We confirmed the expression of OAT1 in OAT1-HEK by RT-PCR and immunocytochemistry. An approximate 20-fold increase in OAT1 compared
to whole kidney lysates was observed. In addition, immunofluorescence revealed that the cells were positive for membrane-localized OAT1
(Figure 1). We then tested the ability of the OAT1-HEK line to uptake a known substrate of OAT1, 5-CF. We observed an uptake ratio of 23:1
relative to the control HEK cells. Moreover, 90% of the OAT1-HEK cells emitted after treatment with the fluorescent 5-CF (Figure 2). The
activity of the OAT1-HEK cells was time- and dose-dependent (Figure 3), and stable over 14 passages (Figure 4). Taken together, these data
indicate that the OAT1-HEK cell line stably expressed a functional OAT1 protein.
We then tested the ability of two inhibitors of OAT1-mediated activity, probenecid and novobiocin, to block 5-CF transport into the OATHEK
cells (Figure 5). We observed that both compounds displayed potent inhibitory effects on 5-CF transport (probenecid IC50=15.9 µM;
novobiocin IC50=8.1 µM), providing further evidence to the usefulness of these cells in toxicological testing.
Figure 1. Confirmation of OAT1 expression in OAT1-HEK by RT-PCR and immunocytochemistry. (A) RT-PCR revealed a ~20-fold increase
in human OAT1 relative to whole kidney lysate, N=3. (B) The RT-PCR product was analyzed by gel electrophoresis and found to be of the
expected size of the human OAT1 sequence. (C) Immunofluorescent staining with a human anti-OAT1 antibody in cultured OAT1-HEK cells
revealed the cells were ~90% positive for membrane localized OAT1.
Figure 2. OAT1 mediated uptake of fluorogenic substrate in OAT1-HEK cells. (A) OAT1-HEK or control cells were incubated with the fluorogenic
substrate 5-CF as described in the materials and methods. Mean RFUs for the control line was <400 while the signal from the OAT1-HEK line
was >9000, N=3. (B) This indicated an uptake ratio of 23 relative to the control, N=3 (middle). (C) Fluorescent microscopy revealed that ~90%
of OAT1-HEK, but not control cells, exhibited uptake and accumulation of 5-CF.
Figure 3. Time- and concentration-dependent uptake of 5-CF in OAT1-HEK cells. OAT1-HEK cells incubated with 5-CF for the indicated times
and concentrations demonstrated a linear increase in RFUs (r2
≥ 0.99) up to 30 minutes, N=4.
Figure 4. OAT1 mediated 5-CF uptake is stable over extended culture in OAT1-HEK cells. 5-CF uptake was determined as described in the
materials and methods and expressed relative to the control line. No significant decrease in uptake was seen up to at least 14 passages (P+
indicates passage number; p= 0.77), N=4.
Figure 5. Dose-dependent inhibition of 5-CF uptake by OAT1 inhibitors probenecid and novobiocin in OAT1-HEK. OAT1-HEK cells were
incubated with the indicated compounds as described in the materials and methods and tested for 5-CF uptake. The results were graphed as
(A) raw signal or as (B) percentage uptake relative to non-treated control cells, N=3.