ABSTRACT
OBJECTIVE: To establish a cell model stably expressing mouse organic anion transporter1( OAT1) in MDCK cells, for the purpose of screening potent OAT1 inhibitors in vitro. METHODS: Recombinant plasmid pcDNA3.1(+) -OAT1 was constructed and transfected into MDCK cells using Lipofectamine™ 2000 reagent. After the process of G418 screening, cells were collected for further validation. Cells were harvested, and the quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) was carried out to test the OAT1 mRNA expression in MDCK-OAT1 cells. The function of the stably transfected cells were validated by the uptake activity of (6-Carboxyfluorescein, 6-CFL),a substrate of OAT1. The inhibitors of OAT1 were selected according to their inhibition activity towards the uptake of 6-CFL into the OAT1-over expressing cells in comparison with the typical inhibitor of OAT1,probenecid. RESULTS: The pcDNA3.1(+) -OAT1 was well conducted. The mRNA expression of OAT1 was significantly higher than that in mock cells; MDCK-OAT1 cells had a significantly high mRNA expression comparing with the mock cells, being 4 862 fold of that in mock cells. The uptake-ability of 6-CFL in MDCK-OAT1 and MDCK-mock cells was obviously different, with a 14.9 fold increase in comparison with mock cells. In the presence of probenecid and several monomers from Chinese herbs, fluorescence values in cell lysates were reduced to varying degrees, and results showed that rhein, luteolin, chrysin and quercetin could significantly inhibited the 6-CFL uptake mediated by hOAT1,with a reduction of more than 80% of the control. CONCLUSION: The aim to establish a cell model which could stably express OAT1 is achieved. Further study could be done using this cell model, for the screening of potential inhibitors of OAT1 from monomers of Chinese herbs,and then could be used as a tool in the research of herb-drug interaction.
ABSTRACT
Aim To investigate the effect of green tea polyphenols(GTP)on serum level of uric acid in potas-sium oxonate (PO)-induced hyperuricemic mice,and explore the potential mechanism.Methods PO and GTP were intragastricly administered to mice for seven consecutive days.Uric acid level in serum was exam-ined.Meanwhile,activity and expressions of xanthine oxidase(XOD)in liver were tested.In additon,ex-pressions of urate transporters including urate-anion transporter (URAT ) 1 , organic anion transporter (OAT)1 and 3 in kidney were analyzed.Results GTP significantly decreased the serum level of uric acid in PO-induced hyperuricemic mice.At the same time, GTP markedly reduced the activity and expression of XOD in liver of hyperuricemic mice.Finally,GTP markedly reduced the expression of URAT1 ,OAT1 and OAT3 in kidney of hyperuricemic mice.Conclusion GTP has the effect of lowering uric acid in PO-induced hyperuricemic mice through both decreasing the uric acid production and increasing uric acid excretion.
ABSTRACT
The organic anion transporters (OAT) have recently been identified. Although the some transport properties of OATs in the kidney have been verified, the regulatory mechanisms for OAT's functions are still not fully understood. The rat OAT1 (rOAT1) transports a number of negatively charged organic compounds between the cells and their extracellular milieu. Caveolin (Cav) also plays a role in membrane transport. Therefore, we investigated the protein-protein interactions between rOAT1 and caveolin-2. In the rat kidney, the expressions of rOAT1 mRNA and protein were observed in both the cortex and the outer medulla. With respect to Cav-2, the expressions of mRNA and protein were observed in all portions of the kidney (cortex < outer medulla = inner medulla). The results of Western blot analysis using the isolated caveolae-enriched membrane fractions or the immunoprecipitates by respective antibodies from the rat kidney showed that rOAT1 and Cav-2 co-localized in the same fractions and they formed complexes each other. These results were confirmed by performing confocal microscopy with immunocytochemistry using the primary cultured renal proximal tubular cells. When the synthesized cRNA of rOAT1 along with the antisense oligodeoxynucleotides of Xenopus Cav-2 were co-injected into Xenopus oocytes, the [14C]p-aminohippurate and [3H]methotrexate uptake was slightly, but significantly decreased. The similar results were also observed in rOAT1 over-expressed Chinese hamster ovary cells. These findings suggest that rOAT1 and caveolin-2 are co-expressed in the plasma membrane and rOAT1's function for organic compound transport is upregulated by Cav-2 in the normal physiological condition.