Yumiko Urakami

Creatinine Transport by Basolateral Organic Cation Transporter hOCT2 in the Human Kidney

AbstractPurpose. 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 creatinine in the human kidney. Methods. Transport of [14C]creatinine by human organic ion transporters (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. Creatinine uptake by hOCT2 was dependent on the membrane potential. The Michaelis constant (Km) for creatinine transport by hOCT2 was 4.0 mM, suggesting low affinity. Various cationic drugs including cimetidine 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.

Gender differences in expression of organic cation transporter OCT2 in rat kidney.

The organic cation transporter (OCT) mediates translocation of various cationic molecules including drugs, toxins and endogenous substances. We examined gender differences in the expression of rat (r) OCT2 in the kidney. Slices and basolateral membrane vesicles of male rat kidney showed a higher transport activity for tetraethylammonium than those of female rat kidney. The expression levels of rOCT2 mRNA and protein in the kidney of males were much higher than those in females. There was no gender difference in mRNA expression of rOCT1 and rOCT3. These findings suggest that rOCT2 is responsible for the gender differences in renal basolateral membrane organic cation transport activity.

Hormonal regulation of organic cation transporter OCT2 expression in rat kidney

Rat (r) OCT2 was identified as the second member of the organic cation transporter (OCT) family, and is predominantly expressed in the kidney. We reported previously that rOCT2 was responsible for the gender differences in renal basolateral membrane organic cation transport activity. As renal rOCT2 expression in males is much higher than that in females, we hypothesized that rOCT2 expression may be under the control of sex hormones. Treatment of male and female rats with testosterone significantly increased the expression levels of rOCT2 mRNA and protein in the kidney, whereas estradiol treatment moderately decreased the expression levels of rOCT2. There was no regulation of renal rOCT1 mRNA expression by testosterone or estradiol. Treatment of male and female rats with testosterone significantly stimulated the tetraethylammonium (TEA) accumulation by renal slices, whereas estradiol treatment caused a decrease in the TEA accumulation by slices from male but not female rats. The present findings suggested that testosterone up‐regulates renal rOCT2 expression and estradiol moderately down‐regulates rOCT2.

cDNA cloning, functional characterization, and tissue distribution of an alternatively spliced variant of organic cation transporter hOCT2 predominantly expressed in the human kidney.

A cDNA coding a novel organic cation transporter, hOCT2-A, was isolated from human kidney. The hOCT2-A cDNA is an alternatively spliced variant of hOCT2 with an insertion of 1169 bp. The open reading frame encodes a 483-amino acid protein that has 81% amino acid identity with hOCT2. From hydropathy analysis, hOCT2-A is predicted to have nine transmembrane domains. hOCT2-A mRNA is expressed mainly in kidney and weakly in brain, liver, colon, skeletal muscle, bone marrow, spinal cord, testis, and placenta. When expressed in HEK293 cells, hOCT2-A stimulated the uptake of tetraethylammonium (TEA) in an electrogenic manner. The transport of TEA by hOCT2-A-transfected cells was saturable with the apparent Km value of 63 microM. hOCT2-A stimulated the uptake of TEA, 1-methyl-4-phenylpyridinium, and cimetidine as well as did hOCT2. The uptake of guanidine and choline by hOCT2-transfected cells also increased markedly but not that by hOCT2-A-transfected cells. The uptake of TEA mediated by hOCT2-A but not by hOCT2 was inhibited significantly by organic cations such as procainamide, N-acetylprocainamide, and levofloxacin, indicating that hOCT2-A differs from hOCT2 in its affinity for several compounds. These findings suggested that hOCT2-A contributes to the renal clearance of endogenous and exogenous organic cations.

Molecular mechanisms of organic cation transport in OCT2-expressing Xenopus oocytes

The molecular mechanisms of organic cation transport by rat OCT2 was examined in the Xenopus oocyte expression system. When extracellular Na+ ions were replaced with K+ ions, uptake of tetraethylammonium (TEA) by OCT2-expressing oocytes was decreased, suggesting that TEA uptake by OCT2 is dependent on membrane potential. Kinetic analysis revealed that the decreased TEA uptake by ion substitution was caused at least in part by decreased substrate affinity. Acidification of extracellular buffer resulted in decreased uptake of TEA, whereas TEA efflux from OCT1- and OCT2-expressing oocytes was not stimulated by inward proton gradient, in consistent with basolateral organic cation transport in the kidney. Inhibition of TEA uptake by various organic cations revealed that apparent substrate spectrum of OCT2 was similar with that of OCT1. However, the affinity of procainamide to OCT1 was higher than that to OCT2. Uptake of 1-methyl-4-phenylpyridinium was stimulated by OCT2 as well as OCT1, but uptake of levofloxacin, a zwitterion, was not stimulated by both OCTs. These results suggest that OCT2 is a multispecific organic cation transporter with the characteristics comparable to those of the basolateral organic cation transporter in the kidney.

Differential localization of organic cation transporters rOCT1 and rOCT2 in the basolateral membrane of rat kidney proximal tubules

Abstract. Organic cation transporters play an important role in the secretion of cationic drugs as well as endogenous cationic metabolites in the renal tubules. Immunoblotting showed the presence of organic cation transporter proteins, rOCT1 and rOCT2, in the rat kidney. By immunofluorescence microscopy, rOCT1 was shown to be concentrated in the proximal tubules in the renal cortex. rOCT2, on the other hand, was rich in the proximal tubules in the outer stripe of the outer medulla. Confocal microscopy revealed that both rOCT1 and rOCT2 were localized to the basolateral membranes of these tubule cells. These findings directly show that rOCT1 and rOCT2 are basolateral membrane proteins and are differentially distributed along the proximal tubules.

Distinct Characteristics of Organic Cation Transporters, OCT1 and OCT2, in the Basolateral Membrane of Renal Tubules

AbstractPurpose. This study was performed to determine the detailed mRNA distribution of organic cation transporters, rOCT1 and rOCT2, along the rat nephron and to distinguish the substrate affinities of these transporters. Methods. The distributions of rOCT1 and rOCT2 mRNA were determined by reverse transcriptase polymerase chain reaction analysis of microdissected nephron segments. Using MDCK cells transfected with rOCT1 or rOCT2 cDNA, the inhibitory effects of various compounds on the uptake of [14C]tetraethylammonium were assessed. Results. rOCT1 mRNA was detected primarily in the superficial and juxtamedullary proximal convoluted tubules, whereas rOCT2 mRNA was detected widely in the superficial and juxtamedullary proximal straight and convoluted tubules, medullary thick ascending limbs, distal convoluted tubule, and cortical collecting duct. The IC50 values for cationic drugs and endogenous cations on [14C]tetraethylammonium uptake across the basolateral membranes in the transfectants indicated that rOCT1 and rOCT2 had similar inhibitor specificity for many compounds but showed moderate differences in the specificity for several compounds, such as 1-methyl-4-phenylpyridinium, dopamine, disopyramide, and chlorpheniramine. Conclusions. rOCT1 and rOCT2 possess similar but not identical multispecificities for various compounds with distinct distributions along the nephron, indicating that the two transporters share physiologic and pharmacologic roles in the renal handling of cationic compounds.