Organic Anion Transproter System Mediates The Transport Of Aristolochic Acid In Tubular Epithelial Cells

BackgroundAristolochic acids(AA) belong to a family of structurally related nitrophenanthrene carboxylic acids.The consumption of herbs containing AA has been associated with severe nephropathy which is called aristolochic acid nephropathy(AAN).AAN is characterized by a paucicelluar progressive and extensive interstitial fibrosis without prominent glomerular lesions and inflammatory cell infiltration.Clinical and in vitro findings have suggested that the proximal tubule was the major site of AA-induced renal injury.We have reported AA-induced cytotoxic and fibrogenic effects on proximal tubule epithelial cells.However the potential pathogenic mechanism of AAN has not been clarified.Could AA enter the proximal tubular epithelial cells? From the structural point of view,AA are classified into a diverse group of organic compounds of low molecular weight.For the entry of these compounds,special membrane proteins such as transporters are required, because they cannot penetrate the lipid bilayer of the cell membrane.In the kidney, organic anion transporters,which are expressed in the apical and basolateral membranes of tubular epithelial cells,are responsible for tubular secretion of organic anions including drugs,toxins and endogenous compounds.The mRNA levels of hOAT1 and hOAT3 were much higher than those of other organic ion transporters in the human kidney cortex.These findings suggest that hOAT1 and hOAT3 play important roles in the tubular uptake of various drugs from the circulation.Under such circumstance,we decided to investigate whether AA could enter the proximal tubular epithelial cells and if organic anion transporters are involved in this process.Objectives1) To investigate whether AA could enter the proximal tubular epithelial cells (HKC).2) To investigate which transporters are involved in the process of AA entering HKC.3) To investigate whether decreased intracellular AA could down-regulate its cytotoxic and fibrogenic effects on HKC.Methods1) Liquid chromatography-tandem mass spectrometry was applied to measure intracellular AA.2) The release of lactate dehedrogenase(LDH) induced by AA in the presence of organic anion transporter inhibitor(probenecid) or organic cation transporter inhibitor(tetraethylammonium) was evaluated.3) After HKC were transiently transfected with pCMV5-HA-hOAT1/3,the cytotoxic effect of AA on HKC was assessed by the release of LDH.4) The effects of probenecid on AA induced mRNA and protein expression of connective tissue growth factor(CTGF) were also determined by real time polymerase chain reaction and Western blots,respectively.Results1) Intracellular AA of HKC,which was not detected in the control group,were detected and measured in the suspension of the broken HKC cells.2) The LDH releasing from HKC stimulated by 60mg/L AA-Na was significantly inhibited by 1 mM probenecid(P＜0.05),but not by 1mM tetraethylammonium.3) The cytotoxic effect of AA on the HKC cells was increased when HKC were transiently transfected with pCMV5-HA-hOAT1/3.4) The up-regulated mRNA and protein expression of CTGF in HKC stimulated by 40mg/L AA-Na was significantly inhibited by 1mM probenecid(P＜0.05) with a inhibition rate of 16%and 21%,respectively.Conclusions1) The results suggest that AA could be transported into HKC through organic anion transporters. 2) AA can be transported into HKC and then exerts its biological response and cytotoxic effect,which can be inhibited by probenecid.3) The cytotoxic effect of AA on HKC was increased with overexpressed hOAT1/3 in a dose-dependend manner.