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Kidney Week

Abstract: FR-PO1125

Proteomics of Laser-Captured Microdissected Glomeruli and Tubulointerstitium Reveals Extracellular Matrix Remodelling of Kidney Allografts with Antibody-Mediated Rejection

Session Information

  • Transplantation: Basic
    November 08, 2019 | Location: Exhibit Hall, Walter E. Washington Convention Center
    Abstract Time: 10:00 AM - 12:00 PM

Category: Transplantation

  • 1901 Transplantation: Basic

Authors

  • Clotet Freixas, Sergi, University Health Network, Toronto, Ontario, Canada
  • McEvoy, Caitriona M., University Health Network, Toronto, Ontario, Canada
  • Batruch, Ihor, University Health Network, Toronto, Ontario, Canada
  • Kotlyar, Max, University Health Network, Toronto, Ontario, Canada
  • Pastrello, Chiara, University Health Network, Toronto, Ontario, Canada
  • Van, Julie Anh Dung, University Health Network, Toronto, Ontario, Canada
  • Bozovic, Andrea, University Health Network, Toronto, Ontario, Canada
  • Kulasingam, Vathany, University Health Network, Toronto, Ontario, Canada
  • Chen, Pei Xuan, University Health Network, Toronto, Ontario, Canada
  • Chruscinski, Andrzej, University Health Network, Toronto, Ontario, Canada
  • John, Rohan, University Health Network, Toronto, Ontario, Canada
  • Konvalinka, Ana, University Health Network, Toronto, Ontario, Canada
Background

Kidney transplantation is the optimal treatment for end-stage kidney disease, but most grafts fail prematurely. Antibody-mediated rejection (AMR) accounts for >50% of graft loss. AMR is caused by antibodies against HLA and non-HLA antigens in two main renal compartments: glomeruli and tubulointerstitium. We hypothesized that compartment-specific proteome alterations may uncover the mechanisms of early antibody-mediated injury.

Methods

We performed laser-capture/microdissection to isolate glomeruli and tubulointerstitium from FFPE kidney biopsies, and subjected samples to proteome analysis. We compared 7 biopsies with AMR with 23 matched ‘non-AMR’ biopsies with T-cell rejection or acute tubular necrosis. Primary human glomerular microvascular endothelial cells (HGMEC) were studied in vitro.

Results

We identified 2026 proteins in glomeruli and 2399 in tubulointerstitium (FDR=0.01). 120 proteins were differentially expressed (p<0.05) in AMR vs. non-AMR glomeruli and 180 in the tubulointerstitium. Proteins involved in HLA-mediated antigen presentation were increased in AMR. Proteins decreased in AMR were basement membrane components, and belonged to processes such as extracellular matrix (ECM) and cytoskeleton. Reduced glomerular protein levels of LAMC1, NPHS1, and PTPRO in AMR was verified by immunostaining. Levels of ECM proteins correlated directly and significantly (R>0.7; p<0.05), suggesting co-regulation in AMR. Protein expression of CCT8 (cytoskeleton dynamics) and CALU (protein folding) significantly and directly correlated with histological features of AMR, namely glomerulitis and peritubular capillaritis (q=0.017). Protein-protein interaction and pathway analysis of our glomerular protein signature revealed enrichment of inflammatory pathways, such as IL-8 signaling. Stimulation of HGMECs with anti-HLA class I antibody increased the secretion of IL-8 and MCP-1 cytokines.

Conclusion

Basement membranes are often remodeled in late chronic AMR and are the targets of non-HLA antibodies, suggesting that our findings may represent early, important alterations in AMR. Targeting early ECM changes in AMR may represent a new therapeutic opportunity.

Funding

  • Government Support - Non-U.S.