Introduction

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OTA is a mycotoxin produced as a secondary metabolite in different species of Aspergillus and Penicillium (Harwig et al., 1983). These species often occur in improperly stored food and animal chow (Delacruz and Bach, 1990; Bauer and Gareis, 1987). OTA is nephrotoxic, carcinogenic (Bauer and Gareis, 1987; Krogh et al., 1974) and has been unmasked as the agents responsible for the Danish porcine nephropathy (Delacruz and Bach, 1990; Krogh et al., 1974). Furthermore, there is epidemic evidence for an important role of OTA in the development of the BEN (Radic et al., 1997; Petkova-Bocharova and Castegnaro, 1991; Kuiper-Goodman and Scott, 1989) and for the increased incidence of urinary tract tumors in areas with high occurrence of OTA (Nikolov et al., 1996). Recent studies in Tunisia indicate that OTA may also play a role in the development of CIN (Maaroufi et al., 1995), a disease similar to BEN (Berndt et al., 1980). Because it is becoming more and more evident that the complete avoidance of OTA exposure to humans is impossible, it is necessary to improve our knowledge of the toxodynamics and toxicokinetics of OTA. Millimolar concentrations of OTA were found to be acutely toxic, leading to inhibition of mRNA synthesis (Meisner and Polsinelli, 1986), inhibition of phenylalanyl-tRNA-synthetase (Creppy et al., 1983; Roth et al., 1989), protein synthesis (Baudrimont et al., 1997) and DNA-damage (Kane et al., 1986). OTA concentrations detected in context with the above mentioned diseases, however, were in the nano- to micromolar range (Kuiper-Goodman and Scott, 1989). Chronic treatment of rats with naturally occurring doses of OTA led to a reduced renal clearance and renal secretion of PAH (Gekle and Silbernagl, 1993; Gekle and Silbernagl, 1994). These data point to an inhibitory effect of OTA on the activity of the renal organic anion transporter in the nano- to micromolar range, because PAH is the classical substrate for this transporter.

The organic anion transport system of the proximal tubule of the kidney plays a crucial role in the excretion of a variety of potentially toxic compounds, such as endogenous metabolites or xenobiotics (Friis, 1991). This is also true for OTA, because it is also a substrate of the organic anion transport system (Gekle et al., 1993; Delacruz and Bach, 1990). The organic anion transport system consists of the basolaterally located organic anion exchanger and a less well characterized transport step across the apical membrane (Dantzler, 1996). The basolateral organic anion exchanger is a tertiary active transport system dependent on the maintenance of the inward-directed Na⁺-gradient to drive uptake of -KG. Subsequently, -KG is exchanged for PAH, or any other competitive organic anion, driving their uptake across the basolateral membrane (Pritchard and Miller, 1992; Dantzler, 1996; Ullrich and Rumrich, 1988; Friis, 1991). Apical secretion of PAH is along its electrochemical gradient and is carrier-mediated (Friis, 1991). The above mentioned transport steps are of major importance for the elimination of xenobiotics or metabolites that are organic anions (Friis, 1991).

To investigate the effect of OTA on the organic anion transporter in more detail and without possible systemic interactions [e.g., renal blood flow (Gekle and Silbernagl, 1993)] we used a proximal tubule-derived cell line (OK cells). The OK cell line is well characterized and an accepted model system to investigate organic anion secretion (Nagai et al., 1995; Hori et al., 1993).

Our data show that incubation of OK cells with nano- to micromolar concentrations of OTA for 1 to 3 days leads to a dose-dependent decrease of basolateral uptake and transepithelial secretion of PAH. Cell viability and other energy-depending transport systems were not or only to a very minor extent affected in this concentration range. Thus, OTA can inhibit its own elimination and the elimination of other xenobiotics in a specific way and by a mechanism that is not disturbing cellular function in general.

	Materials and Methods

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Cell culture. OK cells were obtained from Dr. Biber, Department of Physiology, University of Zurich. Cells were maintained in culture at 37°C in a humidified 5% CO₂, 95% air atmosphere. The growth medium was minimal essential medium, pH 7.4, supplemented with Earl's salts, nonessential amino acids, 10% (v/v) fetal calf serum (Biochrom KG, 12213 Berlin, FRG) and 26 mmol/liter NaHCO₃. Cells were cultured on permeable supports (3-µm pore diameter, Falcon, Becton Dickinson Labware, Franklin Lakes, NJ) for transport measurements. The effective growth area on one permeable support was 4.3 cm²/filter. All studies were performed between passage 60 and 100. The seeding density was 0.4 · 10⁶ cm². The medium was changed every third day and the monolayers were used for experiments 4 to 7 days after reaching confluence.

Transport measurements. The volumes of the apical and basolateral compartment were 1.3 ml and 2.5 ml to avoid hydrostatic pressure differences. Before each experiment, the cells were washed three times with PBS (138 mmol/liter NaCl, 1 mmol/liter NaH₂PO₄, 4 mmol/liter Na₂HPO₄, 4 mmol/liter KCl, 1 mmol/liter MgCl₂, 1 mmol/liter CaCl₂, 5 mmol/liter glucose, pH 7.4). Unless otherwise stated, transport measurements were performed in PBS at pH 7.4 and 37°C for 30 min. The concentrations of the radio-labeled substrates were as follows: [¹⁴C]PAH: 1.5 · 10⁶ mol/liter, [¹⁴C]TEA: 7.5 · 10⁶ mol/liter, [¹⁴C]glutarate: 1.5 · 10⁶ mol/liter, [¹⁴C]cycloleucine: 1.5 · 10⁶ mol/liter, [³H]-OTA: 10⁶ mol/liter. The corresponding concentrations of unlabeled substrates, if used, are mentioned in the text. Correction of paracellular fluxes and measurement of extracellular water space was performed with [³H]mannitol (55 · 10⁹ mol/liter) or [¹⁴C]mannitol (6.2 · 10⁹ mol/liter). At the end of the experiment, the apical and basolateral solutions were collected. Subsequently, the cells were washed twice with ice-cold PBS. Radioactivity of the solutions and the cells was measured using a liquid scintillation counter (Packard Instruments, Frankfurt, Germany).

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Specific binding. To investigate binding of [¹⁴C]PAH to the plasma membrane, cells were incubated with 1.5 · 10⁶ mol/liter [¹⁴C]PAH at 4°C for 60 min. Cells were washed twice with ice-cold PBS and apical and basolateral solutions and cellular compartment were collected separately afterward. Specific binding of [¹⁴C]PAH was calculated as the difference between binding of 1.5 · 10⁶ mol/liter [¹⁴C]PAH (alone or in presence of a certain amount unlabeled PAH) and binding of 1.5 · 10⁶ mol/liter [¹⁴C]PAH in presence of 15 mmol/liter PAH. Kinetic data were calculated as described for transport kinetics.

Amino acid. Protein biosynthesis was determined by the incorporation of [¹⁴C]L-phenylalanine according to Ling et al. (1996). Three days after reaching confluence, cells were incubated with MEM containing 10⁶ mol/liter OTA for 72 hr. 0.15 mBq [¹⁴C]L-phenylalanine was present during the last 12 hr.

Other experimental methods. For determination of K⁺-content (Montrose and Murer, 1996), cells were seeded in 6-well culture dishes. The seeding density and performance of incubation was as mentioned before. The epithelia were washed twice with ice-cold washing buffer (130 · 10³ mol/liter TEA-Cl; 10³ mol/liter MgCl₂; 10³ mol/liter EGTA; 10² mol/liter HEPES; 20 · 10³ mol/liter BaCl₂; pH 7.4). After detaching the cells with perchloric acid (10%), the probes were centrifuged. The supernatant was investigated using flame photometry (Flammenphotometer Eppendorf, Hamburg, Germany). The protein concentrations were determined using the method of Lowry et al. (1951). LDH release was measured according to Roth et al. (1989) at room temperature in a photometer.

Analysis of kinetic data. Kinetic data (affinity, transport maximum) were obtained by fitting the values to the Michaelis-Menten equation according to the least-square-method, using Sigma Plot Software (Jandel Corte Madera, CA). IC₅₀ values were determined according to Delean et al. (1978) by the equation: where V is the transport rate at any given [I], [I] is the concentration of the inhibitor (OTA), V₀ is the transport rate at [I] = 0, and K is the transport rate at maximum inhibition.

Data analysis. Data are presented as mean ± S.E.M., except for V_max, K_m and IC₅₀ which are presented as ±S.D. n is given in the text or in the figures. n is the number of culture plates or filters used to perform the measurements. All data shown contain values from at least two different passages of cells. Statistical significance was determined by unpaired Student's t test. Results were considered statistically different at P < .05. The data were fitted using the least-square-method (Sigma Plot, Jandel Corte Madera, CA).

Materials. [¹⁴C]PAH (55 mCi/mmol), [¹⁴C]TEA (5 mCi/mmol), [¹⁴C]glutarate (55 mCi/mmol), [¹⁴C]cycloleucine (55 mCi/mmol), [¹⁴C]L-phenylalanine (475 mCi/mmol), [¹⁴C]mannitol (56 mCi/mmol), [³H]mannitol (15 mCi/mmol) and [³H]thymidine (6.7 mCi/mmol) were purchased from American Radiolabeled Chemicals Inc., St. Louis, MO. [³H]OTA (6 mCi/mmol) from Moravek Biochemicals Inc. Brea, CA and [¹⁴C]mannitol (51.5 mCi/mmol) from Du Pont De Nemours (Germany) GmbH. If not stated otherwise, all other chemicals were from Sigma.

	Results

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Kinetics of the organic anion transport system. First we determined the kinetic parameters of specific organic anion transport in OK cell epithelia under control conditions. Calculated data were V_max = 578 ± 20 (pmol · cm² · 30 min¹), K_m = 248 ± 21 (µmol/liter) for basolateral uptake (fig. 1A) and V_max = 490 ± 82 (pmol · cm² · 30 min¹), K_m = 36 ± 17 (µmol/liter) for transepithelial secretion (fig. 1B). These data are in good agreement with data published previously (Hori et al., 1993).

View larger version (16K): [in this window] [in a new window]   Fig. 1.   Basolateral uptake (A) and transepithelial secretion (B) of PAH from the basolateral side of OK cell epithelia. Uptake of 1.5 · 106 mol/liter [14C]PAH was determined alone and in presence of 37.7, 75, 150, 375, 750 µmol/liter unlabeled PAH in the basolateral bath. Secretion of 1.5 · 106 mol/liter [14C]PAH was determined alone and in presence of 15, 37.5, 75, 150 µmol/liter unlabeled PAH. Vmax and Km were calculated according to the isotope dilution technique mentioned in "Materials and Methods." Data points for Michaelis-Menten plot were obtained by multiplication of measured V with the amount of total PAH present. Amount of secretion or uptake is plotted against the sum of labeled and unlabeled PAH present. For all data points n is given in parentheses besides the symbol.

Effect of acute OTA exposure. As 99% or more of OTA binds to plasma albumin (Hagelberg et al., 1989; Delacruz and Bach, 1990), we investigated the effect of acute OTA exposure in the absence and presence of BSA in the bath. We have chosen a concentration of 4 g/liter BSA, according to the amount of BSA in the growth medium used in cell culture. Up to 10⁶ mol/liter OTA were added simultaneously with 1.5 · 10⁶ mol/l [¹⁴C]PAH to the bath on the basolateral side of OK cell epithelia in the presence or absence of 4 g/liter BSA. As shown in figure 2, 10⁶ and 10⁵ mol/liter OTA had no acute effect on transepithelial secretion of [¹⁴C]PAH in the presence of BSA, whereas in the absence of BSA, secretion was inhibited by 20 and 80%, respectively. A total of 10⁷ mol/liter OTA exerted no acute effect. Uptake of PAH was affected in the same way as secretion. To test whether this acute effect is due to specific competitive action of free OTA on organic anion transport, we investigated the acute effect of OTA on TEA transport. As shown in figure 3B, up to 10⁵ mol/liter free OTA did not alter basolateral uptake of organic cations in OK cells. Transepithelial secretion of TEA was also not affected (fig. 3A).

View larger version (34K): [in this window] [in a new window]   Fig. 2.   Acute effect of 107, 106 and 105 mol/liter OTA on the transepithelial secretion of 1.5 · 106 mol/liter [14C]PAH through OK cell epithelia in the presence or absence of 4 g/liter BSA. Transepithelial secretion is expressed in percent of control. OTA and/or BSA were given simultaneously with [14C]PAH into the basolateral compartment and secretion was measured for 30 min. n is 3 for all data points.

View larger version (34K): [in this window] [in a new window]   Fig. 3.   Acute effect of 106 and 105 mol/liter free OTA on [14C]TEA transport in OK cells. OTA was given simultaneously with 7.5 · 106 mol/liter [14C]TEA in the basolateral compartment and uptake and secretion was measured for 30 min. n is given in parentheses within the bars.

Intracellular OTA concentration during acute exposure. To estimate the amount of OTA present in the cells during 30 min exposure, uptake of [³H]OTA (10⁶ mol/liter) was determined. The resulting intracellular concentration of [³H]OTA was 1.1 · 10⁶ ± 10⁷ mol/liter (n = 6).

Time course of chronic OTA exposure. As OTA had no effect when applied acutely, we incubated the cells with 10⁶ mol/liter OTA for different time periods. As seen in figure 4, basolateral PAH uptake was already decreased significantly after 24 hr. Maximum inhibition was reached after 48 hr. All subsequent experiments were performed after 72 hr incubation.

View larger version (26K): [in this window] [in a new window]   Fig. 4.   Time course of the effect of incubation with 106 mol/liter OTA on the basolateral uptake of 1.5 · 106 mol/liter [14C]PAH into OK cells. Uptake was measured after 30 min. Uptake is expressed in percent of control, whereby control is uptake in untreated cells. Incubation times were 24, 48 and 72 hr. n is given in parentheses within the bars.

Kinetics and extent of inhibition. Figure 5 shows uptake (fig. 5A) and secretion (fig. 5B) of [¹⁴C]PAH after 72 hr incubation of OK cells with OTA. Determined IC₅₀ values were 6 · 10⁷ ± 2 · 10⁷ mol/liter for transepithelial secretion and 6 · 10⁸ ± 5 · 10⁸ mol/liter for basolateral uptake of PAH. Maximum inhibition was almost 100% for transepithelial secretion and 60% for basolateral uptake. Because 10⁶ mol/liter OTA leads to almost maximum inhibition with virtually no reduction of cell viability (see below), this concentration was used in all subsequent experiments. A total of 10⁵ mol/liter OTA was the highest concentration tested. There, epithelia started to show a massive decrease in tightness, as determined by mannitol fluxes (see below).

View larger version (14K): [in this window] [in a new window]   Fig. 5.   Effect of a 72 hr incubation with different concentrations of OTA on the basolateral uptake (A) and transepithelial secretion (B) of 1.5 · 106 mol/liter [14C]PAH in OK cells. OTA concentrations went from 108 to 105 mol/liter. IC50 values were calculated according to the equation mentioned in the "Materials and Methods" and are given in the text. For all data points n is given in parentheses beside the symbol.

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View this table: [in this window] [in a new window]   TABLE 1 Kinetic parameters of PAH-transport in control OK cells and in OK cells incubated with 106 mol/liter OTA for 72 hr

Specific basolateral binding of PAH. We also investigated specific binding of [¹⁴C]PAH to the basolateral membrane of OK cells. As shown in table 1, 72 hr preincubation with OTA (10⁶ mol/liter) induced a 50% decrease in affinity of specific basolateral binding of PAH. Similar to V_max for basolateral uptake, the maximal basolateral binding was not changed after OTA incubation. Thus, although kinetic data for basolateral uptake were gained in the saturation phase, we conclude that this effect is the same in the initial phase. Secretion of PAH was still linear after 30 min.

OTA concentrations after 72 hr incubation. To determine the amount of OTA remaining within the cells after 72 hr incubation followed by the standard washing procedures, we incubated OK cells with 10⁶ mol/liter [³H]OTA for 72 hr. After the standard washing and equilibrating procedures, the amount of OTA left in the intra- and extracellular compartment was determined. Intracellular OTA concentration was 1.8 · 10⁷ ± 0.2 · 10⁷ mol/liter and the extracellular concentration was 17.4 · 10¹¹ ± 1.4 · 10¹¹ mol/liter (n = 6 for both). No metabolites of OTA were detectable after 72 hr, as determined by high-performance liquid chromatography analysis (data not shown).

Specificity of the OTA effect. To gain information about the specificity of the effect of the 72 hr OTA incubation, we determined the activity of three other transport systems expressed in proximal tubular cells: the apically located transporter for neutral amino acids (Schwegler et al., 1989), the basolaterally located organic cation transporter (Pritchard and Miller, 1992) and the Na⁺/dicarboxylate cotransporter (see below). As shown in figure 6 neither basolateral uptake of glutarate, nor apical uptake of cycloleucine was affected by incubation with 10⁶ mol/liter OTA for 72 hr. Intracellular cycloleucine concentration exceeded the extracellular concentration 4-fold, indicating that cycloleucine accumulates. Accumulation is an active transport process requiring metabolic energy. As intracellular accumulation of cycloleucine did not change after treatment with 10⁶ mol/liter OTA for 72 hr, a general disturbance of the metabolic state of the cells seems to be unlikely. To our surprise, TEA-uptake was reduced to 36 ± 9% of control (n = 7) after 72 hr OTA incubation, whereas transepithelial secretion of TEA was not affected (fig. 7). At the moment we can only speculate about possible reasons for this discrepancy (see "Discussion").

View larger version (33K): [in this window] [in a new window]   Fig. 6.   Effect of 72 hr incubation with 106 mol/liter OTA on the apical uptake of [14C]cycloleucine (A) and basolateral uptake of [14C]glutarate (B) in OK cells. Data are presented as percent of control. Used concentrations were 1.5 · 106 mol/liter [14C]cycloleucine, respectively 1.5 · 106 mol/liter [14C]glutarate. Uptake was determined for 30 min (cycloleucine) respectively 10 min (glutarate). n is given in parentheses within the bars.

View larger version (25K): [in this window] [in a new window]   Fig. 7.   Effect of 72 hr incubation with 106 mol/liter OTA on [14C]TEA transport in OK cells. Basolateral uptake (B) and secretion (A) were determined according to "Materials and Methods." n is given in parentheses within the bars.

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View larger version (25K): [in this window] [in a new window]   Fig. 8.   Effect of preloading OK cells for 10 min with 5 · 103 mol/liter glutarate on basolateral uptake of 1.5 · 106 mol/liter [14C]PAH. Uptake was measured for 5 min in control cells and in OK cells incubated with 106 mol/liter OTA for 72 hr. Preloading of the cells and the transport measurements were performed as mentioned in "Materials and Methods." n is 6 for every bar.

Cell viability. Furthermore, we investigated cell viability and epithelial tightness by determination of protein content, LDH release and mannitol flux. As shown in table 2, incubation with 10⁶ mol/liter OTA for 3 days did not reduce protein content per filter or LDH release. Paracellular permeability, as measured by mannitol fluxes, was only slightly increased by incubation with 10⁶ mol/liter OTA. Cellular K⁺-content was slightly increased. Incorporation of [¹⁴C]L-phenylalanine was not affected by OTA treatment. Incubation with 10⁵ mol/liter OTA for 72 hr led to a massive reduction of protein content and epithelial tightness. These data indicate that treatment with 10⁶ mol/liter OTA for 72 hr does not, or only to a very minor extent, impair general viability and epithelial integrity of OK cells.

Efflux of PAH. Finally we determined whether treatment of OK cells with 10⁶ mol/liter OTA for 72 hr affects [¹⁴C]PAH efflux. As shown in figure 9, OTA treatment led to a reduction of PAH efflux into the apical compartment. Furthermore a greater fraction of PAH remained within the OK cells and was extruded into the basolateral compartment. Thus, as a functional consequence of OTA incubation, transport mechanisms at the basolateral and at the apical side seem to be altered in proximal-tubular cells.

View larger version (29K): [in this window] [in a new window]   Fig. 9.   Effect of 72 hr incubation of OK cells with 106 mol/liter OTA on the efflux of [14C]PAH. Preloading and the efflux experiment were performed as mentioned in "Materials and Methods." Efflux was measured for 10 min at 37°C. After that the content of [14C]PAH in the apical and the basolateral bath, as well as in the cells itself, was measured. The content of [14C]PAH in the OK cell epithelia at the beginning of the efflux was set as 100%. For control values 100% is 2.24 ± 0.23 pmol · cm2 · 30 min1, for cells incubated with OTA 100% is 1.67 ± 0.08 pmol · cm2 · 30 min1. n is 9 for every bar.

	Discussion

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Many of the effects of OTA described previously, were studied in a concentration range which exceeds the naturally occurring one (Kuiper-Goodman and Scott, 1989). The inhibition of phenylalanyl-tRNA-synthetase is reported to occur at concentrations above 10⁴ mol/liter (Baudrimont et al., 1997). DNA-damage is induced by 10⁴ mol OTA per kg body weight (Kane et al., 1986). Natural occurring concentrations of OTA are in the range of 10¹³ to 10⁸ mol/liter in human plasma in Germany (Deutsche Forschungsgemeinschaft, 1990) or up to 10⁵ mol/liter in plasma samples from people in Bulgaria and Yugoslavia (Delacruz and Bach, 1990), countries with areas of high incidence of BEN. The free OTA concentration is about one-hundred times lower as compared to total OTA concentration. It has been shown that the incidence of urinary tract tumors is seven to nine times higher in BEN areas, than it is in nonendemic areas (Nikolov et al., 1996). Because OTA exposure seems to be unavoidable, it is important to investigate effects of OTA in the nano- to micromolar range, to gain more information about the nephrotoxic action during natural exposures. Recent studies (Bruinink et al., 1997) showing toxic effects of OTA in the nanomolar concentration range underline this importance. This is additionally underlined by a study that indicates no relevant inhibition of phenylalanyl-tRNA-synthetase by naturally occurring OTA concentrations in vivo (Roth et al., 1993).

The polyspecific organic anion carrier of the kidney is of crucial importance for the excretion of a variety of potentially toxic compounds such as e.g., metabolites, xenobiotics and drugs (Friis, 1991; Ullrich and Rumrich, 1988; Pritchard and Miller, 1992). OTA itself is a substrate for the organic anion transport system (Sokol et al., 1988; Gekle et al., 1993; Delacruz and Bach, 1990). In vivo studies in rats showed a reduced renal secretion and clearance of PAH after subchronic (6 days) OTA exposure (Gekle and Silbernagl, 1994), indicating that the renal organic anion transport system may be a target of OTA toxicity. Impairment of organic anion secretion by OTA affects kinetics of a variety of substances, thereby possibly leading to indirect toxic effects.

Basolateral uptake and secretion of PAH are clearly saturable in OK cells. The kinetic parameters for uptake and secretion are in the same range as described previously (Hori et al., 1993). Because total estimated cell volume per filter was 2.5 · 10⁶ liter, the intracellular concentration of PAH is about 2.5 · 10⁶ mol/liter. Thus, under our conditions, PAH was accumulated 1.7-fold within the cells, indicating (tertiary) active uptake.

In the absence of proteins, acute exposure to OTA reduced PAH transport. This inhibition was totally abolished by adding 4 g/liter BSA. We used this concentration of BSA, because it corresponds to the concentration in media containing 10% FCS, which are used under standard cell culture conditions as well as when the cells were incubated for 72 hr with OTA. The fact that BSA was able to avoid acute effects of OTA on PAH transport completely, is explained by binding of OTA to BSA (Stojkovic et al., 1984; Chu, 1971). Similar observations have been made for the action of OTA on growth of rat proximal tubular cells (Gekle et al., 1995). Our experiments show, that in presence of BSA, OTA in concentrations up to 10⁵ mol/liter did not acutely influence PAH transport. We conclude that the inhibitory effect of acute OTA application in the absence of BSA is just due to competitive inhibition as suggested by Sokol et al. (1988), because both, OTA and PAH, are substrates for the organic anion transporter (Gekle et al., 1993).

By contrast, the sensitivity of the organic anion transporter to long-term exposure (72 hr) was much higher. The half-maximal inhibitory concentration (IC₅₀) for basolateral uptake was 6 · 10⁸ mol/liter. During evaluation of the data obtained after OTA incubation it is important to keep in mind, that BSA was always present during the incubation period. Thus, the estimated concentrations of free OTA (Stojkovic et al., 1984; Chu, 1971) inducing half-maximal inhibition of PAH transport are ~10¹⁰ mol/liter. Thus, the sensitivity during long-term exposure is at least three orders of magnitude higher as compared to acute exposure in the presence of BSA. Furthermore, the effect of OTA was maximal only after 48 hr. These data are strong evidences that the mechanisms acting during long-term exposure are different from those acting during acute exposure (competitive inhibition). For competitive inhibition of PAH transport, the crucial parameter is the OTA concentration in the extracellular medium. At the beginning of the transport experiments, after standard OTA incubation, the extracellular concentration of OTA is about three orders of magnitude lower than 10⁷ mol/liter. OTA at 10⁷ mol/liter had no effect during acute exposure. Thus, OTA was no longer present in concentrations that can lead to competition with PAH when PAH transport was determined after long-term incubation.

Incubation of OK cells with 10⁶ mol/liter or 10⁵ mol/liter PAH for 3 days did not affect organic anion uptake (91 ± 11, respectively 93 ± 4, values in % of control; n = 3). This indicates that the described effect of OTA is not due to physiological down-regulation of organic anion transport when OK cells are exposed to any substrate of the organic anion carrier. However, the data do not exclude the possibility that PAH at higher concentrations leads to down regulation of the organic anion carrier. As the task of the organic anion transport system of the kidney is to excrete organic anions constitutively, a downregulation would make no physiological sense. Thus, the impairment of organic anion transport observed after long term incubation is a specific pathophysiological property of OTA.

As OTA affects the organic anion transport, it was of interest to see whether OTA also affects other transport systems in the concentration range tested. Because neither the apical uptake of the neutral amino acid cycloleucine nor the basolateral uptake of glutarate was impaired, OTA does not affect transport systems in general. The reduction of TEA uptake is in agreement with data published before (Berndt et al., 1980). Additionally recent data (Wolff et al., 1997) show some identity (33% respectively 32% in amino acid sequence) of the organic anion transporter (OAT1) and the organic cation transporter (OCT1 resp. OCT2). As organic cation transport is not acutely affected by OTA, one can speculate that the mechanism of the long-term action of OTA is related to these sequence identities. However, the organic cation transporter is specific enough to exclude a direct competition of TEA and OTA. This hypothesis will be tested in future investigations. Furthermore, the energy-dependent enrichment of cycloleucine still occurs to the same extent as compared to controls, indicating that OTA does not disturb cell metabolism in general. This hypothesis is also supported by the results from our glutarate-preload experiments. Glutarate is a nonmetabolizable analogue of -ketoglutarate and acts as a counterion for the organic anion transporter (Dantzler, 1996). PAH uptake was stimulated by glutarate in untreated OK-cells. If OTA incubation would lead to a decrease in metabolic activity of the cells and thereby reduce the availability of counterions, glutarate-preload should restore PAH uptake at least in part. Yet, our data show that glutarate-preload had no effect. As already mentioned above, uptake of glutarate was not changed after incubation with OTA. These data indicate that OTA incubation affects the PAH/dicarboxylate-exchanger directly and does not act via a change of driving forces. Otherwise, glutarate-preloading should have stimulated PAH uptake.

The K⁺-content of OK cells was not reduced by OTA, again reinforcing the conclusion that OTA incubation does not act on cellular energy homeostasis. As described above, primary metabolism of the cells seems not to be affected by incubation with 10⁶ mol/liter OTA for 72 hr. Furthermore, this treatment did not reduce the protein content per filter (table 2) or protein synthesis. In fact a slight increase in protein content was observed, which is in agreement with published data showing a hypertrophic effect of OTA (Gekle et al., 1995). OTA incubation did not reduce cell membrane integrity as shown by the lack of effect on LDH release. The slight increase of mannitol flux points at a small decrease in epithelial tightness since mannitol moves only via the paracellular space (Suki and Eknoyan, 1992). By contrast, 72 hr exposure to 10⁵ mol/liter OTA led to 25% decrease in protein content and to a 28% decrease in epithelial tightness. Hence, this concentration seems to mark the transition to unspecific toxic effects as it was observed in rat proximal tubular cells (Gekle et al., 1995).

Affinity of basolateral uptake decreased after OTA incubation (table 1), whereas V_max remained unchanged. This could be due to reduced affinity of the organic anion transporter protein for PAH. Another possible reason could be a reduced accessibility of the transporter for the substrate. Furthermore, the translocation step could be impaired or the dissociation of the substrate from the carrier could be hindered. To gain further information about the mechanism and the site of the OTA effect, we investigated specific binding of PAH to the basolateral membrane of OK cells. Affinity of basolateral binding was reduced to 50% after 72 hr of incubation with 10⁶ mol/liter OTA, whereas maximal binding was not altered. These data are in agreement with the kinetic data for basolateral uptake of PAH mentioned above. Thus, the specific binding sites (i.e., carrier proteins) at the basolateral membrane seem to be affected by OTA. OTA treatment could lead to a reduced synthesis of binding sites. Furthermore, missorting of the protein, in a sense that it is not only directed to the basolateral membrane but also to the apical membrane, could also reduce the number of basolateral binding sites. Because maximal binding and overall protein synthesis were not reduced, we think that these possibilities are highly unlikely. Possible mechanisms leading to reduced affinity include changes in the confirmation of the carrier or the microenvironment surrounding the carrier. In addition OTA could act on the regulation of the organic anion transporter, e.g., by PKC (Takano et al., 1996; Nagai et al., 1997). Yet, as PKC is a general regulatory protein, it seems unlikely that the specific OTA effects described here, are mediated by PKC alone. Future studies will have to unveil a possible involvement of PKC in OTA-induced transport inhibition. Although the precise mechanism of action still has to be unveiled, our data clearly show that OTA affects directly organic anion/dicarboxylate exchange via a decrease of substrate affinity.

OTA incubation reduces organic anion efflux across the apical membrane, and increases the efflux across the basolateral membrane (fig. 9). Thus, OTA seems to also act on transport processes at the apical membrane. This dual effect of OTA could explain the fact that the kinetic parameters of uptake and secretion are altered in a different way by OTA. Additionally, the secretion data are in good agreement with the reduction of organic anion secretion in vivo (Gekle and Silbernagl, 1994).

The renal organic anion transport system is responsible for the blood-to-lumen transport of OTA (Gekle et al., 1993; Delacruz and Bach, 1990). Thus, this mycotoxin delays its own elimination by reducing the activity of this transport system. Our data show that it is the exchange of dicarboxylates and organic anions that is affected by nanomolar concentrations of OTA and not dicarboxylate uptake or the sodium pump. Organic cation uptake, which is mediated by a transporter with similarities in sequence to the organic anion transporter, is also impaired by OTA incubation. Additionally, OTA alters transport of organic anions at the apical membrane. The reduced secretion may also contribute to the reported elimination half-lives of OTA which are in the range of several hundred hours in mammals (Hagelberg et al., 1989). Thus, OTA has a self-enhancing effect. Furthermore, OTA delays the elimination of other substances and thereby may cause indirect toxic action.