ABSTRACT
Objective: To explore the effect of hirudin on hyperuricemia rats and its mechanism. Methods: Male Wistar rats were randomly divided into control group, model group, allopurinol (30 mg/kg) group, hirudin low-, middle- and high-dose (0.2, 0.4, 0.8 g/kg) group. Rats were ig potassium oxonate (0.75 g/kg) to induce hyperglycemia model, once a day for five weeks. And all administration groups were respectively ig corresponding doses of drugs. The level of uric acid in serum and urine of rats were measured by biochemical method; The level of organic anion transporter 1 (OAT1) in kidney was measured by immunohistochemistry; The protein expressions of glucose transporter 9 (GLUT9), OAT1 and urate transporter 1 (URAT1) in kidney were measured by western blotting; The expression levels of GLUT9, OAT1 and URAT1 mRNA in kidney were detected by qRT-PCR. Results: Compared with control group, the level of uric acid in serum and urine of rats in model group was significantly increased (P < 0.01), the expressions of GLUT9, URAT1 mRNA and protein were significantly increased (P < 0.01), the expressions of OAT1 mRNA and protein were significantly decreased (P < 0.01). Compared with model group, the level of uric acid in serum and urine of rats in hirudin group were significantly decreased (P < 0.01), the expressions of GLUT9, URAT1 mRNA and protein were significantly reduced (P < 0.01), the expressions of OAT1 mRNA and protein were significantly increased (P < 0.01). Conclusion Hirudin can reduce the uric acid by regulating the expressions of renal urate transporters OAT1, URAT1 and GLUT9 in hyperuricemia rats.
ABSTRACT
Stellera chamaejasme L. is a traditional Chinese medicine with a long history to treat stubborn skin ulcer, and it also has antiviral and antitumor effects. Neochamaejasmine B (NCB), Neochamaejasmine A (NCA) and Chamaechromone (CMC) are the major components in dried roots of Stellera chamaejasme L.. Our studies suggested that NCB, NCA and CMC are inhibitors of Organic anion transporter 1 (OAT1). OAT1 is encoded by solute carrier family 22 member 6 gene (SLC22A6) in humans and plays a critical role in the organic anion drug uptake and excretion in the kidney. Lamivudine is the typical substrate of OAT1 and is frequently used in combination with other antiviral drugs in clinical antiviral treatments. The aim of this study is to investigate the interaction and its mechanism between these bi-flavone components in Stellera chamaejasme L. and lamivudine via OAT1 both in vitro and in vivo. In vitro, the uptake studies in Madin-Darby canine kidney (MDCK) cells overexpressing OAT1 suggested that NCB inhibited the uptake of 6-CFL and lamivudine.Similar results were obtained for NCA and CMC. NCB was a noncompetitive and competitive inhibitor interaction with OAT1. IC values of NCB, NCA and CMC for inhibiting OAT1-mediated lamivudine transport were 2.46, 8.35 and 0.61 μmol·L, respectively. In vivo, the pharmacokinetic results of lamivudine in rats showed that the mean area under the plasma concentration-time curve (AUC) and maximal plasma concentration (C) of lamivudine after co-administration is increased 2.94-fold and 1.87-fold, respectively, compared to lamivudine administration alone. The results of interactions between lamivudine and these bi-flavone components in Stellera chamaejasme L. extracts via OAT1 in vivo are consistent with studies in vitro. The inhibition of OAT1-mediated uptake of lamivudine by NCB, NCA and CMC is the possible mechanism for Stellera chamaejasme L. extracts improving the oral bioavailability of lamivudine in rats.
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
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.