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.
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.
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.
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.
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These studies were supported by DK093360 (NIH) and BX004285 (VA) to MHW, EB021214 to SR and WHF, and the Vanderbilt O’Brien Kidney Center Cell and Genome Engineering Core (DK114809). The VMC Flow Cytometry Shared Resource is supported by the Vanderbilt Ingram Cancer Center (P30 CA68485) and the Vanderbilt Digestive Disease Research Center (DK058404). Brittany K. Matlock in the VMC Flow Cytometry Shared Resource provided valuable assistance in developing the intracellular staining protocol for high-throughput sampling. Confocal immunofluorescence image collection and data analysis were performed in part through the use of the VUCell Imaging Shared Resource (supported by NIHgrants CA68485, DK20593, DK58404, DK59637 and EY08126) and NIH S10 Grant Number 1S10RR027396-01. Mouse anti-myc antibody was produced by the Vanderbilt Antibody and Protein Resource. The Vanderbilt Antibody and Protein Resource is supported by the Vanderbilt Institute of Chemical Biology and the Vanderbilt Ingram Cancer Center (P30 CA68485).
Conflict of interest
MHW, RAV, WL, and RCW declare no conflicts of interest. SR and WHF are founders of Silicon Kidney.
No human or animal studies were carried out by the authors for this article.
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Wilson, M.H., Veach, R.A., Luo, W. et al. Genome Engineering Renal Epithelial Cells for Enhanced Volume Transport Function. Cel. Mol. Bioeng. 13, 17–26 (2020). https://doi.org/10.1007/s12195-019-00601-3
- Sodium hydrogen exchanger
- MDCK cells