Role of Organic Anion Transporters in the Tubular Transport of Indoxyl Sulfate and the Induction of its Nephrotoxicity

In Vivo Experimental Design

Male Sprague-Dawley rats (Clea, Tokyo, Japan) aged 7 wk were anesthetized, and five sixths of the normal kidney mass was infarcted, which induced CRF in most of the rats (33,34). Two days after the operation, the treated rats showed increased serum concentrations of creatinine (1.0 approximately 1.4 mg/dl), blood urea nitrogen (BUN) (67 approximately 92 mg/dl), and reduced levels of creatinine clearance (0.67 approximately 1.12 ml/min). Sham-operated rats as well as operated ones were given an intraperitoneal infusion of antibiotics (50 to 100 μl of 5000 U/ml penicillin-streptomycin; Life Technologies BRL, New York, NY).

Fourteen rats were paired and assigned to two separate groups: (1) 5/6-NPX rats (n = 7) and (2) 5/6-NPX rats orally administered IS in water daily at a dose of 50 mg/kg body wt and additionally receiving a daily intraperitoneal injection at a dose of 50 mg/kg body wt (n = 7). The administration of IS began at the fifth week after the nephrectomy and continued for 16 wk. At 0, 4, 10, and 16 wk after the initial administration of IS, BP measurement, the serum concentrations of creatinine, IS ,and BUN, the urinary concentrations of protein and IS, and the creatinine clearance levels were determined. BP was measured using the tails of the rats with a pneumatic cuff and a sphygmomanometer for small animals (UR-5000, Ueda Avancer Co., Tokyo, Japan). At the end of the experiments, the rats were sacrificed and the remnant kidneys were removed for immunohistochemical study.

Antibodies

The characterization of the monoclonal anti-IS antibody was described previously (15). There was no positive staining in the renal cortex when the primary antibody was preincubated with IS (data not shown) or when the primary antibody was omitted from the protocol. A rabbit polyclonal antibody was raised against a synthesized polypeptide of the carboxyl terminal of rOAT1 and rOAT3. The specificity of the antibody was described elsewhere (30,32).

Immunohistochemical Studies in 5/6-NPX Rat Kidney

Immunostaining of IS in renal tissue sections (1-μm-thick) was performed by the SABC (streptavidin-biotinylated peroxidase complex) method, as described previously (15,30). For immunohistochemical detection of rOAT1 and rOAT3, the sections were incubated with polyclonal antibodies against rOAT1 (1:2000 dilution) and rOAT3 (1:250 dilution), respectively. The sections were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody against rabbit Ig (Dako, Glostrup, Denmark), and the HRP was detected using a diaminobenzidine (DAB)-H₂O₂ solution.

Cell Culture and Establishment of S2 Cells Stably Expressing rOAT1 and rOAT3 (S2 rOAT1 and S2 rOAT3)

An immortalized cell line derived from the second segment of the proximal tubule (S2) cells was established from the kidney of transgenic mice harboring the simian virus 40 large T-antigen gene as described previously (35,36). S2 cells do not express mouse OATs by RT-PCR (data not shown) and show a faint uptake of para-amino hippuric acid (PAH). Moreover, although protein kinase C activation downregulates estrone sulfate (ES) uptake by S2 rOAT3, other cells derived from the same transgenic mice did not show such regulation (37). Therefore, we assumed this cell line to be appropriate for the transfection. The establishment of S2 rOAT1 and S2 rOAT3 was carried out as described previously (36,37). Briefly, the full-length cDNAs of rOAT1 (18) and rOAT3 (21) were subcloned into pcDNA 3.1(+) (Invitrogen, San Diego, CA). S2 cells were transfected with pcDNA3.1-rOAT1 and pcDNA3.1-rOAT3 coupled with pSV2neo, a neomycin resistance gene, using TfX-50 according to the manufacturer’s instructions. S2 cells transfected with pcDNA3.1 lacking an insert and pSV2neo were designated S2 pcDNA 3.1 and used as a control. The cells were grown in a humidified incubator at 33°C under 5% CO₂ using RITC 80–7 medium (Iwaki Co., Tokyo, Japan) containing 5% fetal bovine serum, 10 μg/ml transferrin, 0.08 U/ml insulin, 10 ng/ml recombinant epidermal growth factor and 400 μg/ml geneticin. The cells were subcultured in a medium containing 0.05% trypsin-EDTA solution (containing 137 mM NaCl, 5.4 mM KCl, 5.5 mM glucose, 4 mM NaHCO₃, 0.5 mM EDTA, and 5 mM HEPES; pH 7.2). Clonal cells were isolated by using a cloning cylinder and screened by determining the optimal substrate for each transporter, i.e., [¹⁴C]PAH (53.1 mCi/mmol; Perkin Elmer Life Sciences, Boston, MA) (18) and [³H]ES (53 Ci/mmol; Perkin Elmer Life Sciences) for rOAT3 (21). Staining of vertical sections of S2 rOAT1 and S2 rOAT3 with polyclonal antibodies against rOAT1 and rOAT3, respectively, showed that the subcellular localization of rOAT1 and rOAT3 proteins was mainly on the cell membrane (36,38). Both the basolateral and apical portions of the membrane showed positive staining. Therefore, the cells were cultured on a solid support for subsequent experiments.

Uptake Experiments in S2 rOAT1 and S2rOAT3

Uptake experiments were performed as described previously (36–38). The cells were seeded in 24-well tissue culture plates at a cell density of 1 × 10⁵ cells/well. After culturing for 2 d, they were washed three times with Dulbecco modified phosphate-buffered saline (D-PBS) solution (containing 137 mM NaCl, 3 mM KCl, 8 mM Na₂HPO₄, 1 mM KH₂PO₄, 1 mM CaCl₂, and 0.5 mM MgCl₂; pH 7.4). They were then preincubated in the same solution at 37°C for 10 min. The cells were thereafter incubated in D-PBS containing 5 μM [¹⁴C]PAH or 50 nM [³H]ES in the absence or presence of IS at 37°C. After incubation, the cells were washed three times with ice-cold D-PBS. The cells in each well were lysed with 0.5 ml of 0.1 M sodium hydroxide, 2.5 ml of aquasol-2 was added, and radioactivity was determined by using a β-scintillation counter (LSC-3100, Aloka, Co., Tokyo, Japan).

Kinetic Analyses

After preincubation as described above, S2 rOAT1 and S2 rOAT3 were incubated in a solution containing [¹⁴C]PAH or [³H]ES at different concentrations in the absence or presence of IS (Sigma, St. Louis, MO) at 37°C for 2 min. On the basis of the level of organic anion uptake under each condition, analyses of Lineweaver-Burk plot as well as Eadie-Hofstee plot were performed as described previously (39). When the inhibition was competitive, Ki values were calculated based on the following equation,

Ki = concentration of IS/[(Km PAH or ES with IS/Km PAH or ES without IS) − 1]

Measurement of Intracellular IS Content

S2 cells were seeded in 6-well tissue culture plates at a cell density of 3 × 10⁵ cells/well, cultured, and preincubated as in the uptake experiments. The cells were thereafter incubated in a solution containing 100 μM IS in the absence or presence of 1 mM probenecid or 1 mM cilastatin at 37°C for 5, 15, and 30 min. After washing the cells with IS-free PBS, the intracellular concentrations of IS were determined using HPLC (40).

Cytotoxicity Assays

Cell viability was assessed using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) assay as described previously (38). S2 cells were incubated in a solution in the absence or presence of IS at 33°C for 24 or 48 h. In addition, to elucidate the protective effect of probenecid on IS-induced cytotoxicity, the cells were incubated in a solution containing IS in the absence or presence of 1 mM probenecid. After the incubation, 1 ml of 0.5% MTT was added to the media. The cells were then further incubated at 33°C for 4 h. After the cells were lysed with isopropanol/HCl solution, the optical density was measured at 570 nm using that at 630 nm as a reference (Beckman, Du-640).

Statistical Analyses

Results are expressed as the means ± SEM. To compare values among the three groups, ANOVA and Fisher’s least significance difference (LSD) test were performed. Results were considered statistically significant when the P value was less than 0.05.

Biochemical Parameters in Vivo

As shown in Table 1, significant increases in systolic BP, serum creatinine, BUN, serum IS, and urinary protein excretion and significant decrease in creatinine clearance level were observed in 5/6-NPX rats compared with sham rats. In addition, 5/6-NPX rats administered with IS showed further increases in serum creatinine, BUN, serum IS, urinary IS excretion, and urinary protein excretion, with a decrease in creatinine clearance being much more predominant than in 5/6-NPX rats. Renal function deteriorated time-dependently until the 16th wk; remnant kidneys from 5/6-NPX rats with or without administration of IS were therefore used for immunohistologic analyses at week 16.

Localization of OAT1, OAT3, and IS in Renal Cortex by Immunohistochemistry

Immunohistochemical analyses were performed to determine the localization of rOAT1, rOAT3, and IS in various sections of the renal cortex. In the control, rOAT1 is localized in the basolateral membrane of the S₂ segment of the proximal tubule but not in the S₁ segment (Figure 2A), whereas rOAT3 is localized in the basolateral membrane of both S₁ and S₂ segments, the thick ascending limb of Henle (TAL), and the distal convoluted tubule (DCT) (Figure 2D) as well as the S₃ segment (data not shown). The 5/6-NPX rats administered with IS showed increased intensity of staining for rOAT3 in the renal cortex as compared with control, and rOAT1 was found to be slightly induced in the S₁ segment. IS was weakly stained in the proximal tubule but not in the glomeruli of control rats. In 5/6-NPX rats, IS was strongly localized in the S₂ segments of proximal tubule and glomerular epithelial cells and weakly localized in the S₁ segment, TAL, and DCT. The 5/6-NPX rats administered with IS showed a marked increase in IS immunoreactivity not only in the S₁ and S₂ segments of the proximal tubule but also in the glomerular epithelial cells, DCT, and TAL with macula densa. These data confirm the localization of IS and demonstrate that IS administration markedly enhances the tubular uptake of IS in the remnant kidney. In DCT and TAL with macula densa, IS staining was found to be colocalized with the staining of rOAT3 but not of rOAT1.

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Figure 2. Immunostaining of rOAT1, rOAT3, and IS in the renal cortex of control rats (Sham; A, D, and G), uremic rats (5/6-NPX; B, E, and H), and uremic rats to which IS was administered (5/6-NPX+IS; C, F, and I). Serial sections from each rat kidney were stained using a polyclonal anti-rOAT1 antibody (A, B, C), a polyclonal anti-rOAT3 antibody (D, E, F), and a monoclonal anti-IS antibody (G, H, I). These figures are representatives of typical samples from seven rats. The S₁ segment of the proximal tubule (stars) was connected to the glomerulus, followed by the S₂ segment. The thick ascending limb of the loop of Henle (TAL) is represented by triangles, the distal convoluted tubule (DCT) by circles, and the macula densa by arrows. Magnification x 150.

Effects of Various Concentrations of IS on rOAT1- and rOAT3-Mediated Organic Anion Uptake

As shown in Figure 3, S2 rOAT1 (A) and S2 rOAT3 (B) exhibited a time-dependent uptake of PAH and ES, respectively. In addition, S2 rOAT1 and S2 rOAT3 exhibited a dose-dependent increase in PAH and ES uptake, respectively (data not shown). Eadie-Hofstee analyses showed that the Michaelis constant (Km) for PAH uptake was 17.6 ± 5.6 μM in S2 rOAT1; for ES uptake, it was 4.30 ± 1.20 μM in S2 rOAT3, values which are similar to those in the Xenopus laevis oocyte expression system (18,21). As shown in Figure 3, IS inhibited the rOAT1-mediated (C) and rOAT3-mediated (D) organic anion uptake in a dose-dependent manner (n = 4; *P < 0.01 versus control). In contrast, IS did not significantly inhibit PAH uptake and/or ES uptake in S2 pcDNA 3.1 (n = 4; NS). As shown in Figure 4, kinetic analyses of the inhibitory effects were performed using Lineweaver-Burk plot (A and C) as well as Eadie-Hosfstee plot (B and D). The results of these kinetic analyses revealed that the mode of inhibition was competitive. The inhibition constant (Ki) values for rOAT1 and rOAT3 were determined to be 34.2 μM (n = 4) and 74.4 μM (n = 4), respectively.

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Figure 3. Effects of various concentrations of IS on rOAT1- and rOAT3-mediated organic anion uptake. (A and B) Time course of organic anion uptake by rOAT1 and rOAT3. S2 rOAT1 (A) and S2 rOAT3 (B) and S2 pcDNA3.1 were incubated in a solution containing 5 μM [¹⁴C]PAH and 50 nM [³H]ES at 37°C during the incubation time up to 15 min. (C and D) Inhibition experiments. S2 rOAT1 (C), S2 rOAT3 (D), and S2 pcDNA3.1 were incubated in a solution containing 5 μM [¹⁴C]PAH and 50 nM [³H]ES at 37°C for 2 min in the absence or presence of various concentrations of IS. The values are expressed as a percentage of organic anion uptake in S2 rOAT1, S2 rOAT3, or S2 pcDNA3.1 in the absence of IS. Each value represents the mean ± SEM of four determinations from one typical experiment. *P < 0.01 versus control.

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Figure 4. Kinetic analysis of the inhibitory effects of IS on rOAT1- and rOAT3-mediated organic anion uptake. S2 rOAT1 (A and B) and S2 rOAT3 (C and D) were incubated at 37°C for 2 min in a solution containing various concentrations of [¹⁴C]PAH and [³H]ES in the absence (•) or presence (▪, 250 μM;□), 1000 μM) of IS. Lineweaver-Burke plot analysis (A and C) and Eadie-Hofstee plot analysis (B and D) were performed. Each value represents the mean ± SEM of four determinations from one typical experiment.

HPLC Determination of IS Content in S2 rOAT1 and S2 rOAT3

To determine whether rOAT1 and rOAT3 mediate the uptake of IS, we measured the intracellular content of IS using HPLC. As shown in Figure 5, S2 rOAT1 (A) and S2 rOAT3 (B) showed a significantly higher intracellular IS content compared with that in S2 pcDNA 3.1 (n = 4; *P < 0.01 versus S2 pcDNA 3.1). In addition, we have examined the effects of organic anion transport inhibitors on IS uptake mediated by rOAT1 and rOAT3. Organic anion transport inhibitors used were probenecid and cilastatin (41). Probenecid and cilastatin dose-dependently inhibited the organic anion uptake by S2 rOAT1 and S2 rOAT3 at concentrations up to 2 mM (data not shown). As shown in Figures 5A and 5B, probenecid and cilastatin at 1 mM significantly inhibited IS uptake mediated by rOAT1 and rOAT3 (n = 4; #P < 0.05 versus S2 rOAT1 or S2 rOAT3; ##P < 0.01 versus S2 rOAT1 or S2 rOAT3). Both probenecid and cilastatin exhibited no significant inhibitory effects on IS uptake by S2 pcDNA 3.1 (data not shown).

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Figure 5. Intracellular IS content in S2 cells determined by HPLC. Confluent S2 rOAT1 (A), S2 rOAT3 (B), and S2 pcDNA 3.1 in six-well plates were incubated in a solution containing 100 μM IS in the absence or presence of 1 mM probenecid or 1 mM cilastatin at 37°C for 2, 5, 15, and 30 min. After washing the cells with a solution, the intracellular IS contents were determined using HPLC. Each value represents the mean ± SEM of three determinations from one typical experiment. *P < 0.01 versus S2 pcDNA3.1; #P < 0.05 versus S2 rOAT1 or S2 rOAT3; ##P < 0.01 versus S2 rOAT1 or S2 rOAT3.

Effects of IS on the Viability of S2 rOAT1 and S2 rOAT3 in the Absence or Presence of Probenecid

We determined whether accumulated IS within the cells induced toxicity in S2 rOAT1 and S2 rOAT3. For this purpose, we examined the effects of various concentrations of IS on the viability of S2 rOAT1, S2 rOAT3, and S2 pcDNA 3.1 using the MTT colorimetric assay. As shown in Figure 6, IS after 24-h culture significantly decreased the viability of S2 rOAT1 (A), but not that of S2 rOAT3 (B), compared with that of S2 pcDNA 3.1 (n = 4; *P < 0.05 and **P < 0.01 versus S2 pcDNA3.1, respectively). In contrast, the viability after 48 hr culture was significantly decreased in both S2 rOAT1 and S2 rOAT3 compared with that of S2 pcDNA3.1 (Figure 6, A and B; n = 4; **P < 0.01 versus S2 pcDNA3.1). To confirm the role of rOAT1 and rOAT3 in the IS-induced nephrotoxicity, we evaluated the effects of probenecid, an organic anion transport inhibitor (41), on the IS-induced decrease in the viability. As shown in Figure 6, 1 mM probenecid significantly reversed the decrease in the viability of S2 rOAT1 (A) and S2 rOAT3 (B) treated with IS (n = 4; #P < 0.05). Addition of 1 mM probenecid alone did not show any significant effects on the cell viability (data not shown).

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Figure 6. Effects of IS on the viability of S2 rOAT1, S2 rOAT3, and S2 pcDNA3.1 in the absence or presence of probenecid. S2 rOAT1 (A), S2 rOAT3 (B), and S2 pcDNA3.1 in 24-well plates were incubated in a solution containing 10, 25, or 50 μM IS in the absence or presence of 1 mM probenecid for 24 or 48 hr. Each value represents the mean ± SEM of four determinations from one typical experiment. *P < 0.05 and **P < 0.01 versus S2 pcDNA3.1; #P < 0.01 versus S2 rOAT1 or rOAT3.