Cellular Membrane Transporters Mediate Hepatic And Renal Transport Of Entecavir

Hepatitis B is an inflammation of the liver disease caused by the hepatitis B virus(HBV),which will cause multiple organ damage,and can be developed into liver cirrhosis(LC),primary liver Cell carcinoma(HCC).The comprehensive treatment,including anti-virus,immune regulation,anti-inflammatory and improve liver function,has been applied clinically,in which the anti-viral has been the key one.Conventional antiviral drugs in the clinical application have their own limitations.Entecavir(ETV)is a new nucleoside analogue with a high antiviral efficacy.After approval in 2005,it has been unanimously recommended as fist-line treatment of hepatitis B.According to its pharmacokinetics and pharmacodynamics,kidney is the main excretory organ,while liver is the target organ.However,it has been confirmed that ETV is a hydrophilic weak base with a pKa value of 10.5(charged to cationic state at physiological pH is 99.9%),and is difficult to pass the membrane by passive diffusion,therefore,we deduced some transporters play important roles in hepatic and renal transport of ETV.Human kidney-specific isoform of the multidrug and toxin efflux extrusion(hMATE2-K)is H+/organic cation antiporter discovered in 2006,co-expressed with MATE1 and ABC efflux protein in the brush border membrane of proximal tubules,assist some uptake transporters expressed in basolateral membrane to transport the cationic compound,some kind of neutral and anionic substances into the urine.According to the structure of ETV,we reasoned that some renal or hepatic transporters,including hMATE2-K,might mediate the transport of ETV.Since the transporters expressed in immortalized liver,kidney cell lines often are at low level or even no expression,the transgenic cells stably expressing transporters become ideal models for study in vitro.Our laboratory has many transgenic cell models,but cell model stably expressing hMATE2-K has not been established,thus we first constructed MDCK?-hMATE2-K cell model,and then studied the renal and hepatic transport mechanism of ETV with the transfected cell models,including MDCK?-hMATE2-K,and mouse primary renal tubular cells and hepatocytes.1.Construction of cell model with stable expression of hMATE2-KTo establish the Madin-Darby canine kidney(MDCK?)cell models with stably-expressing human MATE2-K(hMATE2-K).The hMATE2-K gene was PCR from pCMV-SLC47A2 plasmid.The plasmids pcDNA3.1(+)-MATE2-K was constructed and transfected into MDCK? by lipo2000 followed by hygromycin B screening.Then the selected monoclonal cells verified by classical substrate(MPP+,metformin)accumulation with or without known inhibitors(cimetidine or quinidine,100?M),the mRNA and protein level.The results showed that the mRNA/protein expression was much higher in MDCK?-hMATE2-K than that in mock cells.The uptake of MPP+ and metformin in positive colones was much higher than that in mock cells.The kinetic parameters of substrates in MDCK?-hMATE2-K are close to that in literatures.The above results show that the MDCK?-hMATE2-K is successfully constructed,which can be used to study hMATE2-K mediates drug transport and drug-drug interactions.2.Interaction of ETV with renal transportersThe inhibitory effect of ETV on hMATEs mediated uptake of MPP+was determined in MDCKII-hMATEl,hMATE2-K,and the uptake of the ETV in MDCKII-hMATEs and mock cells was also estimated.The uptake of ETV was significantly higher in MDCKII-hMATEs cells than in mock cells,and the uptake in MDCKII-hMATEs was significantly inhibited by hMATEs inhibitors(cimetidine or quinidine,100?M),suggesting ETV is a substrate of hMATEs.The kinetic parameters of ETV uptake in MDCKII-hMATEs analysed by Eadie-Hofstee showed that the accumulation was an atypical dynamics,under low concentration,both of the Km values were 0.30mM,the Vmaxs were 0.90,3.1nmol/mg protein/min,respectively;while under high concentration,the Km values were 1.5,1.1 mM,respectively,Vmax values were 2.7,6.5nmol/mg protein/min,respectively.We also applied MDCKII stably expressing hOCT2,hOCTN1/2,hMDRl and hMRP2 to study whether other transporters mediated ETV renal transport,and the results showed that ETV was a substrate of hOCT2,hOCTN1/2,and the accumulation of ETV in MDCKII-hOCT2,hOCTNl/2 showed typical dynamics,the Km values were 0.3,2.8 and 2.2mM respectively,the Vmax values were 1.0,4.3 and 3.0nmol/mg protein/min respectively.In addition,we also found that ETV was a substrate of hMDRl and hMRP2.Finally,mouse primary renal tubular cells(mPRTC)were used to confirm which transporters involved in the renal disposition of ETV.The inhibitors of Oct2,Oats,Octnl/2 and Mrp2 obviously affected the intracellular accumulation of ETV in mPRTC.In summary,the renal tubular secretion of ETV may mediate by hOCT2 for uptake into proximal tubular cells and efflux by hMATEs,hMDRl,hMRP2,meanwhile,hOCTNl,2 may contribute to renal reabsorption of ETV.3.Interaction of ETV with hepatic transporterThe primary mouse hepatocytes and transgenic cells stably expressing transporters were applied to study the role of transprters in the liver disposition of ETV.The accumulation in hepatocytes incubated at 37? was obviously higher than that at 4?,probenecid the inhibitor of organic anion transporters(OATs),and cimetidine,the inhibitor of organic cation transporters(OCTs),significantly inhibited the intracellular accumulation of ETV.Meanwhile MK571,the MRPs inhibitor,markedly increased intracellular accumulation.Subquently,MDCKII-hOCT1/3,MDCKII-hENT4 cells were applied to confirm the effect of hOCTl,hOCT3 and hENT4 on liver disposition of ETV.The results showed that ETV was a weak substrate of hOCT3 and hENT4,but not of hOCT1.Above all,these results indicate that hOCT3,hENT4 located in sinusoidal(basolateral)membrane mediate ETV uptake in hepatocytes,while the unphosphorylated ETV would be transported to the bile mediated by hMATE1 and hMRP2 located in bile canaliculi.