University of Southern California / Children's Hospital Los Angeles
Introduction: Primary membranous nephropathy (MN) is a leading cause of nephrotic syndrome in adults worldwide due to the deposition of anti-podocyte-anlibodies against in the glomerular subepithelial space. While complement deposition is thought to play a crucial pathogenic role, the exact effector mechanism of complement in MN is unclear due to the lack of in vitro and in vivo systems that recapitulate human disease. We have developed a novel glomerulus-on-a-chip system (GOAC) using human primary podocytes and glomerular endothelial cells (GEC) and assessed functional response to human MN serum, role of Membrane-attack-complex (MAC) formation and C3a/C3aR1 signaling in MN pathogenesis.
Methods: GOACs were cultured with serum containing either anti-PLA2R+ or THSD7A+ MN patients; sera from healthy individuals were used as control. Functional response was assessed by albumin permeability assay to evaluate permselectivity. Role of MAC and C3a/C3aR1 signaling pathway was assessed by immunofluorescence and functional analysis while mechanisms of action were explored by PCR arrays, Western Blotting and immunostaining. Results were confirmed in vitro using podocytes on which C3aR1 was silenced and in vivo using THSD7A induced MN in balb/c mice.
Results: Following exposure to sera from MN patients, we have confirmed deposition of human lgG on podocytes and formation of MAC complex, accompanied by albumin leakage. MAC inhibition did not prevent albumin leakage while GOAC supplemented with C3aR1 antagonists as well as GOAC using podocytes in which C3aR1 was silenced were able to prevent glomerular filtration damage and albumin leakage. Efficacy of C3aR1 antagonists in preventing proteinuria was confirmed in vivo, substantiating our findings.
Conclusions: We have successfully developed a glomerulus-on-a-chip system that closely mimics the GFB structure and provides a powerful tool for studying renal regenerative and disease mechanisms in proteinuric diseases. Using a combination of in vitro and in vivo models, we showed that C3a/C3aR signaling plays a dominant role in complement-mediated MN pathogenesis.