Aquaporin-1 and pressure-driven water transport across aortic endothelia. Aquaporin-1 expression, distribution and regulation

In this thesis we utilize quantitative immunohistochemistry techniques to identify and show the existence and distribution of Aquaporin-1 (AQP1) on both the luminal and abluminal membranes of whole vessel rat aortic endothelial cells. We also note evidence from the theses of my labmates (T. Nguyen, S. Russell, Y. Xue) that either blocking aquaporins chemically or knocking down their expression reduces endothelial hydraulic conductivity (the ratio of the transmural water flux to the driving pressure difference), both in cultured cell monolayers and in excised whole vessels ex vivo.;Hypertension is a known risk factor for atherosclerosis and we postulate that one external condition that may influence a vessel's AQP expression is its transmural pressure. We confirm this hypothesis in two male rat models: (1) Normotensive Wistar Kyoto rats and their genetically modified hypertensive cousins, the Spontaneously Hypertensive rats (SHR). (2) Non-genetically modified normotensive Sprague-Dawley rats, Sprague-Dawley rats made hypertensive by having undergone the Goldblatts procedure that induces the renin-angiotensin pathway to hypertension and Sprague-Dawleys that have undergone a sham operation. Using quantitative immunohistochemistry we show that endothelial cells from the both groups of chronically hypertensive rats appear to express far more AQP than their normotensive analogues. This evidence, in aggregate, supports the hypothesis that aortic endothelial cells may be able to actively regulate their aquaporin expression in response to chronic hypertensive conditions, which results in partial control of their transmural transport processes.;Water flows as discrete entities through an AQP, however we used the continuum Navier Stokes approach for modeling this flow and calculating its hydraulic conductivity; the results obtained were in close proximity to experimental data and molecular simulations results. We also present simple kinetic models based on the Law of Mass Action to describe molecular transcription and translation processes that describe our observed transmural pressure-induced differential AQP1 expression. This mechanism assumes that changes in transmural pressure impact the transcription factors (TFs) that serve as a regulatory point for the control of gene expression. We first examine the model's steady states for different transmural pressures and compare with data from our lab. From these data we extract certain model kinetic parameters and find ranges for them. We then extend our findings by numerically solving (using Matlab&dotbelow; RTM 7.3.0) the dynamical version of our model. In particular, we predict the time dependence of the concentration of TFs, mRNA and AQP1 in response to step changes in transmural pressure. We also postulate that siRNA against AQP1 mRNA destroys or degrades these mRNAs with second order kinetics, and in the process themselves are consumed. This loss of siRNA subsequently affects gene expression until the siRNA concentration is severely depleted and the mRNA and its protein can recover. We solve for the corresponding dynamics of several cases of AQP1 reduction upon siRNA introduction and its subsequent recovery with siRNA depletion.;Improved understanding of AQP1 regulation by, e.g., transmural pressures, may lead to novel therapies, not just in the case of atherosclerosis, but also for a wide variety of human diseases. (Abstract shortened by UMI.)...