Screening Of Potent Inhibitors Of Organic Anion Transporter 1 And 3 From Natural Compounds And Their Protective Effects On Aristolochic Acidinduced Renal Toxicity

Organic anion transporter 1 and 3（OAT1 and OAT3）are two major organic anion transporters in kidney and play critical roles in the transportation and elimination of various anion substrates and their metabolites.Aristolochic acid I（AAI）can cause severe aristolochic acid nephropathy（AAN）and is a known substrate of OAT1 and OAT3.Studies have shown that probenecid,an OAT inhibitor,can prevent AAN by reducing the accumulation of AAI-DNA adducts in kidney.Therefore,inhibitors of OAT1 and OAT3 may effectively prevent the development of AAN and protect against AAs-induced renal injury.In addition,OAT1 and OAT3 have been considered as the important targets for drug-drug interactions（DDIs）.To date,many studies have focused on the interaction of clinical“western”drugs with OATs,but relatively little is known about the interactions between natural products and OATs.Objective:The aim of this study was to identify OAT1 and OAT3 potent inhibitors from a large natural compound library（270 natural compounds）,to explore the interactions between inhibitors and OAT1/3,and to evaluate their protective effects on AAN.Methods:Fluorescence-based high-throughput screening assay and cell uptake assay were used to screen the inhibitors of OAT1 and OAT3.Compounds（inhibiting 6-CF uptake more than 70%）were selected to further study their IC₅₀ values and inhibitory modes using concentration-dependent inhibition assays.The homology models of OAT1 and OAT3 were established using SWISS-MODEL and the interactions of inhibitors with OAT1 and OAT3 were explored using molecular docking.Wedelolactone and wogonin,two newly identified potent OAT1/3 inhibitors,were used to investigate their effects on pharmacokinetics of AAI in rats as well as their protective effects on renal injury in AAN mice.Results:Of the 270 natural compounds,21 OAT1 inhibitors and 45 OAT3 inhibitors were identified.Further concentration-dependent inhibition studies demonstrated 7potent inhibitors of OAT1 and 10 potent inhibitors of OAT3(IC₅₀ values of<10μM).In vitro studies showed that AAI inhibited 6-CF uptake in both HEK-OAT1 and HEK-OAT3 cells,but resulted in more cytotoxicity in HEK-OAT1 than HEK-OAT3 cells.In vivo studies showed that both wedelolactone and wogonin markedly increased serum AAI concentrations in AAI-treated rats and ameliorated kidney injuries in AAI-treated mice.Conclusion:Potent inhibitors of OAT1/3 are identified from 270 naturally occurring compounds,and their potential interactions with OAT1/3 are evaluated.The present study demonstrates for the first time the promising protective effects of wedelolactone and wogonin on AAN.Our findings will not only aid in understanding natural compound-drug interactions,but also provide a natural source for the drug development to treat AAN.