Electronic Thesis and Dissertation Repository

Thesis Format

Integrated Article

Degree

Doctor of Philosophy

Program

Pathology and Laboratory Medicine

Supervisor

McIntyre, Christopher W

2nd Supervisor

Pasternak, Stephen

Co-Supervisor

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.

Summary for Lay Audience

Hemodialysis (HD) is a lifesaving treatment for those suffering from kidney failure. HD replaces some functions of the kidneys by removing waste products and excess fluid from the blood. However, HD causes negative side effects, such as decreases in blood pressure, damage to blood vessels, and reduced blood flow to various organs (heart, brain, liver, and kidneys). Currently, only specialized, time-intensive, and costly imaging methods are used to monitor and understand this kind of HD based injury. Development of a blood test could provide similar results while also expanding access and reducing cost. Research has found that measuring components of blood called Extracellular Vesicles (EVs) may be valuable to study as markers of HD injury. EVs are tiny fragments of injured or activated cells that may be able to identify blood vessel injury and as a result organ injury. My work focuses on developing and refining an EV based blood test to determine whether it is a valuable tool to use among HD patients. To explore this, I first created and refined an EV based technique for a highly sensitive machine used to analyze EVs. This ensured that we would obtain reliable and consistent results. Next, I exposed human blood vessel cells to HD-related stress and saw that when inflammation conditions were present, smaller EVs are produced. Furthermore, to test the effect of HD we exposed rats to a scaled-down version of HD. Similar to humans, the rats presented with lowered blood pressure and reduced blood flow to their leg muscle during HD. They also had an increase in EV levels in their blood over the same HD treatment. Among HD patients, EV levels also increased over the treatment. We found that depending on the EV levels that patients had at the beginning of their HD treatments, there was a connection with the severity of complications experienced during their HD session. Higher EV levels before treatment were associated with greater decreases in blood pressure and heart injury during HD. Altogether my work has shown that an EV based blood test has the potential to monitor and assess HD induced injury.

Creative Commons License

Creative Commons Attribution-Noncommercial 4.0 License
This work is licensed under a Creative Commons Attribution-Noncommercial 4.0 License

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