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 HO reabsorption. The non-viral 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 transposon vectors. Transgene expression was evaluated 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

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.