New imidazole-coordinated chemotherapeutics with low epithelial toxicity

The new imidazole-coordinated chemotherapeutics with low epithelial toxicity (NICE) presented in this article feature innovative drugs that combine epithelial toxicity comparable with that of carboplatin with novel carrier ligands optimized for DNA interaction. Recent identification of the pivotal role of basolateral organic cation transporters (OCTs) in cisplatin nephrotoxicity by a new model system (electrical resistance breakdown assay) facilitated the search for substances with a favorable organotoxic profile. The assay uses the high transepithelial electrical resistance (TEER) of the C7-clone of Madin-Darby canine kidney (MDCK) cells and the exclusive basolateral expression of OCT2 in these cells. TEER and caspase-3 activity of MDCK-C7-cells grown on microfilter membranes were monitored in response to exposure of either the apical or basolateral plasma membrane to platinum complexes. The impact of complexes on cancer cell lines was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bomide tests. Effects of substituents on pharmacological properties of NICE were systematically investigated by introducing sterically demanding groups as well as electron-donating and electron-withdrawing groups. Derivatives of NICE showed different renal epithelial toxic profiles and effects on cancer cells. NICE were significantly less toxic than cis-or oxaliplatin. The chlorine substituted NICE had no effect on epithelial integrity but markedly cytotoxic activity against amelanotic melanoma cells. Together, side effect targeted screening for new anticancer drugs with the electrical resistance breakdown assay offers an interesting approach for identifying and investigating new compounds. NICE feature the first group of platinum-based cytostatics discovered by using this system for systematic screening of new chemotherapeutics with low renal epithelial toxicity.     

Platinum complex cytotoxicity tested by the electrical resistance breakdown assay.

The electrical resistance breakdown assay provides a novel approach for the quantification of cytotoxic activity of platinum based anticancer drugs. It is a functional assay system for cancer cell invasion that detects nanoscale alterations of an epithelial test barrier prior to microscopic morphometric changes. We measured changes in transepithelial electrical resistance (TEER) of a tight epithelial MDCK-C7 monolayer in response to highly invasive amelanotic melanoma cells (A7-clone) in combination with different platinum complexes (cis-, oxali- and carboplatin). The efficiency of the electrical resistance breakdown assay was compared a standard method for measurement of cytostatic activity, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. The MTT-assay utilizes mitochondrial enzymatic activity to draw conclusions from a functional cell metabolism to the number of living cells in a sample. When human melanoma cells were seeded on top of an electrically tight MDCK-C7 monolayer, electrical leakage occurred within 48 h of co-culture. Electrical resistance breakdown was effectively prevented by cisplatin and its analogs (no significant difference between 100 microM cisplatin and corresponding controls with non-invasive cells). The results of the electrical resistance breakdown and MTT-assay were linearly dependent. Significance of both tests was equivalent, but the electrical resistance breakdown assay gave additional functional information. Compared to oxali- and carboplatin, cisplatin was more effective in preventing TEER-breakdown than reducing the number of tumor cells, giving rise to the assumption that cisplatin can reduce tumor cell number as well as invasiveness. In conclusion the electrical resistance breakdown assay provides a sensitive, continuous and cell-based assay system for the quantification of cancer cell invasiveness and evaluation of chemotherapeutics under physiological conditions.     

Properties and regulation of organic cation transport in freshly isolated human proximal tubules.

The kidney, and more specifically the proximal tubule, is the main site of elimination of cationic endogenous metabolites and xenobiotics. Although numerous studies exist on renal organic cation transport of rat and rabbit, no information is available from humans. Therefore, we examined organic cation transport and its regulation across the basolateral membrane of isolated human proximal tubules. mRNA for the cation transporters hOCT1 and hOCT2 as well as hOCTN1 and hOCTN2 was detected in these tubules. Organic cation transport across the basolateral membrane of isolated collapsed proximal tubules was recorded with the fluorescent dye 4-(4-dimethylamino)styryl-N-methylpyridinium (ASP(+)). Depolarization of the cells by rising extracellular K(+) concentration to 145 mm reduced ASP(+) uptake by 20 +/- 5% (n = 15), indicating its electrogeneity. The substrates of organic cation transport tetraethylammonium (K(i) = 63 microm) and cimetidine (K(i) = 11 microm) as well as the inhibitor quinine (K(i) = 2.9 microm) reduced ASP(+) uptake concentration dependently. Maximal inhibition reached with these substances was approximately 60%. Stimulation of protein kinase C with 1,2-dioctanoyl-sn-glycerol (DOG, 1 microm) or ATP (100 microm) inhibited ASP(+) uptake by 30 +/- 3 (n = 16) and 38 +/- 13% (n = 6), respectively. The effect of DOG could be reduced with calphostin C (0.1 microm, n = 7). Activation of adenylate cyclase by forskolin (1 microm) decreased ASP(+) uptake by 29 +/- 3% (n = 10). hANP (10 nm) or 8-bromo-cGMP (100 microm) also decreased ASP(+) uptake by 17 +/- 3 (n = 9) or 32 +/- 5% (n = 10), respectively. We show for the first time that organic cation transport across the basolateral membrane of isolated human proximal tubules, most likely mediated via hOCT2, is electrogenic and regulated by protein kinase C, the cAMP- and the cGMP-dependent protein kinases.     

Mutation of amino acid 475 of rat organic cation transporter 2 (rOCT2) impairs organic cation transport

Protein sequence alignments revealed one amino acid position, where organic cation transporters (OCTs, aspartate (D) at position 475 of rOCT2) and organic anion transporters (OATs, arginine (R) at position 466 of rOAT1) are charged oppositely. To address the impact of this amino acid for protein function we cloned rat organic cation transporter 2 (rOCT2), the renal electrogenic cation transporter of the basolateral side of proximal tubule cells. Site-directed mutagenesis was used to generate rOCT2-D475R (rOCT2-mut). Heterologous expression of rOCT2 wild-type (rOCT2-wt) in A6 cells resulted in a significant uptake of the fluorescent organic cation 4-(4-dimethylaminostyryl)-N-methylpyridinium (ASP(+)). Accordingly, rOCT2-wt-transfected COS 7 cells showed an almost fourfold uptake of 25 microM [(14)C]-TEA, whereas rOCT2-mut did not exhibit any uptake of [(14)C]-TEA. These data indicate that rOCT2 transports both ASP(+) and TEA and that aspartate at position 475 of rOCT2 plays a critical role in transport function.     

Organic cation uptake by a cultured renal epithelium

Several organic cations are actively transported by proximal renal tubules by mediated processes across both the apical and basolateral cell membranes. In order to evaluate this transport system in a cultured renal epithelium, uptake of 3H-tetraethylammonium (TEA) across the apical membrane was measured in LLCPK1 cells, a cell line with several characteristics of proximal tubules. 3H-TEA progressively entered these cells and reached a near-steady state by 30 min. Three-minute uptake was saturable with an apparent Vmax of 1,669 +/- 129 fmoles/micrograms DNA and apparent Km of 34.0 +/- 3.4 microM. 3H-TEA uptake was inhibited by an excess of nonradioactive TEA, other organic cations, sodium azide, and hypothermia. An alkaline external pH was associated with greater 3H-TEA uptake than an acid pH. However, efflux of 3H-TEA from cells was not appreciably affected by changes in external pH. Preincubation of cells in acid or alkaline media did not affect uptake. Alteration of cell pH by ammonium chloride addition or removal had little effect on 3H-TEA uptake. Finally, uptake of 3H-TEA was not accelerated by preloading cells with an excess of nonradioactive TEA. These results indicate that intact LLCPK1 cells possess a mechanism(s) in their apical membranes for the mediated transport of a prototypic organic cation. The mechanism(s) involved in this transport is uncertain. However, neither organic cation/proton nor organic cation/organic cation exchange appears to be the predominant process.     

Cimetidine transport in rat renal brush border and basolateral membrane vesicles

The transport of cimetidine by rat renal brush border and basolateral membrane vesicles has been studied in relation to the transport system of organic cation. Cimetidine inhibited [3H]tetraethylammonium uptake by basolateral membrane vesicles in a dose dependent manner, and the degree of the inhibition was almost the same as that by unlabeled tetraethylammonium. In contrast, cimetidine inhibited the active transport of [3H]tetraethylammonium by brush border membrane vesicles more strongly than unlabeled tetraethylammonium did. In agreement with the transport mechanism of tetraethylammonium in brush border membranes, the presence of an H+ gradient ([H+]i greater than [H+]o) induced a marked stimulation of cimetidine uptake against its concentration gradient (overshoot phenomenon), and this concentrative uptake was inhibited by unlabeled tetraethylammonium. These results suggest that cimetidine can share common carrier transport systems with tetraethylammonium in renal brush border and basolateral membranes, and that cimetidine transport across brush border membranes is driven by an H+ gradient via an H+-organic cation antiport system.     

Differential toxicity as a result of apical and basolateral treatment of LLC-PK1 monolayers with S-(1,2,3,4,4-pentachlorobutadienyl)glutathione and N-acetyl-S-(1,2,3,4,4-pentachlorobutadienyl)-L-cysteine

Monolayers of LLC-PK1 cells, a cell line with features typical of proximal tubular epithelial cells, were treated at the apical and basolateral side with S-(1,2,3,4,4-pentachlorobutadienyl)glutathione (PCBD-GSH) and N-acetyl-S-(1,2,3,4,4-pentachlorobutadienyl)-L-cysteine (PCBD-NAC). Apical treatment with PCBD-GSH (greater than 20 microM) resulted in cytotoxicity, which could be inhibited by acivicin and aminooxyacetic acid (AOAA), inhibitors of gamma-glutamyltranspeptidase (gamma GT) and beta-lyase respectively. In contrast apical treatment with PCBD-NAC was only toxic at high concentrations (greater than 850 microM), and this effect could hardly be inhibited by AOAA. Basolateral treatment of confluent LLC-PK1 monolayers, grown on porous membranes, with PCBD-GSH gave a much smaller response than apical treatment, consistent with the fact that gamma GT is predominantly present at the apical side. Basolateral treatment even with high concentrations of PCBD-NAC (1.1 mM) did not show an increase in cytotoxicity when compared to the effect after apical treatment. These results suggest the absence of an organic anion transporter, by which these conjugates in vivo are transported into the cells from the basolateral side. This supposition was substantiated in a study of transcellular transport of the model ions tetraethyl ammonium (TEA) and para-aminohippurate (PAH), in LLC-PK1 monolayers, grown as indicated above. No active PAH transport could be demonstrated, whereas an active TEA transport was present. The absence of an organic anion transporter limits the usefulness of LLC-PK1 cells for the study of nephrotoxicity of compounds, like PCBD-NAc, needing this transport to enter the cells. However, the finding of an active basolateral organic cation transporter, together with the presence of gamma GT, dipeptidase and beta-lyase, makes this system especially interesting for testing all compounds that use this transporter or these enzymes in order to elicit toxicity.     

Carrier-mediated transport systems of tetraethylammonium in rat renal brush-border and basolateral membrane vesicles

Transport of [3H]tetraethylammonium, an organic cation, has been studied in brush-border and basolateral membrane vesicles isolated from rat kidney cortex. Some characteristics of carrier-mediated transport for tetraethylammonium were demonstrated in brush-border and basolateral membrane vesicles; the uptake was saturable, was stimulated by the countertransport effect, and showed discontinuity in an Arrhenius plot. In brush-border membrane vesicles, the presence of an H+ gradient ( [H+]i greater than [H+]o) induced a marked stimulation of tetraethylammonium uptake against its concentration gradient (overshoot phenomenon), and this concentrative uptake was completely inhibited by HgCl2. In contrast, the uptake of tetraethylammonium by basolateral membrane vesicles was unaffected by an H+ gradient. Tetraethylammonium uptake by basolateral membrane vesicles was significantly stimulated by a valinomycin-induced inside-negative membrane potential, while no effect of membrane potential was observed in brush-border membrane vesicles. These results suggest that tetraethylammonium transport across brush-border membranes is driven by an H+ gradient via an electroneutral H+-tetraethylammonium antiport system, and that tetraethylammonium is transported across basolateral membranes via a carrier-mediated system and this process is stimulated by an inside-negative membrane potential.     

The organic cation transporters rOCT1 and hOCT2 are inhibited by cGMP

The electrogenic cation transporters OCT1 and OCT2 in the basolateral membrane of renal proximal tubules mediate the first step during secretion of organic cations. Previously we demonstrated stimulation and change of selectivity for rat OCT1 (rOCT1) by protein kinase C. Here we investigated the effect of cGMP on cation transport by rOCT1 or human OCT2 (hOCT2) after expression in human embryonic kidney cells (HEK293) or oocytes of Xenopus laevis. In HEK293 cells, uptake was measured by microfluorimetry using the fluorescent cation 4-(4-(dimethyl-amino)styryl)-N-methylpyridinium iodide (ASP + ) as substrate, whereas uptake into Xenopus laevis oocytes was measured with radioactively labelled cations. In addition, ASP +-induced depolarizations of membrane voltages (Vm) were measured in HEK293 cells using the slow whole-cell patch-clamp method. Incubation of rOCT1-expressing HEK293 cells for 10 min with 100 mM 8-Br-cGMP reduced initial ASP + uptake by maximally 78% with an IC50 value of 24 +/- 16 mM. This effect was not abolished by the specific PKG inhibitor KT5823, indicating that a cGMP-dependent kinase is not involved. An inhibition of ASP + uptake by rOCT1 in HEK293 cells was also obtained when the cells were incubated for 10 min with 100 mM cGMP, whereas no effect was obtained when cGMP was given together with ASP +. ASP + (100 mM)-induced depolarizations of Vm were reduced in the presence of 8-Br-cGMP (100 mM) by 44 +/- 11% (n = 6). Since it could be demonstrated that [3H]cGMP is taken up by an endogeneous cyanine863-inhibitable transporter, the effect of cGMP is probably mediated from inside the cell. Uptake measurements with [14C]tetraethylammonium and [3H]2-methyl-4-phenylpyridinium in Xenopus laevis oocytes expressing rOCT1 performed in the absence and presence of 8-Br-cGMP showed that cGMP does not interact directly with the transporter. The data suggest that the inhibition mediated by cGMP observed in HEK293 cells occurs most likely via a mammalian cGMP-binding protein that interacts with OCT1-2 transporters.     

Human Cytomegalovirus Glycoprotein B Contains Autonomous Determinants for Vectorial Targeting to Apical Membranes of Polarized Epithelial Cells

We previously reported that human cytomegalovirus (CMV) glycoprotein B (gB) is vectorially transported to apical membranes of CMV-infected polarized human retinal pigment epithelial cells propagated on permeable filter supports and that virions egress predominantly from the apical membrane domain. In the present study, we investigated whether gB itself contains autonomous information for apical transport by expressing the molecule in stably transfected Madine-Darby canine kidney (MDCK) cells grown on permeable filter supports. Laser scanning confocal immunofluorescence microscopy and domain-selective biotinylation of surface membrane domains showed that CMV gB was transported to apical membranes independently of other envelope glycoproteins and that it colocalized with proteins in transport vesicles of the biosynthetic and endocytic pathways. Determinants for trafficking to apical membranes were located by evaluating the targeting of gB derivatives with deletions in the lumen, transmembrane (TM) anchor, and carboxyl terminus. Derivative gB(Δ717-747), with an internal deletion in the luminal juxtamembrane sequence that preserved the N- and O-glycosylation sites, retained vectorial transport to apical membranes. In contrast, derivatives that lacked the TM anchor and cytosolic domain (gBΔ646-906) or the TM anchor alone (gBΔ751-771) underwent considerable basolateral targeting. Likewise, derivatives lacking the entire cytosolic domain (gBΔ772-906) or the last 73 amino acids (gBΔ834-906) showed disrupted apical transport. Site-specific mutations that deleted or altered the cluster of acidic residues with a casein kinase II phosphorylation site at the extreme carboxyl terminus, which can serve as an internalization signal, caused partial missorting of gB to basolateral membranes. Our studies indicate that CMV gB contains autonomous information for apical targeting in luminal, TM anchor, and cytosolic domain sequences, forming distinct structural elements that cooperate in vectorial transport in polarized epithelial cells.     

Impaired organic ion transport in proximal tubules of rats with Heymann nephritis

Organic ion transport across the basolateral membrane of proximal tubules was measured by means of the tissue slice technique in each of the four different stages of Heymann nephritis. Impairment of both organic anion and cation transport was detected early in Stage 2, and became more severe in Stage 3 of Heymann nephritis. The decreased transport function was associated with extensive damage to proximal tubule cells, including loss of brush border microvilli and basal infoldings. Despite these abnormalities of structure and function, oxygen consumption of proximal tubule cells remained essentially normal. Partial recovery of organic cation transport was noted late in Heymann nephritis (Stage 4). Recovery of the cation transport function was associated with a partial restoration of brush border microvilli and basal infoldings to proximal tubule cells. However, organic anion transport remained depressed throughout the entire course of disease. Impairment of organic ion transport in rats with Heymann nephritis appeared to result from damage to basolateral membrane transport elements rather than general deterioration of the metabolic machinery of proximal tubule cells. Decreased organic cation transport appeared to be the consequence of a reduction in the number of carrier sites, a phenomenon that could have resulted from decreased membrane surface area. However, the depression of organic anion transport was associated with decreased substrate affinity of the anion carrier, indicating that qualitative, rather than quantitative changes, were primarily responsible for that defect. Specific antibody-mediated damage to the anion transport elements in basolateral membranes of proximal tubules is postulated to occur in Heymann nephritis.     

Distinct mechanisms of zinc uptake at the apical and basolateral membranes of caco-2 cells.

Zinc uptake mechanisms at the apical and basolateral membrane borders of caco-2 cells were examined. This human-derived cell line possesses many morphological and functional characteristics of absorptive small intestinal cells. By day 14, confluent and well-differentiated monolayers were formed when the cells were grown on porous polycarbonate filters. Labelled zinc was placed on the apical or basal side of the monolayer and its uptake by the cells, as well as its transport across the monolayer, were measured. Zinc uptake by the cells from the apical side was found to be a saturable process (Kt = 41 microM; Vmax = 0.3 nmols/cm2/10 min) with a diffusional term at higher concentrations (1.0 sec/cm). Apical uptake was not affected by metabolic inhibitors or potential zinc ligands. Zinc uptake from the basolateral side was concentration dependent (Kd = 1.3 sec/cm) and was partially inhibited (30%) by ouabain and vanadate, suggesting that the (Na-K)-ATPase on the basolateral membrane is involved in the serosal uptake of zinc by the cell. Transport of zinc across the monolayers from the apical or basolateral compartment was concentration dependent and was not affected by metabolic inhibitors. Zinc transport from the basolateral side was greater than 2-fold greater than apical transport. Hence, separate mechanisms can be distinguished with respect to zinc uptake at the apical and basolateral membranes of caco-2 cells.     

Localisation of Alanine uptake by cultured renal epithelial cells (LLC-PK1) to the basolateral membrane

Alanine uptake by LLC-PK¹ cells has previously been demonstrated to be almost exclusively sodium dependent. We here confirm that when the cells are grown on an impermeable substratum there is a marked fall in uptake as confluence is reached. By applying an autoradiographic technique to visualize transported alanine, it is clear, however, that even in subconfluent cultures there is marked cellular inhomogeneity with regard to uptake, which takes place predominantly in those cells at the periphery of growing islands and not those at the interior. In contrast, when cells are grown on permeable substrata, a uniform distribution of silver grains is found. In two other types of experiment, we found that when confluent cell monolayers on an impermeable support were treated briefly with a chelating agent or suspended by mechanical treatment, there was a marked increase per cell in sodium-dependent alanine uptake and in ouabain-sensitive potassium uptake. We conclude that the apparent fall in alanine uptake as cells reach confluence on an impermeable support is due to masking of transport sites, which are predominantly, if not exclusively, located at the basolateral membrane.     

Establishment of renal cell lines derived from S2 segments of the proximal tubule.

The aim of this study was to establish epithelial cell lines derived from defined nephron segments. Primary cultures were prepared from dissected proximal S2 segments of the rabbit kidney, and grown in monolayers. Immortalization was observed after nuclear microinjection of the cells with simian virus 40 DNA and resulted in the development of cell lines of epithelial morphology. These cell lines were maintained in culture for at least 24 passages, then cells were frozen. One of the cell lines, the RKPC-2, was selected and further characterized. RKPC-2 cells formed domes on impermeable supports, indicating fluid and solute transport. RKPC-2 cells formed continuous monolayers of low transepithelial resistance on collagen-coated filters. They were able to accumulate tetraethylammonium, an organic cation; however, no significant transcellular transport could be measured. We conclude that this cell line which shows characteristics of epithelial cells has maintained certain properties of intact proximal tubules, in particular the capacity to accumulate organic cations.     

Differentiation of a clone isolated from the HT29 cell line: polarized distribution of histocompatibility antigens (HLA) and of transferrin receptors

The HT29 cell line, derived from a human colon adenocarcinoma, is able to differentiate if galactose replaces glucose in the culture medium. We have isolated a clone (HT29-18) from this cell line which displays differentiated properties of the parent cell line. HT29-18 cells grown in glucose-containing medium form multiple layers of round cells without specific cell-cell adhesion. In contrast, when grown in galactose-containing medium, they form a monolayer with tight junctions and exhibit a well differentiated brush border at their apical membrane, which faces the culture medium. The polarized properties of HT29-18 cells grown in galactose-containing medium were demonstrated by immunofluorescent techniques with antibodies against 2 plasma membrane proteins. Class I histocompatibility antigens (HLA) and transferrin receptors, 2 well characterized integral membrane proteins, are uniformly distributed on the cell surface of undifferentiated HT29-18 cells, but acquire a polarized distribution during differentiation, localized on the basolateral membranes and absent from the apical surface. Binding of 125I-labeled transferrin was used to determine transferrin receptor distribution on apical and basolateral membranes. Functional tight junctions in the differentiated cultures were demonstrated, as the monolayer was impermeable to a permeation dye (ruthenium red) as well as to antibodies. The sealing of these tight junctions is, as in vivo, Ca++-dependent as they could be opened by a short incubation in Ca++-free medium.     

Molecular mechanisms of organic cation transport in OCT2-expressing Xenopus oocytes

The molecular mechanisms of organic cation transport by rat OCT2 was examined in the Xenopus oocyte expression system. When extracellular Na+ ions were replaced with K+ ions, uptake of tetraethylammonium (TEA) by OCT2-expressing oocytes was decreased, suggesting that TEA uptake by OCT2 is dependent on membrane potential. Kinetic analysis revealed that the decreased TEA uptake by ion substitution was caused at least in part by decreased substrate affinity. Acidification of extracellular buffer resulted in decreased uptake of TEA, whereas TEA efflux from OCT1- and OCT2-expressing oocytes was not stimulated by inward proton gradient, in consistent with basolateral organic cation transport in the kidney. Inhibition of TEA uptake by various organic cations revealed that apparent substrate spectrum of OCT2 was similar with that of OCT1. However, the affinity of procainamide to OCT1 was higher than that to OCT2. Uptake of 1-methyl-4-phenylpyridinium was stimulated by OCT2 as well as OCT1, but uptake of levofloxacin, a zwitterion, was not stimulated by both OCTs. These results suggest that OCT2 is a multispecific organic cation transporter with the characteristics comparable to those of the basolateral organic cation transporter in the kidney.     

Characterization of tetraethylammonium uptake across the basolateral membrane of the Drosophila Malpighian (renal) tubule

Basolateral transport of the prototypical type I organic cation tetraethylammonium (TEA) by the Malpighian tubules of Drosophila melanogaster was studied using measurements of basolateral membrane potential (V(bl)) and uptake of [(14)C]-labeled TEA. TEA uptake was metabolically dependent and saturable (maximal rate of mediated TEA uptake by all potential transport processes, reflecting the total transport capacity of the membrane, 0.87 pmol.tubule(-1).min(-1); concentration of TEA at 0.5 of the maximal rate of TEA uptake value, 24 muM). TEA uptake in Malpighian tubules was inhibited by a number of type I (e.g., cimetidine, quinine, and TEA) and type II (e.g., verapamil) organic cations and was dependent on V(bl). TEA uptake was reduced in response to conditions that depolarized V(bl) (high-K(+) saline, Na(+)-free saline, NaCN) and increased in conditions that hyperpolarized V(bl) (low-K(+) saline). Addition of TEA to the saline bathing Malpighian tubules rapidly depolarized the V(bl), indicating that TEA uptake was electrogenic. Blockade of K(+) channels with Ba(2+) did not block effects of TEA on V(bl) or TEA uptake indicating that TEA uptake does not occur through K(+) channels. This is the first study to provide physiological evidence for an electrogenic carrier-mediated basolateral organic cation transport mechanism in insect Malpighian tubules. Our results also suggest that the mechanism of basolateral TEA uptake by Malpighian tubules is distinct from that found in vertebrate renal tubules.     

Expression and function of TETRAN, a new type of membrane transporter

In contrast to transport across basolateral membranes, the mechanism governing transport of organic anions across the luminal membranes of proximal tubules has remained unclear. We recently found Tetracycline transporter-like protein (TETRAN), a human ortholog of yeast Tpo1p that can transport anionic Non-steroidal anti-inflammatory drugs (NSAIDs). In this study, we examine the expression and function of TETRAN. TETRAN mRNA is expressed in various human tissues, including kidney. When overexpressed in cultured cells, TETRAN was predominantly localized on cytoplasmic membranes. Immunohistochemical analysis of human and mouse kidney tissue showed that TETRAN was expressed at the luminal membranes of proximal tubules. Overexpression of TETRAN in cultured cells facilitated the uptake of organic anions such as indomethacin (a NSAID) and fluorescein. The results suggest that TETRAN is a novel human organic anion transporter, and that it serves as a transporter for some NSAIDs and various other organic anions at the final excretion step.     

Uptake properties of lamivudine (3TC) by a continuous renal epithelial cell line

The purpose of this study was to characterize the renal uptake properties of the cytidine analog and antiretroviral agent 3TC. The uptake of radiolabelled 3TC was measured at 37 degrees C in a continuous porcine renal epithelial cell line (i.e., LLC-PK1 cells) grown as a monolayer on an impermeable support. 3TC (5 microM) uptake (37 degrees C) by the monolayer cells was saturable (Km = 1.2 +/- 0.2 mM) but not significantly altered by various dideoxynucleoside analog drugs, nucleosides, and nucleoside transport inhibitors, suggesting that a nucleoside transporter is not involved in 3TC uptake. A number of endogenous organic cation probes and inhibitors significantly reduced 3TC uptake by the monolayer cells. Quinine, trimethoprim (TMP), and tetraethylammonium (TEA) inhibited 3TC uptake in a dose dependent manner with IC50 values of 0.6 mM, 0.63 mM, and 1.9 mM, respectively. In turn, the uptake of the typical organic cation substrate TEA was inhibited by high concentrations of 3TC. An outwardly directed proton gradient significantly increased the uptake of 3TC by the monolayer cells, suggesting the involvement of a proton exchange process. Conversely, in the presence of monensin, a Na+/H+ ionophore, the uptake of 3TC was significantly reduced. These results suggest that the uptake of 3TC by a cultured renal epithelium may be mediated by an organic cation-proton exchanger. The observed clinical interaction between 3TC and trimethoprim may be explained by competition for a common renal organic cation tubular transporter.