Development of an Extracellular Vesicle-Based Biomarker of Hemodialysis Induced Vascular Injury
Abstract
Hemodialysis (HD) is a life-saving treatment for severe chronic kidney disease but results in ischemic organ injury which contributes to an increased risk of cardiovascular (CV) disease, stroke, and death. HD-induced ischemic injury is monitored using complex imaging-based approaches which are not suitable for routine use. Currently there is no reliable blood-based biomarker that is comparable to findings seen from imaging-based studies and is also suitable for clinical practice. However, biomarkers of growing interest pertaining to HD-induced microcirculatory injury, are circulating endothelial and platelet derived Extracellular Vesicles (EVs), which are known to directly reflect cellular activation and apoptosis. Therefore, the purpose of this thesis was to optimize and validate nanoscale flow cytometry (nFC) for EV-based analysis to determine the utility of EVs as biomarkers of HD-induced vascular injury through using in vitro, in vivo, and observational patient-based studies. Our results showed that through refining pre-analytical guidelines, the nFC is an appropriate methodology for EV enumeration, characterization, and linear detection of EVs between approximately 100 and 1,000 nm. Utilizing this sensitive methodology, we found that the uremic milieu has minimal impact on EV levels, though inflammatory stress caused by lipopolysaccharide resulted in an increase in small sized (<500nm) endothelial EVs. To further understand the direct impact of HD and its potential influence on EVs (concentration and size), we used a rodent model of HD and applied intravital microscopy to observe muscle perfusion during HD. These experiments demonstrated that HD causes hypo-perfusion of muscle and hemodynamic instability, and an increase in endothelial and platelet EVs over HD. To translate to a clinical context, we used observational patient-based studies and observed that EV levels increased over HD. Additionally, we found that Pre-HD EV levels correlated with important measures of HD-induced injury such as ultrafiltration rate, hypotension, and cardiac contractile function. Altogether, my thesis describes a comprehensive approach which determines and evaluates an EV based assay for HD-induced vascular injury. Our overall findings are promising and warrant further refinement of an EV-based approach to monitor and risk assess patients receiving HD.