Deficiency in the organic cation transporters 1 and 2 (Oct1/Oct2 [Slc22a1/Slc22a2]) in mice abolishes renal secretion of organic cations

Abstract

The polyspecific organic cation transporters 1 and 2 (Oct1 and -2) transport a broad range of substrates, including drugs, toxins, and endogenous compounds. Their strategic localization in the basolateral membrane of epithelial cells in the liver, intestine (Oct1), and kidney (Oct1 and Oct2) suggests that they play an essential role in removing noxious compounds from the body. We previously showed that in Oct1(-/-) mice, the hepatic uptake and intestinal excretion of organic cations are greatly reduced. Since Oct1 and Oct2 have extensively overlapping substrate specificities, they might be functionally redundant. To investigate the pharmacologic and physiologic roles of these proteins, we generated Oct2 single-knockout and Oct1/2 double-knockout mice. Oct2(-/-) and Oct1/2(-/-) mice are viable and fertile and display no obvious phenotypic abnormalities. Absence of Oct2 in itself had little effect on the pharmacokinetics of tetraethylammonium (TEA), but in Oct1/2(-/-) mice, renal secretion of this compound was completely abolished, leaving only glomerular filtration as a TEA clearance mechanism. As a consequence, levels of TEA were substantially increased in the plasma of Oct1/2(-/-) mice. This study shows that Oct1 and Oct2 together are essential for renal secretion of (small) organic cations. A deficiency in these proteins may thus result in increased drug sensitivity and toxicity.

FIG. 1.

Targeted disruption of the Oct2 gene by homologous recombination. (A) In structures of the wild-type and mutant alleles, exons are indicated by closed boxes (exons are not drawn to scale). In the targeting construct, exon 1 was replaced with an inverted (as indicated with an arrow) pgk-neo cassette. Only relevant restriction sites are indicated. For Southern analysis, 5′ and 3′ probes were used on ScaI (5′)- and HindII (3′)-digested genomic DNA. Sizes of diagnostic restriction fragments for wild-type and targeted alleles are indicated by double-headed arrows (drawn to scale). (B) Schematic representation (drawn to scale) of the Oct1-3 (Slc22a1-3) gene cluster localized on mouse chromosome 17 based on the physical map of the mouse genome (7).

FIG. 2.

Oct1 and Oct2 RNA and protein analysis. (A) Northern analysis of total RNA from kidneys of wild-type and Oct2^−/− mice. Oct2 and Igf2r bands originate from the same gel, and their sizes are indicated. (B) Immunodetection of Oct1 in the livers and kidneys of wild-type and Oct1/2^−/− mice. A polyclonal antibody raised against rat Oct1 (which cross-reacts with mouse Oct1) was used on crude membrane fractions of liver (20 μg per lane) and kidney (10 μg per lane). The same blot was incubated with a monoclonal antibody raised against rat cytochrome P450 3a (Cyp3a; expressed only in the liver), which was used as a protein loading control. Molecular size markers are indicated in kilodaltons on the right. Part of these data was previously published (13).

FIG. 3.

Concentrations of TEA in plasma and its renal excretion in Oct1^−/− versus Oct1/2^−/− mice. Levels of radioactivity in Oct1^−/− and Oct1/2^−/− mice at 60 min after intravenous injection of [¹⁴C]TEA (0.2 mg/kg). (A) Levels of [¹⁴C]TEA in plasma. (B) Percentage of dose of [¹⁴C]TEA excreted in urine. Urine was collected from the bladder. *, P < 0.05; **, P < 0.01.

FIG. 4.

Steady-state pharmacokinetics of TEA. Steady-state levels of [¹⁴C]TEA in wild-type, Oct1^−/−, Oct2^−/−, and Oct1/2^−/− mice are shown. [¹⁴C]TEA was continuously infused at a rate of 37 ng/h with intraperitoneally implanted micro-osmotic pumps. (A) Steady-state levels of [¹⁴C]TEA in plasma. (B) Ratios of [¹⁴C]TEA concentrations in urine and plasma. (C) Ratios of [¹⁴C]TEA concentrations in liver and plasma. (D) Ratios of [¹⁴C]TEA concentrations in the kidneys and plasma. Results are means ± SD (n = 4). *, P < 0.05; **, P < 0.01 (compared to wild-type values).

FIG. 5.

CL_inulin and clearance of TEA in wild-type versus Oct1/2^−/− mice. (A) Plasma [¹⁴C]inulin concentration-versus-time curves of wild-type and Oct1/2^−/− mice. Levels of radioactivity in plasma were determined at 5, 10, 20, 30, 40, 50, and 60 min after intravenous administration of 25 mg of [¹⁴C]inulin per kg. Results are means ± SD (n = 5). (B) Plasma [¹⁴C]TEA concentration-versus-time curves of wild-type and Oct1/2^−/− mice. Levels of radioactivity in plasma were determined at 2.5, 5, 10, 20, 30, 40, 50, and 60 min after intravenous administration of 0.2 mg of [¹⁴C]TEA per kg. Results are means ± SD (n = 5 to 7). (C) CL_renal of TEA in wild-type and Oct1/2^−/− mice. CL_renal was calculated by dividing the amount of TEA excreted in the urine over 60 min by the plasma AUC_(0-60). The estimated GFR was approximately 21 ml/h for both genotypes and is indicated with a dashed line. **, P < 0.01.