Establishment And Application Of Cell Models With Stable Expression Of HOCTN1/2

OBJECTIVES: To establish cell models with stable expression of human carnitine/organic cation transporter 1 or 2( hOCTN1 or hOCTN2) in MDCK cells, and then applied the models to study the interaction of sulpiride, a psychotropic drug, with hOCTN1/2. Additionally, the interactions of sulpiride with other transporters high expressed in human kidney, such as OCT2, MATE1 and MATE2-K were also studied.METHODS:(1)The hOCTN1 or hOCTN2 cDNA was cloned from human kidney by PCR using specific primer, and then MDCK cells were transfected with recombinant plasmid pcDNA3.1(+)-hOCTN1 or pcDNA3.1(+)-hOCTN2. Several stable transfected clones were obtained after G418 screening. MDCK-h OCTN1 or MDCK-hOCTN2 clones were then exposed to ergothioneine or mildronate, good substrate of hOCTN1 or hOCTN2 respectively, for cellular uptake assay to identify the best candidates. The m RNA expression, transport function and Michaelis-Menten kinetics were studied to comfirm authenticity of cell models.(2)To determine whether sulpiride is inhibitor of hOCTN1 or hOCTN2, the uptake of ergothioneine or mildronate, with or without the presence of sulpiride, was studied using the cell models established in our study. While to determine whether sulpiride is substrate of hOCTN1 or hOCTN2, the cellular accumulation of sulpiride in MDCK-hOCTN1 or MDCK-hOCTN2 cells and MDCK empty vector cells was performed, with or without inhibitors.(3) To predict potential interaction between sulpiride and organic cation transporters functionally expressed in human kidney, the cellular accumulation of sulpiride were also conducted in MDCK empty vector cells and constructed cells models, including MDCK or CHO cells models transfected with different transporters of human organic cation transporter 2( hOCT2), human multidrug and toxin extrusion transporter 1 and 2K( hMATE1/2-K) and human peptide transporter 2(PEPT2).RESULTS:(1) The cellular accumulation of ergothioneine in MDCK-hOCTN1 cells or mildronate in MDCK-hOCTN2 cells was 122 and 108 folds of the mock cells, respectively. The kinetic parameters, Km and Vmax of ergothioneine, mediated by MDCK-hOCTN1, were 8.19 ± 0.61 μmol/L and 1427 ± 49 pmol/mg protein/min, while Km and Vmax of mildronate by MDCK-h OCTN2 were 52.3 ± 4.3 μmol/L and 2454 ± 64 pmol/mg protein/min. The above results demonstrated that cell models with good stable hOCTN1 and hOCTN2 functions were established successfully, which can be applied to study interactions with compounds and h OCTN1/2.(2) Sulpiride was found to markedly inhibit hOCTN1/2-mediated ergothioneine or mildronate transport in a concentration-dependent manner. Furthermore, The cellular accumulation of sulpiride in MDCK-hOCTN1/2 cells were significantly higher than that in mock cells, which could be inhibited by hOCTN1/2 inhibitors, suggesting sulpiride was substrate of hOCTN1/2.(3) The cellular accumulation of sulpiride in cell models with high expression of hOCT2, hMATE1 or hMATE2-K was obviously higher than that of mock cells, however, no marked difference was found in CHO cells with high expression of PEPT2 and mock cells. In particular, the hMATE2-K-mediated transport of sulpiride was higher than other transporters, while hOCTN1-mediated transport of sulpiride was as high as that of hMATE1 following hMATE2-K, whereas the affinity of sulpiride with hMATE1/2-K was markedly higher than hOCT2 or hOCTN1/2.CONCLUSIONS:(1) Cell models with good stable hOCTN1 and hOCTN2 functions were established successfully, which can be applied to study interactions with drugs and transporters of hOCTN1 and hOCTN2.(2) Membrane transporter hOCT2, localized at the basolateral membrane of renal tubular epithelial cells, together with hOCTN1/2 and hMATE1/2-K, localized at the apical membrane, would be responsible for renal secretion and systemic elimination of sulpiride. On the other hand, hOCTN1/2 might play quite a role in renal reabsorption of sulpiride.(3) Competition between sulpiride and endogenous compounds for hOCTN1/2-mediated reabsorption could possibly occur in vivo.(4) Tubular luminal pH range might possibly play a role in renal disposition of sulpiride. Furthermore, inhibitors of hOCT2, hOCTN1/2, hMATE1/2-K could contribute at least in part to the altered disposition of sulpiride in kidney.