Development of a bioartificial kidney (BAK) as a renal transport model

DMPK (Drug Metabolism and Pharmacokinetics) studies are essential for drug development but 90% of potential compounds are rejected in clinical trials, thus increasing the cost and time required for any drug to enter the market. Current in vitro models utilise 2D cell cultures that do not accurately represent the in vivo environment resulting in altered cell functions. Therefore, new models are needed that incorporate mechanical and structural conditions in a cell culture in order to mimic the in vivo environment. The aim of this project is to develop a hollow fibre based BAK (Bioartificial Artificial Kidney) device as a 3D cell culture model, similar to the proximal tubule physiology and study the active transport of renal transporter specific compounds and substrates to accurately predict renal clearance.

Firstly, polymer biomaterials were produced and characterised in terms of cell adhesion, viability and function. PVDF (Polyvinylidene fluoride) polymers have shown be excellent biomaterials for cell adhesion without the use of any coatings while cell viability was not affected. Furthermore, HEK-OCT2 and MDCK-Mdr1a cells attached on the PVDF flat membranes were able to uptake their specific substrates in similar ratios as cells attached on TCP (Tissue culture plastic). However, PVDF proved to be an unsuitable material for HF (Hollow Fibre) production by using a conventional wet spinning process. PVDF HFs were not homogeneous with large pores making them unsuitable for the BAK device. As an alternative, commercially polypropylene based HFs (P1LX) were chosen instead.

Secondly, fluorescence-based assays were developed to assess the functionality of the three main drug transporters involved in the renal transport of pharmaceutical compounds (P-gp, BCRP and OCT2). Primary RPTECs and cell lines were utilised to compare cell type variability while fluorescent substrates were used as an alternative to standard compounds. Fluorescent substrates alternatives Rho123, MTX-FITC and ASP+ were identified as suitable for studying the functions of P-gp, BCRP and OCT2 renal transporters, respectively.

Finally, custom made BAK devices that could house a single HF were designed and produced that would allow for media perfusion both in the HF lumen and ECS (Extracapillary space). The renal similar basement membrane matrix coating Geltrex was used to enhance cell attachment on the HF membrane and flow rate of 3 μL/min was applied to expose cells to a physiological shear stress. MDCK cells were able to form a tight barrier under flow conditions after 7 days and were able to maintain it for at least 3 days. Human cells (HEK and RPTECs) however were not able to reach confluency within the HF and thus only uptake transport was studied.

Cells grown in a 3D environment within the BAK device had different gene expression profiles compared to 2D grown cells. Most notably, the upregulation of the microvilli marker CD-133 and the downregulation of the renal transporters P-gp and BCRP while active transport of fluorescent substrates was significantly reduced. OCT2 expression and function on the other hand remained unchanged. These results demonstrate the differences between the function of cells grown in 2D static and 3D dynamic environments.

This is a progress towards the development of a new renal model that incorporates a physiological environment to aid the improvement and efficiency of DMPK studies.