Kidney Week

Abstract: TH-PO020

An In Vitro Model of Idiopathic Membranous Nephropathy Reveals PLA2R- and Complement-Dependent Pathways of Podocyte Injury

Session Information

• Complement Your Knowledge of Kidney Disease
  November 02, 2017 | Location: Hall H, Morial Convention Center
  Abstract Time: 10:00 AM - 10:00 AM

Category: Glomerular

• 1001 Glomerular: Basic/Experimental Immunology and Inflammation

Authors

• Haddad, George, University of Zurich, Zurich, Switzerland

George Haddad,

• Kistler, Andreas D., None, Winterthur, Switzerland

Andreas D. Kistler,

Background

Idiopathic membranous nephropathy (iMN) is an autoimmune kidney disease that usually manifests as nephrotic syndrome through damage of podocytes and leads to progressive renal failure in a significant proportion of patients. Recently, the target antigen of autoantibodies in the majority of patients with iMN has been identified as the phospholipase A₂ receptor (PLA₂R). The definitive proof for pathogenicity of PLA₂R antibodies, however, is still lacking. Furthermore, mechanisms of podocyte injury remain elusive, although sublytic complement injury has been proposed. In this study, we aim to develop an in vitro model for iMN to determine downstream mechanisms of anti-PLA2R-antibody mediated injury to podocytes.

Methods

PLA₂R expression levels in conditionally immortalized human podocytes were modulated by infection with a lentivirus vector carrying FLAG-tagged full length human PLA2R or by siRNA-mediated knock down. These cells were then pretreated with sera from PLA₂R-positive iMN patients or control sera and subsequently, human complement was added. Cell lysates were collected and analyzed by qPCR, Western blot, and IF.

Results

Podocytes overexpressing PLA₂R treated with a high-titer (1:1000) PLA2R antibody positive sera and complement in sublytic concentration resulted in decrease synaptopodin and NEPH1 expression with a noticeable synaptopodin rearrangement. The complement sublytic effect on podocytes is likely to involve the activation of the lectin pathway and C3aR1 and C5aR1 signaling as knock down of these receptors rescued synaptopodin and NEPH1. Synaptopodin and NEPH1 degradation appeared to occur via two independent pathways that require cysteine and aspartate proteases, respectively.

Conclusion

Podocyte injury by iMN serum and sublytic complement includes synaptopodin and NEPH1 degradation. In addition, we have developed an in vitro assay to specifically assess the complement-dependent podocytopathic effect of iMN sera that will allow to screen for protective compounds.