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Am J Physiol Renal Physiol 293: F391-F397, 2007. First published April 11, 2007; doi:10.1152/ajprenal.00376.2006
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Cl–-dependent upregulation of human organic anion transporters: different effects on transport kinetics between hOAT1 and hOAT3

Harumasa Ueo, Hideyuki Motohashi, Toshiya Katsura, and Ken-ichi Inui

Department of Pharmacy, Kyoto University Hospital, Faculty of Medicine, Kyoto University, Kyoto, Japan

Submitted 18 September 2006 ; accepted in final form 8 April 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Chloride ion has a stimulatory effect on the transport of organic anions across renal basolateral membranes. However, the exact mechanisms at molecular levels have been unclear as of yet. Human organic anion transporters hOAT1 and hOAT3 play important roles in renal basolateral membranes. In this study, the effects of Cl– on the activities of these transporters were evaluated by using HEK293 cells stably expressing hOAT1 or hOAT3 (HEK-hOAT1 or HEK-hOAT3). The uptake of p-[14C]aminohippurate by HEK-hOAT1 and [3H]estrone sulfate by HEK-hOAT3 was greater in the presence of Cl– than in the presence of SO42– or gluconate. Additionally, the uptake of various compounds by HEK-hOAT1 and HEK-hOAT3 was significantly higher in the Cl–-containing medium than the gluconate-containing medium, suggesting that the influences of Cl– are not dependent on substrate and that Cl– directly stimulates the functions of hOAT1 and hOAT3. The substitution of gluconate with Cl– did not change the Km value for the uptake of p-[14C]aminohippurate by HEK-hOAT1 but caused an approximately threefold increase in the maximal uptake rate (Vmax) value. On the other hand, replacement of gluconate with Cl– decreased the Km value for the uptake of [3H]estrone sulfate and cefotiam by HEK-hOAT3 to about one-third, while it did not change the Vmax value. In summary, Cl– upregulates the activities of both hOAT1 and hOAT3, but its effects on transport kinetics differ between these transporters. It was suggested that Cl– participates in the trans-location process for hOAT1, and the substrate recognition process for hOAT3.

renal secretion; basolateral membrane; SLC22A; HEK293

THE ORGANIC ANION TRANSPORT systems in renal proximal tubules play important physiological roles in the excretion of a wide variety of anionic compounds, including endogenous substances, xenobiotics, and their metabolites, into the urine. These systems are highly effective and mediate the tubular secretion of various drugs. Tubular secretion in the proximal epithelia consists of two processes, uptake at the basolateral membrane and efflux at the brush-border membrane. Numerous studies have been performed regarding these transport systems (2, 7, 15).

Chloride ion is the most abundant anion in the blood and is involved in various physiological processes, including the regulation of cell volume, regulation of intracellular pH, and maintenance of blood osmolality. In our previous study (8), the effects of Cl– on the basolateral transport of organic anions in the proximal tubules were investigated using rat renal basolateral membrane vesicles. The substitution of Cl– on both sides of the vesicles with SCN– or SO42– decreased the uptake of p-aminohippurate into the vesicles, suggesting that Cl– plays an important role in the organic anion transport systems in basolateral membranes.

Several organic anion transporters have been identified in renal basolateral membranes (3, 6). The mRNA levels of human organic anion transporter (hOAT)-1 and hOAT3 are much higher than those of other organic ion transporters in the human kidney cortex, and both transporters are located at the basolateral membranes (12). These findings indicated that hOAT1 and hOAT3 play important roles in the tubular uptake of various drugs from the circulation. However, little is known about the influences of Cl– on hOAT1 and hOAT3 and their relation to renal drug secretion.

The purpose of this study is to clarify whether Cl– affects hOAT1 and hOAT3 activities. Using cells expressing these transporters, we found differences in Cl–-dependent regulation between hOAT1 and hOAT3.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. p-[Glycyl-1-14C]aminohippurate (1.9 GBq/mmol) was purchased from NEN Life Science Products (Boston, MA). [6,7-3H(N)]estrone sulfate, ammonium salt (2.1 TBq/mmol), was from PerkinElmer Life Sciences (Boston, MA). [N-methyl-3H]cimetidine (451 GBq/mmol) was from Amersham Biosciences (Uppsala, Sweden). [3',5',7'-3H(N)]methotrexate, disodium salt (851 GBq/mmol), and [3H(G)]ochratoxin A (666 GBq/mmol) were from Moravek Biochemicals (Brea, CA). [14C]captopril (115 MBq/mmol) was kindly provided by Sankyo (Tokyo, Japan). Cefotiam was from Takeda Chemical Industries (Osaka, Japan). Probenecid and aspartate aminotransferase were from Sigma Chemical (St. Louis, MO). Malate dehydrogenase was from Toyobo (Osaka, Japan). NADH was from Nacalai Tesque (Kyoto, Japan). 6-Methoxy-N-(3-sulfopropyl)quinolinium (SPQ) was from Biotium (Hayward, CA). All other chemicals used were of the highest purity available.

Uptake of radiolabeled compounds by HEK293 cells stably expressing hOAT1 or hOAT3. According to our previous report (21), uptake experiments were carried out using HEK293 cells stably expressing hOAT1 (HEK-hOAT1) or hOAT3 (HEK-hOAT3). Cells transfected with empty vector (HEK-pBK) were used as control cells. HEK-pBK, HEK-hOAT1, and HEK-hOAT3 were seeded on poly-D-lysine-coated 24-well plates at a density of 2 x 105 cells/well. At 48 h after seeding, the cells were used for the uptake experiments.

In the experiments on the effects of extracellular anions on the uptake of p-[14C]aminohippurate and [3H]estrone sulfate by HEK-hOAT1 and HEK-hOAT3, respectively, the composition of the incubation medium was as follows (in mM): 3 KCl, 1 CaCl2, 0.5 MgCl2, 5 D-glucose, 5 HEPES, and the salt indicated [145 NaCl, 145 NaBr, 72.5 Na2SO4, or 145 Na gluconate for each incubation medium (pH 7.4)]. In the experiments on the effects of extracellular Cl–, the composition of Cl–-free [Cl–(–)] incubation medium was as follows (in mM): 145 Na gluconate, 3 K gluconate, 1 Ca gluconate, 0.5 MgSO4, 5 D-glucose, and 5 HEPES (pH 7.4). And the composition of Cl–-containing [Cl–(+)] incubation medium was as follows (in mM): 145 NaCl, 3 KCl, 1 CaCl2, 0.5 MgCl2, 5 D-glucose, and 5 HEPES (pH 7.4). After preincubation of the cells with 0.2 ml of Cl–(+) incubation medium at 37°C for 10 min, the medium was replaced with 0.2 ml of each incubation medium containing test compounds. At the end of the incubation, the medium was aspirated, and then cells were washed two times (3 times for the uptake of ochratoxin A) with 1 ml of ice-cold incubation medium.

In the experiments on the effects of Cl– at various concentrations, for which intracellular and extracellular Cl– were equilibrated, the composition of Cl–(–) high-K medium was as follows (in mM): 25 Na gluconate, 120 K gluconate, 1 Ca gluconate, 0.5 MgSO4, 5 D-glucose, and 5 HEPES (pH 7.4). And the composition of Cl–(+) high-K medium was as follows (in mM): 25 NaCl, 120 KCl, 1 CaCl2, 0.5 MgCl2, 5 D-glucose, and 5 HEPES (pH 7.4). The high-K media containing each concentration of Cl– were made by mixture of these solutions. After preincubation of the cells with 0.2 ml of high-K medium containing each concentration of Cl–, 5 µM nigericin, and 10 µM tributyltin at 37°C for 10 min, the cells were incubated with 0.2 ml of each medium containing 5 µM nigericin, 10 µM tributyltin, and test compound for 1 min. At the end of the incubation, the medium was aspirated, and then cells were washed two times with 1 ml of ice-cold Cl–(+) incubation medium.

After uptake was finished, the cells were lysed in 0.25 ml of 0.5 N NaOH solution, and the radioactivity in aliquots was determined in 3 ml of ACSII (Amersham International, Buckingham Shire, UK). The protein contents of the solubilized cells were determined by the method of Bradford (1) using the Bio-Rad Protein Assay kit (Bio-Rad, Hercules, CA) with bovine {gamma}-globulin as a standard.

Uptake of cefotiam by HEK293 cells stably expressing hOAT1 or hOAT3. The uptake of cefotiam was examined as described previously (21) with some modifications. Briefly, HEK-pBK, HEK-hOAT1, and HEK-hOAT3 were seeded on poly-D-lysine-coated 35-mm dishes at a density of 1 x 106 cells/dish. At 48 h after seeding, the cells were used for the uptake experiments. The composition of the incubation medium was as described above. After preincubation of the cells with 1 ml of Cl–(+) incubation medium at 37°C for 10 min, the medium was replaced with 1 ml of each incubation medium containing cefotiam. At the end of the incubation, the medium was aspirated, and then cells were washed three times with 2 ml of ice-cold incubation medium. To measure the accumulation of cefotiam, the cells were scraped and homogenized with 0.5 ml of distilled water. Protein levels were determined with 5 µl of the homogenate. For measuring the amount of cefotiam, 50 µl of distilled water and 10 µl of phosphoric acid were added to 0.45 ml of the homogenate, and the solution was mixed for 30 s. Then, 0.5 ml of the sample was loaded onto an Oasis HLB cartridge (Waters, Milford, MA) preconditioned with 1 ml each of methanol and distilled water. The column was washed with 1 ml of 5% methanol, and cefotiam was eluted from the column with 1 ml of methanol. The eluate was evaporated dry at 45–50°C and resuspended in 130 µl of distilled water. The solution was filtered through a 0.45-µm polyvinylidene fluoride filter. The concentration of cefotiam was measured using a high-performance liquid chromatograph (LC-10AT, LC-10AD; Shimadzu, Kyoto, Japan) equipped with a UV spectrophotometric detector (SPD-10AV, SPD-10A, Shimadzu) under the following conditions: column, Zorbax ODS column, 4.6-mm inside diameter x 250 mm (Du Pont, Wilmington, DE); mobile phase, 30 mM phosphate buffer (pH 6.5) in methanol at 78:22; flow rate, 0.8 ml/min; wave length, 254 nm; injection volume, 50 µl; temperature, 40°C.

Measurement of intracellular {alpha}-ketoglutarate. Intracellular {alpha}-ketoglutarate ({alpha}-KG) concentration was determined by the fluorimetric method of Williamson and Corkey (23). Briefly, HEK-pBK, HEK-hOAT1, and HEK-hOAT3 cells were seeded on poly-D-lysine-coated 35-mm dishes at a density of 1 x 106 cells/dish. At 48 h after seeding, the cells were used for the experiments. The cells were incubated with 0.5 ml of 3% HClO4 for 30 min on ice to extract intracellular {alpha}-KG and to denature protein. After incubation, the buffer was transferred into a microtube and neutralized by 62 µl of 3 M NaOH. The amount of extracted {alpha}-KG in the neutralized buffer was measured by an enzymatic analysis. The conversions of {alpha}-KG and aspartate to glutamate and oxaloacetate, respectively, were catalyzed by aspartate aminotransferase. The oxaloacetate was then converted to malate by malate dehydrogenase. The associated conversion of NADH to NAD+ was determined fluorimetrically (excitation, 355 nm; emission, 460 nm) at 37°C with MIthras LB940 (Berthold Technologies, Bad Wildbad, Germany).

Measurement of intracellular Cl– concentration. Intracellular concentration of Cl– was determined by the use of the Cl–-sensitive fluorophore SPQ (4, 11). HEK-pBK cells were seeded on a 96-well polystyrene plate at a density of 4 x 104 cells/well, and at 48 h after seeding, the cells were used for the experiments. The composition of solution 1 was as follows (in mM): 106 NaCl, 24 Na gluconate, 5 K gluconate, 2 CaCl2, 2 MgCl2, 5 D-glucose, and 5 HEPES (pH 7.4). And the composition of KSCN medium was as follows (in mM): 145 KSCN, 1 CaCl2, 0.5 MgCl2, 5 D-glucose, and 5 HEPES (pH 7.4). According to a previous report (4), SPQ was loaded into the cells by incubation in a 1:1 mixture of solution 1 and distilled water containing 5 mM SPQ at 23°C for 4 min. Then, the cells were washed two times with 0.2 ml of Cl–(–) high-K medium and incubated with 0.1 ml of high-K medium containing each concentration of Cl–, 5 µM nigericin, and 10 µM tributyltin at 37°C. During the incubation, SPQ fluorescence was measured (excitation, 355 nm; emission, 460 nm) with MIthras LB940. The background signal due to cell and instrument autofluorescence was obtained by incubation of the cells with 0.1 ml of KSCN medium containing 5 µM valinomycin, which has been shown to quench 99% of SPQ fluorescence (4). Data were fitted to the Stern-Volmer quenching equation: F0/FCl = 1 + Kq[Cl–], where F0 is the fluorescence in the absence of Cl–, FCl is the fluorescence in the presence of each concentration of Cl–, Kq is the Stern-Volmer constant, and [Cl–] is the concentration of Cl–.

Statistical analysis. Data were analyzed statistically using a nonpaired t-test. Multiple comparisons were performed using Scheffé's test. Probability values of <5% were considered significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Effects of extracellular anions on the uptake of p-[14C]aminohippurate and [3H]estrone sulfate by HEK-hOAT1 and HEK-hOAT3, respectively. The effects of extracellular anions on the transport of organic anions via hOAT1 and hOAT3 were examined, using p-[14C]aminohippurate and [3H]estrone sulfate, respectively, as model substrates. As shown in Fig. 1, the uptake of p-[14C]aminohippurate by HEK-hOAT1 with each anion was stimulated in the following order: Cl– > Br– > SO42– > gluconate. The rank order observed in the uptake of [3H]estrone sulfate by HEK-hOAT3 was Cl– = Br– > SO42– > gluconate.


Figure 1
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  		Fig. 1. Effects of extracellular anions on the uptake of p-[14C]aminohippurate by HEK-hOAT1 (A) and [3H]estrone sulfate by HEK-hOAT3 (B). HEK-pBK, HEK-hOAT1, and HEK-hOAT3 were incubated with 5 µM p-[14C]aminohippurate (A) or 10 nM [3H]estrone sulfate (B) in the presence of 145 mM Cl– (open column), 145 mM Br– (black column), 72.5 mM SO42– (shaded column), or 145 mM gluconate (hatched column) at 37°C for 1 min. Each column represents the mean ± SE of 3 monolayers from a typical experiment. hOATs, human organic anion transporters; HEK-hOAT1, HEK293 cells stably expressing hOAT1; HEK-hOAT3, HEK293 cells stably expressing hOAT3; HEK-pBK, cells transfected with empty vector. *P < 0.05, significantly different from Cl–.

 
Effects of extracellular Cl– on the uptake of various compounds by HEK-hOAT1 and HEK-hOAT3. In the subsequent experiments, the Cl–(–) incubation medium, where Cl– was substituted with gluconate, and the Cl–(+) incubation medium were used. The uptake of p-[14C]aminohippurate, [3H]estrone sulfate, [3H]cimetidine, [3H]methotrexate, [14C]captopril, [3H]ochratoxin A, and cefotiam by HEK-hOAT1 and HEKhOAT3 was evaluated in the absence or presence of extracellular Cl–(Table 1). The uptake of p-[14C]aminohippurate, [3H]cimetidine, [3H]methotrexate, [14C]captopril, and [3H]ochratoxin A by HEK-hOAT1 was significantly greater in the presence than absence of Cl–. The uptake of p-[14C]aminohippurate, [3H]estrone sulfate, [3H]cimetidine, [3H]methotrexate, [3H]ochratoxin A, and cefotiam by HEK-hOAT3 was significantly higher in the Cl–-containing medium than in Cl–-free medium. However, the uptake of [14C]captopril by HEK-hOAT3 was not affected. The reason for this could be as follows: as shown in Table 1, the uptake of [14C]captopril by HEK-pBK was significantly reduced when gluconate was replaced with Cl–, indicating that the increase in the transporter-mediated uptake caused by Cl– was masked.


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  		Table 1. Effects of extracellular Cl– on the uptake of various compounds by HEK-hOAT1 and HEK-hOAT3

 
Characterization of the effects of Cl– on hOAT1 and hOAT3. Figure 2 shows the time course of uptake of p-[14C]aminohippurate and [3H]estrone sulfate by HEK-hOAT1 and HEK-hOAT3, respectively, in the absence or presence of extracellular Cl–. The uptake of p-[14C]aminohippurate and [3H]estrone sulfate by HEK-hOAT1 and HEK-hOAT3, respectively, was greater in the Cl–(+) incubation medium than the Cl–(–) medium throughout.


Figure 2
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  		Fig. 2. Effects of extracellular Cl– on the time course of the accumulation of p-[14C]aminohippurate in HEK-hOAT1 (A) and [3H]estrone sulfate in HEK-hOAT3 (B). A: HEK-pBK ({triangleup}, {circ}) and HEK-hOAT1 ({blacktriangleup}, bullet) were incubated in Cl–-free ({triangleup}, {blacktriangleup}) or Cl–-containing (bullet, {circ}) incubation medium with 5 µM p-[14C]aminohippurate at 37°C for specified periods. B: HEK-pBK ({triangleup}, {circ}) and HEK-hOAT3 ({blacktriangleup}, bullet) were incubated in Cl–-free ({triangleup}, {blacktriangleup}) or Cl–-containing ({circ}, bullet) incubation medium with 10 nM [3H]estrone sulfate at 37°C for specified periods. Each point represents the mean ± SE of 3 monolayers from a typical experiment.

 
OAT1 and OAT3 are organic anion/dicarboxylate exchangers (18, 19), and intracellular {alpha}-KG, endogenous dicarboxylate, is involved in the uptake of organic anions by HEK-hOAT1 and HEK-hOAT3. Therefore, we examined the effect of extracellular Cl– on the intracellular concentration of {alpha}-KG. When HEK-pBK, HEK-hOAT1, and HEK-hOAT3 were incubated with Cl–(+) incubation medium, the intracellular concentrations of {alpha}-KG were not changed compared with cells incubated with Cl–(–) incubation medium (Fig. 3).


Figure 3
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  		Fig. 3. Effect of extracellular Cl– on the intracellular concentration of {alpha}-ketoglutarate ({alpha}-KG). After incubation in Cl–-free (open column) or Cl–-containing (black column) incubation medium at 37°C for 1 min, intracellular concentration of {alpha}-KG was determined. Each column represents the mean ± SE of 3 monolayers from a typical experiment.

 
Next, the effects of extracellular Cl– at various concentrations on the uptake of p-[14C]aminohippurate and [3H]estrone sulfate by HEK-hOAT1 and HEK-hOAT3, respectively, were examined. The concentration of extracellular Cl– was adjusted by replacing Cl– with gluconate. The uptake of p-[14C]aminohippurate and [3H]estrone sulfate by HEK-hOAT1 and HEK-hOAT3, respectively, was increased with the elevation in the concentration of extracellular Cl– (Fig. 4).


Figure 4
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  		Fig. 4. Effects of extracellular Cl– at various concentrations on the uptake of p-[14C]aminohippurate by HEK-hOAT1 (A) and [3H]estrone sulfate by HEK-hOAT3 (B). HEK-hOAT1 (A) and HEK-hOAT3 (B) were incubated with 5 µM p-[14C]aminohippurate and 10 nM [3H]estrone sulfate, respectively, in incubation medium containing various concentrations of Cl– at 37°C for 1 min. The values were obtained by subtracting the values for the uptake in HEK-pBK from those in HEK-hOAT1 or HEK-hOAT3. The concentration of Cl– was adjusted by replacing Cl– with gluconate. Each point represents the mean ± SE of 3 monolayers from a typical experiment.

 
Moreover, the effects of Cl– at various concentrations on the uptake of p-[14C]aminohippurate and [3H]estrone sulfate by HEK-hOAT1 and HEK-hOAT3, respectively, were examined when there were no concentration gradients of Cl– across plasma membranes. We adjusted the Cl– concentration by substituting gluconate for it. The concentration of intracellular Cl– was kept equal to that of extracellular Cl– by using a high-K medium, the K+/H+ exchange ionophore nigericin, and the Cl–/OH– exchange ionophore tributyltin (4, 11). The high-K medium in the presence of nigericin clamps internal pH at the external pH and strongly depolarizes cell membrane potential. The addition of tributyltin to pH-clamped cells results in equal intracellular and extracellular Cl– concentrations. At first, we confirmed the change of internal Cl– concentration using the Cl–-sensitive fluorophore SPQ (4, 11). Chloride ion quenches the fluorescence of SPQ, and this compound is used to determine intracellular Cl–. The change of fluorescence induced by medium containing each concentration of Cl– was fitted to the Stern-Volmer quenching equation (Fig. 5A). There was a linear correlation between change in fluorescence and Cl– concentration, indicating that intracellular concentration of Cl– was changed in parallel with extracellular concentration. Figure 5, B and C, shows that the uptake of p-[14C]aminohippurate and [3H]estrone sulfate by HEK-hOAT1 and HEK-hOAT3, respectively, was increased in proportion to Cl– concentration. Although we simultaneously measured the intracellular {alpha}-KG level in HEK-pBK at each Cl– concentration, {alpha}-KG level was not affected by Cl– concentration (data not shown).


Figure 5
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  		Fig. 5. A: the change of intracellular Cl– concentration induced by high-K medium containing various concentrations of Cl–, nigericin, and tributyltin. After loading with SPQ, HEK-pBK were incubated in high-K medium containing various concentrations of Cl–, 5 µM nigericin, and 10 µM tributyltin at 37°C. During the incubation, SPQ fluorescence was measured. The change of fluorescence was fitted to the Stern-Volmer quenching equation (see MATERIALS AND METHODS for further details). The ordinate (F0/FCl) is the total fluorescence measured in the absence of Cl– divided by that measured in the presence of each concentration of Cl–. B and C: effects of Cl– at various concentrations on the uptake of p-[14C]aminohippurate by HEK-hOAT1 and [3H]estrone sulfate by HEK-hOAT3 under the conditions in which intracellular and extracellular Cl– are equilibrated. HEK-hOAT1 (B) and HEK-hOAT3 (C) were incubated with 5 µM p-[14C]aminohippurate and 10 nM [3H]estrone sulfate, respectively, in high-K medium containing various concentrations of Cl–, 5 µM nigericin, and 10 µM tributyltin at 37°C for 1 min. The values were obtained by subtracting the values for the uptake in HEK-pBK from those in HEK-hOAT1 or HEK-hOAT3. The concentration of Cl– was adjusted by replacing Cl– with gluconate. Each point represents the mean ± SE of 3 or 4 monolayers from a typical experiment.

 
Furthermore, the concentration-dependent uptake of p-[14C]aminohippurate by HEK-hOAT1 (Fig. 6) and [3H]estrone sulfate by HEK-hOAT3 (Fig. 7) was assessed in the absence or presence of extracellular Cl–. With the use of a nonlinear least squares regression analysis, kinetic parameters were calculated according to the Michaelis-Menten equation in three separate experiments. Apparent Michaelis-Menten constants (Km) and maximal uptake rate (Vmax) values are summarized in Table 2. Substitution of gluconate with Cl– did not change the Km value for the uptake of p-[14C]aminohippurate by HEK-hOAT1 but caused an approximate threefold increase in the Vmax value. On the other hand, replacement of gluconate with Cl– decreased the Km value for the uptake of [3H]estrone sulfate by HEK-hOAT3 to about one-third but did not change the Vmax value. In addition, we evaluated the influence of Cl– on the concentration-dependent uptake of cefotiam by HEK-hOAT3 (Fig. 8). The Km values for the uptake of cefotiam by HEK-hOAT3 in Cl–-free and in Cl–-containing medium were 1,702.0 and 552.3 µM, respectively. The Vmax values for the uptake in Cl–-free and in Cl–-containing medium were 146.4 and 186.9 pmol·mg protein–1·15 min–1, respectively. These results demonstrated that, similar to the results for the uptake of [3H]estrone sulfate by HEK-hOAT3, the Km value was decreased to approximately one-third for the uptake of cefotiam by HEK-hOAT3, while the Vmax value was little affected.


Figure 6
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  		Fig. 6. Effect of extracellular Cl– on the concentration-dependent uptake of p-[14C]aminohippurate by HEK-hOAT1. HEK-hOAT1 were incubated in Cl–-free (A) or Cl–-containing (B) incubation medium with various concentrations of p-[14C]aminohippurate in the absence (bullet) or presence ({circ}) of unlabeled 5 mM p-aminohippurate at 37°C for 1 min. Each point represents the mean ± SE of 3 monolayers from a typical experiment.

 

Figure 7
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  		Fig. 7. Effect of extracellular Cl– on the concentration-dependent uptake of [3H]estrone sulfate by HEK-hOAT3. HEK-hOAT3 were incubated in Cl–-free (A) or Cl–-containing (B) incubation medium with various concentrations of [3H]estrone sulfate in the absence (bullet) or presence ({circ}) of unlabeled 1 mM estrone sulfate at 37°C for 1 min. Each point represents the mean ± SE of 3 monolayers from a typical experiment.

 

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  		Table 2. Effects of extracellular Cl– on Km and Vmax values for the uptake of p-aminohippurate by HEK-hOAT1 and estrone sulfate by HEK-hOAT3

 

Figure 8
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  		Fig. 8. Effect of extracellular Cl– on the concentration-dependent uptake of cefotiam by HEK-hOAT3. HEK-hOAT3 were incubated in Cl–-free (A) or Cl–-containing (B) incubation medium with various concentrations of cefotiam in the absence (bullet) or presence ({circ}) of 1 mM probenecid at 37°C for 15 min. Each point represents the mean ± SE of 3 monolayers from a typical experiment.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In the human kidney, organic anion transport systems play critical roles in the excretion of organic anions. It has been described that the activities of these transport systems are regulated by inorganic anions. Chloride ion is one of the most abundant anions in the human body and has been demonstrated to affect renal tubular transport of organic anions (8, 13, 14). However, this phenomenon has not been sufficiently studied at the molecular level. In this study, we showed that both hOAT1 and hOAT3 were upregulated by Cl–, but the effects of Cl– on transport kinetics were different between hOAT1 and hOAT3.

Schmitt and Burckhardt (14) reported that the substitution of Cl– with Br– did not affect the uptake of p-aminohippurate by bovine renal basolateral membrane vesicles, while the replacement of Cl– with SO42– or gluconate decreased the uptake. Pritchard (13) also demonstrated that replacing Cl– with SO42– or gluconate decreased the uptake of p-aminohippurate by rat renal basolateral membrane vesicles. Therefore, Br– is an effective substitute for Cl– in basolateral organic anion transport, while SO42– and gluconate are poor substitutes for Cl–. In the present study, the replacement of Cl– with Br– had little effect on the uptake of p-[14C]aminohippurate by HEK-hOAT1, while accumulation of p-[14C]aminohippurate was decreased to one-half to one-third by replacing Cl– with SO42– or gluconate. Therefore, p-aminohippurate uptake by hOAT1 is highly dependent on Cl–. Br– could maintain the uptake as a substitute for Cl–, but SO42– and gluconate could not. Our results are consistent with previous studies, suggesting that hOAT1 is involved in the Cl–-dependent regulation of the basolateral transport of p-aminohippurate in renal proximal tubules.

In our previous examination (8), the uptake of p-aminohippurate by rat renal basolateral membrane vesicles was found to depend on the concentration of Cl–, and the same result was obtained by Pritchard (13). In the present study, the uptake of p-[14C]aminohippurate by HEK-hOAT1 and [3H]estrone sulfate by HEK-hOAT3 was dependent on the concentration of Cl– and was not saturated at 0–150 mM. Physiologically, the Cl– concentration in blood ranges from 95 to 105 mM in healthy individuals (10). However, in patients with diseases such as acidosis and alkalosis, the plasma concentration of Cl– can be in the range of 78–128 mM (17). On the basis of the present results, it is suggested that the activities of hOAT1 and hOAT3 are not saturated and altered with the change in the concentration of Cl–. In addition, information is now available about the movement of Cl– at the basolateral membranes of proximal tubules. Ishibashi et al. (9) demonstrated that a Na+-dependent Cl–/HCO3– exchanger plays a dominant role in the efflux of Cl– at the basolateral membranes. It is implied that the Cl– transport system controls the concentration of Cl– around hOAT1 and hOAT3 and cooperates with hOAT1 and hOAT3. Soleimani et al. (16) indicated that the basolateral Cl–/base exchange activity in proximal tubular cells decreased under acidic conditions and increased under alkalotic conditions. Thus it is probable that the functions of hOAT1 and hOAT3 are affected under pathological conditions such as metabolic acidosis and alkalosis.

Several studies have examined the effects of Cl– on the transport of organic anions at basolateral membranes (8, 13, 14), but only p-aminohippurate was used as a substrate. In the current study, the uptake of various compounds by hOAT1 and hOAT3 was increased by Cl–. These results indicated that Cl– affects both hOAT1 and hOAT3, and these effects are not dependent on the substrates. It has been shown that hOAT1 and hOAT3 are involved in the basolateral uptake of various drugs including diuretics, antiviral drugs, and cephalosporin antibiotics (5, 20, 21). Therefore, it is implied that the basolateral uptake of these drugs from blood circulation into tubular cells is regulated by Cl–.

It has been shown that OAT1 and OAT3 are organic anion/dicarboxylate exchangers (18, 19), and organic anion transport into the cells by these transporters is undertaken in exchange for endogenous dicarboxylate, {alpha}-KG. Thus intracellular concentration of {alpha}-KG is a major determinant of the transport activities of hOAT1 and hOAT3. In the current study, we showed that Cl– had no effect on intracellular {alpha}-KG concentration. It was suggested that the change of intracellular concentration of {alpha}-KG is not associated with the effects of Cl– on the functions of hOAT1 and hOAT3.

Previously, we demonstrated that p-aminohippurate uptake by rat renal basolateral membrane vesicles was stimulated by the increase in Cl– concentration, when the concentration of Cl– in the vesicles was equal to that on the outside (8). In the current study, the uptake of p-aminohippurate by HEK-hOAT1 and estrone sulfate by HEK-hOAT3 was dependent on extracellular Cl– concentration, and these phenomena were also observed when there were no concentration gradients of Cl– across plasma membranes. Therefore, it is reasonable to assume that Cl– outside of the cells, i.e., the same side as the substrate, upregulates the activities of hOAT1 and hOAT3.

Subsequently, the influences of extracellular Cl– on the kinetic parameters of transport via hOAT1 and hOAT3 were examined. When gluconate was substituted with Cl–, the Vmax value for the uptake of p-aminohippurate by hOAT1 was increased about threefold without a significant effect on the Km value. On the other hand, under the same conditions, the Km values for the hOAT3-mediated uptake of estrone sulfate and cefotiam were decreased to approximately one-third without any effect on Vmax values. These results suggested that Cl– participates in the trans-location rate for the uptake of p-aminohippurate by hOAT1 and in the substrate affinity for the uptake of estrone sulfate and cefotiam by hOAT3. The substrate recognition of hOAT1 is different from that of hOAT3. For example, hOAT1 efficiently transports the nucleotide antivirals adefovir, cidofovir, and tenofovir (22), whereas hOAT3 transports cephalosporin antibiotics (21). Therefore, at basolateral membranes, Cl– affects Vmax values for the uptake of nucleotide antivirals or Km values for the uptake of cephalosporin antibiotics. Although how Cl– affects the activities of hOAT1 and hOAT3 is unclear, it was indicated that the transport activities of hOAT1 and hOAT3 are upregulated in a different manner by Cl–.

In summary, Cl–-dependent regulation of hOAT1 and hOAT3 was examined in detail for the first time. It was suggested that Cl– is involved in the regulation and maintenance of organic anion secretion in renal proximal tubules. In addition, the effects of Cl– on transport kinetics differ between hOAT1 and hOAT3, suggesting that Cl– participates in the trans-location process for hOAT1 and the substrate recognition process for hOAT3.


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This work was supported in part by a grant-in-aid for Research on Advanced Medical Technology from the Ministry of Health, Labor and Welfare of Japan, by a grant-in-aid for Scientific Research from the Ministry of Education, Science, Culture and Sports of Japan, and by the 21st Century COE program "Knowledge Information Infrastructure for Genome Science." H. Ueo was supported as a teaching assistant by the 21st Century Center of Excellence program "Knowledge Information Infrastructure for Genome Science."


   FOOTNOTES
 

Address for reprint requests and other correspondence: K. Inui, Department of Pharmacy, Kyoto University Hospital, Sakyo-ku, Kyoto 606-8507, Japan (e-mail: inui{at}kuhp.kyoto-u.ac.jp)

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.


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