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Previous Article | Next Article 
Molecular and Cellular Biology, November 2003, p. 7902-7908, Vol. 23, No. 21
0270-7306/03/$08.00+0 DOI: 10.1128/MCB.23.21.7902-7908.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
Division of Experimental Therapy, The Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands
Received 15 May 2003/ Returned for modification 21 July 2003/ Accepted 5 August 2003
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ABSTRACT |
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INTRODUCTION |
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In rodents, Oct1 (Slc22a1) is expressed in the liver, kidney, and small intestine (11, 13, 27), whereas in humans, OCT1 is primarily expressed in the liver (6). Oct2 (Slc22a2) has a substrate specificity similar to that of Oct1 but is predominantly expressed in the kidney in both rodents and humans (10). Immunohistochemical studies with rats have demonstrated that Oct1 is localized at the sinusoidal (basolateral) membrane of hepatocytes in the liver, whereas in the kidney, Oct1 and Oct2 are both localized at the basolateral membrane of epithelial cells of the proximal tubules (15, 21, 31). The broad substrate specificity and strategic localization of Oct1 and Oct2 in the major excretory organs suggest that these proteins are essential in the removal of cationic toxins and waste products from the body via the liver, kidney, and intestine.
Previously, we generated Oct1-/- mice and showed that absence of Oct1 resulted in greatly reduced hepatic uptake and direct intestinal excretion of substrate organic cations, indicating that Oct1 plays an essential role in the disposition of organic cations by the liver and intestine (13, 34). Despite its high expression in the kidney, the exact role of Oct1 in the renal elimination of organic cations remained unclear. In Oct1 knockout mice, loss of Oct1 from the liver and intestine resulted in increased excretion of drugs via the kidney. Loss of Oct1 from the kidney did not appear to affect the renal elimination of organic cations. This might be due to compensation by redundant transporters such as Oct2 (13).
In this study, we generated Oct2 knockout mice and Oct1/2 double-knockout mice to study the respective and possibly overlapping roles of Oct2 and Oct1 in physiology and pharmacology. We show here that the renal secretion of the model organic cation TEA was completely abolished in Oct1/2-/- mice, resulting in substantially increased levels in plasma. These findings indicate that Oct1 and Oct2 together play an essential role in the renal secretion of organic cations and that a deficiency in these transporters may result in increased drug sensitivity and/or toxicity.
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MATERIALS AND METHODS |
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Materials. [14C]TEA (55 Ci/mol) was from American Radiolabeled Chemicals, Inc. (St. Louis, Mo.); TEA (tetraethylammonium chloride) was from Fluka Chemie AG (Buchs, Switzerland); [3H]MPP+ (N-[methyl-3H]-1-methyl-4-phenylpyridinium acetate; 82 Ci/mmol) was from NEN Life Science Products, Inc. (Boston, Mass.); MPP+ iodide was from Research Biochemicals International (Natick, Mass.); methoxyflurane (Metofane) was from Medical Developments Australia Pty. Ltd. (Springvale, Victoria, Australia); [14C]inulin (6.7 Ci/mol) was from Amersham Life Science; anti-rat cytochrome P450 3a1 (monoclonal) was from Oxford Biomedical Research, Inc. (Oxford, Mich.); donkey anti-rabbit immunoglobulin (Ig), F(ab')2 fragment, was from Amersham Pharmacia Biotech; goat anti-mouse Ig was from DAKO (Glostrup, Denmark). All other compounds were reagent grade.
Cloning of 129/OLA Oct2 genomic DNA and construction of the targeting vector.
Mouse Oct2 genomic DNA sequences were cloned from a 129/OLA-derived genomic library constructed in bacteriophage 
GEM12. A genomic sequence containing exon 1 was identified and cloned into the pGEM5 vector (Promega). From this construct, fragments were subcloned into the pGEM3 vector (Promega), resulting in replacement of a 2.3-kb StuI-Asp718 fragment containing exon 1 with a 1.9-kb pgk-neo cassette in reverse transcriptional orientation (Fig. 1A). Deletion of exon 1 resulted in removal of the start codon and of sequences encoding putative TMD1 and the large extracellular loop located between putative TMD1 and -2.
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Southern analysis of ES cells and generation of chimeric mice. Correct targeting in G418-resistant clones was verified by Southern analysis with 5' and 3' Oct2-specific probes. Hybridization of HindII-digested genomic DNA with the 3' probe resulted in a wild-type band of 9.9 kb and a mutant band of 6.6 kb (probes and fragment sizes are indicated in Fig. 1A). Hybridization of ScaI-digested genomic DNA with a 5' probe resulted in a wild-type band of 16.1 kb and a mutant band of 5.9 kb. Absence of additional pgk-neo cassettes inserted elsewhere in the genome was confirmed by hybridization with a neo-specific probe (data not shown). Chimeric mice were generated by microinjection of two independently targeted ES cell clones into blastocysts. By this approach, two independent Oct2-/- and Oct1/2-/- mouse lines were established.
Clinical-chemical analysis of plasma. Standard clinical chemistry analyses of plasma were performed on a Hitachi 911 analyzer to determine levels of bilirubin, alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, creatinine, urea, Na+, K+, Ca2+, Cl-, phosphate, total protein, and albumin.
Histopathological analysis. Complete necropsies were performed on adult and aged mice of both sexes. For microscopic examination, tissues were fixed in 4% phosphate-buffered formalin, embedded in paraffin, sectioned at 4 µm, and stained with hematoxylin and eosin in accordance with standard procedures.
RNase protection analysis. Total RNA was isolated from mouse tissues by use of Trizol reagent (Life Technologies, Inc. [GIBCO BRL], Rockville, Md.), in accordance with the manufacturer's instructions. RNase protection assays were performed as described previously (13, 26), with 10 µg of total RNA per sample. A mouse probe for Oct2 was made by cloning a 1,147-nucleotide (nt) PCR fragment (positions 457 to 1603 relative to the translation start) into the pGEM-T vector. After linearization with EcoRI, a 246-nt antisense RNA probe was generated by transcription with SP6 RNA polymerase, yielding a protected probe fragment of 197 nt. The mouse Gapdh probe was described previously (26).
Northern analysis. Northern blot assays were performed in accordance with standard procedures. Blots were hybridized with a 617-nt probe for mouse Oct2 (positions 989 to 1605 relative to the translation start). The same blot was rehybridized with an Igf2r probe (cDNA covering exons 3 to 6) to check the amount and integrity of the RNA loaded.
Western analysis. Crude membrane fractions were prepared as previously described (23). Protein concentrations were determined with the Bradford protein assay (Bio-Rad Laboratories, Munich, Germany). Proteins were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose (HybondECL; Amersham Pharmacia Biotech). The filters were blocked for 1 h at room temperature with TBS-T (100 mM Tris [pH 7.6], 150 mM NaCl, 0.1% [wt/vol] Tween 20) with 5% skim milk powder. Incubation with the affinity-purified polyclonal antibody (ab1) (21) against rat Oct1 (dilution of 1:5,000) or with an anti-Rat cytochrome P450 3a1 (Cyp3a1) monoclonal antibody (dilution of 1:1,000) was performed at 4°C overnight (in TBS-T containing 5% skim milk powder). Antibodies were detected by incubation of the blot with horseradish peroxidase-conjugated donkey anti-rabbit (for Oct1 detection) or goat anti-mouse (for Cyp3a detection) IgG for 1 h at room temperature in TBS-T containing 5% skim milk powder. Antibody binding was visualized with the ECL Western blotting detection system (Amersham Pharmacia Biotech).
Pharmacokinetic experiments. For intravenous drug administration, 5 µl of drug solution per g of body weight was injected into the tail vein of mice lightly anesthetized with methoxyflurane. Animals were sacrificed at the indicated time points by axillary bleeding after anesthesia with methoxyflurane. Urine was collected from the bladder, and organs and tissues were removed and homogenized in a 4% (wt/vol) bovine serum albumin solution. Where applicable, intestinal content was separated from intestinal tissue before homogenization. Levels of radioactivity in homogenates were determined by liquid scintillation counting. Continuous-infusion experiments were performed with intraperitoneally implanted micro-osmotic pumps with a pumping rate of 1 µl/h, a capacity of 100 µl, and a duration of 3 days (Alzet 1003D; Alza Corporation, Palo Alto, Calif.). For implantation, mice were first anesthetized with methoxyflurane, a median incision was made in the abdomen, and pumps were intraperitoneally inserted. Subsequently, the musculoperitoneal layer and skin were closed with a silk suture (size 5/0; Perma-hand silk; Ethicon, Norderstedt, Germany). Finally, mice received an intravenous bolus injection (5 µl/g of body weight) of the same drug solution in order to accelerate the point at which the distribution equilibrium was achieved. In all cases, a steady state was achieved within 1 day as determined by repeated sampling of plasma radioactivity. Two days after implantation, mice were sacrificed and steady-state drug levels were determined in plasma, tissues, and urine.
Determination of CLrenal of TEA. Mice, anesthetized with a combination of ketamine (100 mg/kg) and xylazine (6.7 mg/kg), received intravenous [14C]TEA (0.2 mg/kg), and blood samples (of about 40 µl) were taken from the tail at 2.5, 5, 10, 20, 30, 40, 50, and 60 min. Urine was collected from the bladder after 60 min. Renal clearance (CLrenal) was determined with the following equation: CLrenal = TEAurine (0-60)/AUC(0-60). Where TEAurine (0-60) and AUC(0-60) are the cumulative urinary excretion of TEA up to 60 min and the area under the plasma concentration-time curve from 0 to 60 min as calculated by use of the linear trapezoidal rule, respectively.
Determination of GFR.
The glomerular filtration rate (GFR) was determined by measuring the clearance of inulin (CLinulin). Mice, anesthetized with a combination of ketamine (100 mg/kg) and xylazine (6.7 mg/kg), received intravenous [14C]inulin (25 mg/kg), and blood samples (of about 40 µl) were taken from the tail at 5, 10, 20, 30, 40, 50, and 60 min. The GFR was determined with the following equation: GFR = CLinulin = Dose/AUC(0-
). Where AUC(0-) is the area under the plasma concentration-time curve extrapolated to infinity with the MW/Pharm software package (24). For calculation of the estimated GFR (in milliliters per hour), the following equation was used: GFR = 0.036 · BW0.74 ± 0.15, where BW is body weight (12).
Statistical analysis. All values are given as means ± standard deviations (SD). The two-tailed unpaired Student t test was used to assess the significance of differences between two sets of data. Differences were considered to be statistically significant when P was <0.05.
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RESULTS |
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Mice received intravenous [14C]TEA (0.2 mg/kg), and after 20 min, levels of radioactivity were measured in plasma, organs, feces, and urine (Table 1). No significant differences were observed in the distribution and excretion of TEA between Oct2-/- and wild-type mice, except for the brain, in which levels were reduced with borderline significance in the Oct2-/- mice (P = 0.046). The excretion of TEA in both the Oct2-/- and wild-type mice was mainly via CLrenal (nearly 40% of the dose in 20 min), and a substantial amount accumulated in the liver (about 10% of the dose in 20 min), which corresponded well to our previous findings obtained with wild-type mice (13). From these results, it can be concluded that absence of Oct2 in itself has little effect on the pharmacokinetics of TEA, suggesting that its absence can be compensated for by other transporters in the kidney.
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Renal secretion of TEA is abolished in Oct1/2-/- mice. The CLrenal of a drug is the result of glomerular filtration and tubular secretion and reabsorption. This means that when there is net tubular secretion of a drug, the CLrenal of that drug is greater than the GFR. As the CLrenal of TEA is primarily mediated via secretion, it is an excellent substrate with which to study this process (4, 22, 30). To establish the contribution of Oct1 and Oct2 to the secretion of TEA, we determined the CLrenal of TEA in Oct1/2-/- and wild-type mice and compared it with the GFR. First, we determined the GFR in Oct1/2-/- and wild-type mice by measuring the CLinulin. [14C]inulin was administered intravenously at a dose of 25 mg/kg, and plasma concentration profiles were determined (Fig. 5A). The GFR (see Materials and Methods) was not significantly different between Oct1/2-/- and wild-type mice and corresponded well to the estimated GFR calculated from the mean body weights (12) (Table 2). Next, we determined the CLrenal of TEA after intravenous administration of 0.2 mg of [14C]TEA per kg. The elimination of TEA from the plasma and the CLrenal of TEA (see Materials and Methods) were substantially decreased in Oct1/2-/- mice compared to those in wild-type mice (Fig. 5B and C). The ratio of the CLrenal of TEA to the GFR was about 2.4 for wild-type mice, which is comparable to what has been found by others (4, 22, 30). In Oct1/2-/- mice, the ratio of the CLrenal of TEA to the GFR was reduced to about 1, indicating that the net tubular secretion of TEA was completely abolished in these mice.
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DISCUSSION |
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Many of the polyspecific drug transporters that have been identified and characterized to date are expressed in the liver and kidney. In the past decade, knockout mouse models for several of these transporters have been generated to elucidate their respective physiologic and pharmacologic functions. Whereas a clear pharmacologic function of these transporters could often be demonstrated in the liver, their exact in vivo role in the renal excretion of compounds remained essentially unclear (13, 14, 26, 28). The complexity of renal drug elimination, which involves passive filtration at the glomerulus, tubular secretion, and reabsorption, makes it intrinsically difficult to study this process in vivo. In contrast, hepatobiliary elimination is a relatively simple process, just involving the uptake of a compound from the bloodstream at the sinusoidal membrane of hepatocytes and its subsequent excretion at the canalicular membrane into the bile.
In our previous study, we showed that mice with a deficiency in Oct1 displayed greatly reduced hepatic uptake and direct intestinal excretion of substrate organic cations, demonstrating that Oct1 plays an essential role in the disposition of organic cations by the liver and intestine (13, 34). On the basis of our present study, it appears that the absence of Oct2 in itself has no immediately apparent physiologic or pharmacologic consequences. This is consistent with the possibility that Oct1 and Oct2 are mutually redundant in the kidney, the primary organ where both transporters are coexpressed. However, although Oct1 and Oct2 have similar substrate specificities, they are not identical (31, 32), and a deficiency in either Oct1 or Oct2 may already be sufficient to impair the renal secretion of some substrates. Moreover, in contrast to rodents, which express both Oct1 and Oct2 in the kidney, humans only express OCT2 (10). Therefore, a deficiency in OCT2 in humans might have effects on renal elimination similar to those of a deficiency in both Oct1 and Oct2 in mice.
Unexpectedly, we found that at steady-state levels in plasma, the relative accumulation of TEA in the kidney was decreased about twofold in both Oct1 and Oct2 knockout mice compared with wild-type mice, whereas at the same time the plasma and urinary concentrations of TEA were not different (Fig. 4). This can be explained when the transcellular transport rate of TEA through proximal tubular cells is primarily limited by the efflux processes across the apical membrane in wild-type, Oct1-/-, and Oct2-/- mice. In that case, loss of either Oct1 or Oct2 from the basolateral membrane of proximal tubular cells will reduce the accumulation of TEA in tubular cells but will not necessarily reduce the net transcellular transport rate and secretion. This observation is consistent with the data of Schäli et al. (25) that suggested a rate-limiting role for the apical membrane in the secretion of TEA by renal proximal tubules. That the effect is almost exactly twofold is probably determined by a fortuitous combination of the infusion rate and transport characteristics of Oct1 and Oct2. The similar effects of either Oct1 or Oct2 deficiency indicate that under the conditions applied, the TEA transport capacities of renal tubular Oct1 and Oct2 are virtually identical. In Oct1/2-/- mice, the relative accumulation of TEA in the kidney was reduced even more, to about 10% of that in wild-type mice, and TEA secretion was (virtually) abolished. Apparently, when both Oct1 and -2 are absent, the transcellular transport of TEA across the proximal tubular cells is primarily limited by passage over the basolateral membrane, resulting in drastically decreased accumulation and net secretion.
The organic cation transporters may also be important for the transport of natural toxins or endogenous compounds. Many studies in vitro have shown that OCT1 and OCT2 can transport physiologically relevant endogenous compounds such as monoamine neurotransmitters (e.g., adrenaline, noradrenaline, dopamine), choline, and guanidine, suggesting that absence of these transporters might have physiologic consequences (1, 9, 10). The fact that our knockout mice are apparently healthy and show no signs of abnormal physiology indicates that these transporters are not absolutely essential and may possibly be compensated for by redundant transporters. It should be noted, however, that many physiologic aberrations only become apparent under specific or extreme conditions that may not occur under the relatively controlled conditions of our animal facility. For example, we have shown previously that mice with a deficiency in another polyspecific drug transporter, the breast cancer resistance protein (Bcrp1/Abcg2), are hypersensitive to a dietary phototoxin that is only sporadically present at toxic levels in the mouse diet (14).
Recently, several groups have reported the occurrence of polymorphisms in the human OCT1 and OCT2 genes, some of which have been shown to result in severely reduced transport activity (17, 19). Genetic deficiencies for these genes may have both positive and negative consequences for drug therapy. In the case of an OCT1 deficiency, reduced uptake of drugs into the liver may result in a decreased efficacy of drugs that have their therapeutic action in the liver. On the other hand, reduced uptake of drugs into the liver could be beneficial for drugs that have adverse effects in the liver or need hepatic metabolic activation. The latter is exemplified by the antidiabetic drug metformin, which has been shown to have reduced toxicity in Oct1-/- mice (33, 34). Unlike rodents that express both Oct1 and Oct2 in the kidney, humans express only OCT2 (10). Therefore, it is likely that the Oct1/2-/- mouse model better reflects the effect on renal function of an OCT2 deficiency in humans than the Oct2-/- mouse model. On the basis of our findings, we expect that humans with a deficiency in OCT2 will have impaired renal elimination of some drugs and that this may result in increased exposure to these drugs. It will therefore be of interest to determine whether polymorphisms in the human OCT1 and OCT2 genes also correlate with altered drug pharmacokinetics in patients. If our findings can indeed be extrapolated to humans, these knockout mouse models will provide powerful tools for predicting and explaining drug sensitivity and toxicity, which may ultimately result in improved drug therapy.
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ACKNOWLEDGMENTS |
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This work was supported in part by grant NKI 97-1434 (to A. H. Schinkel) from the Dutch Cancer Society.
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FOOTNOTES |
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