Transport of Paraquat by Isolated Renal Proximal Tubular Segments from Rabbits

Abstract: Paraquat is a non-selective herbicide, which induces lung, liver and kidney damage in mammalian species. Because paraquat is mainly eliminated by the kidneys, the induced kidney damage may suppress excretion and enhance toxicity of paraquat in other organs. Since proximal tubules appear to be the target segment of the nephron, this study focuses on transport of paraquat by isolated proximal tubular segments from rabbits. Proximal tubules were isolated using a combined magnetic iron perfusion and collagenase method. Incubations were carried out at 25° under 100% oxygen or nitrogen for varying times at different concentrations of paraquat. Proximal tubules accumulated paraquat by a slow process, which was non-saturable in the concentration range (0.1–5 μM) examined. Tubular excretion of cations involves transport across both basolateral and luminal membranes of the cell. The basolateral uptake of paraquat was inhibited by low temperature, low medium pH and quinine. In contrast to quinine, tetraethylammonium enhanced paraquat accumulation probably by trans-stimulating the basolateral uptake. Incubation under nitrogen enhanced paraquat accumulation possibly by reducing the transport out of the cell at the luminal membrane. Thus, this study shows that proximal tubules accumulate paraquat by an active process related to the cation transport mechanism.     

Effect of lamivudine on uptake of organic cations by rat renal brush-border and basolateral membrane vesicles

The effect of lamivudine on uptake of a representative organic cation, tetraethylammonium (TEA), by rat renal brush-border membrane vesicles (BBMV) and basolateral membrane vesicles (BLMV) has been investigated. The pH-driven uptake of TEA by BBMV (pHin = 6.0, pHout = 7.5) was inhibited by lamivudine. The IC50 value (concentration resulting in 50% inhibition) for the concentration-dependent effect of lamivudine on TEA uptake by BBMV after 30 s was 2668 microM whereas IC50 values for cimetidine and trimethoprim were < 2.5 microM and < 25 microM, respectively. The early uptake of TEA by BLMV was also reduced significantly by lamivudine. The IC50 value for the concentration-dependent effect of lamivudine on uptake of TEA by BLMV at 30 s was > 25 mM, whereas the IC50 values for cimetidine and trimethoprim were 2116 microM and 445 microM, respectively. These findings suggest that compared with other cationic drugs, such as trimethoprim and cimetidine, lamivudine is a weak inhibitor of organic cation transport into the tubules by the brush-border and basolateral membranes of renal epithelial cells. It is unlikely lamivudine will have any significant effect on the excretion of co-administered cationic drugs by the renal tubules.     

New clues for nephrotoxicity induced by ifosfamide: preferential renal uptake via the human organic cation transporter 2

Anticancer treatment with ifosfamide but not with its structural isomer cyclophosphamide is associated with development of renal Fanconi syndrome leading to diminished growth in children and bone problems in adults. Since both cytotoxics share the same principal metabolites, we investigated whether a specific renal uptake of ifosfamide is the basis for this differential effect. First we studied the interaction of these cytotoxics using cells transfected with organic anion or cation transporters and freshly isolated murine and human proximal tubules with appropriate tracers. Next we determined changes in membrane voltage in proximal tubular cells to understand their differentiated nephrotoxicity. Ifosfamide but not cyclophosphamide was significantly transported into cells expressing human organic cation transporter 2 (hOCT2) while both did not interact with organic anion transporters. This points toward a specific interaction of ifosfamide with hOCT2, which is the main OCT isoform in human kidney. In isolated human proximal tubules ifosfamide also interacted with organic cation transport. This interaction was also seen in isolated mouse proximal tubules; however, it was absent in tubules from OCT-deficient mice, illustrating the biological importance of this selective transport. Ifosfamide decreased the viability of cells expressing hOCT2, but not that of control cells. Coadministration of cimetidine, a known competitive substrate of hOCT2, completely prevented this ifosfamide-induced toxicity. Finally, ifosfamide but not cyclophosphamide depolarized proximal tubular cells. We propose that the nephrotoxicity of ifosfamide is due to its selective uptake by hOCT2 into renal proximal tubular cells, and that coadministration of cimetidine may be used to prevent ifosfamide-induced nephrotoxicity.     

Characterization of renal excretion mechanism for a novel diuretic, M17055, in rats

M17055 was developed as a novel diuretic that inhibits both Na(+), K(+), and 2Cl(-) cotransport at the thick ascending Henle's loop and Na(+) reuptake at the distal tubule. It is secreted at the renal proximal tubules. The purpose of the present study was to characterize the renal excretion mechanism of M17055. We used the renal cortical slices and brush border membrane vesicles (BBMVs) to investigate the transport mechanisms across the basolateral and brush border membranes, respectively. M17055 uptake by rat renal slices increased with time and was saturable. Several organic anions including probenecid, para-aminohippurate (PAH), and estrone-3-sulfate, decreased M17055 uptake. The uptake of M17055 was also observed into HEK293 cells expressing rat OAT1, and was inhibited by PAH. M17055 uptake by BBMVs was time-dependent, saturable, osmolarity-sensitive, and inhibited by several organic anions, but not by PAH. These results suggest that plural organic anion transport systems are involved in M17055 transport via both basolateral and brush border membranes of proximal tubule epithelial cells, a part of the renal uptake being mediated by OAT1.     

P-glycoprotein- and mrp2-mediated octreotide transport in renal proximal tubule

1. Transepithelial transport of a fluorescent derivative of octreotide (NBD-octreotide) was studied in freshly isolated, functionally intact renal proximal tubules from killifish (Fundulus heteroclitus). 2. Drug accumulation in the tubular lumen was visualized by means of confocal microscopy and was measured by image analysis. Secretion of NBD-octreotide into the tubular lumen was demonstrated and exhibited the all characteristics of specific and energy-dependent transport. Steady state luminal fluorescence averaged about five times cellular fluorescence and was reduced to cellular levels when metabolism was inhibited by NaCN. 3. NBD-octreotide secretion was inhibited in a concentration-dependent manner by unlabelled octreotide, verapamil and leukotriene C(4) (LTC(4)). Conversely, unlabelled octreotide reduced in a concentration dependent manner the p-glycoprotein (Pgp)-mediated secretion of a fluorescent cyclosporin A derivative (NBDL-CS) and the mrp2-mediated secretion of fluorescein methotrexate (FL-MTX). 4. This inhibition was not due to impaired metabolism or toxicity since octreotide had no influence on the active transport of fluorescein (FL), a substrate for the classical renal organic anion transport system. 5.The data are consistent with octreotide being transported across the brush border membrane of proximal kidney tubules by both Pgp and mrp2.     

Characterization of MPP+ secretion across human intestinal Caco-2 cell monolayers: role of P-glycoprotein and a novel Na(+)-dependent organic cation transport mechanism

1. In the kidney, a number of transport proteins involved in the secretion of permanently charged organic cations have recently been cloned. To evaluate the possible similarities between intestine and kidney in the handling of organic cations we investigated the transport of 1-methyl-4-phenylpyridinium (MPP+) across monolayers of intestinal Caco-2 cells. MPP+ is a prototypic substrate of the cloned organic cation transporters hOCT1 and hOCT2. 2. In Caco-2 cell monolayers, the basolateral to apical flux of MPP+ was significantly greater than the apical to basolateral flux, consistent with net secretion of MPP+. 3. Net secretion of MPP+ was abolished by addition of either 10 microM cyclosporin A or 100 microM verapamil to the apical membrane. In contrast, secretion of MPP+ was unaffected by addition of either TEA (2 mM) or decynium-22 (2 microM) to either apical or basolateral membranes. These results suggest that MPP+ secretion is mediated primarily by P-glycoprotein located at the apical membrane. We found no evidence of a role for hOCT1 or hOCT2 in the secretion of MPP+. 4. In addition to net secretion of MPP+, we found evidence of a Na(+)-dependent MPP+ uptake mechanism at the apical membrane of Caco-2 cells. 5. Na(+)-dependent MPP+ uptake was sensitive to inhibition by the organic cations; decynium-22 (2 microM), TEA (2 mM) and cimetidine (5 mM) but not by carnitine, guanidine or proline. 6. These results suggest that net secretion of MPP+ across the apical membrane of Caco-2 cells is a function of the relative contributions of MPP+ secretion mediated by P-glycoprotein and MPP+ absorption mediated by a novel Na(+)-dependent transport mechanism.     

Transport of cimetidine across the basolateral membrane of rabbit kidney proximal tubules: interaction with organic anions.

Since in whole animal studies and in man the renal clearance of cimetidine was prolonged by the coadministration of probenecid, the aim of the present study was to examine the interaction of the organic base cimetidine with the organic anion transport system at the basolateral membrane of isolated non-perfused rabbit proximal tubules. S2 segments of proximal tubules were incubated at 37.5 degrees C with 3H-cimetidine (2 x 10(-7) mol/l) or 3H-PAH (4 x 10(-6) mol/l, as a marker for the organic anion transport system) and 14C-inulin (marker for the extracellular space) for 25 min to achieve a steady state. Afterwards, a nonradiolabelled substance was added to the bath, and the change of the cellularly stored radioactivity was measured at 5-min intervals. Probenecid (5 x 10(-5) mol/l) decreased the cellular amount of 3H-cimetidine to 26% of the control value. At this concentration, furosemide and Na2SO4 had no effect. At a concentration of 10(-3) mol/l, these substances reduced the cellular 3H-cimetidine uptake to 33% (furosemide) and 57% (Na2SO4) of the control value. 10(-4) and 10(-3) mol/l succinate diminished the steady-state uptake of cimetidine to 77% and 53% of the control value, respectively. On the other hand, cimetidine (10(-3) mol/l) decreased the cellular uptake of 3H-PAH to 52% of the control value. N1-methylnicotinamide (5 x 10(-5) mol/l and 10(-3)mol/l) had no effect on the steady-state uptake of 3H-PAH. These results indicate that the organic base cimetidine, besides its high affinity for the cation transporter, also interacts with the organic anion transport system at the basolateral membrane of rabbit proximal tubules.     

Renal organic anion transport system: a mechanism for the basolateral uptake of mercury-thiol conjugates along the pars recta of the proximal tubule

The basolateral handling of 20 microM inorganic mercury (Hg(2+)), in the form of mercuric conjugates of cysteine (Cys), N-acetylcysteine (NAC), or glutathione (GSH), was studied in isolated perfused S2 segments of the rabbit proximal tubule. One of the primary aims of the present study was to determine in a direct manner whether basolateral uptake of Hg(++) occurs in the pars recta of the proximal tubule and, more importantly, whether the p-aminohippurate-sensitive (PAH) organic anion transport system is involved in this process. Basolateral uptake and accumulation of Hg(++) occurred when the basolateral membrane of the tubular segments was exposed to mercuric conjugates of Cys, NAC, or GSH. Net basolateral uptake of Hg(++) was more than twice as great in the tubules exposed to mercuric conjugates of Cys or NAC than in the tubules exposed to mercuric conjugates of GSH, indicating that mercuric conjugates of Cys or NAC are transported more efficiently than mercuric conjugates of GSH. When PAH (1 mM) was added to the basolateral compartment (bath) surrounding a perfused S2 segment, the net uptake of Hg(++) (in the form of the mercuric conjugates) was reduced by 60-70%. In addition, when glutarate (4 mM), a transportable substrate for both the sodium-dependent dicarboxylate transporter and the dicarboxylate/organic anion exchanger (OAT1), was added to the basolateral compartment, there was a significant reduction in the uptake and accumulation of Hg(++) in the form of mercuric conjugates of Cys. Overall, these data indicate that Hg(++), in the form of biologically relevant mercuric conjugates of Cys, NAC, or GSH, is taken up significantly at the basolateral membrane of pars recta segments of the proximal tubule, and this uptake is mediated mainly by the actions of the PAH-sensitive organic anion transport system.     

Regulation of the renal basolateral transport system for organic anions by calcium channel blockers.

Calcium channel blockers have been reported to exert multiple effects on renal tubular function. Therefore, the effects of the calcium channel blockers nifedipine and verapamil on the cellular uptake of tritiated para -aminohippuric acid (PAH) into microdissected non-perfused rabbit kidney S2 proximal tubule segments were investigated to study a possible influence of calcium channel blockers on renal PAH transport. Since the tubules were entirely collapsed, the accumulated radioactivity overwhelmingly reflects the transport across the basolateral membrane. Tubular PAH accumulation was increased by 1 and 10 microm verapamil, and by 1 microm nifedipine, but it was unaffected by lower or higher concentrations of these calcium channel blockers. The increase in PAH accumulation caused by 1 microm nifedipine or 10 microm verapamil was independent from changes in intracellular Ca(2+) and inhibited by staurosporine (1 and 10 n m) a potent inhibitor of protein kinase C (PKC). Our results indicate that the calcium channel blockers nifedipine and verapamil increase the transport of organic anions across the basolateral membrane of proximal tubules, probably by an activation of PKC. Furthermore, the increased tubular PAH transport may disturb the estimation of the renal PAH-clearance.     

Characterization of rabbit primary proximal tubule kidney cell cultures grown on Millicell-HA membrane filters

With the aim of developing a kidney cell culture system that can be used to assess renal toxicity in vivo, freshly isolated rabbit proximal tubules were plated on Millipore cellulose filters mounted in plastic inserts (Millicell-HA). DNA synthesis peaked on day 6 of culture and cells reached confluency by days 12–14. The integrity of the monolayer was confirmed by exclusion of [¹⁴C]inulin and cell viability demonstrated by linearity of protein synthesis over a 24-hr period. In confluent cultures, the organic anion, [¹⁴C]p-aminohippuric acid (PAH) and cation [¹⁴C]tetraethylammonium bromide (TEA) were shown to be transported from the basolateral to the apical side at a rate 5–6 times greater than that from the apical to basolateral side during the first 60 min of exposure. Probenecid decreased PAH transport by 60% and N-methylnicotinamide and quinine inhibited TEA transport by 40 and 56%, respectively. Uptake of [¹⁴C]-methylglucopyranoside into the cells was three times greater when label was added to the apical side than when label was added to the basolateral side. Apical uptake of glucose was sodium dependent and inhibited by more than 90% with phloridzin. Thus, kidney proximal tubule cells in the filter insert culture system display functional polarity which appears to mimic function in vivo and may be useful for examining mechanisms of nephrotoxicity.     

Effects of inorganic divalent cations on the renal basolateral transport system for organic anions.

Recent work demonstrated that the heavy metal ion Cd2+ increases the transport of p-aminohippuric acid (PAH) across the basolateral membrane of microdissected non-perfused rabbit kidney S2 proximal tubule segments. Usually, such ions induce damage of various renal transport systems, therefore the effects of divalent metal ions Zn2+, Co2+ and Ni2+ on this transporter were investigated. Addition of Ni2+ or Zn2+ to the bathing solution leads to a significant reduction of basolateral PAH transport, with IC50 values of 2 x 10(-5) and 10(-6) M, respectively, whereas Co2+ failed to inhibit PAH accumulation. Simultaneous incubation with thrombin (10(-9)M), which is known to increase [Ca2+]i, abolished the effects of the divalent ions. Our results indicate that Ni2+ and Zn2+ reduce cellular PAH uptake. Because Ni2+ and Zn2+ are calcium channel blockers, these effects are probably due to a reduction of [Ca2+]i by an interaction of these metals with binding sites in the calcium channel, whereas Co2+ does not affect these binding sites. This finding is supported by the fact that thrombin abolished the cation effects.     

Molecular cloning, functional characterization and tissue distribution of rat H+/organic cation antiporter MATE1

Purpose Transport characteristics and tissue distribution of the rat H⁺/organic cation antiporter MATE1 (multidrug and toxin extrusion 1) were examined.Methods Rat MATE1 cDNA was isolated by polymerase chain reaction (PCR) cloning. Transport characteristics of rat MATE1 were assessed by HEK293 cells transiently expressing rat MATE1. The mRNA expression of rat MATE1 was examined by Northern blot and real-time PCR analyses.Results The uptake of a prototypical organic cation tetraethylammonium (TEA) by MATE1-expressing cells was concentration-dependent, and showed the greatest value at pH 8.4 and the lowest at pH 6.0–6.5. Intracellular acidification induced by ammonium chloride resulted in a marked stimulation of TEA uptake. MATE1 transported not only organic cations such as cimetidine and metformin but also the zwitterionic compound cephalexin. MATE1 mRNA was expressed abundantly in the kidney and placenta, slightly in the spleen, but not expressed in the liver. Real-time PCR analysis of microdissected nephron segments showed that MATE1 was primarily expressed in the proximal convoluted and straight tubules.Conclusions These findings indicate that MATE1 is expressed in the renal proximal tubules and can mediate the transport of various organic cations and cephalexin using an oppositely directed H⁺ gradient.     

Carrier-mediated uptake of cisplatin by the OK renal epithelial cell line

The purpose of this study was to investigate whether transport of cis-diamminedichloroplatinum II (cisplatin) across renal epithelial cell line OK cells is mediated by the organic cation transport system. OK cell monolayers cultured on permeable membranes were incubated with 100 microM cisplatin on the apical or basolateral side, and the cellular accumulation and the transport of cisplatin across the monolayer were measured. The accumulation from the basolateral medium and the basolateral-to-apical transport of cisplatin were higher than the accumulation from the apical medium and the apical-to-basolateral transport, respectively. The cell monolayers were incubated with different concentrations of cisplatin (0.02 approximately 3 mM) in the basolateral medium. The relationship between the cisplatin concentrations in the medium and in the cells revealed that cisplatin accumulation tended to be saturable. The basolateral-to-apical transport of cisplatin was increased when the pH of the apical medium was decreased, with a concomitant decrease in the accumulation of cisplatin. Coincubation of cisplatin with tetraethylammonium (TEA), a typical substrate for the organic cation transporter, significantly decreased the accumulation and transport of cisplatin from the basolateral medium. These results suggest that the uptake and basolateral-to-apical transport of cisplatin are mediated by not only simple diffusion but also by the organic cation transport system.     

Modulatory effects of hormones,drugs, and toxic events on renal organic anion transport

The human body is exposed continuously to a wide variety of exogenous compounds, many of which are anionic compounds. In addition, products of phase II biotransformation reactions are negatively charged, viz. glucuronides, sulfate esters, or glutathiones. Renal transport of organic anions is an important defense mechanism of the organism against foreign substances. The combination of the rate of uptake and efflux and the intracellular disposition of organic anions in the proximal tubule determines the intracellular concentration and the nephrotoxic potential of a compound. Modulation of organic anion secretion is observed after exposure of proximal tubules to various hormones, and the subsequent receptor-mediated response is signaled by protein kinases. Transport of anionic compounds across the basolateral as well as the luminal membrane is modified by activation or inhibition of protein kinases. Protein kinase C activation reduces the uptake of organic anions mediated by the organic anion transporter 1 (OAT1/Oat1) and Oat3 and reduces Mrp2-mediated efflux. In addition, activation of protein kinase C has been shown to inhibit transport by the organic anion transporting polypeptide 1 (Oatp1) across the luminal membrane. Additional protein kinases have been implicated in the regulation of organic anion transport, and the role of nuclear factors in xenobiotic excretion is an emerging field. The physiological regulation of organic anion transporters may also be influenced by exogenous factors, such as exposure to xenobiotics and cellular stress. This commentary discusses the current knowledge of endogenous and exogenous influences on renal anionic xenobiotic excretion.     

Cellular energetics and glutathione status in NRK-52E cells: toxicological implications

Cellular energetics and redox status were evaluated in NRK-52E cells, a stable cell line derived from rat proximal tubules. To assess toxicological implications of these properties, susceptibility to apoptosis induced by S-(1,2-dichlorovinyl)-L-cysteine (DCVC), a well-known mitochondrial and renal cytotoxicant, was studied. Cells exhibited high activities of several glutathione (GSH)-dependent enzymes, including gamma-glutamylcysteine synthetase, GSH peroxidase, glutathione disulfide reductase, and GSH S-transferase, but very low activities of gamma-glutamyltransferase and alkaline phosphatase, consistent with a low content of brush-border microvilli. Uptake and total cellular accumulation of [14C]alpha-methylglucose was significantly higher when cells were exposed at the basolateral as compared to the brush-border membrane. Similarly, uptake of GSH was nearly 2-fold higher across the basolateral than the brush-border membrane. High activities of (Na(+)+K(+))-ATPase and malic dehydrogenase, but low activities of other mitochondrial enzymes, respiration, and transport of GSH and dicarboxylates into mitochondria were observed. Examination of mitochondrial density by confocal microscopy, using a fluorescent marker (MitoTracker Orange), indicated that NRK-52E cells contain a much lower content of mitochondria than rat renal proximal tubules in vivo. Incubation of cells with DCVC caused time- and concentration-dependent ATP depletion that was largely dependent on transport and bioactivation, as observed in the rat, on induction of apoptosis, and on morphological damage. Comparison with primary cultures of rat and human proximal tubular cells suggests that the NRK-52E cells are modestly less sensitive to DCVC. In most respects, however, NRK-52E cells exhibited functions similar to those of the rat renal proximal tubule in vivo.     

Transporter-mediated renal handling of nafamostat mesilate

Nafamostat mesilate (NM) is a serine-protease inhibitor that is rapidly eliminated from the circulation and accumulated in the kidney. This study was conducted to characterize the mechanism of NM transport in the kidney because a serious side effect of NM-induced hyperkalemia may be related to accumulation of NM in the kidney. Measurements of uptake of NM in vivo by the kidney uptake index (KUI) method and of transport in an in vitro-cultured LLC-PK1 cell system suggested the involvement of an organic cation transporter (OCT). To clarify the involvement of OCTs located in the basolateral membrane of proximal tubules, we evaluated NM transport by OCTs expressed in Xenopus laevis oocytes. The IC(50) values of NM on [(14)C]TEA ([(14)C]tetraethylammonium) uptake by rOCT1, rOCT2, and hOCT2 were 50, 0.5, and 20 microM, respectively, and NM was concluded to be a substrate of OCTs. To investigate the transport of NM across the brush-border membrane, we examined the uptake of NM into brush-border membrane vesicles (BBMVs) isolated from rat renal cortex. NM was taken up into the BBMVs, and the uptake was decreased by unlabeled NM and temperature, implying that a transporter(s) is also involved in NM transport across the apical membrane. NM was not a substrate of hOCTN1, hOCTN2, or P-gp, implying the involvement of some unknown transporter(s). Thus, renal accumulation of NM can be explained by the involvement of the basolateral OCTs, though the influence of the apical membrane transporter remains to be clarified.     

Renal organic cation transporters mediated cadmium-induced nephrotoxicity

The involvement of renal organic cation transporters (OCTs) in cadmium transport was investigated in Chinese hamster ovary (CHO-K1) cells stably and singly transfected with rabbit (rb)OCT1 and rbOCT2, in murine isolated renal proximal tubule, and in intact kidney following bilateral ureteral ligation of rat. Cadmium inhibited uptake of [³H]-tetraethylammonium (TEA), a substrate of rbOCT1 and rbOCT2, via both transporters with half maximal inhibitory concentration (IC₅₀) of cadmium for rbOCT1- and rbOCT2-mediated TEA uptake of 96 ± 5 μM and 207 ± 12 μM, respectively. Cadmium similarly inhibited [³H]-TEA uptake in isolated non perfused renal proximal tubules. Cadmium accumulation in the transfected CHO-K1 cells was significantly higher than that of parent cells and this could be attenuated by TEA. In addition, cadmium accumulation in whole kidney was also reduced by TEA administration. Furthermore, exposure of the rbOCT1- and rbOCT2-expressing CHO-K1 cells to cadmium led to cytotoxicity, which could be prevented by TEA treatment. Taken together, this study provides for the first time, evidence showing OCT1 and OCT2 mediating cadmium transport across the basolateral membrane into the renal proximal tubular cells.     

NH4+ modulates renal tubule amantadine transport independently of intracellular pH changes

A bicarbonate-dependent organic cation transporter, unique from rOCT1 and rOCT2, primarily mediates amantadine uptake into renal proximal tubules. We examined whether intracellular pH regulates bicarbonate-dependent amantadine transporter function in these tubules. NH(4)Cl treatment resulted in immediate intracellular alkalinization of tubules for up to 30s followed by gradual acidification that was maximal at 5min. Proximal tubule amantadine uptake was similarly inhibited (60%) by NH(4)Cl during both the early intracellular alkalinization and later acidification phases. Sodium propionate treatment resulted in immediate intracellular acidification of proximal tubules without inhibiting amantadine uptake. NH(4)Cl inhibition of bicarbonate-dependent amantadine uptake was dose-dependent, competitive and sex-dependent. NH(4)Cl, NH(4)NO(3), (NH(4))(2)SO(4) and (NH(4))(2)HPO(4) inhibited amantadine uptake into proximal tubules similarly. NH(4)Cl also stimulated efflux of amantadine and tetraethylammonium from preloaded proximal tubules, suggesting mediation of a facilitated process. These data suggest the potential for direct modulation of organic cation transporters by NH(4)(+) in rat kidney proximal tubules.     

Epithelial Transport of Anthelmintic Ivermectin in a Novel Model of Isolated Proximal Kidney Tubules

Purpose. The mechanism of excretion of the anthelmintic drug ivermectin was investigated in a novel experimental model of functionally intact proximal tubules isolated from a teleost fish (Fundulus heteroclitus). Methods. Secretion into the lumens of freshly isolated proximal tubules was studied by means of confocal laser scanning microscopy and digital image analysis using ivermectin and fluorescent labelled ivermectin (BODIPY-ivermectin; BI) as substrates. Results. The tubular cells rapidly accumulated BI from the medium and attained steady state within 25 minutes. Luminal fluorescence in the steady state was 5-7 times higher as compared to intracellular fluorescence. The secretion of BI into the tubular lumens was inhibited in a dose dependent manner by unlabelled ivermectin and inhibitors of the renal excretory membrane pump p-glycoprotein, namely SDZ PSC-833 and verapamil, but not by leukotriene C₄, a substrate of the renal export protein mrp2. Accumulation inside the tubular cells was not affected by the added inhibitors. Ivermectin inhibited the renal secretion of the fluorescent cyclosporin derivative NBDL-CS, a substrate of p-glycoprotein, but not the secretion of the mrp2-substrate fluorescein-methotrexate, nor the secretion of fluorescein, a substrate of the classical renal organic anion transporter. Conclusions. The data are consistent with BI and ivermectin interacting in teleost kidney tubules exclusively with p-glycoprotein, but not with one of the other known excretory transport systems. In addition, the studies demonstrate that freshly isolated functionally intact kidney tubules from killifish are a useful tool to differentiate the substrate specificity of renal transport systems with respect to drug elimination.     

Evaluation of air-interfaced Calu-3 cell layers for investigation of inhaled drug interactions with organic cation transporters in vitro

A physiologically pertinent in vitro model is urgently needed for probing interactions between inhaled drugs and the organic cation transporters (OCT) in the bronchial epithelium. This study evaluated OCT expression, functionality, inhibition by common inhaled drugs and impact on formoterol transepithelial transport in layers of human bronchial epithelial Calu-3 cells grown at an air-liquid interface. 21 day old Calu-3 layers expressed OCT1, OCT3, OCTN1 and OCTN2 whereas OCT2 could not be detected. Quantification of the cellular uptake of the OCT substrate ASP(+) in presence of inhibitors suggested several OCT were functional at the apical side of the cell layers. ASP(+) uptake was reduced by the bronchodilators formoterol, salbutamol (albuterol), ipratropium and the glucocorticoid budesonide. However, the OCT inhibitory properties of the two β(2)-mimetics were suppressed at therapeutically relevant concentrations. The absorptive permeability of formoterol across the cell layers was enhanced at a high drug concentration shown to decrease ASP(+) uptake by ～50% as well as in presence of the OCT inhibitor tetraethylammonium (TEA). Secretory transport was unaffected by the drug concentration but was reduced by TEA. Our data indicate air-interfaced Calu-3 layers offer a low-cost in vitro model suitable for assessing inhaled drug-OCT interactions in the bronchial epithelium.