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

Supplementary Information Files for Development of a hollow fibre-based renal module for active transport studies<br>Understanding 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, diferences 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 fbre (Plasmaphan P1LX, 3M) that serves as a porous scafold and compared to conventional Transwell cell-based bidirectional transport studies. In addition, a constant fow 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 fuorescence microscopy while epithelial barrier integrity under shear stress was shown to be achieved by day 7. qPCR results have shown signifcant 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 fvefold 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 efciency compared to Transwell cell cultures while efux of the P-gp-specifc substrates Hoechst and Rhodamine 123 was decreased. These results further support the efect of the microenvironment and fuidic 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.<br>