Atenolol Renal Secretion Is Mediated by Human Organic Cation Transporter 2 and Multidrug and Toxin Extrusion Proteins

Abstract

Atenolol is a b-blocker widely used in the treatment of hypertension. Atenolol is cleared predominantly by the kidney by both glomerular filtration and active secretion, but the molecular mechanisms involved in its renal secretion are unclear. Using a panel of human embryonic kidney cell lines stably expressing human organic cation transporter (hOCT) 1-3, human organic anion transporter (hOAT) 1, hOAT3, human multidrug and toxin extrusion protein (hMATE) 1, and hMATE2-K, we found that atenolol interacted with both organic cation and anion transporters. However, it is transported by hOCT1, hOCT2, hMATE1, and hMATE2-K, but not by hOCT3, hOAT1, and hOAT3. A detailed kinetic analysis coupled with absolute quantification of membrane transporter proteins by liquid chromatographytandem mass spectrometry revealed that atenolol is an excellent substrate for the renal transporters hOCT2, hMATE1, and hMATE2-K. The K m values for hOCT2, hMATE1, and hMATE2-K are 280 6 4, 32 6 5, and 76 6 14 mM, respectively, and the calculated turnover numbers are 2.76, 0.41, and 2.20 s 21 , respectively. To demonstrate unidirectional transepithelial transport of atenolol, we developed and functionally validated a hOCT2/hMATE1 double-transfected Madin-Darby canine kidney cell culture model. Transwell studies showed that atenolol transport in the basal (B)-to-apical (A) direction is 27-fold higher than in the A-to-B direction, whereas its B-to-A/A-to-B transport ratio was only 2 in the vectortransfected control cells. The overall permeability of atenolol in the B-to-A direction in hOCT2/hMATE1 cells was 44-fold higher than in control cells. Together, our data support that atenolol tubular secretion is mediated through the hOCT2/hMATEs secretion pathway and suggest a significant role of organic cation transporters in the disposition of an important antihypertensive drug.

Impact of Substrate-Dependent Inhibition on Renal Organic Cation Transporters hOCT2 and hMATE1/2-K-Mediated Drug Transport and Intracellular Accumulation

“…In vitro transport and inhibition studies were performed as previously described (Duan and Wang, 2010;Yin et al, 2015). Briefly, transporterexpressing and control HEK-293 cells were seeded in poly-D-lysinecoated plates and allowed to grow for 2-3 days to reach 80-90% confluence.…”

“…Transporter-expressing cells generally showed transport activities much higher (∼10-to 30-fold) than control HEK-293 cells for a probe substrate (e.g., metformin or MPP 1 ) (Yin et al, 2015). Both transporter-expressing and control HEK-293 cells were maintained in Dulbecco's modified Eagle's medium (high glucose) supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 IU/ml penicillin, 100 mg/ml streptomycin, and 150 mg/ml hygromycin B. Vectors (pcDNA3.11 and pcDNA3.1/Hygro(1)) and hOCT2/ hMATE1 double-transfected Madin-Darby canine kidney (MDCK) cell lines were previously established in our laboratory (Yin et al, 2015). MDCK cells were maintained in minimum essential medium supplemented with 10% fetal bovine serum, 500 mg/ml G418, and 200 mg/ml hygromycin B.…”

“…Its impact on intracellular drug accumulation, which is an important predictor for drug-induced nephrotoxicity, is also unknown. In this study, we investigated the influence of probe substrate choice on predicting transporter-mediated DDIs using metformin and a newly identified drug substrate of hOCT2 and hMATEs, atenolol (Yin et al, 2015). The overall impact of substrate-dependent inhibition on tubular secretion and intracellular accumulation was also investigated using a double-transfected cell culture model of renal OC secretion.…”

“…Cell Lines and Cell Culture. Control (pcDNA5-transfected) and Flp-In human embryonic kidney (HEK)-293 cell lines stably expressing hOCT2, hMATE1, and hMATE2-K were previously established and maintained in Dulbecco's modified Eagle's medium Yin et al, 2015). Transporter-expressing cells generally showed transport activities much higher (∼10-to 30-fold) than control HEK-293 cells for a probe substrate (e.g., metformin or MPP 1 ) (Yin et al, 2015).…”

“…Control (pcDNA5-transfected) and Flp-In human embryonic kidney (HEK)-293 cell lines stably expressing hOCT2, hMATE1, and hMATE2-K were previously established and maintained in Dulbecco's modified Eagle's medium Yin et al, 2015). Transporter-expressing cells generally showed transport activities much higher (∼10-to 30-fold) than control HEK-293 cells for a probe substrate (e.g., metformin or MPP 1 ) (Yin et al, 2015). Both transporter-expressing and control HEK-293 cells were maintained in Dulbecco's modified Eagle's medium (high glucose) supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 IU/ml penicillin, 100 mg/ml streptomycin, and 150 mg/ml hygromycin B. Vectors (pcDNA3.11 and pcDNA3.1/Hygro(1)) and hOCT2/ hMATE1 double-transfected Madin-Darby canine kidney (MDCK) cell lines were previously established in our laboratory (Yin et al, 2015).…”