Date of Award
8-2013
Document Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
Legacy Department
Bioengineering
Committee Chair/Advisor
Nagatomi, Jiro
Committee Member
Dean , Delphine
Committee Member
Purves , Todd
Committee Member
Yao , Hai
Abstract
A majority of men and women aged 40 and over experience lower urinary tract symptoms, including urgency, incontinence, and frequency, which often affect the individual's quality of life. Although often considered a simple structure, the bladder is a complex system with sophisticated sensory and motor feedback mechanisms that allow for the sensation of fullness and pain, reflexive responses to bladder filling, and conscious control over the time and place of micturition. Although disruptions of these sensory mechanisms are believed to cause certain lower urinary tract dysfunctions, the specific mechanisms involved in sensing bladder fullness, distension, tension, or pressure at the cellular level have yet to be fully elucidated.
The work presented here has been undertaken to determine if the epithelial cells that line the urinary tract are mechanosensitive to hydrostatic pressure, which may serve as a potential bladder sensing mechanism. This project explored the hypothesis that urothelial cell sensitivity to hydrostatic pressure is transduced as part of (1) a calcium signaling response, (2) a volume change due to modified ion flux, or (3) an inflammatory response through activation of caspase-1. Changes in these cellular parameters can potentially modulate cellular responses to bladder filling, including bladder surface area increase and neurotransmitter release for communication with afferent nerves. These potential responses were examined using fluorescence imaging techniques, including novel imaging methods and equipment, which enabled the measurement of intracellular calcium concentration, cell volume, and inflammasome activation in live urothelial cells under physiological and pathophysiological pressures while subjected to various pharmacological agents.
Previous in vitro studies in our lab have found that urothelial cells release adenosine triphosphate (ATP) upon the application of an increased hydrostatic pressure without stretch, and this effect was dependent upon extracellular calcium. Therefore, we first investigated the effects of increased hydrostatic pressure on intracellular calcium concentration of urothelial cells via live-cell calcium imaging experiments. We have shown that a detectable increase in intracellular calcium concentration does not occur in either primary rat urothelial cells or UROtsa cells, an immortalized human urothelial cell line. Thus, ATP is likely not released from urothelial cells as part of a calcium signaling process upon changes in hydrostatic pressure.
We further tested the hypothesis that a hydrostatic pressure stimulus induces a cell volume change in urothelial cells due to modified ion flux across the cell membrane. Both 3D volume reconstruction of confocal image stacks and real-time fluorescence intensity imaging with a volume sensitive dye were performed on urothelial cells while under physiological levels of hydrostatic pressure. We have shown that cell volume increases that exceed our system sensitivity likely do not occur due to increased hydrostatic pressure under the experimental conditions of this study.
Finally, we explored whether physiological or pathophysiological levels of hydrostatic pressure initiate an inflammatory response through activation of caspase-1, which potentially could serve as a bladder sensing mechanism or as part of the inflammation cascade experienced in the case of Bladder Outlet Obstruction. Results of imaging experiments to measure real-time caspase-1 activation, however, failed to show a measurable change induced by a 1-hour exposure to increased hydrostatic pressure of up to 40 cmH2O.
Cumulatively, these results show that a release of ATP from urothelial cells in response to hydrostatic pressure likely does not occur due to a short-term increase in cytoplasmic calcium concentration or membrane stretch due to cell swelling. Additionally, observed increases in caspase-1 activation do not appear to be due to short term cellular responses of urothelial cells to increased hydrostatic pressure without stretch. Although the mechanism responsible for hydrostatic pressure sensing was not identified, it is hoped that the improved understanding of these mechanosensory mechanisms provided by this research, as well as the experimental techniques developed to investigate them, may ultimately lead to more specific drug targets to treat lower urinary tract symptoms, as well as treatments for other potentially pressure-related diseases, including glaucoma, hypertension, and bone loss.
Recommended Citation
Champaigne, Kevin, "Ion Channel-mediated Hydrostatic Pressure Mechanotransduction in Urothelial Cells" (2013). All Dissertations. 1167.
https://open.clemson.edu/all_dissertations/1167