Fluorescent organic cations for human OCT2 transporters screening: uptake in CHO cells stably expressing hOCT2

The aim of this study was to assess the suitability of amiloride, rhodamine 6G and rhodamine 123 as non-radioactive substrates for characterizing hOCT2 using CHO cells. The uptake characteristics of these compounds were compared in wild-type (WT) and human organic cation transporter 2 (hOCT2)-stably transfected Chinese Hamster Ovary (CHO) cells. All the compounds were accumulated by the CHO-hOCT2 cells. Intracellular uptake of the compounds was higher in CHO cells stably-expressing hOCT2 compared to the WT. The uptake was concentration–dependent and saturable (except for rhodamine 123). The affinities of the compounds for the hOCT2 (in descending order) were: amiloride (Km = 72.63 12.02 μM) > rhodamine 6 G (Km = 82.47 29.15 μM). Uptake of amiloride in transfected cells was pH -dependent and significantly inhibited by hOCT2 inhibitors (quinine, verapamil and quinidine). Based on our kinetic data and other considerations, we recommend the use of amiloride for characterizing hOCT2 transporters

Mechanisms and control of intestinal and renal sulfate secretion in marine teleosts

Marine teleosts, which maintain their plasma osmotic pressure below that of the surrounding marine environment, continually imbibe seawater to counteract dehydration. Seawater contains high levels of SO42− (25 mM), and excess SO42− is acquired through ingestion. Renal excretion, primarily through proximal tubular secretion, removes this SO42− burden. While it is generally accepted that the gastrointestinal tract is the avenue for excess absorption, the exact location and mechanism is not known. Data presented in this thesis shows that while the anterior intestine passively absorbs SO4 2−, an active secretory component dependent on metabolism and the plasma membrane Na+ gradient is also present. Active secretion is facilitated by an electroneutral brush border SO42− /Cl− exchanger and is abolished by satiety (i.e., full stomach). It is hypothesized that active intestinal SO4 2− secretion functions in maintenance of SO4 2− homeostasis and water absorption. ^ A basolateral SO42−/OH− exchanger and a brush border anion exchanger with affinities for both luminal HCO3− and Cl− facilitate active renal tubular SO42− secretion. Tubular SO42− secretion is dependent on carbonic anhydrase (CA) and is stimulated by cortisol. To further examine this relationship, the effect of cortisol on CA-dependent SO42− secretion by primary monolayer cultures of flounder proximal tubule cells (fPTCs) was examined. Cortisol was found to increase tubular CA enzymatic activity, CA isoform II protein expression, and CA-dependent SO4 2− secretion. Thus, regulation of CA expression may provide cortisol with a level of control over renal tubular SO4 2− secretion. ^ The uptake of SO42− across the basolateral membrane of proximal tubule cells is dependent on the plasma membrane Na + gradient and facilitated by SO42−/OH − exchange prompting an investigation of the role of Na +/H+ exchange (NHE) and the effect of interstitial acidification (metabolic acidosis). Metabolic acidosis stimulated tubular SO42− secretion in fPTCs and the intact animal. In fPTCs, the stimulation caused by metabolic acidosis was dependent on CA and brush border Na+/H+ exchange (NHE) activity. NHE isoforms 1, 2, and 3 were identified in proximal tubule cells, and induction of one or all of these NHEs may be required for enhanced renal tubular SO 42− secretion during metabolic acidosis.

Incubation Time Influences Organic Anion Transporter 1 Kinetics In Vitro and Renal Clearance Predictions for Para-Aminohippurate

Examined was the effect of in vitro incubation time on ligand interactions with organic anion transporter 1 (OAT1), and how the time chosen influences renal clearance (CLR) predictions for para-aminohippurate (PAH). Transport studies were performed with Chinese hamster ovary cells expressing OAT1 (CHO-OAT1). The Mechanistic Kidney model (MechKiM) within the Simcyp Simulator (v15, Certera, Inc) was used for pharmacokinetic predictions. Increasing in vitro incubation time led to a decrease in the maximal transport rate and intrinsic uptake clearance (CLint) for PAH, with CLint values ranging 11-fold when using incubation times ranging from 15 sec (CLint,15s) to 45 min (CLint,45min). There was also a significant effect of time on the Michaelis constant. Inhibition potency of five drugs against PAH transport was tested using incubation times of either 15 sec or 10 min. There was no effect of time on inhibition potency for omeprazole or furosemide. Indomethacin was less potent at 10 min, whereas probenecid (~2-fold) and telmisartan (~7-fold) were more potent when using the longer incubation. Pre-treatment of CHO-OAT1 cells with telmisartan for increasing periods of time (30 sec – 30 min) using multiple concentrations (10 nM – 200 nM) showed that its inhibitory effect is time-sensitive. A MechKiM model was developed for PAH using the PAH CLint obtained at 15 sec, in vitro-in vivo extrapolation, and parameter estimation of the OAT1 relative activity factor. Using these parameters, the simulated plasma concentration-time profile, CLR and cumulative urinary excretion-time profile of PAH agreed reasonably well with reported data. Next we used the CLint values obtained from studies using incubation times greater than 15 sec (up to 45 min). The simulated CLR of PAH was sensitive to the time-associated CLint value used, with CLR values 2.7-fold higher when using CLint,15s vs. CLint,45min. The simulated percentages of the PAH dose excreted in the urine after 2 h were 100% (CLint,15sec) vs. 81% (CLint,45min). Simulations predicted a maximum intracellular PAH concentration in tubule cells that was 3.4-fold higher when using CLint,15s vs. CLint,45min. These data show that in vitro incubation time influences transport kinetics and predictions of the involvement of drug transport in pharmacokinetics

2012. Expression of organic anion transporter 2 in the human kidney and its potential role in the tubular secretion of guanine-containing antiviral drugs. Drug Metab. Dispos

Abstract The organic anion transporters 1 and 3 (OAT1 and OAT3), and organic cation transporter 2 (OCT2) are important for renal tubular drug secretion. In contrast, evidence for OAT2 expression in the human kidney is limited, and its role in renal drug transport is unknown. Both mRNA (real-time polymerase chain reaction) and protein (Western blotting) for OAT2 was detected in renal cortex from eight donors, and interindividual variability in protein levels was 3-fold. OAT2 protein in the renal cortex was localized (by immunohistochemistry) to the basolateral domain of tubules, as were OAT1 and OAT3. The absolute abundance of OAT2 mRNA was similar to OAT1 mRNA, 3-fold higher than OCT2 mRNA, but 10-fold lower than OAT3 mRNA. A previous observation that OAT2 transports cyclic guanosine monophosphate (cGMP) led us to examine if acyclovir, ganciclovir and penciclovir are OAT2 substrates; they are guanine-containing antivirals that undergo active tubular secretion. Transport of the antivirals into human embryonic kidney cells was stimulated 10-to 20-fold by expression of OAT2, but there was little to no transport of the antivirals by OAT1, OAT3 or OCT2. The K m values for acyclovir, ganciclovir and penciclovir transport were 94 µM, 264 µM and 277 µM, respectively, and transport efficiencies were relatively high (6 to 24 µl·min -1 ·mg protein -1 ). This study provides definitive evidence for the expression of OAT2 in the human kidney, and is the first to demonstrate that OAT2, compared to OAT1, OAT3 or OCT2, has a preference for antiviral drugs mainly eliminated in the urine via active secretion

Incubation Time Influences Organic Anion Transporter 1 Kinetics and Renal Clearance Predictions

Accurate predictions of drug uptake transporter involvement in renal excretion of xenobiotics require determination of in vitro transport kinetic parameters under initial-rate conditions. The purpose of the present study was to determine how changing the incubation time from initial rate to steady state influences ligand interactions with the renal organic anion transporter 1 (OAT1), and the impact of the different experimental conditions on pharmacokinetic predictions. Transport studies were performed with Chinese hamster ovary cells expressing OAT1 (CHO-OAT1) and the Simcyp Simulator was used for physiological-based pharmacokinetic predictions. Maximal transport rate and intrinsic uptake clearance (CLint) for PAH decreased with increasing incubation time. The CLint values ranged 11-fold with incubation times spanning from 15 s (CLint,15s, initial rate) to 45 min (CLint,45min, steady state). The Michaelis constant (Km) was also influenced by the incubation time with an apparent increase in the Km value at longer incubation times. Inhibition potency of five drugs against PAH transport was tested using incubation times of either 15 s or 10 min. There was no effect of time on inhibition potency for omeprazole or furosemide, whereas indomethacin was less potent, and probenecid (~2-fold) and telmisartan (~7-fold) more potent with the longer incubation time. Notably, the inhibitory effect of telmisartan was reversible, albeit slowly. A pharmacokinetic model was developed for PAH using the CLint,15s value. The simulated plasma concentration-time profile, renal clearance, and cumulative urinary excretion-time profile of PAH agreed well with reported clinical data, and the PK parameters were sensitive to the time-associated CLint value used in the model

Organic anion transporter 2 transcript variant 1 shows broad ligand selectivity when expressed in multiple cell lines

Organic anion transporter 2 (OAT2) is likely important for renal and hepatic drug elimination. Three variants of the OAT2 peptide sequence have been described – OAT2 transcript variant 1 (OAT2-tv1), OAT2 transcript variant 2 (OAT2-tv2) and OAT2 transcript variant 3 (OAT2-tv3). Early studies helping to define the ligand selectivity of OAT2 failed to identify the variant used, and the studies used several heterologous expression systems. In preliminary studies using OAT2-tv1, we failed to observe transport of several previously identified substrates, leading us to speculate that ligand selectivity of OAT2 differs with variant and/or heterologous expression system. The purpose was to further investigate the ligand selectivity of the OAT2 variants expressed in multiple cell types. We cloned OAT2-tv1 and OAT2-tv2, but were unsuccessful at amplifying mRNA for OAT2-tv3 from human kidney. OAT2-tv1 and OAT2-tv2 were individually expressed in human embryonic kidney (HEK), Madin-Darby Canine Kidney (MDCK) or Chinese hamster ovary (CHO) cells. mRNA for OAT2-tv1 and OAT2-tv2 was demonstrated in each cell type transfected with the respective construct, indicating their expression. OAT2-tv1 trafficked to the plasma membrane of all three cell types, but OAT2-tv2 did not. OAT2-tv1 transported penciclovir in all three cell types, but failed to transport para-aminohippurate, succinate, glutarate, estrone-3-sulfate, paclitaxel or dehydroepiandrosterone sulfate – previously identified substrates of OAT2-tv2. Not surprising given its lack of plasma membrane expression, OAT2-tv2 failed to transport any of the organic solutes examined, including penciclovir. Penciclovir transport by OAT2-tv1 was sensitive to large (e.g., cyclosporine A) and small (e.g., allopurinol) organic compounds, as well as organic anions, cations and neutral compounds, highlighting the multiselectivity of OAT2-tv1. The potencies with which indomethacin, furosemide, cyclosporine A and cimetidine inhibited OAT2-tv1 are in good agreement with previous studies using this variant, but inconsistent with studies using OAT2 with an unidentified sequence. This study shows that organic molecules with diverse physicochemical properties interact with OAT2-tv1, making it a likely site of drug interactions. Many previously identified substrates of OAT2 are not transported by OAT2-tv1, suggesting that variant and/or expression system may contribute. Future work should establish the expression pattern and ligand selectivity of OAT2-tv3