of PLA2R1 in patients with idiopathic MN and next generation sequencing of the genomic loci of the identified PLA2R1 and HLA-DQA1 alleles. Fine mapping of the GWAS results utilizing newest HLA sequencing data and sequencing data from our well-defined MN cohort will provide unparalleled insights into the role of the HLA locus.

The pathogenic effects of epitopes and gene variants will be tested in vitro and in vivo. The effects of PLA2R1 variants on cell trafficking, membrane expression and epitope presentation will be assessed in transfected podocyte cell cultures. The cell alterations induced by antibody binding to newly identified podocyte epitopes will be analysed in the presence or the absence of complement Podocyte-specific transgenic mice will be generated for the antigens of interest, starting with mice with inducible expression of PLA2R1 (to avoid podocyte senescence), to establish new MN experimental models. We will also test whether immunisation with the immunodominant peptides can induce MN in mice.

Characterization of T-cell epitopes, T-cell regulation and triggering events involved in disease initiation, remission and recurrence
Specific T-cell frequencies will be monitored by FACS and ELISPOT in patients treated or untreated, in relation to anti-PLA2R1 levels, and peptide binding to T-cells and to HLA will be explored .The Treg/Theff/Th17 balance will be investigated at disease onset, remission, relapse and under treatment, and the biomarker profile of these cells will be determined in the blood at the protein (Luminex), mRNA (transcriptome), and miRNA levels.

Development of proprietary assays for the identified biomarkers and assessment of their diagnostic and predictive values: towards a new ontology
ELISA, and protein/antigen, peptide/epitope and gene chips will be developed, the ultimate goal being the construction of MN-specific DNA and peptide arrays which will allow personalized diagnostic and therapy. Urinary bi-omarkers of disease stage will be sought by proteomics, miRNomics and metabolomics in whole urine and exosomes in a carefully selected population of patients and compared with other diseases (SRNS, HUS). We will determine the diagnostic and predictive value (outcome, response to treatment) of the whole set of biomarkers in large cohorts of patients. Bioinformatics expertise will be instrumental in clustering the data and providing both reference -omics profile for each category of patients, and for the development of appropriate preventive (grafted patients) or therapeutic personalised interventions.