Secretion Of Organic Cations Research Articles

Multidrug And Toxin Extrusion's (MATE) Role in Renal Organic Cation Secretion

The molecular identity of the luminal element of renal organic cation (OC) secretion is suspected to include two novel homologous transport proteins, Multidrug And Toxin Extruders 1 & 2K (MATE1 and MATE2K); they share anticipated characteristics of luminal OC transport, including expression in the apical membrane of human renal proximal tubule (RPT) cells and support of OC/H+ exchange. Here we investigated aspects of the multispecificity of human MATE1 (hMATE1) and MATE2K (hMATE2K). Stable transfection in Chinese Hamster Ovary cells resulted in Kt values for transport of 1‐methyl‐4‐phenylpyridinium (MPP) of 7.6 μM (MATE1) 5.2 μM (MATE2K). Inhibition by tetraethylammonium produced IC50 values of 52 μM and 26 μM for hMATE1 and hMATE2K, respectively. Preliminary data shows that cimetidine is a potent inhibitor of MPP, with IC50s of 1 μM (MATE1) and 3μM (MATE2K). Increasing extracellular H+ concentration cis inhibited hMATE1‐mediated MPP transport with an IC50 value of 15.4 nM (pH of 7.8). Immunohistochemical analysis of paraffin embedded human kidney cortex samples confirmed that hMATE1 and hMATE2K are located in the apical membrane of RPT cells and initial evidence suggests a heterogeneous distribution of both transporters in the cortical RPTs. In conclusion, the selectivities and distribution of MATE1 and MATE2K support the contention that they play a key role in OC secretion in human RPT. (F31DK084769)

Functional Characteristics of Two Human MATE Transporters: Kinetics of Cimetidine Transport and Profiles of Iinhibition by Various Compounds

Human multidrug and toxin extrusion protein 1 (hMATE1) and hMATE2-K are organic cation/H+ antiporters that have recently been identified and suggested to be involved in the renal brush border secretion of various organic cations. Information about functional characteristics of them has been accumulating, but still insufficient to fully understand their functions and respective roles. The present study was conducted to help clarify them. The cDNA of hMATE1 was isolated from human brain cDNA by RT-PCR and hMATE2-K cDNA was from human kidney cDNA. HEK293 cells were stably transfected with hMATE1 and hMATE2-K, and the cellular uptakes of [3H]cimetidine and [14C]tetraethylammonium (TEA) were evaluated. It was first found that both hMATE1 and hMATE2-K can transport cimetidine with high affinities, indicated by small Michaelis constants of 8.00 mM and 18.18 mM, respectively. These were much smaller than those for TEA (366 mM and 375 mM, respectively, for hMATE1 and hMATE2-K). Subsequent investigation using cimetidine as a probe substrate into the profiles of inhibition of the two hMATEs by various compounds indicated that they are similar in principle but different to some extent in substrate recognition, reflecting the modest differences in amino acid sequences between them. In fact, cimetidine transport by hMATE1 was correlated to that by hMATE2-K, which is 65% similar to hMATE1, but not as good as to that by rat MATE1, which is 86% similar. Cimetidine was demonstrated to be a high affinity substrate of both hMATEs. Subsequent evaluation of the inhibition of hMATEs by various compounds indicated no major difference in function or role between hMATE1 and hMATE2-K.

MATE1 has an external COOH terminus, consistent with a 13-helix topology

The mammalian members of the Multidrug And Toxin Extruder family, i.e., MATE1 and MATE2-K, are suspected of mediating the luminal step in renal secretion of organic cations. The 1,000+ prokaryotic/fungal/plant MATE family members are predicted to have 12 transmembrane helices (TMHs), whereas MATE1/2-K appear to have an additional (13th) COOH-terminal helix. Here, we determined whether rabbit MATE1 has an external COOH terminus, consistent with the presence of 13 TMHs. A V5 epitope tag at the COOH terminus of MATE1 was freely accessible to external V5 antibody, whereas tags at the NH(2) terminus, or at sites of truncation within the long cytoplasmic loop between predicted TMHs 12 and 13, were only accessible to the V5 antibody following permeabilization of the membrane. The truncated mutants that lacked TMH13 still retained transport activity, indicating that the terminal helix was not necessary for transport function. Cells that expressed a mutant lacking only TMH13 displayed similar K(t) and J(max) values to those of the full-length protein, although when normalized to protein expressed at the plasma membrane, the transport rate of the mutant was <10% that of full-length MATE1. An effectively cysteine-less MATE1 mutant (Delta13Cys) was functional and refractory to reaction with the impermeant marker of accessible cysteine residues, maleimide-PEO(2)-biotin. Delta13Cys mutants with an added cysteine residue at the truncation sites within the terminal cytoplasmic loop reacted with maleimide biotin only after permeabilization of the membrane, whereas a mutant with a cysteine residue at the COOH terminus was freely accessible to maleimide biotin. These data are consistent with a mammalian MATE topology that includes 13 TMHs and indicate that the terminal TMH, although not necessary for transport function, may influence the turnover characteristics of the transporter.

Regulatory Role of Testosterone in Organic Cation Transport: in Vivo and in Vitro Studies

The renal proximal tubule (RPT) plays a crucial role in organic cation (OC) secretion and has a major impact on pharmacokinetics of OC drugs. Secretory transport is vectorial. Thus, it involves transporters located at both basolateral and apical membranes. Although sex hormones have been shown to regulate OC transport, there is little data on the effect of testosterone on OC secretion in a whole animal. Therefore, we determined the clearance of tetraethylammonium (TEA), a model OC substrate, in intact and castrated male mice. Castration significantly decreased renal TEA secretion by 30%, and testosterone supplementation returned TEA secretion to control levels in castrated mice. The mechanism of this effect was further examined in isolated mouse renal proximal tubules (mRPT). TEA uptake in isolated mRPT from castrated mice was reduced by 36%. This effect was reversed in tubules from castrated mice supplemented with testosterone. Kinetic analysis of [(3)H]-TEA uptake in isolated mRPT showed a decreased V(max) with no change in K(m), implying that the decrease in transport rate was caused by lowering in the number of transporters in castrated mice rather than a change in transporter affinity. Quantitative real time polymerase chain reaction (real time PCR) revealed that organic cation transporter (OCT)2 is the major TEA transporter in male mice. Moreover, OCT2 mRNA level was significantly reduced after castration. Castrated mice also showed a modest increase in organic cation/carnitine transporter 1 (OCTN1) mRNA level, indicating that testosterone may also regulate apical OCTN1 expression. These data suggest that testosterone regulates transepithelial transport of OC through modulation of OCT2 expression in male mice.

Functional effects of protein sequence polymorphisms in the organic cation/ergothioneine transporter OCTN1 (SLC22A4)

OCTN1 is a multispecific transporter of organic cations and zwitterions, including several clinically important drugs as well as the antioxidant ergothioneine. OCTN1 is highly expressed in the kidney, where it is thought to aid in active secretion of organic cations, and may facilitate the active reabsorption of ergothioneine. Genetic variation in OCTN1 may help to explain interindividual variability in the pharmacokinetics of many cationic or zwitterionic drugs. We screened for human genetic variants in the OCTN1 coding region by direct sequencing in a large sample (n=270) of ethnically diverse healthy volunteers. Six protein sequence-altering variants were identified, including five-amino-acid substitutions and one nonsense mutation. Two of the variants, T306I and L503F, were polymorphic, occurring at frequencies of 37 and 19%, respectively, in the total sample. Allele frequencies are varied by ethnicity. In biochemical assays, two of the variants (D165G and R282X) resulted in complete loss of transport function, and one variant (M205I) caused a reduction in activity to approximately 50% of the reference sequence protein. One variant, L503F, showed altered substrate specificity; this variant occurred at particularly high allele frequency (42%) in the European-American participants in our sample. Subcellular localization and ergothioneine inhibition kinetics were similar among the common amino-acid sequence variants of OCTN1. The common OCTN1-L503F variant may explain a significant amount of population variation in the pharmacokinetics of OCTN1 substrate drugs. The rare loss-of-function variants provide a rational tool for studying the importance of ergothioneine in humans in vivo.

Inorganic mercury interacts with cysteine residues (C451 and C474) of hOCT2 to reduce its transport activity

Human organic cation transporter 2 (hOCT2) is essential for the renal tubular secretion of many toxic organic cations. Previously, of the cysteines (C437, C451, C470, and C474) that occur within transmembrane helices that comprise the hydrophilic cleft (proposed site of substrate binding), only C474 was accessible to maleimide-PEO(2)-biotin (hydrophilic thiol-reactive reagent), and covalent modification of this residue caused lower transport rates (Pelis RM, Zhang X, Dangprapai Y, Wright SH, J Biol Chem 281: 35272-35280, 2006). Thus it was hypothesized that the environmental contaminant Hg(2+) (as HgCl(2)) would interact with C474 to reduce hOCT2-mediated transport. Uptake of [(3)H]tetraethylammonium (TEA) into Chinese hamster ovary cells stably expressing hOCT2 was reduced in a concentration-dependent manner by HgCl(2), with an IC(50) of 3.9 +/- 0.11 microM. Treatment with 10 microM HgCl(2) caused a sixfold reduction in the maximal rate of TEA transport but did not alter the affinity of hOCT2 for TEA. To determine which cysteines interact with Hg(2+), a mutant with all four cleft cysteines converted to alanines (quadruple mutant), and four variants of this mutant, each with an individual cysteine restored, were created. The quadruple mutant was less sensitive to HgCl(2) than wild-type, whereas the C451- and C474-containing mutants were more sensitive than the quadruple mutant. Consistent with the HgCl(2) effect on transport, MTSEA-biotin only interacted with C451 and C474. These data indicate that C451 and C474 of hOCT2 reside in the aqueous milieu of the cleft and that interaction of Hg(2+) with these residues causes reduced TEA transport activity.

Drugdrug interactions involving membrane transporters in the human kidney

The kidneys play a critical role in the elimination of xenobiotics. Factors affecting the ability of the kidney to eliminate drugs may result in marked changes in the pharmacokinetics of a given compound. Drugdrug interactions due to competitive inhibition of renal organic anion or cation secretion systems have been noticed clinically for a long time. However, our understanding of the physical sites of interactions, that is, the specific transport proteins that the interacting drugs act on, has just begun very recently. This review summarises the latest progress in molecular identification and functional characterisation of major drug transporters in the human kidney. In particular, the review focuses on relating cloned renal drug transporters to clinically observed drugdrug interactions. The authors opinion on the current status and future directions of research in these areas is also offered.

Transport of drugs in the kidney by the human organic cation transporter, OCT2 and its genetic variants

The human organic cation transporter 2 (OCT2, SLC22A2) is a multispecific transporter of organic cations, including many clinically used drugs. OCT2 is primarily responsible for the uptake of organic cations across the basolateral membrane of renal tubular epithelial cells and is considered a major transporter in the active secretion of organic cations in the kidney. Uptake of organic cations by OCT2 is driven by the inside-negative membrane potential and is pH-sensitive. Regulation of OCT2 at the transcriptional level by steroid hormones and at the protein level by various protein kinases has been described. Several human genetic variants in the coding region of OCT2 have been identified and functionally characterized, including both polymorphic and rare variants. A variety of structurally diverse compounds have been shown to interact with OCT2, including endogenous compounds, drugs, and dietary supplements.

This Month's Highlights

### Drug Disposal: Possible Source of Biologic Variation. Toxicity of many drugs is related to plasma levels, which are affected by renal clearance. Organic anion and cation secretion is an important role of the kidney and accounts for renal clearance of many endogenous compounds and also of

Deficiency in the Organic Cation Transporters 1 and 2 (Oct1/Oct2 [Slc22a1/Slc22a2]) in Mice Abolishes Renal Secretion of Organic Cations

The polyspecific organic cation transporters 1 and 2 (Oct1 and -2) transport a broad range of substrates, including drugs, toxins, and endogenous compounds. Their strategic localization in the basolateral membrane of epithelial cells in the liver, intestine (Oct1), and kidney (Oct1 and Oct2) suggests that they play an essential role in removing noxious compounds from the body. We previously showed that in Oct1(-/-) mice, the hepatic uptake and intestinal excretion of organic cations are greatly reduced. Since Oct1 and Oct2 have extensively overlapping substrate specificities, they might be functionally redundant. To investigate the pharmacologic and physiologic roles of these proteins, we generated Oct2 single-knockout and Oct1/2 double-knockout mice. Oct2(-/-) and Oct1/2(-/-) mice are viable and fertile and display no obvious phenotypic abnormalities. Absence of Oct2 in itself had little effect on the pharmacokinetics of tetraethylammonium (TEA), but in Oct1/2(-/-) mice, renal secretion of this compound was completely abolished, leaving only glomerular filtration as a TEA clearance mechanism. As a consequence, levels of TEA were substantially increased in the plasma of Oct1/2(-/-) mice. This study shows that Oct1 and Oct2 together are essential for renal secretion of (small) organic cations. A deficiency in these proteins may thus result in increased drug sensitivity and toxicity.

Distinct transport activity of tetraethylammonium from l-carnitine in rat renal brush-border membranes

We investigated the contribution of the Na+/l-carnitine cotransporter in the transport of tetraethylammonium (TEA) by rat renal brush-border membrane vesicles. The transient uphill transport of l-carnitine was observed in the presence of a Na+ gradient. The uptake of l-carnitine was of high affinity (Km=21 μM) and pH dependent. Various compounds such as TEA, cephaloridine, and p-chloromercuribenzene sulfonate (PCMBS) had potent inhibitory effects for l-carnitine uptake. Therefore, we confirmed the Na+/l-carnitine cotransport activity in rat renal brush-border membranes. Levofloxacin and PCMBS showed different inhibitory effects for TEA and l-carnitine uptake. The presence of an outward H+ gradient induced a marked stimulation of TEA uptake, whereas it induced no stimulation of l-carnitine uptake. Furthermore, unlabeled TEA preloaded in the vesicles markedly enhanced [14C]TEA uptake, but unlabeled l-carnitine did not stimulate [14C]TEA uptake. These results suggest that transport of TEA across brush-border membranes is independent of the Na+/l-carnitine cotransport activity, and organic cation secretion across brush-border membranes is predominantly mediated by the H+/organic cation antiporter.

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.

Molecular and physiological evidence for multifunctionality of carnitine/organic cation transporter OCTN2.

OCTN2 is an Na(+)-dependent transporter for carnitine, which is essential for fatty acid metabolism, and its functional defect leads to fatal systemic carnitine deficiency (SCD). It also transports the organic cation tetraethylammonium (TEA) in an Na(+)-independent manner. Here, we studied the multifunctionality of OCTN2, by examining the transport characteristics in cells transfected with mouse OCTN2 and in juvenile visceral steatosis (jvs) mice that exhibit a SCD phenotype owing to mutation of the OCTN2 gene. The physiological significance of OCTN2 as an organic cation transporter was confirmed by using jvs mice. The embryonic fibroblasts from jvs mice exhibited significantly decreased transport of [(14)C]TEA. Pharmacokinetic analysis of [(14)C]TEA disposition demonstrated that jvs mice showed decreased tissue distribution and renal secretory clearance. In transport experiments using OCTN2-expressing cells, TEA and carnitine showed mutual trans-stimulation effects in their transport, implying a carnitine/TEA exchange mechanism. In addition, Na(+) affected the affinity of carnitine for OCTN2, whereas Na(+) is unlikely to be involved in TEA transport. This is the first molecular and physiological demonstration of the operation of an organic cation transporter in renal apical membrane. The results are consistent with the physiological coupling of carnitine reabsorption with the secretion of organic cations.

Regulation of renal tubular secretion of organic compounds

Information on the molecular basis underlying organic anion and cation transport in renal tubules has expanded in recent years with the identification and characterization of numerous transporters. However, little is known about the regulation of this transport. Both English and Russian language studies dealing with the regulation of organic ion transport by the kidney have been reviewed. This review summarizes the literature on the physiological and pharmacological aspects of the regulation of organic ion transport, linking this information where possible to underlying transport mechanisms. Current models of the tubular secretion of organic anions and cations are reviewed. Factors that inhibit or enhance tubular secretion of xenobiotics are described, and their influence on proximal tubule cell transport and function is discussed. Important roles for substrate stimulation, the adrenergic nervous system, numerous hormones, P-glycoprotein, and protein kinase C activity have been identified. Despite considerable advances in the understanding of basic transport pathways and mechanisms involved in the tubular secretion of organic compounds, there is still relatively little information on the regulation of this transport. Studies combining the techniques of integrative and cell physiology and molecular biology will provide significant new insights into the pathways regulating the tubular transport of these compounds.

ROCT2 is a basolateral potential-driven carrier, not an organic cation/proton exchanger.

The driving forces mediating tetraethylammonium (TEA) transport were systematically assessed in Xenopus oocytes and MDCK cells expressing organic cation transporter (OCT) 2 cloned from rat kidney (rOCT2). In rOCT2 cRNA-injected oocytes, uptake of [14C]TEA was saturable, with an estimated Michaelis constant (Km) of 393 microM, and was specifically inhibited by organic cations. Furthermore, TEA uptake demonstrated two distinct components, one that was potential sensitive and one that was pH sensitive. When membrane potential was intact, TEA uptake was largely unaffected by changes in medium pH; when the oocyte membrane was depolarized (K+ in = out = 102.5 mM, plus valinomycin), decreasing external medium pH significantly reduced TEA uptake. Consistent with the potential sensitivity of uptake, electrophysiological analysis of rOCT2-injected oocytes demonstrated movement of positive charge into the oocyte upon TEA addition. To further evaluate the nature of the pH effect and assess the properties of rOCT2 in a renal epithelium, rOCT2 was introduced into MDCK cells. A stably transfected single cell clone (MDCK-rOCT2) showed mediated, potential-sensitive, pH-sensitive TEA uptake (Km = 48 microM). TEA efflux from preloaded MDCK-rOCT2 cells was stimulated by externally applied (trans) tetramethylammonium but was trans-inhibited by H+ (external pH 5.4). The effect of external H+ was to modulate rOCT2-mediated transport. Thus rOCT2 is a potential-driven transporter, not an organic cation/H+ exchanger, consistent with a physiological role in the basolateral entry step in renal organic cation secretion.

Drug secretion systems in renal tubular cells: functional models and molecular identity.

For the last several decades, functional characterization of renal drug handling has provided the conceptual framework of drug transport, especially drug secretion by the renal tubular cells. Functional models have been postulated for the two distinct groups of drugs with regard to their ionization characteristics at physiological pH (i.e., organic cations and organic anions). Organic cations are predicted to cross the basolateral membranes through a potential-sensitive uptake system along an inside-negative potential gradient, with secretion of organic cations into urine across apical membranes appearing to occur via a proton-organic cation antiport system. Organic anions are predicted to enter the cells against an electrical-potential gradient through basal membranes via an organic anion-dicarboxylic acid exchanger. Secretion of organic anions into urine across apical membranes is less well characterized, but the presence of an organic anion exchanger and a potential-driven transporter is hypothesized. Recent data obtained using molecular biology techniques have helped to elucidate molecular identity of each system postulated in these models. Novel drug transport proteins have also been discovered that were not part of the original organic cation/anion model, such as P-glycoprotein for hydrophobic neutral and cationic compounds. Moreover, the search for homologous genes of these transporters has led to the discovery of previously unknown transporter proteins, whose function has yet to be identified. Integral roles of these transport proteins in overall tubular handling of drugs remain to be determined.

A nucleoside-sensitive organic cation transporter in opossum kidney cells.

Renal secretion of organic cations and anions are pleiotropic, active processes in mammals. Some nucleosides such as deoxyadenosine (dAdo), 2-chlorodeoxyadenosine, and azidothymidine are secreted by human and rodent kidneys. Previous work (J. A. Nelson, J. F. Kuttesch, Jr., and B. H. Herbert. Biochemical Pharmacology 32: 2323-2327, 1983) indicated a role for the classic organic cation transporter (OCT) in the secretion of the dAdo analog, 2'-deoxytubercidin, by mouse kidney. Using [14C]tetraethylammonium bromide ([14C]TEA) as a substrate, we tested several renal cell lines for a nucleoside-sensitive OCT. American opossum kidney proximal tubule cells (OK) express a cimetidine-sensitive and metabolic-dependent ability to efflux TEA. Other classic OCT inhibitors and several nucleosides also inhibit TEA efflux by these cells in a manner reflecting structural specificity for the carrier. Inhibition of OCT by nucleosides is not a universal feature of OCTs, since TEA transport mediated by cloned rat kidney OCT2 in the Xenopus laevis oocyte system was not inhibited by the same nucleosides. In conclusion, OK cells appear to possess an OCT that may also transport some nucleosides by a novel carrier.

Molecular pharmacology of organic cation transporters in kidney.

The homeostasis of endogenous organic cations, such as choline and N P-methylnicotinamide, monoamine neurotransmitters, cationic drugs, such as cimetidine, morphine, quinine and amantadine, and of cationic xenobiotics is controlled by reabsorption and excretion in the small intestine, by metabolic conversion and excretion in the liver, and by reabsorption and excretion in the kidney [1–6]. In the kidney organic cations may be ultrafiltrated in the glomeruli and reabsorbed or secreted in renal tubules. Hydrophilic cations are readily ultrafiltrated, however, more hydrophobic cations are bound to plasma membrane proteins that may not permeate the filtration barrier. Secretion and reabsorption of organic cations have been described in renal proximal tubules but may also occur in distal tubules or collecting ducts [7–11]. These processes have been functionally characterized by measurements using intact kidneys or tissue slices [10, 12–14], by microperfusion experiments using proximal tubules [7, 9, 15], by uptake measurements using isolated tubules or cells [8, 11, 16, 17], and by uptake measurements using membrane vesicles from proximal tubules [18–28]. In luminal and basolateral membranes of proximal tubules different transport processes have been demonstrated, although all the involved transport systems may not have been identified by these measurements because a variety of different polyspecific organic cation transporters with overlapping substrate specificity are expressed [5]. After the cloning of the first organic cation transporter rOCT1 from a rat kidney library [29], an increasing number of homologous cation transporters have been identified and it has been shown that the renal anion transporters also belong to this new protein family. The functional characterization of the expressed transporters and their immunohistochemical localization in the kidney has begun [30–35]. This review describes the molecular structure of renal organic cation transporters and homologous gene products. The transport properties and renal localization of the cloned cation transporters are summarized and their presumed role in renal cation handling of endogenous and exogenous cations is discussed.

Cellular and molecular mechanisms of renal tubular secretion of organic anions and cations

A wide variety of endogenous organic ions and xenobiotics are secreted into the urine via organic anion and cation transport systems, expressed in brush-border and basolateral membranes of renal tubular cells. Using membrane vesicles isolated from the kidney, cultured renal epithelial cells, isolated renal tubules, and slices of renal cortex, extensive studies have been done regarding the mechanisms of renal tubular secretion of organic ions. Basolateral entry of organic anions is mediated by the organic anion/dicarboxylate exchange system, whereas apical extrusion of organic anions from epithelial cells is mediated by an anion exchanger and/or by membrane potential-sensitive transport systems. Studies using membrane vesicles have made clear the fact that the basolateral transport of organic cations is stimulated by inside-negative membrane potential, whereas the transport of organic cations in brush-border membranes is achieved by a proton gradient. Trasport studies using cultured renal epithelial cells have shown other aspects of organic ion transport, such as regulatory mechanisms for transcellular transport of orgnaic anions and cations. The recent development of molecular techniques has greatly advanced our understanding of the molecular aspects of various transport processes. In 1994, a cDNA clone encoding the prototype organic cation transporter was isolated from rat kidney. Within the last 3 years, several organic anion and cation transporters in the kidney have been identified by different cloning techniques. In this review, we describe the mechanisms mediating renal tubular secretion of organic anions and cations, including recent topics in this area.

Hepatobiliary and intestinal clearance of amphiphilic cationic drugs in mice in which both mdr1a and mdr1b genes have been disrupted.

1. We have used mice with homozygously disrupted mdr1a and mdr1b genes (mdr1a/1b (-/-) mice) to study the role of the mdr1-type P-glycoprotein (P-gp) in the elimination of cationic amphiphilic compounds from the body. These mice lack drug-transporting P-gps, but show no physiological abnormalities under laboratory conditions and have normal bile flow. 2. 3H-labelled cationic drugs were administered intravenously (i.v.) to mice as a single bolus dose and the disposition of the studied cationic drugs was investigated by focusing on drug secretion into bile, intestinal lumen and urine. 3. Hepatobiliary secretion of the investigated cationic drugs was profoundly reduced in mice devoid of the mdr1-type P-gps. In fact, the cumulative biliary output, measured during 1 h, of the small type 1 compounds tri-butylmethyl ammonium (TBuMA) and azidoprocainamide methoiodide (APM), as well as of the more bulky type 2 cationic drug vecuronium, was reduced by at least 70% in the mdrla/lb (-/-) mice compared to wild-type. 4. The intestinal secretion of TBuMA, APM and vecuronium was also profoundly reduced in mdrla/lb (-/-) mice compared to wild-type mice. The absence of the mdrl-type P-gp resulted in virtual elimination of intestinal secretion of TBuMA and APM (>90% reduced as compared to wild-type (P=0.0001 and 0.0022, respectively)). The intestinal secretion of the type 2 cation drug vecuronium was reduced by 58% (P=0.0004) compared to the wild-type mice. 5. Increased renal clearances of both the type 1 compounds TBuMA and APM and also of the type 2 cationic compound vecuronium in the mdrla/lb (-/-) mice were observed. Furthermore, the balance between hepatic, intestinal and renal clearances of small type 1 organic cations clearly shifted towards a predominant role for renal clearance. Increased renal clearance may be explained by (over)expression of additional mechanisms for renal organic cation secretion, alternatively they may also point to an as yet undefined role of P-glycoprotein in kidney physiology and renal secretory function. 6. We conclude that the elimination from the body of a broad spectrum of cationic amphiphilic drugs via liver and intestine, is largely dictated by the activity of mdrl-type P-glycoproteins.