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Abstract: SA-OR28

C3a/C3aR1 Signaling as a Crucial Pathogenic Mechanism in Membranous Nephropathy

Session Information

Category: Glomerular Diseases

  • 1302 Glomerular Diseases: Immunology and Inflammation

Authors

  • Da Sacco, Stefano, Children's Hospital of Los Angeles, Los Angeles, California, United States
  • Zhang, Qi, Children's Hospital of Los Angeles, Los Angeles, California, United States
  • Bin, Sofia, Icahn School of Medicine at Mount Sinai, New York, New York, United States
  • Petrosyan, Astgik, Children's Hospital of Los Angeles, Los Angeles, California, United States
  • Sanchez Russo, Luis F., Icahn School of Medicine at Mount Sinai, New York, New York, United States
  • Villani, Valentina, Children's Hospital of Los Angeles, Los Angeles, California, United States
  • Gentile, Micaela, Icahn School of Medicine at Mount Sinai, New York, New York, United States
  • De Filippo, Roger E., Children's Hospital of Los Angeles, Los Angeles, California, United States
  • Lemley, Kevin V., Children's Hospital of Los Angeles, Los Angeles, California, United States
  • Perin, Laura, Children's Hospital of Los Angeles, Los Angeles, California, United States
  • Cravedi, Paolo, Icahn School of Medicine at Mount Sinai, New York, New York, United States
Background

Primary membranous nephropathy (MN) is a leading cause of nephrotic syndrome in adults worldwide caused by the deposition of anti-podocyte-antibodies 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 can faithfully 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 exposed to anti-PLA2R+ sera from MN patients; sera from healthy individuals were used as control. Functional response was assessed by albumin permeability assay to evaluate permselectivity. Role of PLA2R1, IgGs, complement, MAC and C3a/C3aR1 signaling pathway were assessed by immunofluorescence, western blotting and functional analysis while mechanisms of action were explored by PCR arrays, proteomics 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 confirmed lgG deposition, complement activation and MAC formation accompanied by albumin leakage. PLA2R silencing on podocytes, IgG neutralization as well as complement inactivation successfully prevented injury while leakage still occurred following MAC inhibition. GOAC supplemented with C3aR1 antagonists as well as GOAC using podocytes in which C3aR1 was silenced were able to counteract glomerular filtration damage and prevent albumin leakage and lessen oxidative stress in podocytes. Efficacy of C3aR1 antagonists in preventing proteinuria was confirmed in vivo, substantiating our findings.

Conclusion

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.

Funding

  • NIDDK Support