Kidney Week

Abstract: TH-PO008

Modeling Glomerular Complement Activity In-Vitro Using a Novel Human Glomerular Endothelial Cell Assay

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

• 1005 Clinical Glomerular Disorders

Authors

• Ruan, Qin, Regeneron Pharmaceuticals, Tarrytown, New York, United States

Qin Ruan,

• Devalaraja-Narashimha, Kishor B., Regeneron Pharmaceuticals, Tarrytown, New York, United States

Kishor B. Devalaraja-Narashimha,

• Mao, Shu, Regeneron Pharmaceuticals, Tarrytown, New York, United States

Shu Mao,

• Broden, Joshua Reid, Regeneron Pharmaceuticals, Tarrytown, New York, United States

Joshua Reid Broden,

• Morton, Lori, Regeneron Pharmaceuticals, Tarrytown, New York, United States

Lori Morton,

• Macdonnell, Scott, Regeneron Pharmaceuticals, Tarrytown, New York, United States

Scott Macdonnell,

Background

Reproducible methods for evaluating inhibitory effects of drug candidates on complement activation are essential for preclinical development and potential clinical translation. Due to the complexity of complement activation pathways, an in-vitro assay should use relevant cells and endpoints to the given therapeutic indication. Here, using primary human glomerular endothelial cells (HGECs), we validated a complement C3 & C5 deposition model for use evaluating the blocking activity of C5 monoclonal antibodies (mAb).

Methods

Resting or adenosine 5′-diphosphate (ADP)-activated confluent HGECs were incubated for 4 hours with 50% normal human serum, C3 or C5 depleted human serum, or pretreated with anti-C5 mAbs. Thereafter, HGECs were fixed with BD Cytofix, stained with ThermoFisher anti-human C3/C3b or Abcam anti-human C5b-9 antibodies and complement deposits were analyzed (9 sites per well in duplicated wells) by ImageXpress.

Results

C3 and C5b-9 deposition was observed on ADP-activated HGECs exposed to normal human serum but not on non-activated HGECs (C3: 1.5x10⁷±1.0x10⁷; C5: 7.9x10⁶±6.6x10⁶, P<0.05 vs non-ADP-activated HGEC). The deposition of C3 and C5b-9 were significantly reduced on ADP-activated HGEC exposed to C3 or C5 depleted serum (C3: 3.3x10⁵±4.8x10⁴; C5: 1.5x10⁶±6.0x10⁵, P<0.05). Addition of a blocking anti-C5 mAb significantly reduced normal human serum derived C5b-9 deposition onto ADP-activated HGEC, deposition was comparable to C5 depleted sera (C5 mAb: 1.02x10⁶±6.0x10⁵, Control mAb 3.7x10⁶±1.6x10⁶, P<0.05 vs. control mAb).

Conclusion

These data demonstrate utility of an in vitro primary human glomerular endothelial cell assay to model complement C3 & C5 deposition. In addition to in-vitro screening, this assay offers potential as a translational model to evaluate anti-complement strategies in renal disease using patient derived serum samples.

Funding

• Commercial Support –