Theoretical and Experimental Evaluations of the Impact of Hemodynamic flow (Hydrostatic Pressure) on hepatic clearance

The proposed project involves investigating in hepatocytes the impact of hemodynamic flow on the exocytosis and related activity of OATP transporters involved in hepatic uptake. This will involve: (a) culturing hepatocytes either with or without hemodynamic flow conditions using the IBIDI flow chamber connected to a peristaltic pump (Year 1). The dimensions of the flow chamber provided by the manufacturer will allow us to determine exactly the stress imposed to cells as a function of the debit. (b) measuring the expression levels of OATPs for each condition using immunohistochemistry and all cell western blotting (Year 1). This will determine whether the hemodynamic flow has any impact on OTAPs expression over time. It is expected that at least week-long experiments will be performed. These results will be confirmed by immunohistochemistry (see below). (c) determining the membrane location of OATPs (internalized/intracellular or exposed to the plasmalemma) using immunohistochemistry and membrane fractionation with gradient gels (Year 2). We will concentrate on using primary antibodies of OATPs targeting the extracellular part of transporters to allow us a double labelling of OATPs outside (step1:without membrane permeabilisation) and inside (step2: after step 1 and following membrane permeabilisation). Membrane fractionation will be performed using the density gradient ultracentrifugation (DGUC) method that is widely used for physical isolation of cellular membranes including vesicles, endoplasmic reticulum (ER), Golgi apparatus, endosomes, and lipid rafts. (d) Kinetic uptake-activity of known OATP drug substrates will be measured for each condition using state of the art UPLC-MS/MS and compared with the in vivo situation (Year 2 - 6 months Vertex placement). This technique combines the physical separation capabilities of liquid chromatography with the mass analysis capabilities of mass spectrometry. (e) investigating the morphology of membrane organelles using specific fluorescent dyes (Year 3). We expect that the application of the hemodynamci flow will impact on organelle structures over time remodelling the membrane trafficking. A number of markers exist and are available commercially for labelling intracellular compartments. (f) finally, a physical/mathematical model based on hydrostatic pressure changes related to the hemodynamic flow will be developed to explain and predict the impact of hemodynamic flow on hepatocyte function (Year 3-4).