We confirmed the expression of OAT1, OCT2, or OAT3 in RPTEC/TERT1 by RT-PCR. The expression of OAT1 or OCT2 was verified by western blot. In addition, immunofluorescence revealed that the cells were positive for membrane-localized OAT1, OCT2, or OAT3 (Figure 2). Further, the clonal OAT1, OCT2, or OAT3 cells retain important characteristics of the parental counterpart RPTEC/TERT1 cell; dome formation and normal expression of CD13 and E-cadherin were observed in OAT1, OCT2, and OAT3 positive clones (Figure 3).
Next, we tested the ability of OAT1- and OAT3-expressing clones to uptake 6-CF, a known substrate of OAT1 and OAT3. We observed significant uptake ratios relative to the parental cells. Further, the uptake activity of OAT1- and OAT3-expressing clones was observed to be dose dependent (Figure 4). Similarly, we tested OCT2-expressing cells to uptake EAM-1, a known substrate of OCT2. As in the OAT1- and OAT3-expressing cells, we observed significant, concentration-dependent uptake ratios in OCT2-expressing cells relative to the parental cells (Figure 4).
We then tested the ability of various organic ion transporter inhibitors to block substrate transport into OAT1, OCT2, and OAT3-expressing cells. We observed that the compounds probenecid and novobiocin were able to block 6-CF (Probenecid: IC50=16.91 μM; novobiocin: IC50=59.17 μM) transport into RPTEC/TERT1-OAT1 (Figure 5). Similarly, probenecid and novobiocin blocked 6-CF (Probenecid: IC50=49.02 μM; novobiocin: IC50=70.90 μM) uptake into RPTEC/TERT1-OAT3 (Figure 5). In the same regard, the compounds cimentidine and quintinin were able to block EAM-1 (Cimentidine: IC50=93.5 μM; quintinin: IC50=59.5 μM) entry into RPTEC/TERT1-OCT2 (Figure 5). The IC50 values that were observed in these experiments were similar to those reported in other competitive inhibition studies using the test compounds.4-8
Over the course of this project we generated and tested three new hTERT-immortalized, renal SLC protein models. The new cell models displayed: appropriate transporter protein expression and localization; key proximal tubule cell biologies; and potent and specific transporter activity. Thus these new SLC protein-expressing cell models are robust tools for toxicological testing.
Figure 2. Characterization of the RPTEC/TERT1 SLC transporter cells. RT-PCR data demonstrates the presence of (A) OAT1, (B) OCT2, or (C) OAT3 mRNA in the respective cell lines. Immunoblot demonstrates the presence of (D) OAT1 and (E) OCT2 protein expression in the respective cell lines. Immunofluorescence demonstrates expression and plasma membrane localization (F) OAT1, (G) OCT2, or (H) OAT3, in the respective cell lines, but not the parental line. Original magnification, x20.
Figure 3. The RPTEC/TERT1 transporter cells retain the most important characteristics of the parental cells. RPTEC/TERT1 SLC transporter cells were subjected to immunostaining and dome formation assays. (A) OAT1, OCT2, and OAT3 clones display the same renal epithelial growth pattern as parental RPTEC/TERT1 cells. (B) The renal epithelial markers CD13 and E-cadherin are expressed in both parental RPTEC/TERT1 cells
and in the OAT1, OCT2, and OAT3 lines. (C) Dome formation is not compromised in OAT1-, OCT2-, and OAT3- expressing cells, as demonstrated by the formation of dome-like structures (arrows) caused by solute transport across an intact epithelial barrier.
Figure 4. SLC transporter-expressing cells display potent substrate uptake activity. Solute uptake activity of (A) OAT1- and (C) OAT3-expressing cells was assessed using 6-CF as a substrate. Solute uptake activity of (B) OCT-2 was assessed using EAM-1 as a substrate. As expected, uptake increases with increasing substrate concentration in solute transporter-expressing cells but not in parental RPTEC/TERT1 cells (n=3; error bars indicate standard deviation), indicating that the observed transport is due to OAT1, OCT2, or OAT3 expression.
Figure 5. SLC transporter-expressing cells demonstrate highly specific uptake activities. OAT1-expressing cells were exposed to increasing concentrations of the known OAT inhibitors (A) probenecid and (B) novobiocin while 6-CF concentration and uptake time were held constant at 3 μM and 20 minutes, respectively. OCT2-expressing cells were exposed to increasing concentrations of the known OCT2 inhibitors (C) cimetidine and (D) quinitin while EAM-1 concentration and uptake time were held constant at 5 μM and 20 minutes, respectively. OAT3-expressing cells were exposed to increasing concentrations of the known OAT inhibitors (E) probenecid and (F) novobicin while 6-CF concentration and uptake time were held constant at 3 μM and 20 minutes, respectively. The resulting inhibition curves indicate that OAT1, OCT2, and OAT3 transport activity was substrate specific when overexpressed in RPTEC/TERT1 cells (n=3; error bars indicate standard deviation).