Date of Award
5-2024
Document Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
Department
Bioengineering
Committee Chair/Advisor
Jiro Nagatomi
Committee Member
Sarah Harcum
Committee Member
J. Todd Purves
Committee Member
F. Monty Hughes
Abstract
Bladder outlet obstruction (BOO) is a prevalent urological condition and can be characterized by the presence of lower urinary tract symptoms (LUTS), such as hesitancy, weak stream, and nocturia. The main cause of BOO is a partial blockage of the urethra, which results in elevated voiding pressure, high storage pressure, and tissue ischemia, which are thought to be the triggers of bladder inflammation and subsequent fibrosis. Often patients do not seek treatment until the conditions get severe. Thus, it would be ideal to have an early diagnostic tool to detect changes in the bladder due to BOO. Extracellular vesicles (EVs) found in urine are an attractive option as they may contain biomarkers that can be collected non-invasively. Urothelial cells (UCs) lining the bladder lumen are subjected to hypoxia and elevated pressure and contribute to bladder inflammation. In addition, these stimuli may also influence EV and exosome release and bring forth changes in the cargo of these EVs. Although studies have implicated pressure and hypoxia as contributing factors to BOO, the role of these stimuli in the development of BOO pathology has yet to be elucidated. In addition, the impacts of these stimuli on UC release of EVs and cargo changes has yet to be explored.
The objective of the current study was to elucidate the role of hypoxia in the initiation of inflammation in UCs and to reveal how pressure and hypoxia lead to changes in exosomal cargo. The objectives were achieved through the following aims: 1) characterize a two-enzyme system for creating an in vitro hypoxic environment, 2) quantify the urothelial cell response to hypoxia and identify inflammasome activation mechanisms and 3) characterize exosome release from UCs in response to pressure and hypoxia. The novel use of enzymes to create hypoxic environments in vitro enabled us to investigate the effects of hypoxia on UCs. In addition, a custom bioreactor was used to apply pressure cycles to UCs.
From this study, we demonstrated that hypoxia leads to inflammation in a time-dependent manner. Furthermore, hypoxia-induced inflammation was primarily driven through the ROS/TXNIP/NLRP3 pathway. Pressure and hypoxia were found to stimulate the release of exosomes from urothelial cells. Through RNA Seq analysis of EV cargo, pressure led to changes in genes expressed and non-coding RNAs. The findings from this study offer new therapeutic target candidates for BOO and can aid in the development of a non-invasive diagnostic tool.
Recommended Citation
Hudson, Britney, "An in Vitro Investigation of Urothelial Cell Function in Response to Hypoxia and Pressure in Relation to Bladder Outlet Obstruction" (2024). All Dissertations. 3550.
https://tigerprints.clemson.edu/all_dissertations/3550