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Establishment and Characterization of a Kidney Drug Interaction Model by Stably Expressing hOAT1 in HEK 293T/17 Cells

Poster
White, green and blue kidney glomeruli.

SOT 2016

New Orleans , Louisiana, United States

February 28, 2016

Abstract

In vivo studies have shown that kidney membrane transporters play a key part in drug disposition and renal clearance. One such transporter is OAT1 (SLC22A6), which is critical for maintaining homeostasis of endogenous substances. This makes OAT1 a good transporter to assay for drug interactions with the kidney. Unfortunately, primary cells lose OAT1 expression in culture, and transiently expressed OAT1 has great variations between production lots, which make data hard to interpret. In our study we have generated HEK 293T/17 cells that stably overexpress the OAT1 gene driven by the human elongation factor-1 alpha (EF1α) promoter. After confirming the mRNA expression by RT-PCR, we performed immunostaining that indicated OAT1 is correctly trafficked to the membrane. Most importantly, we validated that the overexpressed OAT1 transporter has normal transport activities by using 5-carboxyfluorescein (5-CF) and para-aminohipurate (PAH; data not shown) uptake assays, and that the uptake can be inhibited by the well-known inhibitors probenecid and novobiocin. Both inhibitors responded in a dose dependent manner for 5-CF uptake with IC50 values between 5-16 μM. Even at higher passages, the cell line retained the functionality of OAT1. Overall, our data has shown that this modified cell line is a very useful in vitro tool for testing regulation of OAT1 membrane transporter activity in kidney cells.

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