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

Lead Research Organisation: University of Nottingham
Department Name: School of Veterinary Medicine and Sci


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).


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Studentship Projects

Project Reference Relationship Related To Start End Student Name
BB/M016374/1 30/09/2015 29/09/2019
1644977 Studentship BB/M016374/1 30/09/2015 29/09/2019 Lewis Burton
Description Application of pressure to porcine liver cells and human derived cell lines cultured in a laboratory increases the expression and production of enzymes involved in drug metabolism.
Application of hydrostatic pressure to human hepatocytes significantly increased the expression of certain drug metabolising enzymes. This results in ~2-3-fold increase in the clearance of a probe substrate in in vitro studies. The typical under-prediction of drug clearance falls within the range of 2-3-fold. This results in increased metabolism could explain some of the under predictions seen when cells cultured in vitro are used to predict drug metabolism seen in vivo. Thus, applying pressure could lead to more accurate translations from in vitro work to in vivo and reduce the use of animals in pre-clinical drug discovery.
Exploitation Route 1. Expanding the range of pressure applied.
2. Applying pressure to cells in 3D culture.
3. looking at the effect of non-metabolising genes
Sectors Pharmaceuticals and Medical Biotechnology

Description DMDG 46th Annual meeting 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact 10 Postgraduate students studying in the area were chosen for free places with travel expenses covered, to present their research as a poster and 2 minute oral presentation to a collection of both industrial and academics in the field. The intended outcome was for the next generation of DMPK scientists to interact with current industrial and academic leaders in the area and share ideas.
Year(s) Of Engagement Activity 2017