Creatinine Transport by Basolateral Organic Cation Transporter hOCT2 in the Human Kidney Yumiko Urakami, 1 Naoko Kimura, 1 Masahiro Okuda, 1 and Ken-ichi Inui 1,2 Received January 17, 2004; accepted March 15, 2004 Purpose. Creatinine is excreted into urine by tubular secretion in addition to glomerular filtration. The purpose of this study was to clarify molecular mechanisms underlying the tubular secretion of cre- atinine in the human kidney. Methods. Transport of [ 14 C]creatinine by human organic ion trans- porters (SLC22A) was assessed by HEK293 cells expressing hOCT1, hOCT2, hOCT2-A, hOAT1, and hOAT3. Results. Among the organic ion transporters examined, only hOCT2 stimulated creatinine uptake when expressed in HEK293 cells. Cre- atinine uptake by hOCT2 was dependent on the membrane potential. The Michaelis constant (K m ) for creatinine transport by hOCT2 was 4.0 mM, suggesting low affinity. Various cationic drugs including ci- metidine and trimethoprim, but not anionic drugs, markedly inhibited creatinine uptake by hOCT2. Conclusion. These results suggest that hOCT2, but not hOCT1, is responsible for the basolateral membrane transport of creatinine in the human kidney. KEY WORDS: creatinine; glomerular filtration rate; hOCT2; or- ganic cation transporter; tubular secretion. INTRODUCTION In the proximal tubules of mammalian kidney, organic ion transporters limit or prevent the toxicity of organic anions and cations by actively secreting these substances from the circulation into the urine (1–5). We isolated a second member of the organic cation transporter (OCT) family, rat (r) OCT2 (6), showing 67% amino acid identity to rOCT1 (7). Func- tional studies using Xenopus oocytes (6–10) and transfected mammalian cells (11–13) as expression systems suggested that rOCT1 and rOCT2 transport various structurally unrelated cations in a voltage-dependent fashion. rOCT1 and rOCT2 possess similar but not identical specificities for various cat- ionic compounds. Both rOCT1 and rOCT2 protein were lo- calized in the basolateral membrane of renal tubular cells (14,15), although the distributions of these transporters along the nephron were distinct (13). To date, three distinct genes encoding human organic cation transporters have been identified including hOCT1, hOCT2, and hOCT3 (5). In addition, we identified hOCT2-A, an alternatively spliced variant of hOCT2, expressed in the human kidney, with different transport characteristics from that of hOCT2 (16). We also demonstrated that the mRNA level of hOCT2 was the highest in the human kidney among organic cation transporters examined, suggesting hOCT2 to be the dominant organic cation transporter in the human kid- ney (17). In contrast, hOCT1 is mainly transcribed in the liver, suggesting that hOCT1 is responsible for the hepatic uptake of organic cations (18–19). Although characterization of or- ganic cation transport by hOCT2 have been done, intrinsic roles of hOCT2 in the disposition of physiological substances have not been clarified. It is established that creatinine, a catabolic product of creatine, is eliminated predominantly into urine. Creatinine can also be secreted via the renal tubules in addition to the glomerular filtration, however, the molecules mediating tu- bular secretion of creatinine in the human kidney have not been identified. Because organic ion transporters recognize a wide variety of ionic compounds, thereby mediate tubular secretion of organic ions, we measured creatinine transport by organic ion transporters (SLC22A), hOCT1, hOCT2, hOCT2-A, hOAT1, and hOAT3, to assess the involvement of these transporters in the tubular secretion of creatinine. MATERIALS AND METHODS Cell Culture HEK293 cells (ATCC CRL-1573), a transformed cell line derived from human embryonic kidney, were cultured in complete medium consisting of Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum in an atmosphere of 5% CO 2 /95% air at 37°C. For uptake experiments, the cells were seeded onto poly-D-lysine-coated 24-well plates at a density of 2.0 × 10 5 cells per well. The cell monolayers were used at day 3 of culture for uptake experiments. In this study, HEK293 cells between the 68th and 89th passages were used. Transfection pCMV6-XL4 plasmid vector (OriGene Technologies, Rockville, MD, USA) DNA containing hOCT1, hOCT2, hOCT2-A, hOAT1, and hOAT3 cDNA, and pBK-CMV vec- tor (Stratagene, La Jolla, CA, USA) were purified using Mar- ligen High Purity Plasmid-Prep Systems (Invitrogen, Carls- bad, CA, USA). The day before the transfection, HEK293 cells were seeded onto poly-D-lysine-coated 24-well plates at a density of 2.0 × 10 5 cells per well. The cells were transfected with 0.8 g of total plasmid DNA per well using Lipofect- AMINE 2000 (Invitrogen) according to the methods de- scribed previously (16). At 48 h after transfection, the cells were used for uptake experiments. To construct a transfectant stably expressing hOCT2, HEK293 cells were transfected with 0.8 g of total plasmid DNA (pCMV6-XL4: pBK-CMV vector  2:1) per well. At 24 h after transfection, the cells split between 1:15 and 1:30 were cultured in complete me- dium containing G418 (0.5 mg/ml) (Wako Pure Chemical, Osaka, Japan). Then 14 to 21 days after transfection, single colonies were picked out. G418-resistant colonies were ana- lyzed by RT-PCR for the expression of hOCT2 mRNA. Uptake Experiments Using HEK293 Transfectants Cellular uptake of cationic and anionic compounds using HEK293 cells was measured with monolayer cultures grown 1 Department of Pharmacy, Kyoto University Hospital, Faculty of Medicine, Kyoto University, Kyoto 606-8507, Japan. 2 To whom correspondence should be addressed. (e-mail: inui@kuhp. kyoto-u.ac.jp) ABBREVIATIONS: hOCT, human organic cation transporter; GFR, glomerular filtration rate; MPP, 1-methyl-4-phenylpyridinium; NMN, N 1 -methylnicotinamide; PAH, p-aminohippuric acid; TEA, tetraethylammonium. Pharmaceutical Research, Vol. 21, No. 6, June 2004 (© 2004) Research Paper 976 0724-8741/04/0600-0976/0 © 2004 Plenum Publishing Corporation