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Abstract: TH-PO791

Anti-PLA2R Modulation of Human Podocytes: An In Vitro Model to Investigate the Glomerular Basement Membrane (GBM) in Membranous Nephropathy (MN)

Session Information

Category: Glomerular Diseases

  • 1201 Glomerular Diseases: Fibrosis and Extracellular Matrix

Authors

  • Fresquet, Maryline, University of Manchester, Manchester, United Kingdom
  • Brenchley, Paul E., Manchester Royal Infirmary, Manchester, United Kingdom
  • Lennon, Rachel, University of Manchester, Manchester, United Kingdom
Background

80% of MN patients have IgG autoantibodies to the phospholipase A2 receptor (PLA2R) expressed on podocytes. IgG-PLA2R immune complexes (IC) deposit in the GBM which over time shows expansion with associated proteinuria and consequent nephrotic syndrome. “Spikes” seen on silver stained sections represent formation of new GBM matrix between the deposits. How anti-PLA2R modulates podocyte, IC accumulation in the GBM and the molecular processes that control this GBM dysregulation remain unclear. Hypothesis: Anti-PLA2R treatment of podocytes in vitro will alter cell signaling and matrix composition/deposition.

Methods

In order to identify pathways involved in extracellular matrix (ECM) compositional changes in MN, we performed RNA sequencing and mass spectrometry (MS) analysis. Differentiated human podocytes over-expressing PLA2R were treated with protein G purified human anti-PLA2R from 10min up to 24h. Total RNA was sequenced and differential gene expression analysis was performed between the treated and control groups (P<0.05). For the MS experiment, podocytes were daily challenged with anti-PLA2R over 7 days. Cells were denuded and the deposited matrix analysed. Identified proteins were integrated with the human matrisome database and peptides quantified using Maxquant software.

Results

TGFβ and Hippo signaling pathways were upregulated in the treated podocytes. EGR1, a transcription regulator of target genes involved in inflammatory and tissue damage processes was significantly increased (11fold) 1 hour post treatment. CTGF and CYR61 genes were also upregulated (6 and 3fold) suggesting activation of the YAP/TAZ pathway, component of the Hippo cascade. This pathway maintains podocytes homeostasis, integrity of the GBM and increases expression of matrix genes. Preliminary MS data of the treated podocytes ECM revealed a decrease in laminin and collagen proteins and an increase in ECM regulators and proteoglycans.

Conclusion

Anti-PLA2R can modulate podocyte matrix production in vitro. Our study revealed an early activation of YAP/TAZ pathway in cultured podocytes when treated with anti-PLA2R and a decrease in the main proteins of the GBM at a later stage. Candidate pathways will need to be further validated in order to decipher the mechanism of GBM organisation and remodelling in MN.

Funding

  • Private Foundation Support