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Kidney Week

Abstract: FR-PO1070

Functional Consequences of Complement Activation on Vascular Endothelial Cells - Results of a Pilot RNA Seq Study

Session Information

Category: Glomerular Diseases

  • 1202 Glomerular Diseases: Immunology and Inflammation


  • Jacobs, Erin Nicole, University of Toronto, Toronto, Ontario, Canada
  • Riedl Khursigara, Magdalena, The Hospital for Sick Children, Toronto, Ontario, Canada
  • Ortiz, Carolina, Hospital for Sick Children, Toronto, Ontario, Canada
  • Licht, Christoph, The Hospital for Sick Children, Toronto, Ontario, Canada

Complement dysregulation on vascular endothelial cells (ECs) causes EC injury and leads to thrombotic microangiopathy (TMA), such as atypical hemolytic uremic syndrome (aHUS). As 50% of aHUS causing mutations are unknown, there is clinical need to find additional pathways and genes involved in the TMA phenotype. Here, we report the results of an unbiased RNA seq approach to investigate complement-induced responses of ECs on a molecular level.


Complement activation on ECs was induced via sensitization, activating the classical complement pathway, on blood outgrowth endothelial cells (BOECs) from healthy controls. RNA was isolated 1 hour after treatment. A stranded paired end RNA seq library was used, quality assessed with FASTQC software, and adapters trimmed using Trim Galore. Manual analysis of regulated genes was carried out with a cutoff of fold change > |1.5|. Gene function was determined based on literature analysis (pubmed; genecards). Functional assays included neutrophil adhesion in a microfluidic chamber (Bioflux), wound closure assay, measurement of transendothelial resistance with a voltohmmeter and IF staining for VE-Cadherin.


RNA seq revealed three major findings, (i) upregulation of genes involved in leukocyte adhesion (e.g. ICAM, E-selectin); (ii) regulation of genes collectively resulting in loss of cytoskeletal re-arrangement (e.g. downregulation of RhoA activators and upregulation of ROCK 1 inhibitors); and (iii) downregulation of genes involved in cell adhesion (e.g. VE-Cadherin). Functional relevance of these findings was confirmed by complement-induced (i) EC neutrophil adhesion (Bioflux); (ii) defective EC migration (wound closure assay); and (iii) decreased transendothelial resistance and VE-Cadherin staining (voltohmmeter and IF). Importantly, all these effects were reversible when complement activation was blocked, using complement inactivated serum.


Complement activation on ECs results in an array of molecular responses functionally resulting in (i) neutrophil adhesion, (ii) defective cell migration, and (iii) loss of cell-cell adhesion. Detailed mechanism and their link to the clinical phenotype of TMA remains to be elucidated in future research.


  • Government Support - Non-U.S.