Podocyte cell communication through microRNA-containing exosomes and autophagy in membranous glomerulonephritis

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Primary membranous glomerulonephritis (MGN) is caused by autoantibodies binding to podocyte antigens. The most frequent autoantibody detectable in MGN is the anti-phospholipase A2 receptor antibody (PLA2R-ab). However, it remains unknown how autoantibodies reach the subepithelial space in MGN because the glomerular filtration barrier is usually impermeable for antibodies. The glomerular filtration barrier is composed of specialized glomerular endothelial cells (GEC), the GBM and podocytes. Nephronectin (NPNT) is a podocyte extracellular matrix protein regulated by GEC-derived miR-192. Recently, we could show that knockdown of Npnt in zebrafish induces edema, proteinuria, podocyte effacement and structural changes in the GBM resembling MGN. Furthermore, NPNT was downregulated in patients with MGN.In this project, we would like to investigate the role of cell-cell communication through miR containing exosomes and autophagy in MGN. We speculate that GEC-derived miR-192 is packed in exosomes and crosses the GBM to reach podocytes where it is taken up by podocyte filopodia and down regulates NPNT. We want to study effects of miRs packed in exosomes in podocyte monoculture, in our 3D glomerular co-culture model and analyze functional aspects of miR-based glomerular cell-cell communication in various zebrafish models, including a transgenic zebrafish line with fluorescent autophagosomes and in vivo morpholinos to knock down genes essential for autophagy. We would like to interfere pharmacologically with exosome release and study the relationship between autophagy, exosome release and exosome uptake that will be studied with innovative methods such as pH-dependent fluorescent exosome labelling in vivo and in vitro, autophagy flux measurements, as well as scanning ion conductance microscopy (SICM) and SEM. We would like to investigate the role of NPNT regulating miR-192 not only in exosome- and miR-mediated cell-cell communication, but also in the context of autophagy and endolysosomal pathways in podocytes in vivo and in vitro. In this context, we will evaluate urinary miR-192 as non-invasive biomarker for MGN.The relationship between the extracellular protein NPNT and autophagy has not been investigated so far. This aspect will be studied in a cell culture setup, but also in our podocyte-specific Npnt knockout mice. We will investigate how podocyte specific knockout of Npnt facilitates autoantibody passage in a THSD7A mouse model for MGN and in addition, we will us the transgenic PLA2R mouse to investigate the role of Npnt, miRs and autophagy in MGN. Finally, a potential link between diabetic nephropathy and MGN through miRs and NPNT will be addressed in podocyte-specific Npnt knock out mice treated with Streptozotocin.We hope that this research will help us to better understand the pathophysiological role of miRs and autophagy in MGN with potential relevance for non-invasive biomarker and therapeutic targets for future clinical use.