Renal Drug Transporters – Clinical Significance and Role in Drug-Drug Interactions

Thesis (Ph.D.)--University of Washington, 2016-07Drug transporters play an important role in drug disposition and can be major determinants of drug pharmacokinetics, pharmacodynamics and toxicity. In contrast to drug metabolizing enzymes, which mainly concentrate in the liver and small intestine, drug transporters are expressed ubiquitously throughout human body. In human kidney, drug transporters are primarily expressed in the basolateral and apical membranes of proximal tubule cells and often work in tandem to secrete therapeutic drugs and their metabolites into tubular lumen. Furthermore, transporter-mediated drug-drug interactions (DDIs) have become a significant clinical concern as they can adversely impact drug disposition, efficacy, and toxicity. The overall goal of my research project is to understand the clinical significance of renal drug transporters and their role in drug-drug interactions. An analysis of the top 200 prescribed drugs in U.S. and their DDI studies suggests that there is considerable potential for clinically significant renal DDI to occur among commonly used drugs. Potent drug transport inhibitors elicit significant interactions in vivo that approach maximums predicted from in vitro-derived inhibition kinetics. However, high-magnitude renal DDIs in the clinical setting appear to be rare, which may partly be due to a lack of potent in vivo inhibitors, low fraction of net secretion of victim drugs, and compensating transport pathways for the victim drug. Our analysis also identified two widely used antihypertensive drugs, atenolol and hydrochlorothiazide that are cleared predominantly by the kidney but the molecular mechanisms involved in its renal secretion are virtually unknown. Using a panel of HEK cell lines stably expressing major renal drug transporters and absolute quantification of membrane transporter proteins by LC-MS/MS, we found that atenolol is an excellent substrate for the renal organic cation transporter 2 (hOCT2), multidrug and toxin extrusion proteins 1 and 2-K (hMATE1 and 2-K). It can inhibit but not be transported by renal organic anion transporters 1 and 3 (hOAT1 and hOAT3). We also demonstrate unidirectional transepithelial transport of atenolol in an hOCT2/hMATE1 double-transfected MDCK cell culture model. Our data suggest that renal secretion of atenolol is mediated by hOCT2/hMATEs pathway. On the other hand, hydrochlorothiazide was identified to be a substrate of both organic cation transporters hOAT1 and hOAT3 and organic anion transporters hOCT2 and hMATE2-K. However, hOCT2 and hMATE2-K showed much lower affinity and transport efficiency for hydrochlorothiazide than hOAT1 and hOAT3. Combined with findings from other investigators, we propose that renal tubular secretion of hydrochlorothiazide is mediated by two parallel pathways, with hOATs/multidrug resistance-associated protein 4 (hMRP4) being the major one and hOCT2/hMATE2-K being a minor pathway. Our in vitro inhibition studies using potent hOAT1/3 inhibitors also suggest that caution should be taken when hydrochlorothiazide is co-prescribed with potent hOAT1/3 inhibitors, as renal secretion is not only important for its elimination, it may also play a role in its efficacy. Emerging evidence suggests that renal hOCT2 and hMATE1/2-K exhibit substrate-dependent inhibition but the impact on renal drug secretion and intracellular accumulation is unknown. Regulatory agencies recently developed guidelines for transporter-mediated DDI risk assessment but the potential influence of probe substrate choice on prediction is not clear. Using drug substrates, we showed that inhibition of the renal basolateral organic cation transporter 2 by classic clinical inhibitors is highly substrate-dependent, leading to strikingly different DDI predictions. In contrast, inhibition of the apical multidrug and toxin extrusion proteins is less affected by substrate choice. Using a cell culture model simulating tubular secretion, we further demonstrate that substrate-dependent inhibition can shift the major substrate-inhibitor interacting site between apical and basolateral transporters, leading to different effects on intracellular drug accumulation. These findings revealed the complex and dynamic nature of substrate-inhibitor interactions in multispecific drug transporters. In summary, this dissertation research has evaluated the clinical significance of renal DDIs, demonstrated the important role of renal transporters in the clearance and efficacy of two widely used antihypertensive drugs and highlighted the necessity of considering substrate-dependent inhibition in predicting transporter-mediated DDI