Abstract: FR-PO106
Inhibition of BRD4 Leads to Reduced Activated Neutrophils and Adhesion to Endothelium After Ischemia-Reperfusion Injury
Session Information
- AKI: Mechanisms - Inflammation/Sepsis/Remote Injury
November 08, 2019 | Location: Exhibit Hall, Walter E. Washington Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Acute Kidney Injury
- 103 AKI: Mechanisms
Authors
- Reid, Shelby, University of Toronto, Toronto, Ontario, Canada
- Fine, Noah A., University of Toronto, Toronto, Ontario, Canada
- Bhosle, Vikrant K., The Hospital for Sick Children, Toronto, Ontario, Canada
- Robinson, Lisa, The Hospital for Sick Children, Toronto, Ontario, Canada
- Scholey, James W., University of Toronto, Toronto, Ontario, Canada
Background
Following ischemia reperfusion injury (IRI), pro-inflammatory cascades partly dependent on NFkB, lead to neutrophil recruitment to the site of injury. Neutrophil accumulation then contributes to tissue injury through the release of pro-inflammatory cytokines, reactive oxygen species and proteases. We hypothesize that blockade of NFkB with the BRD4 inhibitor, MS417, will reduce neutrophil recruitment to the kidney following IRI.
Methods
Male C57BL/6 mice were treated with 1μM of MS417 for 7 days before unilateral ischemia was performed followed by reperfusion for 24 hours. Kidney tissue, blood and bone marrow was collected for neutrophil analysis using a custom flow cytometry panel. For in vitro studies, freshly isolated neutrophils from healthy volunteers were labeled with Calcein AM and incubated with MS417 media for 1 hour. HUVEC were activated by TNFα for 4 hours and then incubated with neutrophils and allowed to adhere for 30 minutes. Non-adherent cells were removed and neutrophil adhesion was quantified using a fluorescent plate reader.
Results
Absolute neutrophil counts increased in the bone marrow (p<0.0001 vs sham) and blood (p<0.04 vs sham) 24 hours after IRI and there was a trend towards increased neutrophils in the kidney (p=0.12 vs sham). Primed neutrophils (upregulated CD66a expression) did not increase in the bone marrow and blood at 24 hours, but increased in the kidney (p=0.06 vs sham). BRD4 inhibition reversed IRI induction of neutrophil counts in the bone marrow (p<0.0001 vs IRI) and blood (p<0.03 vs IRI), and in the kidney (trend). In contrast, BRD4 inhibition did not lead to significant decrease in primed neutrophils in the bone marrow and blood, but did reduce primed neutrophils in the kidney (p<0.03 vs IRI), reversing the effects of IRI alone. In vitro studies showed that TNFα increased neutrophil adhesion to HUVEC (p<0.0001 vs control). Pre-treatment of neutrophils with the BRD4 inhibitor led to a significant decrease in neutrophil adhesion (p<0.0001 vs TNFα–activated HUVEC).
Conclusion
BRD4 inhibition blocked neutrophil upregulation of the adhesion receptor, CD66a, neutrophil adhesion to activated endothelial cells, and recruitment of neutrophils to the kidney, following IRI. BRD4 inhibition may represent a therapeutic approach to limiting IRI in the kidney.