Development of a hollow fibre-based renal module for active transport studies

Abstract

AbstractUnderstanding the active transport of substrates by the kidney in the renal proximal convoluted tubule is crucial for drug development and for studying kidney diseases. Currently, cell-based assays are applied for this this purpose, however, differences between assays and the body are common, indicating the importance of in vitro–in vivo discrepancies. Several studies have suggested that 3D cell cultures expose cells to a more physiological environments, thus, providing more accurate cell function results. To mimic the renal proximal tubule, we have developed a custom-made renal module (RM), containing a single polypropylene hollow fibre (Plasmaphan P1LX, 3M) that serves as a porous scaffold and compared to conventional Transwell cell-based bidirectional transport studies. In addition, a constant flow of media, exposed cells to a physiological shear stress of 0.2 dyne/cm2. MDCK-Mdr1a cells, overexpressing the rat Mdr1a (P-gp) transporter, were seeded onto the HF membrane surface coated with the basement membrane matrix Geltrex which facilitated cell adhesion and tight junction formation. Cells were then seeded into the HF lumen where attachment and tight junction formation were evaluated by fluorescence microscopy while epithelial barrier integrity under shear stress was shown to be achieved by day 7. qPCR results have shown significant changes in gene expression compared to cells grown on Transwells. Kidney injury marker such as KIM-1 and the hypoxia marker CA9 have been downregulated, while the CD133 (Prominin-1) microvilli marker has shown a fivefold upregulation. Furthermore, the renal transporter P-gp expression has been downregulated by 50%. Finally, bidirectional assays have shown that cells grown in the RM were able to reabsorb albumin with a higher efficiency compared to Transwell cell cultures while efflux of the P-gp-specific substrates Hoechst and Rhodamine 123 was decreased. These results further support the effect of the microenvironment and fluidic shear stress on cell function and gene expression. This can serve as the basis for the development of a microphysiological renal model for drug transport studies.