Browsing by Author "Groves, C. E."

Organic anion (OA) transport mediates accumulation of the zwitterionic nephrotoxic cysteine S-conjugates S-dichlorovinylcysteine (DCVC) and S-chlorotrifluoroethylcysteine (CTFC) in the rabbit renal proximal tubule (RPT). Although these cysteine conjugates are nephrotoxic to the human RPT, neither the role of OA transport nor the specific OA transport pathway(s) involved in cysteine conjugate accumulation are known. Since the OAT1 transporter has the characteristics of para-aminokippurate (PAH) transport that closely correlate to the native RPT, we examined the interaction of DCVC, CTFC, and the nontoxic benzothiazolylcysteine (BTC) with PAH transport mediated by human OAT1 and rabbit Oat1 expressed in Chinese hamster ovary and COS7 heterologous expression systems, respectively. Although the K-m values for PAH uptake by hOAT1 and rbOat1 (8.9 +/- 3.6 and 20.7 +/- 8 muM, respectively) were 5- to 10-fold less than the K-m for peritubular PAH transport into rabbit RPT, the IC50 values for DCVC, CTFC, and BTC inhibition of PAH uptake mediated by either hOAT1 or rbOat1 were similar between these two transporters and to the IC50 values for these conjugates measured in rabbit RPT. The IC50 for inhibition of hOAT1- and rbOat1-mediated PAH uptake by the hydrophobic conjugate BTC was more than 5- fold lower than the IC50 values seen with DCVC and CTFC, suggesting that hydrophobicity increases the affinity of OAT1 for cysteine conjugates. Finally, preloading cells transfected with hOAT1 with BTC significantly trans-stimulated the uptake of PAH, consistent with the conclusion that BTC and, hence, other cysteine S-conjugates are substrates for hOAT1.

We compared the characteristics of several cloned rabbit organic electrolyte (OE) transporters expressed in cultured cells with their behavior in intact rabbit renal proximal tubules (RPT) to determine the contribution of each to basolateral uptake of the weak acid ochratoxin A (OTA) and the weak base cimetidine (CIM). The activity of organic anion transporters OAT1 and OAT3 proved to be distinguishable because OAT1 had a high affinity for PAH (K(t) of 20 muM) and did not support estrone sulfate (ES) transport, whereas OAT3 had a high affinity for ES (K(t) of 4.5 muM) and a weak interaction with PAH (IC(50) > 1 mM). In contrast, both transporters robustly accumulated OTA. Intact RPT also accumulated OTA, with OAT1 and OAT3 each responsible for similar to50%: ES and PAH each reduced uptake by similar to50%, and the combination of the two eliminated mediated OTA uptake. The weak base CIM was transported by OAT3 (K(t) of 80 muM) and OCT2 (K(t) of 2 muM); OCT1 had a comparatively low affinity for CIM, and CIM uptake by OAT1 was equivocal. Intact RPT accumulated CIM, with TEA and ES reducing CIM uptake by 20 and 75%, respectively, suggesting that OAT3 plays a quantitatively more significant role in CIM uptake in the early proximal tubule than OCT1/2. In single S2 segments of RPT, ES and TEA each blocked similar to50% of CIM uptake. Thus the fractional contribution of different OE transporters to renal secretion is influenced by their affinity for substrate and relative expression level in RPT.