Introduction

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The nonpeptide angiotensin II receptor antagonists represent a new class of drugs with therapeutic use in hypertension and possibly other cardiovascular disorders (MacFayden and Reid, 1994). Losartan was the first of this group of compounds to be approved for the management of hypertension (Johnston, 1995). As a class, the angiotensin II receptor antagonists are highly protein bound and are anions at physiological pH (Csajka et al., 1997). The metabolism and elimination of the angiotensin II receptor antagonists are varied. For example, some angiotensin II receptor antagonists are extensively metabolized in the liver, generating active and/or inactive metabolites, which are eliminated in the bile or by the kidney (e.g., losartan and irbesartan) (Csajka et al., 1997), whereas others undergo little metabolism and are excreted in the bile and urine largely as unchanged drug (e.g., eprosartan) (Cox et al., 1996). In most cases, however, some proportion of the administered dose appears unchanged in the urine (Csajka et al., 1997), suggesting that these molecules may be secreted by the renal organic anion transporter. Evidence that these molecules may interact with renal organic anion transport comes from studies showing that losartan increased uric acid excretion and lowered plasma levels of uric acid in both healthy subjects (Nakashima et al., 1992) and hypertensive patients (Tsunoda et al., 1993). Subsequent studies demonstrated that losartan and, to a much lesser extent, EXP3174, the active metabolite of losartan, and eprosartan inhibited urate/anion exchange in proximal tubule brush border membrane vesicles from rat (Edwards et al., 1996) and human (Roch-Ramel et al., 1997) kidney.

Although the above studies showed that losartan could interact with organic anion reabsorption, little direct evidence exists as to whether losartan and other angiotensin II receptor antagonists can interact with organic anion secretion and whether this class of compounds is secreted by the renal organic anion transporter. Therefore, the purposes of this study were to examine the interaction of angiotensin II receptor antagonists, principally losartan, with renal organic anion secretion and to characterize the transport of [³H]losartan across the proximal tubule.

	Materials and Methods

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Preparation and Perfusion of Isolated Tubules. Isolated tubules were perfused in vitro according to previously published methods (Edwards and Grantham, 1983). Male New Zealand White rabbits (2.5-3 kg) were anesthetized with i.v. pentobarbital, and a kidney was removed. Thin slices were cut and stored in chilled dissection solution consisting of 14 mM KCl, 44 mM K₂HPO₄, 14 mM KH₂PO₄, 8 mM NaHCO₃, 160 mM sucrose, and 0.1% BSA, pH 7.4. Proximal tubule S2 segments were dissected, transferred to a temperature-controlled chamber, and mounted onto micropipettes. In most experiments, the tubules were bathed and perfused with Dulbecco's modified Eagle's medium (DMEM), supplemented with 25 mM NaHCO₃ and equilibrated to pH 7.4 with 95% O₂/5% CO₂. In some experiments, the bath and perfusion solutions consisted of a HEPES-buffered solution that contained 116 mM NaCl, 25 mM HEPES, 5 mM KCl, 2 mM NaH₂PO₄, 1 mM MgSO₄, 1.8 mM CaCl₂, 10 mM sodium acetate, 8.3 mM glucose, and 5 mM alanine, pH 7.4.

(1)

(2)

Chemicals. [³H]PAH (specific activity, 1.28 Ci/mmol) and [³H]losartan (DuP 753; specific activity, 43.9 Ci/mmol) were obtained from New England Nuclear (Boston, MA). Losartan, eprosartan, valsartan, and irbesartan were provided by the Department of Medicinal and Synthetic Chemistry (SmithKline Beecham Pharmaceuticals, King of Prussia, PA). All other reagents were from Sigma Chemical Co. (St. Louis, MO).

	Results

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Initial experiments were conducted to determine whether losartan could cis-inhibit PAH transport as would be expected if an organic anion competed for the same transporter as PAH. As shown in Fig. 1, losartan rapidly and reversibly inhibited PAH secretion by isolated perfused proximal tubules. In the presence of 10 µM [³H]PAH, J_PAH during the control period was 382 ± 4.9 fmol/min/mm. The addition of 10 µM losartan to the bath significantly (p = .0003) inhibited PAH secretion by approximately 40% to 232 ± 3.5 fmol/min/mm. On removal of losartan, PAH secretion returned to control values: 359 ± 9.6 fmol/min/mm. The inhibitory effect of losartan, as well as the other nonpeptide angiotensin II receptor antagonists (eprosartan, valsartan, and irbesartan), was concentration dependent (Fig. 2). The concentrations of the antagonists needed to inhibit PAH secretion by 50% were 15 ± 0.5 µM for losartan, 11 ± 2.3 µM for eprosartan, 3 ± 0.6 µM for valsartan, and 17 ± 2.2 µM for irbesartan. Valsartan was significantly (p < .05) more potent than the other angiotensin II receptor antagonists in inhibiting PAH secretion.

View larger version (20K): [in this window] [in a new window]   Fig. 1.   Time course of the reversible inhibition of [3H]PAH secretion (JPAH) by losartan in the proximal tubule. During the control and recovery periods, the bath contained 10 µM [3H]PAH. Losartan (10 µM) was added to the bath at the indicated time (10-30 min). Results are the mean ± S.E.M. of four tubules.

View larger version (21K): [in this window] [in a new window]   Fig. 2.   Effects of losartan, eprosartan, valsartan, and irbesartan on [3H]PAH secretion (JPAH). Concentration of [3H]PAH was 10 µM. Results are expressed as a percent of control JPAH, which was 365 ± 35 fmol/min/mm. Each point is the mean ± S.E.M. of three or four tubules.

The previous experiments demonstrated that losartan could inhibit PAH secretion, suggesting that it interacted with the PAH transporter. To determine whether losartan itself was transported, additional experiments examined the transport of [³H]losartan. The addition of 10 µM [³H]losartan to the bath resulted in a J_LOS of 598 ± 54 fmol/min/mm. Probenecid, a classic inhibitor of the organic anion transporter, produced a concentration-dependent inhibition of losartan secretion (Fig. 3), with half-maximal inhibition occurring at a concentration of 17.9 ± 4.0 µM. PAH was much less effective in inhibiting [³H]losartan secretion, with an IC₅₀ value of 0.9 ± 0.2 mM. Figure 4 shows the relationship between the bath concentration of [³H]losartan and J_LOS. The bath-to-lumen transport of [³H]losartan was clearly saturable. Double reciprocal plots revealed an apparent affinity (K_m) of 12.3 ± 1.8 µM and a maximal transport rate (V_max) of 1490 ± 22 fmol/min/mm (n = 3). For comparison, similar experiments were performed with [³H]PAH (Fig. 5). Apparent K_m and V_max values for PAH were 88.5 ± 10.7 µM and 3939 ± 561 fmol/min/mm, respectively, values similar to those previously reported for J_PAH in the rabbit S2 segment (Shimomura et al., 1981). The apparent K_m value and the V_max value for losartan were significantly different from the corresponding values for PAH (p = .002 and p = .01, respectively). The data shown in Figs. 4 and 5 represent total bath-to-lumen fluxes of PAH and losartan, which are composed of both active and passive components. In preliminary experiments, we measured the secretion of PAH and losartan over the same concentration range as shown in Figs. 4 and 5 in the presence of 5 mM probenecid to block active secretion (Shimomura et al., 1981). Under these conditions, both PAH and losartan passive fluxes were similar and never exceeded 6% of the total flux at any substrate concentration, and therefore, we did not correct for the passive component.

View larger version (17K): [in this window] [in a new window]   Fig. 3.   Effects of probenecid and PAH on [3H]losartan secretion (JLOS). Concentration of [3H]losartan was 10 µM. Results are expressed as a percent of control JLOS, which was 598 ± 10.6 fmol/min/mm. Each point is the mean ± S.E.M. of four tubules.

View larger version (13K): [in this window] [in a new window]   Fig. 4.   [3H]Losartan secretion (JLOS) as a function of bath losartan concentration. Top, JLOS was measured in the presence of increasing concentrations of losartan added to the bath (5-500 µM). Bottom, double reciprocal plot of the data. Apparent Km and Vmax values were 12.3 ± 1.8 µM and 1490 ± 22 fmol/min/mm, respectively (n = 3).

View larger version (13K): [in this window] [in a new window]   Fig. 5.   [3H]PAH secretion (JPAH) as a function of bath PAH concentration. Top, JPAH was measured in the presence of increasing concentrations of PAH added to the bath (5-500 µM). Bottom, double reciprocal plot of the data. Apparent Km and Vmax values were 88.5 ± 10.7 µM and 3939 ± 561 fmol/min/mm, respectively (n = 3).

In four tubules, the lumen-to-bath flux of [³H]losartan was measured. When perfused with 50 µM losartan, the lumen-to-bath flux amounted to 54 ± 11.1 fmol/min/mm and was unaffected by 1 mM probenecid (53.5 ± 3.3 fmol/min/mm). This represented only 4.6% of the bath-to-lumen flux of losartan measured at the same concentration (1164 ± 77 fmol/min/mm).

According to the current model of organic anion secretion in the proximal tubule (Shimada et al., 1987; Pritchard, 1988), PAH and other organic anions enter the cell across the basolateral membrane in exchange for -ketoglutarate (-KG) moving out of the cell down its concentration gradient. The intracellular concentration of -KG is maintained by intracellular synthesis as well as Na⁺-coupled entry of the dicarboxylate into the cell. Accordingly, exposure of the cells to -KG should stimulate the uptake and secretion of an organic anion if it is transported by the organic anion/-KG exchanger. To determine whether losartan transport was affected by -KG, J_LOS was measured in tubules exposed to increasing concentrations of -KG added to the bath. For these experiments, we used a bicarbonate-free HEPES-buffered solution in which the effects of -KG are more readily demonstrable (Shpun et al., 1995). As shown in Fig. 6, both PAH and losartan secretion were stimulated by the addition of -KG to the bathing medium. In the absence of -KG, both J_PAH and J_LOS were markedly reduced compared with values obtained in previous experiments using DMEM: 59.6 ± 7.8 versus 412 ± 19.9 fmol/min/mm for PAH and 95.9 ± 4.8 versus 683 ± 50.2 fmol/min/mm for losartan. Significant (p < .05) stimulation of PAH secretion occurred at concentrations of -KG of 10 µM and above, whereas stimulation of losartan secretion was not observed until a concentration of 30 µM -KG. At 10 µM -KG, J_PAH values (505 ± 91 fmol/min/mm) were similar to those obtained in DMEM, whereas 100 µM -KG was required to increase J_LOS (616 ± 34.4 fmol/min/mm) to values obtained in DMEM.

View larger version (25K): [in this window] [in a new window]   Fig. 6.   Effect of -KG on JPAH and JLOS. Tubules were perfused and bathed with a HEPES-based buffer in the presence of increasing concentrations of -KG added to the bath. Concentrations of [3H]PAH and [3H]losartan were 10 µM. Results are expressed as a percent of control secretion in the absence of added -KG and were 59.6 ± 7.8 fmol/min/mm for PAH and 95.9 ± 4.8 fmol/min/mm for losartan. Results are mean ± S.E.M. of three tubules for each anion. *p < .05 versus control.

	Discussion

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The renal organic anion transporter or transporters are responsible for the transepithelial movement of a large number of endogenous substances, as well as numerous xenobiotics (Moller and Sheikh, 1983). The present study is the first to characterize the renal tubule secretion of a nonpeptide angiotensin II receptor antagonist, a class of compounds with increasing importance in the treatment of hypertension (MacFayden and Reid, 1994). The results of this study demonstrate that [³H]losartan is secreted by the proximal tubule in a manner similar to the prototypical organic anion, PAH. This conclusion is based on the observations that losartan and PAH cis-inhibited the secretion of each other, that [³H]losartan secretion was saturable and was inhibited by probenecid, and that [³H]losartan secretion was stimulated by the presence of -KG. No evidence for a significant reabsorptive flux of losartan was obtained.

Despite these similarities, there were quantitative differences in the characteristics of secretion of these two anions across the proximal tubule; chief among these was the much higher affinity of the transporter for losartan (12.3 µM) than for PAH (88.5 µM) and the difference in V_max values. This was also reflected in the more effective inhibition of PAH secretion by losartan than inhibition of losartan secretion by PAH. Furthermore, PAH secretion appeared to be more sensitive to the effects of -KG than did losartan. These latter experiments were conducted in a bicarbonate-free, HEPES-based buffer in which the stimulatory effects of -KG on organic anion transport are more readily apparent (Shpun et al., 1995). This is presumably due to a reduction in cellular ATP levels and tricarboxylic cycle intermediates in proximal tubules in the absence of bicarbonate (Shpun et al., 1995). This is reflected in the markedly reduced rate of PAH secretion in the absence of bicarbonate compared with that in the presence of bicarbonate observed in this and other studies (Dantzler and Evans, 1996). In these metabolically compromised tubules, the addition of exogenous -KG causes a marked increase in organic anion uptake by increasing -KG/organic anion countertransport as well as cellular metabolism in general (Shpun et al., 1995). Similar to observations made in basolateral membrane vesicles (Pritchard, 1988) and renal slices (Pritchard, 1990), -KG produced a biphasic effect on J_PAH. At concentrations up to 30 µM, -KG caused a concentration-dependent increase in J_PAH. This likely reflects both cis- and trans-stimulation of PAH/-KG exchange, as was also observed for fluorescein secretion in the proximal tubule (Welborn et al., 1998). The J_PAH values at 30 µM -KG were similar to those obtained in experiments performed in bicarbonate-containing media. However, at 100 µM -KG, J_PAH was reduced relative to values obtained at 30 µM, possibly reflecting competition with PAH for binding to the transporter or to saturation of the Na⁺/dicarboxylate cotransporter and a reduction in the in>out -KG gradient that drives the -KG/PAH exchanger (Pritchard, 1990). In contrast, losartan secretion was not stimulated until exogenous concentrations of -KG reached 30 µM and 100 µM -KG was required for J_LOS values to approach those obtained in bicarbonate-containing buffer. Differences in sensitivity of organic anion transport to -KG have also been observed between urate and PAH transport in pig basolateral membrane vesicles (Werner and Roch-Ramel, 1991). This difference, together with differences in Cl dependence and the rank order potency for inhibition of PAH and urate uptake by various organic anions, led the authors to suggest that in the pig kidney, urate and PAH uptakes occur via similar but separate transporters (Werner and Roch-Ramel, 1991). Whether the differences in PAH and losartan transport observed in the present study represent interaction with different transporters or simply kinetic differences awaits further investigation.

As a class, the angiotensin II receptor antagonists have high affinity for organic anion transport in the kidney. For example, losartan is 7- to 10-fold more potent than probenecid in inhibiting urate uptake in rat and human brush border membranes (Edwards et al., 1996; Roch-Ramel et al., 1997) and is uricosuric in humans (Nakashima et al., 1992; Tsunoda et al., 1993). In the present study, losartan, eprosartan, and irbesartan inhibited PAH transport with a potency similar to published values for probenecid (~15 µM; Dantzler et al., 1995), whereas valsartan was approximately 5-fold more potent than probenecid. The high affinity of this class of compounds for the organic anion secretory pathway, if confirmed in humans, suggests that these compounds may interfere with the renal excretion of other anionic drugs. However, at the present time, we are not aware of any such reports.