Investigating Alternative Mechanisms of Injury in Membranous Nephropathy in a Human Glomerulus-On-A-Chip System
Qi Zhang, PhD1, Paolo Cravedi, MS2, Astgik Petrosyan, PhD3, Valentina Villani, PhD1, Paola Aguiari, PhD1, Roger De Filippo, MD1, Laura Perin, PhD1, Paolo Cravedi, MD2, Stefano Da Sacco, PhD3.
1Children's Hospital Los Angeles, los angeles, CA, USA, 2Mount Sinai, New York, NY, USA, 3Children's Hospital Los Angeles, Los Angeles, CA, USA.
BACKGROUND: Membranous nephropathy (MN) is a major cause of nephrotic syndrome in adults due to anti-podocyte antibodies depositing in the glomerular subepithelial space. Despite complement deposition and membrane-attack-complex (MAC) formation being considered crucial in its pathogenesis, our glomerulus-on-a-chip system (GOAC) showed that vitronectin-mediated MAC inhibition did not prevent albumin leakage, indicating an alternative injury mechanism. We tested this hypothesis using in vitro and in vivo methods.
METHODS: GOACs were generated using human primary podocytes and glomerular endothelial cells and cultured with serum from anti-PLA2R+ patients or healthy controls. Albumin permeability was used to evaluate permselectivity. The roles of PLA2R1, IgG, complement, MAC, and C3a/C3aR1 were assessed through various techniques while mechanisms of action were explored using PCR arrays, proteomics, and immunostaining. The effects of MN serum on podocytes were also assessed.
RESULTS: Exposure to MN patient sera confirmed human lgG deposition on PLA2R-expressing podocytes and MAC complex formation with albumin leakage. MAC inhibition did not prevent albumin leakage while GOACs supplemented with C3aR1 antagonists or using C3aR1-silenced podocytes lessened oxidative stress in podocytes, counteracted glomerular filtration damage, and prevented albumin leakage. C3aR1 antagonists were effective in preventing proteinuria in THSD7A-induced MN in mice.
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.
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