Development of Highly Sensitive and Specific in vitro Renal Solute Carrier (SLC) Uptake Cell Models Using Genetically Modified Human Renal Proximal Tubule Epithelial Cells for Renal Drug Transporter Interaction Studies
Society of Toxicology 2018
San Antonio, Texas, United StatesMarch 12, 2018
The disposition and clearance of drugs by the kidney relies largely on a well-characterized subset of membrane transport pumps collectively known as solute carrier (SLC) proteins. Among the SLC family proteins, OAT1, OCT2, and OAT3 are considered most important transporters in kidney tissue and are recommended by the FDA, ITC, and EMA as targets for drug-drug interaction studies. Therefore, there is a large demand for in vitro kidney transporter models that have normal human kidney origin, functioning transporters, clinical predictability, and consistent data output for drug interaction studies. Unfortunately, primary renal epithelial cells lose OAT1, OCT2, and OAT3 transporter expression quickly in culture. Transiently expressing these transporters in primary renal epithelial cells yields large variations between experimental models, making data hard to interpret. Current cell line-based models are available using MDCK, CHO, U2OS, or other lines, which either do not have human kidney tissue origin or are themselves cancer lines, significantly compromising clinical predictability.
In our study, we have generated kidney transporter cell models using well-characterized hTERT-immortalized primary Renal Proximal Tubule Epithelial Cells that stably overexpress either the OAT1, OCT2, or OAT3 gene. After confirming the SLC mRNA expression for each gene by RT-PCR, we performed immunostaining that showed that OAT1, OCT2, and OAT3 are correctly trafficked to the plasma membrane. Notably, those clones show typical epithelial morphology, functionality, and expression of the appropriate epithelial and kidney tissue specific markers. Most importantly, we verified that the overexpressed transporters have normal transport activities using 6-CF and EAM-1 uptake assays in a high throughput format. We also show that uptake of these compounds are blocked in a dose dependent manner by well-known SLC inhibitors, indicating that the overexpressed kidney transporters are functioning as expected.
Overall, our data demonstrates that these modified renal epithelial cell lines maintain kidney transporter expression over time and provide physiologically relevant high sensitive and specific data regarding human kidney transporter functions. These models are consistent and reliable and can be very valuable tools for high throughput kidney drug toxicity screening to test the effects of exogenous compounds on renal membrane transporter function.
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