Localization of organic cation transporters OCT1 and OCT2 in rat kidney

Ulrich Karbach1,*, Jörn Kricke2,*, Friederike Meyer-Wentrup1, Valentin Gorboulev1, Christopher Volk1, Dominique Loffing-Cueni3, Brigitte Kaissling3, Sebastian Bachmann2, and Hermann Koepsell1

1 Institute of Anatomy of the Bayerische Julius-Maximilians-Universität, 97070 Würzburg; 2 Institute of Anatomy, Charité, 10098 Berlin, Germany; and 3 Institute of Anatomy, 8051 Zürich, Switzerland


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
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Renal excretion and reabsorption of organic cations are mediated by electrogenic and electroneutral organic cation transporters, which belong to a recently discovered family of polyspecific transporters. These transporters are electrogenic and exhibit differences in substrate specificity. In rat, the renal expression of the polyspecific cation transporters rOCT1 and rOCT2 was investigated. By in situ hybridization, significant amounts of both rOCT1 and rOCT2 mRNA were detected in S1, S2, and S3 segments of proximal tubules. By immunohistochemistry, expression of the rOCT1 protein was mainly observed in S1 and S2 segments of proximal tubules, with lower expression levels in the S3 segments. At variance, rOCT2 protein was mainly expressed in the S2 and S3 segments. Both transporters were localized to the basolateral cell membrane. Neither rOCT1 nor rOCT2 was detected in the vasculature, the glomeruli, and nephron segments other than proximal tubules. The data suggest that rOCT1 and rOCT2 are responsible for basolateral cation uptake in the proximal tubule, which represents the first step in cation secretion.

polyspecific cation transporter; organic cation transporter family; gene expression; kidney; immunohistochemisty; in situ hybridization


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

POLYSPECIFIC TRANSPORTERS in the kidney are responsible for the secretion and reabsorption of drugs, xenobiotics, and organic endogenous compounds (for review, see Ref. 26). Organic drugs and some endogenous cations are mainly secreted in the proximal tubule by a two-step procedure: electrogenic uptake at the basolateral membrane and cation release at the luminal membrane that is mediated by an electroneutral proton cation antiporter. Some cations like choline may be also reabsorbed at low plasma concentrations (1). This process involves a potential dependent cation transporter at the luminal membrane of the proximal tubule (31). The first polyspecific organic cation transporter (rOCT1) was cloned in 1994 from rat kidney (13). rOCT1 was the first member of a large family of polyspecific transporters that includes organic cation transporters, organic anion transporters, and transporters for zwitterionic compounds (for reviews, see Refs. 18 and 19). To date, three subtypes of polyspecific cation transporters named OCT1, OCT2, and OCT3 have been cloned from different species (11-13, 15, 17, 24, 28, 32). These transporters are transcribed in the kidney and in other organs such as liver and brain. They mediate electrogenic uptake of various relatively small organic cations, including choline, tetraethylammonium, 1-methyl-4-phenylpyridinium, N1-methylnicotinamide, and monoamine neurotransmitters. They are inhibited by a variety of larger, more hydrophobic cations that are not transported by the OCTs (23). In contrast, these cations may be translocated by polyspecific transporters of the OATP family and by multidrug resistance proteins of the MDR and MRP families (for review, see Ref. 22). Transport properties of the three OCT subtypes that are preserved in all species are electrogenicity, uniport activity, reversibility of transport direction, and independence from Na+. The precise substrate specificity profiles, however, are subtype and species specific (11, 13, 17, 19). Whereas the basolateral localization and function of OCT1 in proximal renal tubules are generally accepted, there is controversy concerning the membrane localization and functional role of OCT2 (for review, see Ref. 19). On the basis of a comparison of Michaelis-Menten constant (Km) values of low-affinity substrates observed in our in vitro studies with in vivo data, we first suggested the localization of rOCT1 at the basolateral membrane of renal proximal tubules (13). Later, we showed that rOCT1 is localized at the sinusoidal membrane in hepatocytes (20), and Inui and collaborators (30) reported that rOCT1 was associated with basolateral membranes that had been isolated from rat kidney cortex (30). rOCT2 has been discussed as a luminal transporter on the basis of functional data comparing the substrate specificity of porcine OCT2 expressed in human embryonic kidney (HEK-293) cells with luminal uptake of cultivated renal epithelial cells (LLC-PK1) from pig (12, 14). After expression in Madin-Darby canine kidney (MDCK) cells, however, both rOCT1 and rOCT2 have been localized to the basolateral membrane (30). In the present paper we demonstrate that both rOCT1 and rOCT2 are localized at the basolateral membrane of renal proximal tubules, showing different levels of expression in different segments of the proximal tubule.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Synthesis of digoxigenin-labeled cRNA. cDNA fragments of rOCT1 and rOCT2 were amplified by using the following primers: for rOCT1 (13), 5'CCT GGG GAG GAG GAA ATC 3' nucleotides 1630-1647 (forward) and 5'GGT AGT TCA TTT GGA ACC TG 3' nucleotides 1812-1831 (reverse); for rOCT2 (9), 5'GAA AGG GCA TCA TTG CTG 3' nucleotides 1710-1727 (forward) and 5'ATG TTG TGG TGG AGA AGG 3' nucleotides 1992-2009 (reverse). The PCR products were cloned into Sma I site of pBluescript SKII. The plasmid with the rOCT1 insert was linearized with BamH 1 (sense) or EcoR I (antisense), and digoxigenin-labeled cRNA was synthesized by using T7 or T3 RNA polymerase for sense and antisense transcripts, respectively. The plasmid carrying the rOCT2 fragment was linearized with EcoR I (sense) or BamH I (antisense), and digoxigenin-labeled cRNA was produced by using T3 or T7 RNA polymerase for sense and antisense transcripts, respectively.

Tissue preparation and in situ hybridization. Sprague-Dawley rats (body wt 300-350 g), which had free access to standard laboratory diet and tap water, were anesthetized with 30 mg pentobarbital sodium/kg body wt. The kidneys were removed directly or after retrograde perfusion. The retrograde perfusion was performed with buffered solutions containing 3% (wt/vol) paraformaldehyde, followed in some cases by PBS that was adjusted to 800 mosmol/kgH2O with sucrose (16). The removed kidneys were rapidly frozen in isopentane or propane, which were cooled by liquid nitrogen and sectioned in a cryostat. Cryosections (5-8 µm thick) from perfusion-fixed (rOCT1) or unfixed kidneys (rOCT2) were thawed on silanized glass slides, postfixed (rOCT1) or fixed (rOCT2) by 5-min incubation with PBS containing 4% (wt/vol) paraformaldehyde, and stored for 1 day in 70 (rOCT1) or 100% ethanol (rOCT2). After rehydration in PBS, the slides were either acetylated with 0.1 M triethanolamine HCl, pH 8.0, containing 0.25% acetic anhydride (rOCT1) or incubated for 5 min in 20 mM HCl and washed for 10 min with 2× standard sodium citrate (SSC; 150 mM NaCl, 15 mM sodium citrate; rOCT2).

Different protocols were followed to optimize the hybridrization of rOCT1 (3) and rOCT2 (27). For rOCT1, the hybridization was performed with 10 µg/ml of the antisense or sense riboprobe for 18 h at 40°C in 20 mM Tris · HCl, pH 7.6, containing 0.3 M NaCl, 10% (vol/vol) Denhardt's solution, 0.1 mg/ml salmon sperm DNA, and 10% (wt/vol) dextransulfate. The slides were washed several times for 30 min, each at 47°C, with 2× SSC, 1× SSC plus 50% formamide (FA), 0.4× SSC plus 50% FA, and 0.2× SSC plus 50% FA. Then they were washed at room temperature for 20 min with 0.5× SSC, 10 min with 0.2× SSC, and 5 min with 100 mM Tris · HCl, pH 7.5, 150 mM NaCl. For rOCT2, hybridization was performed with 0.1 mg/ml of the antisense or sense riboprobe for 18 h at 55°C in 4× SSC containing 10% (vol/vol) Denhardt's solution, 50% FA, and 0.9 g/ml yeast tRNA. In this case, washing was performed for 30 min at room temperature with 2× SSC and for 30 min at 55°C with 2× SSC containing 50% FA. Thereafter, nonhybridized RNA was digested by 30-min incubation at 37°C with 40 µg/ml of RNase A in 1 M Tris · HCl, pH 7.5, 0.4 M NaCl, and 50 mM EDTA, and the section was incubated for 1 h at 60°C with 1 M Tris · HCl, pH 7.5, 0.4 M NaCl, and 50 mM EDTA.

After hybridization of rOCT1 and rOCT2, nonspecific antibody binding sites were blocked by 30-min incubation at room temperature with 100 mM Tris · HCl, pH 7.5, 150 mM NaCl containing 2% (vol/vol) sheep serum, 0.5% (wt/vol) bovine serum albumin, and 0.1% (wt/vol) Triton X-100. The sections were incubated with alkaline phosphatase-conjugated anti-digoxigenin antibody from goat (Boehringer Mannheim), and the alkaline phosphatase reaction was visualized as described earlier (3). Specificity of the detection protocol was tested with the respective sense RNA probes, and by reactions of the rOCT1 antisense probe with rOCT2 expressed in HEK-293 cells and of the rOCT2 antisense probe with rOCT1 expressed in HEK-293 cells (8).

Antibodies and cell lines. The generation, affinity purification, and characterization of the antibodies directed against rOCT1 or rOCT2 that were used in this study have been described earlier (20). Ab1 is a polyclonal antibody raised in rabbits against amino acids 524-542 of rOCT1 and does not cross-react with rOCT2 in Western blots. Ab2 is a polyclonal antibody raised in rabbits against amino acids 319-334 of rOCT2 and does not cross-react with rOCT1 in Western blots. For affinity purification the antigenic peptides of ab1 and ab2 were immobilized on EPOXY-activated 6B-Sepharose (Sigma, Munich, Germany) or on SulfoLink coupling gel from Pierce (Rockford, IL), respectively. The preparation and characterization of HEK-293 cells stably expressing rOCT1 and hOCT2 have been described earlier (7, 8). By using the same transfection procedure and selection protocol, HEK-293 cells were generated that stably express rOCT2. Tracer uptake measurements showed that these cells express highly active uptake of 1-methyl-4-phenylpyridinium, which was inhibited by 50 µM cyanine-863.

Membrane preparations. Brush-border membranes from rat kidney cortex were isolated by the Ca2+ precipitation method (10) and the basolateral membranes by differential centrifugation and fractionation on a Percoll gradient (6). The purity of the preparations was tested by measuring alkaline phosphatase and Na+-K+-ATPase activity as marker enzymes for apical and basolateral membranes, respectively (6). Compared with the homogenate, the specific activity of alkaline phosphatase in our brush-border membrane preparations was increased more than 10-fold, whereas the specific Na+-K+-ATPase activity in the preparation of basolateral membranes was increased more than 7-fold.

Western blotting and immunohistochemistry. For Western blotting, brush-border membranes or basolateral membranes from rat kidney were resolved on discontinuous, denaturing, and reducing SDS polyacrylamide gels and transferred by semidry blotting to nitrocellulose membranes (20). The membranes were blocked for 1 h at 22°C with PBS containing 1% (wt/vol) Tween 20 and 2% (wt/vol) skim-milk powder (Sigma-Aldrich Chemie, Steinheim, Germany). Incubation with the affinity-purified antibodies dissolved in PBS containing 1% Tween 20 and 0.5% skim-milk powder was performed for 1 h at room temperature. The concentrations of the affinity-purified antibodies corresponded to a 1:5,000 (ab1) or 1:1,000 (ab2) dilution of the antiserum. Antibody binding was detected with peroxidase-labeled goat anti-rabbit IgG antibody (DAKO Diagnostika, Hamburg, Germany) and with chemiluminescence (Amersham Buchler, Braunschweig, Germany) (20). The molecular weights were estimated by using a prestained protein ladder from GIBCO-BRL (Karlsruhe, Germany).

Immunohistochemistry was performed on 2- to 5-µm-thick cryosections. The sections were prepared from perfusion-fixed kidneys as described above. For control reactions, cryosections were taken from pellets of HEK-293 cells that stably expressed rOCT1, rOCT2, or hOCT2 and were fixed with 3% paraformaldehyde. Incubation with primary antibodies was performed for 1 h at room temperature in the presence of PBS containing 2% (wt/vol) skim-milk powder and 0.5% (vol/vol) Triton X-100. Compared with Western blotting, 5-10 times higher antibody concentrations were used. To test the immunohistochemical reactions for specificity, 100 µg/ml of the respective antigenic peptides or of nonrelated peptides were added to the antibody solutions and incubated for 30 min at room temperature, before they were applied to the sections. In some experiments, antigen retrieval was attempted by incubating the sections for 5 min with 1% (wt/vol) SDS as described by Brown and coworkers (2, 5), by a 10-min incubation of the sections with 1 N HCl, 1 N NaOH, 1% (wt/vol) Triton X-100, 0.1% (wt/vol) saponin, 0.5 mg/ml trypsin, or by microwave treatment (29). After incubation with the primary antibodies, the sections were washed three times with PBS, and bound antibody was detected by incubation in the same buffers containing goat anti-rabbit IgG antibodies labeled with indocarbocyanin (Dianova, Hamburg, Germany). The secondary antibodies were diluted 1:250. After washing, the sections were embedded in an aqueous embedding solution (DAKO) containing 3% (wt/vol) diazobicyclooctane. The specificity of the antibody reactions was verified as follows: 1) labeling was abolished when the antibodies were preabsorbed with their respective antigenic peptides but not with nonrelated peptides; 2) in Western blots ab1 did not react with plasma membrane proteins from Xenopus laevis oocytes expressing rOCT 2 but reacted readily when rOCT1 was expressed (20); 3) in Western blots ab2 did not react with plasma membrane proteins from oocytes expressing rOCT1 but reacted when rOCT2 was expressed (20); 4) ab1 did not show an immunohistochemical reaction with frozen and fixed pellets of HEK-293 cells expressing rOCT2 but reacted when rOCT1 was expressed; and 5) in rat liver ab2 did not reveal immunohistochemical staining of hepatocytes that express rOCT1 but reacted with proximal tubules in rat kidney .

Materials. Diazobicyclooctane was obtained from Sigma and goat anti-rabbit IgG antibody conjugated with peroxidase from DAKO Diagnostika. The other chemicals and antibodies were described earlier (3, 20, 25).


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Distribution of rOCT1 mRNA as determined by in situ hybridization. The cRNA probe used for in situ hybridization of rOCT1 had 47% nucleotide identity with rOCT2 (24), 40% nucleotide identity with rOCT3 (17), and 58% identical nucleotides with human OCT2 (hOCT2) (11). To evaluate the specificity under the employed hybridization conditions we performed in situ hybridization on HEK-293 cells that stably express rOCT1 (8), rOCT2, or hOCT2 (7) under the same conditions used for the kidney sections. The probe did not cross-hybridize in situ with rOCT2 or hOCT2. Because the cRNA probe has less overall nucleotide identity to rOCT3 than to rOCT2 and does not contain stretches of more than five identical nucleotides with rOCT3, a cross-hybridization with rOCT3 and with the other known, less homologous members of the OCT1 family (19) is highly improbable. Figure 1a shows an overview micrograph of the in situ hybridization with an rOCT1 anti-sense probe on a cryosection of a perfusion-fixed kidney. The coronary section shows that rOCT1 message is unevenly distributed in proximal tubules throughout the renal cortex and the outer stripe of the outer medulla. The strongest hybridization signals were observed in the pars rectae segments of proximal tubules within the cortical medullary rays and outer stripe of outer medulla, consistent with a localization in late S2 and S3 segments. Figure 2 shows an in situ hybridization from the outer cortex at larger magnification. No significant signal could be detected in glomeruli, interlobular arteries, cortical thick ascending limbs of Henle's loops, distal convoluted tubules, connecting tubules, and cortical collecting ducts. However, a strong reaction was observed in S2 and S3 segments, and a less intense reaction in the S1 segments of the proximal tubules.


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Fig. 1.   Overview of the distribution of rat organic cation transporter (rOCT1) mRNA and protein in rat kidney. Coronary sections of a perfusion-fixed kidney. a: In situ hybridization with an rOCT1-specific antisense cRNA probe. No reaction was observed with the respective sense mRNA. b: Immunohistochemistry with the affinity-purified antibody ab1 directed against rOCT1, employing antigen retrieval by incubating the sections for 5 min with 1% (wt/vol) SDS. No labeling was observed after blocking of ab1 with the antigenic peptide (data not shown). Dashed lines, borders between medullary rays (R) and cortical labyrinth and between outer stripe (OS) and inner stripe (IS) of the outer medulla. Bar = 250 µm.



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Fig. 2.   Distribution of rOCT1 mRNA in rat kidney cortex at higher resolution. A midcortical area of a coronary section is shown that was hybridized as described in Fig. 1a. *, S1 segments of proximal tubules; +, S2 segments; open circle , S3 segments of proximal tubules; , distal tubules, connecting tubules, and cortical collecting ducts; G, glomerulus; IA, interlobular artery. Bar = 50 µm.

Immunohistochemical localization of rOCT1 protein. The affinity-purified antibody ab1 against a COOH-terminal peptide of 19 amino acids was used to determine the distribution and membrane localization of rOCT1 protein. This antibody does not cross-react with the known members of the OCT family. By Western blotting (20) and by immunohistochemistry on sections from pellets of HEK-293 cells that stably express rOCT2, ab1 did not cross-react with rOCT2 although the antigenic peptide contains 11 amino acids that are identical in rOCT2. The homologous regions of the other family members (19) contain only six (rOCT3) or fewer than four (rOCTN1, rOCTN2, rOAT1, rOAT2) identical amino acids. The immunoreactivity (IR) of ab1 is shown in Fig. 1b, and at higher magnifications in Figs. 3 and 4. The sections in Figs. 1b, 3c, and 4 were treated for 5 min with 1% (wt/vol) SDS for antigen retrieval (2, 5). Whereas antigen retrieval with HCl, NaOH, Triton X-100, saponin, trypsin, or microwave treatment was unsuccessful, the IR of ab1 was significantly increased by the SDS treatment. IR for rOCT1 was exclusively detected in proximal tubules. A strong IR was observed in S1 and S2 segments. When the sections were treated with SDS, a weaker but still significant IR was also detected in the S3 segments (Figs. 1b and 3c). The expression level of rOCT1 in S1 segments appears to be higher than in S2 segments because proximal tubules in the cortical labyrinth showed a stronger IR for rOCT1 than those in the medullary rays (Fig. 1b). Using the transitions between glomeruli and proximal tubules as a criterion, rOCT1 IR could be localized to the early S1 segments of superficial (not shown), midcortical (Fig. 3a), and juxtamedullary nephrons (Fig. 3b). At the transition between outer stripe and inner stripe of the outer medulla, rOCT1 IR was localized in the late S3 segment (Fig. 3c). It can be seen that rOCT1 protein has the same distribution as the message of rOCT1, i.e., segments S1, S2, and S3 of the proximal tubule, but the relative abundance of rOCT1 mRNA vs. protein within these segments is different. The highest concentration of the mRNA was found in S2 segments and the highest concentration of the protein in S1 segments (Fig. 1). Figures 3, a and b, and 4 show that the IR of rOCT1 in the proximal tubules was localized to the basolateral membrane.


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Fig. 3.   Immunohistochemical localization of rOCT1 in rat kidney cortex and outer stripe of outer medulla. Cryosections of perfusion fixed rat kidney from middle cortex (a), deep cortex (b), or the outer stripe (OS)-inner stripe (IS) transition (c) are shown, which were reacted with ab1. In c, the section was pretreated with SDS for antigen retrieval. Glomeruli (G) and early S1 segments of proximal tubules (S1) are shown in a and b. In c, the OS, IS, and transitions of 2 S3 segments to descending thin limbs of Henle's loops are indicated. Antibody staining of the proximal tubules was competed away by preincubation of the antibody with excess peptide antigen (not shown). Bars = 50 µm.



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Fig. 4.   Localization of rOCT1 immunoreactivity in basolateral membrane of proximal tubules. A 2-µm-thick cryosection from midcortex of a perfusion fixed kidney was treated with SDS and reacted with ab1. a: Antibody staining. b: Interference contrast micrograph. Shown are S2 segments and S3 segments of proximal tubules and a distal tubule (D). Bar = 50 µm.

Distribution of rOCT2 mRNA as determined by in situ hybridization. For in situ hybridization of rOCT2, a 3' probe with 41% identity to rOCT1 and <36% identity to rOCT3 or the other family members was used. Because our in situ hybridization attempts with perfusion-fixed sections were unsuccessful, we used native cryosections that were fixed with acetone. Similar to rOCT1, the message of rOCT2 was detected in proximal renal tubules throughout the renal cortex and in the outer stripe of the outer medulla (Fig. 5). The hybridization signal of rOCT2 in the proximal tubules was less intense in the cortex than in the outer stripe. No significant hybridization signal was found in glomeruli, thick ascending limbs of Henle's loops, distal tubules, and collecting ducts. The specificity of hybridization was confirmed by the lack of hybridization in liver where rOCT1 is expressed and gave a positive hybridization signal with our rOCT1-specific cRNA probe (data not shown). Hybridization with rOCT3 can be excluded because our cRNA probe for rOCT2 hybridization did not contain stretches with more than five nucleotides identical with rOCT3.


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Fig. 5.   Distribution of rOCT2 mRNA in rat kidney. Cryosections of untreated kidneys were fixed with acetone and hybridized with rOCT2-specific antisense (a and b) or sense cRNA probes (c). a and c: Sections from outer cortex. b: Sections from outer medulla. Dashed line, border between OS and IS. Bar = 50 µm.

Immunohistochemical localization of rOCT2 protein. The distribution of rOCT2 protein was investigated with affinity-purified antibody ab2. Ab2 is directed against an intracellular 16-amino acid peptide of rOCT2 (20), which contains 7 identical amino acids with rOCT1 and 4 (rOCT3) or fewer identical amino acids with other known members of the OCT family (19). No cross-reactivity with rOCT1 was detected by Western blots on plasma membranes of X. laevis oocytes that express rOCT1 (20) and by immunohistochemistry on rat liver (data not shown). With ab2, strong IR was detected with proximal tubules in the medullary rays and the outer stripe (Fig. 6). The IR of ab2 could not be increased significantly by the procedures for antigen retrieval described in METHODS, including the incubation with SDS (compare Figs. 6, a-c, and 7, a and b). Similar to rOCT1, rOCT2 was exclusively localized in proximal tubules. No significant IR of ab2 above background was detected in glomeruli, limbs of Henle, distal tubules, collecting ducts, and blood vessels. In the medullary rays, IR of ab2 localized rOCT2 to the straight portions of the S2 segments (Fig. 6, a and b). The localization of rOCT2 in the S3 segments is evident from the IR of ab2 with proximal tubules in the outer stripe (Fig. 6, a and c). Late S3 segments could be identified at their transitions to the thin descending limbs of Henle's loop (see Fig. 6c, for example). In the proximal tubules, rOCT2 protein was clearly localized to the basolateral membrane (Figs. 6 and 7). In S1 segments of proximal tubules it was difficult to detect IR of ab2 above background. However, in some S1 segments faint IR of ab2 was detected at the basolateral membrane (see arrows in Fig. 6b). Thus our data suggest that rOCT2 is transcribed in all segments of the proximal tubule but that rOCT2 protein is mainly expressed in the S2 and S3 segments.


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Fig. 6.   Distribution of rOCT2 protein in rat kidney. A coronary section from a perfusion-fixed rat kidney was analyzed for IR of ab2 without SDS pretreatment. a: An overview micrograph extending from the renal capsule through the cortex and OS to the IS of the outer medulla. Two areas are identified that are presented at higher magnifications in b and c. b: Segments of 2 medullary rays. Arrows, faint immunostaining at basolateral membranes of S1 segments. c: Area from the OS-IS transition. *, Glomeruli; filled white squares and circles, S1 segments of proximal tubules and distal tubules, respectively; arrowhead, transition between an S3 segment and the thin descending limb of Henle's loop. The basolateral staining of proximal tubules in the OS and in medullary rays was competed away by preincubation of the antibody with excess of antigenic peptide (not shown). Bars: 250 µm (a), 50 µm (b), 20 µm (c).



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Fig. 7.   Immunostaining of rOCT2 with and without antigen retrieval by SDS. Cryosections of perfusion fixed rat kidney were either incubated for 5 min with 1% (wt/vol) SDS dissolved in PBS (a and b) or with PBS alone (c). Staining of the sections with ab2 was performed in parallel to the section shown in Fig. 6. The immunoreaction was similar without and with SDS pretreatment. a and b: Micrographs from deep cortex. The area indicated in a is enlarged in b. c: Micrograph of a part of a medullary ray where basolateral staining of S2 segments can be visualized at high magnification. *, Glomerulus; filled white squares, S1 segments; filled white circle, a distal tubule; , collecting duct. All areas shown are immunonegative. Bars: 50 µm (a), 20 µm (b), 25 µm (c).

Membrane localization of rOCT1 and rOCT2 protein determined by Western blotting. To confirm the membrane localization of rOCT1 and rOCT2 by an additional approach, and to investigate renal plasma membranes for cross-reactive proteins, we isolated brush-border and basolateral membranes from renal cortex and probed them with antibodies ab1 and ab2 (Fig. 8). The rOCT1-specific antibody ab1 and the rOCT2-specific antibody ab2 both labeled proteins at the basolateral membrane with apparent molecular masses of 60-70 kDa (Fig. 8). Renal brush-border membranes showed no IR for ab1 and ab2. The data confirm the basolateral localization of rOCT1 and rOCT2 and show the absence of cross-reactive proteins in renal plasma membranes.


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Fig. 8.   Western blot analysis of basolateral and brush-border membranes (m) from rat kidney. Renal brush-border membranes and renal basolateral membranes (10 µg/lane) were separated on SDS polyacrylamide gels. Western blots were developed with ab1 or ab2. A peroxidase-coupled secondary antibody was used, and the reaction was visualized by chemiluminescence. Mr, marker.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

To understand the function of organic cation transporters in the kidney it is necessary to localize the different transporters within the nephron and to determine their localization at the luminal or basolateral plasma membrane. The present study investigates the mRNA and protein distribution of the two electrogenic cation transporters rOCT1 and rOCT2 in adult rat kidney. We show that both rOCT1 and rOCT2 are mainly expressed in proximal tubules and that both transporters have an overlapping but quantitatively different protein distribution. High levels of rOCT1 protein were detected in S1 and S2 segments, and lower levels in the S3 segment. In contrast, rOCT2 protein was mainly localized to the S2 and the S3 segments, and only trace amounts of rOCT2 protein could be detected in the S1 segment. Because in situ hybridization revealed a more even distribution within the various segments of the proximal tubule of mRNAs of rOCT1 and rOCT2, these transporters may undergo differential posttranscriptional regulation, which has been discussed for rOCT1 in the liver (20). The distribution of rOCT2 mRNA observed in our in situ hybridization experiments conflicts with an earlier report, in which rOCT2 mRNA was exclusively assigned to the S3 segment of the proximal tubule by in situ hybridization using an RNase-digested 1.6-kb mRNA fragment of rOCT2 (14). Because the specificity of this probe was not documented, but rOCT1 and rOCT2 contain an NH2-terminal, 260-nucleotide stretch with 97% identity plus several 100 nucleotide stretches with ~80% identity, this probe may not have been specific for rOCT2.

An important finding of the present paper is that both rOCT1 and rOCT2 protein were localized to the basolateral membrane of proximal tubules. The basolateral localization of rOCT1 in the proximal tubule was previously hypothesized (13) and parallels with the immunohistochemical localization of rOCT1 to the sinusoidal membranes in the liver (20). The basolateral localization is also supported by transport measurements after expression of rOCT1 in polarized MDCK cells and by Western blots on isolated luminal and basolateral renal plasma membranes (30). The membrane localization of rOCT2 has been a matter of controversy (14, 19, 30). Urakami and co-workers (30) proposed a basolateral localization because they observed an increased basal-to-luminal cation flux in MDCK cells that were transfected with rOCT2. In contrast, Gründmann et al. (14) speculated that rOCT2 may be a luminal transporter because they had observed some similarity between the affinity of cationic inhibitors of luminal cation uptake into LLC-PK1 cells and cation uptake mediated by the cloned porcine organic cation transporter, pOCT2 (12). The present study clearly shows that rOCT2 protein is located exclusively at basolateral membranes of rat renal proximal tubules. We cannot exclude species differences in the nephron distribution and membrane localization, but at present, no direct evidence for such differences has been reported. There is also a hypothetical possibility that rOCT2 may be redistributed to the luminal membrane when the proximal tubule changes from cation secretion to cation reabsorption (1, 4), but this has not been demonstrated experimentally.

Employing in situ hybridization and immunohistochemistry in human kidneys, we previously obtained data that suggested that hOCT2 was expressed in distal tubules and located at the luminal membrane (11). Because in rats rOCT2 protein could not be detected in any nephron segment outside the proximal tubule, more detailed studies with human kidney are required to exclude the possibility that our previous localization of hOCT2 in the distal tubule represents cross-reactivity with a closely related transporter subtype or a splice variant in the distal tubule that has not been identified. It should be emphasized that we cannot exclude from our previous experiments that also in human kidney OCT2 is localized in basolateral membranes of proximal tubules. In our previous study with the human kidney we used sections where the proximal tubules were collapsed, and recent experiments with rat kidneys showed that the basolateral localization of rOCT1 or rOCT2 was not detected by using immersion-fixed sections with collapsed proximal tubules (unpublished observations). Discussing the apparent differences in the distribution of OCT2 in rats and humans, one must keep in mind that only relatively high levels of message and protein are detected by in situ hybridization and immunohistochemistry so that negative results with these methods do not exclude low levels of expression. For example, employing PCRs with reverse transcribed mRNAs from microdissected nephron segments, we detected rOCT2 mRNA in distal tubules that were microdissected from rat kidneys (data not shown). Thus we must consider the possibility that under certain physiological conditions higher levels of rOCT2 may be expressed in distal tubules from the rat. In this context we would like to state that even a simultaneous basolateral location of OCT2 in the proximal tubule and a luminal location in the distal tubule are not necessarily contradictory. Transcellular cation movement is determined by specificities and rates of the engaged transporters on both cell sides. For cation secretion in the proximal tubule, OCT2 in the basolateral membrane may cooperate with a luminal transporter that may determine the effective selectivity for secretion. rOCT2 in the luminal membrane of the distal tubule may mediate the first step in the reabsorption of some cations. For example dopamine, which is produced in the proximal tubule and released into the tubular fluid, may be reabsorbed by rOCT2 in the distal tubule (21). The presence of different competing cations on the cis-sides and of different stimulatory or inhibitory cations on the trans-sides may differentially determine the in vivo selectivity of OCT2 in the basolateral membrane of the proximal tubule and in the luminal membrane of the distal tubule.

It is a challenge for future experiments to investigate the combined action of basolateral and luminal cation transporters during transcellular cation movements. In the present study the basolateral localization of OCT1 and OCT2 in renal proximal tubules from adult rats has been clearly demonstrated. The localization of these transporters in other species and after treatment with cationic drugs is considered an interesting matter for further investigation.


    ACKNOWLEDGEMENTS

The authors thank Michael Christof for preparing the figures, Eva Engel and Irina Schatz for technical assistance, and Aida Akhoundova for preparing HEK-293 cells that stably express rOCT2.


    FOOTNOTES

*  U. Karbach and J. Kricke contributed equally to this work.

These studies were supported by the Deutsche Forschungsgemeinschaft, SFB 487, Grant A4.

Address for reprint requests and other correspondence: H. Koepsell, Anatomisches Institut der Universität, Koellikerstr. 6, 97070 Würzburg, Germany (E-mail: Hermann{at}Koepsell.de).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 20 September 1999; accepted in final form 8 June 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Acara, M, and Rennick B. Regulation of plasma choline by the renal tubule: bidirectional transport of choline. Am J Physiol 225: 1123-1128, 1973[ISI][Medline].

2.   Alper, SL, Stuart-Tilley AK, Biemesderfer D, Shmukler BE, and Brown D. Immunolocalization of AE2 anion exchanger in rat kidney. Am J Physiol Renal Physiol 273: F601-F614, 1997[Abstract/Free Full Text].

3.   Bachmann, S, LeHir M, and Eckardt KU. Co-localization of erythropoietin mRNA and ecto-5'-nucleotidase immunoreactivity in peritubular cells of rat renal cortex indicates that fibroblasts produce erythropoietin. J Histochem Cytochem 41: 335-341, 1993[Abstract/Free Full Text].

4.   Besseghir, K, Pearce LB, and Rennick B. Renal tubular transport and metabolism of organic cations by the rabbit. Am J Physiol Renal Fluid Electrolyte Physiol 241: F308-F314, 1981[ISI][Medline].

5.   Brown, D, Lydon J, McLaughlin M, Stuart-Tilley A, Tyszkowski R, and Alper S. Antigen retrieval in cryostat tissue sections and cultured cells by treatment with sodium dodecyl sulfate (SDS). Histochem Cell Biol 105: 261-267, 1996[ISI][Medline].

6.   Burckhardt, G. Sodium-dependent dicarboxylate transport in rat renal basolateral membrane vesicles. Pflügers Arch 401: 254-261, 1984[ISI][Medline].

7.   Busch, AE, Karbach U, Miska D, Gorboulev V, Akhoundova A, Volk C, Arndt P, Ulzheimer JC, Sonders MS, Baumann C, Waldegger S, Lang F, and Koepsell H. Human neurons express the polyspecific cation transporter hOCT2, which translocates monoamine neurotransmitters, amantadine, and memantine. Mol Pharmacol 54: 342-352, 1998[Abstract/Free Full Text].

8.   Busch, AE, Quester S, Ulzheimer JC, Gorboulev V, Akhoundova A, Waldegger S, Lang F, and Koepsell H. Monoamine neurotransmitter transport mediated by the polyspecific cation transporter rOCT1. FEBS Lett 395: 153-156, 1996[ISI][Medline].

9.   Busch, AE, Quester S, Ulzheimer JC, Waldegger S, Gorboulev V, Arndt P, Lang F, and Koepsell H. Electrogenic properties and substrate specificity of the polyspecific rat cation transporter rOCT1. J Biol Chem 271: 32599-32604, 1996[Abstract/Free Full Text].

10.   Evers, C, Haase W, Murer H, and Kinne R. Properties of brush border vesicles isolated from rat kidney cortex by calcium precipitation. Membr Biochem 1: 203-219, 1978[ISI][Medline].

11.   Gorboulev, V, Ulzheimer JC, Akhoundova A, Ulzheimer-Teuber I, Karbach U, Quester S, Baumann C, Lang F, Busch AE, and Koepsell H. Cloning and characterization of two human polyspecific organic cation transporters. DNA Cell Biol 16: 871-881, 1997[ISI][Medline].

12.   Gründemann, D, Babin-Ebell J, Martel F, Örding N, Schmidt A, and Schömig E. Primary structure and functional expression of the apical organic cation transporter from kidney epithelial LLC-PK1 cells. J Biol Chem 272: 10408-10413, 1997[Abstract/Free Full Text].

13.   Gründemann, D, Gorboulev V, Gambaryan S, Veyhl M, and Koepsell H. Drug excretion mediated by a new prototype of polyspecific transporter. Nature 372: 549-552, 1994[ISI][Medline].

14.   Gründemann, D, Köster S, Kiefer N, Breidert T, Engelhardt M, Spitzenberger F, Obermüller N, and Schömig E. Transport of monoamine transmitters by the organic cation transporter type 2, OCT2. J Biol Chem 273: 30915-30920, 1998[Abstract/Free Full Text].

15.   Gründemann, D, Schechinger B, Rappold GA, and Schömig E. Molecular identification of the corticosterone-sensitive extraneuronal catecholamine transporter. Nature Neurosci 1: 349-352, 1998[ISI][Medline].

16.   Kaissling, B, and LeHir M. Functional morphology of kidney tubules and cells in situ. Methods Enzymol 191: 265-289, 1990[Medline].

17.   Kekuda, R, Prasad PD, Wu X, Wang H, Fei YJ, Leibach FH, and Ganapathy V. Cloning and functional characterization of a potential-sensitive, polyspecific organic cation transporter (OCT3) most abundantly expressed in placenta. J Biol Chem 273: 15971-15979, 1998[Abstract/Free Full Text].

18.   Koepsell, H. Organic cation transporters in intestine, kidney, liver, and brain. Annu Rev Physiol 60: 243-266, 1998[ISI][Medline].

19.   Koepsell, H, Gorboulev V, and Arndt P. Molecular pharmacology of organic cation transporters in kidney. J Membr Biol 167: 103-117, 1999[ISI][Medline].

20.   Meyer-Wentrup, F, Karbach U, Gorboulev V, Arndt P, and Koepsell H. Membrane localization of the electrogenic cation transporter rOCT1 in rat liver. Biochem Biophys Res Commun 248: 673-678, 1998[ISI][Medline].

21.   Mühlbauer, B, and Osswald H. Feeding but not salt loading is the dominant factor controlling urinary dopamine excretion in conscious rats. Naunyn-Schmiedeberg's Arch Pharmacol 346: 469-471, 1992[ISI][Medline].

22.   Müller, M, and Jansen PLM Molecular aspects of hepatobiliary transport. Am J Physiol Gastrointest Liver Physiol 272: G1285-G1303, 1997[Abstract/Free Full Text].

23.   Nagel, G, Volk C, Friedrich T, Ulzheimer JC, Bamberg E, and Koepsell H. A reevaluation of substrate specificity of the rat cation transporter rOCT1. J Biol Chem 272: 31953-31956, 1997[Abstract/Free Full Text].

24.   Okuda, M, Saito H, Urakami Y, Takano M, and Inui KI. cDNA cloning and functional expression of a novel rat kidney organic cation transporter, OCT2. Biochem Biophys Res Commun 224: 500-507, 1996[ISI][Medline].

25.   Reinhardt, J, Veyhl M, Wagner K, Gambaryan S, Dekel C, Akhoundova A, Korn T, and Koepsell H. Cloning and characterization of the transport modifier RS1 from rabbit which was previously assumed to be specific for Na+-D-glucose cotransport. Biochim Biophys Acta 1417: 131-143, 1999[ISI][Medline].

26.   Roch-Ramel, F, Besseghir K, and Murer H. Renal excretion and tubular transport of organic anions and cations. In: Handbook of Physiology. Bethesda, MD: Am. Physiol. Soc, 1992, sect. 8, vol. II, chapt. 48, p. 2189-2262.

27.   Schmitt, A, Asan E, Püschel B, and Kugler P. Cellular and regional distribution of the glutamate transporter GLAST in the CNS of rats: nonradioactive in situ hybridization and comparative immunocytochemistry. J Neurosci 17: 1-10, 1997[Abstract/Free Full Text].

28.   Schweifer, N, and Barlow DP. The Lx1 gene maps to mouse chromosome 17 and codes for a protein that is homologous to glucose and polyspecific transmembrane transporters. Mammal Gen 7: 735-740, 1996[ISI][Medline].

29.   Shi, SR, Key ME, and Kalra KL. Antigen retrieval in formalin-fixed, paraffin-embedded tissues: an enhancement method for immunohistochemical staining based on microwave oven heating tissue sections. J Histochem Cytochem 39: 741-748, 1991[Abstract].

30.   Urakami, Y, Okuda M, Masuda S, Saito H, and Inui KI. Functional characteristics and membrane localization of rat multispecific organic cation transporters, OCT1 and OCT2, mediating tubular secretion of cationic drugs. J Pharmacol Exp Ther 287: 800-805, 1998[Abstract/Free Full Text].

31.   Wright, SH, Wunz TM, and Wunz TP. A choline transporter in renal brush-border membrane vesicles: energetics and structural specificity. J Membr Biol 126: 51-65, 1992[ISI][Medline].

32.   Zhang, L, Dresser MJ, Gray AT, Yost SC, Terashita S, and Giacomini KM. Cloning and functional expression of a human liver organic cation transporter. Mol Pharmacol 51: 913-921, 1997[Abstract/Free Full Text].


Am J Physiol Renal Fluid Electrolyte Physiol 279(4):F679-F687
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