Genome Engineering Renal Epithelial Cells for Enhanced Volume Transport Function.

INTRODUCTION: Bioengineering an implantable artificial kidney (IAK) will require renal epithelial cells capable of reabsorption of salt and water. We used genome engineering to modify cells for improved Na+/H+ exchange and H2O reabsorption. The non-viral piggyBac transposon system enables genome engineering cells to stably overexpress one or more transgenes simultaneously.
METHODS: We generated epitope-tagged human sodium hydrogen exchanger 3 (NHE3) and aquaporin-1 (AQP1) cDNA expressing piggyBac transposon vectors. Transgene expression was evaluated via western blot and immunofluorescence. Flow cytometry analysis was used to quantitate transporter expression in a library of genome engineered clones. Cell surface biotinylation was used evaluate surface protein localization. Blister formation assays were used to monitor cellular volumetric transport.
RESULTS: piggyBac enabled stable transposon integration and overexpression of cumate-inducible NHE3 and/or constitutively expressing AQP1 in cultured renal (MDCK) epithelial cells. Cell surface delivery of NHE3 and AQP1 was confirmed using cell surface biotinylation assays. Flow cytometry of a library of MDCK clones revealed varying expression of AQP1 and NHE3. MDCK cells expressing AQP1 and cumate-inducible NHE3 demonstrated increased volumetric transport.
CONCLUSIONS: Our results demonstrate that renal epithelial cells an be genome engineered for enhanced volumetric transport that will be needed for an IAK device. Our results lay the foundation for future studies of genome engineering human kidney cells for renal tubule cell therapy. © This is a U.S. government work and its text is not subject to copyright protection in the United States; however, its text may be subject to foreign copyright protection 2019.