Characterisation and establishment of the chPLA2R1 mouse model and the identification of pathomechanisms

Abstract

Membranous nephropathy (MN) is the most common cause of nephrotic syndrome in adults, with a very heterogeneous patient outcome. Primary MN is a rather slowly progressive and chronic disease that is defined by the accumulation of immune deposits at the glomerular basement membrane. These immune deposits are formed through autoantibodies targeting surface proteins of podocytes like phospholipase A 2 receptor 1 (PLA2R1) or thrombospondin type 1 domain-containing 7A (THSD7A). Binding of these autoantibodies will subsequently lead to the binding of complement factors and ultimately result in podocyte damage. However, the exact pathomechanism of how the antibodies damage the podocytes remained largely unknown and the role of the complement system in disease initiation and progression is discussed controversially. To dissect the detailed pathomechanisms and the role of the complement system, a suitable animal model is essential. Since PLA2R1 is not expressed on podocytes in mice, transgenic mouse lines had to be generated. Previously two such mouse models were established, both with some major limitations. One model is based on a mouse line that expresses murine PLA2R1 in poddocytes. These mice exhibit a normal phenotype and will only get sick after passive transfer of the anti-PLA2R1 antibodies. The other model is based on a mouse line expressing human PLA2R1 on the podocytes. These mice will develop autoantibodies from birth on, thus exhibiting a very rapid and exerbating phenotype. Both mouse lines can not resemble the rather slow development of MN. In an effort to generate a mouse model with an active and inducible diease onset, we sought to develop a chimeric mouse strain. We hypothesised that the first three human head domains are immunogenic enough to provoke a robust disease onset, while the backbone of murine C-type lectin-like domains (CTLD) 2 to 8 will maintain the tolerance towards the chimeric protein, preventing the spontaneous formation of autoantibodies. In vivo characterisation of the chimeric PLA2R1 mice, showed that the knock-in of chimeric PLA2R1 does not interfere with a healthy podocyte morphology and behavior. Upon immunisation with hPLA2R1 the mice develop an antigen-specific and immune-mediated disease. Additionally, the mice showed the typical clinical pattern in morphology regarding mIgG and complement deposition. To better understand the role of the complement system in disease progression, we generated a mouse line expressing the chPLA2R1 with a simultaneous, global knock-out of complement component 3 (C3). After immunisation, these showed an attenuated course of disease. Onset of proteinuria was delayed and less severe, accompanied by a longer overall survival. These experiments showed that the complement system plays a role in the pathogenesis of this model of experimental PLA2R1 associated MN. However, the mice still got sick, indicating that also complement independent mechanisms play a role in disease initiation and progression. To gain insight into potential pathomechanisms, single nucleus RNA sequencing analysis revealed ephrin type-A receptor (Epha6) and receptor tyrosine kinase Mer (MerTK) as relevant genes for disease progression. Based on the snRNA-Seq results pathway analysis showed predicted activation of pathways involving actin-cytoskeleton remodeling and receptor tyrosine kinase signaling in diseased mice.