The multispecific organic anion transporter (OAT) family

Organic anion transporters play important roles in the elimination of a variety of endogenous substances, xenobiotics and their metabolites from the body. During the last decade, molecular cloning has identified several families of multispecific organic anion transporters mediating the renal and hepatic elimination of organic anions and, most recently, the OAT (organic anion transporter) family, the founding member of which (OAT1) is the basolateral p-aminohippurate (PAH) transporter in the renal proximal tubule. So far, four isoforms have been identified. OATs are membrane proteins with 12 putative membrane-spanning domains and function as sodium-independent exchangers or facilitators. OATs show weak structural similarity to organic cation transporters (OCTs) and OCTN/carnitine transporters. OATs are multispecific organic anion transporters, the substrates of which include both endogenous (e.g. cyclic nucleotides, prostaglandins, urate, dicarboxylates) and exogenous anions (various anionic drugs and environmental substances). All members of the OAT family are expressed in the kidney, while some are also expressed in the liver, brain and placenta. The OAT family represents the renal secretory pathway for organic anions and is also involved in the distribution of organic anions in the body.     

CATs and HATs: the SLC7 family of amino acid transporters

The SLC7 family is divided into two subgroups, the cationic amino acid transporters (the CAT family, SLC7A1–4) and the glycoprotein-associated amino acid transporters (the gpaAT family, SLC7A5–11), also called light chains or catalytic chains of the hetero(di)meric amino acid transporters (HAT). The associated glycoproteins (heavy chains) 4F2hc (CD98) or rBAT (D2, NBAT) form the SLC3 family. Members of the CAT family transport essentially cationic amino acids by facilitated diffusion with differential trans-stimulation by intracellular substrates. In some cells, they may regulate the rate of NO synthesis by controlling the uptake of l-arginine as the substrate for nitric oxide synthase (NOS). The heterodimeric amino acid transporters are, in contrast, quite diverse in terms of substrate selectivity and function (mostly) as obligatory exchangers. Their selectivity ranges from large neutral amino acids (system L) to small neutral amino acids (ala, ser, cys-preferring, system asc), negatively charged amino acid (system x_c^–) and cationic amino acids plus neutral amino acids (system y⁺L and b^0,+-like). Cotransport of Na⁺ is observed only for the y⁺L transporters when they carry neutral amino acids. Mutations in b^0,+-like and y⁺L transporters lead to the hereditary diseases cystinuria and lysinuric protein intolerance (LPI), respectively.     

Quantitative Fermentbestimmungen in roten Blutzellen

Zusammenfassung 1. Es wurde in gewaschenen Erythrocyten von Gesunden der Gehalt an Aldolase, Trioseisomerase, Phosphoglyceraldehyddehydrogenase, Milchsäuredehydrogenase und Hämiglobinreduktasesystem bestimmt.2. Die gefundenen Fermentaktivitäten wurden auf die Zellzahl, den Hämoglobingehalt und das Erythrocyteneinzelvolumen berechnet.3. Unter Zugrundelegung der Umsatzzahlen für kristalline Fermente wurde der Anteil der einzelnen Gärungsfermentproteine am Gesamteiweiß und Nichthämoglobineiweiß der Erythrocyten bestimmt und mit den entsprechenden Daten des Muskels verglichen.4. Im Erythrocyten und Reticulocyten wurden weder löslichel--Glycerophosphatdehydrogenase (Baranowski-Enzym) noch strukturgebundenel--Glycerophosphatdehydrogenase (Green-Enzym) gefunden.l--Glycerophosphat war ebenfalls nicht nachweisbar.5. Die begrenzenden Reaktionsstufen der Glykolyse im Erythrocyten sind nach unseren Fermentuntersuchungen die Aldolase- und die oxydierende Gärungsfermentreaktion.     

Roles of organic anion transporters (OATs) in renal proximal tubules and their localization

Organic anions (OAs) are secreted in renal proximal tubules in two steps. In the first step, OAs are transported from the blood through basolateral membranes into proximal tubular cells. The prototypical substrate for renal organic anion transport systems, para-aminohippurate (PAH), is transported across basolateral membranes of proximal tubular cells via OAT1 (SLC22A6) and OAT3 (SLC22A8) against an electrochemical gradient in exchange for intracellular dicarboxylates. In the second step, OAs exit into urine through apical membranes of proximal tubules. This step is thought to be performed by multidrug efflux transporters and a voltage-driven organic anion transporter. However, the molecular nature and precise functional properties of these efflux systems are largely unknown. Recently, we characterized an orphan transporter known as human type I sodium-phosphate transporter 4, hNPT4 (SLC17A3), using the Xenopus oocyte expression system. hNPT4 acts as a voltage-driven efflux transporter (“human OATv1”) for several OAs such as PAH, estrone sulfate, diuretic drugs, and urate. Here, we describe a model for an OA secretory pathway in renal tubular cells in which OAs exit cells and enter the tubular lumen via hOATv1 (hNPT4). Additionally, hOATv1 functions as a common renal secretory pathway for both urate and drugs, indicating that hOATv1 may be a leak pathway for excess urate that is reabsorbed via apical URAT1 to control the intracellular urate levels. Therefore, we propose a molecular mechanism for the induction of hyperuricemia by diuretics: the diuretics enter proximal tubular cells via basolateral OAT1 and/or OAT3 and may then interfere with the NPT4-mediated apical urate efflux in the renal proximal tubule.     

Glutamate cycling may drive organic anion transport on the basal membrane of human placental syncytiotrophoblast

Key points

• The placenta removes waste products, drugs and environmental toxins from the fetal circulation and two of the transport proteins responsible for this are OAT4 and OATP2B1 localised to the basal membrane of placental syncytiotrophoblast.
• We provide evidence that OAT4 and OATP2B1 mediate glutamate efflux when expressed in Xenopus oocytes and that in the perfused placenta, bromosulphothalein (an OAT4 and OATP2B1 substrate) stimulates glutamate efflux.
• Furthermore the efflux of glutamate can only be seen in the presence of aspartate, which will block glutamate reuptake by the placenta, consistent with cycling of glutamate across the basal membrane.
• We propose that glutamate efflux down its transmembrane gradient drives placental uptake via OAT4 and OATP2B1 from the fetal circulation and that reuptake of glutamate maintains this driving gradient.

Abstract

The organic anion transporter OAT4 (SLC22A11) and organic anion transporting polypeptide OATP2B1 (SLCO2B1) are expressed in the basal membrane of the placental syncytiotrophoblast. These transporters mediate exchange whereby uptake of one organic anion is coupled to efflux of a counter‐ion. In placenta, these exchangers mediate placental uptake of substrates for oestrogen synthesis as well as clearing waste products and xenobiotics from the fetal circulation. However, the identity of the counter‐ion driving this transport in the placenta, and in other tissues, is unclear. While glutamate is not a known OAT4 or OATP2B1 substrate, we propose that its high intracellular concentration has the potential to drive accumulation of substrates from the fetal circulation. In the isolated perfused placenta, glutamate exchange was observed between the placenta and the fetal circulation. This exchange could not be explained by known glutamate exchangers. However, glutamate efflux was trans‐stimulated by an OAT4 and OATP2B1 substrate (bromosulphothalein). Exchange of glutamate for bromosulphothalein was only observed when glutamate reuptake was inhibited (by addition of aspartate). To determine if OAT4 and/or OATP2B1 mediate glutamate exchange, uptake and efflux of glutamate were investigated in Xenopus laevis oocytes. Our data demonstrate that in Xenopus oocytes expressing either OAT4 or OATP2B1 efflux of intracellular [¹⁴C]glutamate could be stimulated by conditions including extracellular glutamate (OAT4), estrone‐sulphate and bromosulphothalein (both OAT4 and OATP2B1) or pravastatin (OATP2B1). Cycling of glutamate across the placenta involving efflux via OAT4 and OATP2B1 and subsequent reuptake will drive placental uptake of organic anions from the fetal circulation.

Abbreviations

BM

basal membrane

BSP

bromosulphothalein

DHEAS

dehydroepiandrosterone‐3‐sulfate

OAT

organic anion transporter

OATP

organic anion transporting polypeptide

PAH

para‐aminohippuric acid

     

Asymmetry in the transport of lactate by basolateral and brush border membranes of rat kidney cortex

The uptake ofl(+)lactate into rat renal cortical brush border (BBV) and basolateral (BLV) membrane vesicles, isolated through differential centrifugation and free flow electrophoresis, were studied using a rapid filtration technique. In contrast to the lactate transport into the BBV, that into the BLV: 1) was found to proceed only towards equilibrium, 2) showed Na⁺-independent coupling of the influx ofl(+)lactate and the efflux ofl(+) but not to the efflux ofd(–)lactate, 3) was not inhibited byd(–)lactate, 2-thiolactate or 3-phenyl-lactate, but 4) was inhibited by 3-thiolactate and -hydroxybutyrate and 5) was accelerated by changes in inwardly directed ionic gradients or by increases in cation conductance both of which led to increased intravesicular positivity. The latter changes had the opposite effect on the uptake ofl(+)lactate by BBV. Thus, while thel(+)lactate transport system present in BBV showed the characteristics of Na-dependent electrogenic cotransport system, that in the BLV was consistent with a carrier mediated Na-independent, facilitated diffusion system.     

Assignment of the gene locus for human α-l-fucosidase to chromosome 1 by analysis of somatic cell hybrids

The -l-fucosidases (EC 3.2.1.51) from human and mouse cells could be separated by isoelectric focusing of neuraminidase-treated cell extracts in acrylamide slab gels. Fourteen hybrid clones derived from the fusion of mouse and human cultured fibroblasts and 37 hybrid clones derived from the fusion of human long-term lymphoid lines with mouse RAG cells were tested for expression of human -l-fucosidase. A strong correlation between the expression of the human enzyme and the presence or absence of human chromosome 1 was found. The presence of human -l-fucosidase in clones scored as positive by isoelectric focusing was confirmed by Ouchterlony double immunodiffusion against IgG from rabbits immunized with purified human -l-fucosidase. It is concluded that the structural gene locus for human -l-fucosidase is located on chromosome 1.     

Branched-chain aminotransferase deficiency in Chinese hamster cells complemented by two independent genes on human chromosomes 12 and 19

Branched-chain aminotransferase (BCT) catalyzes the reversible transamination of the branched-chain -keto acids to the branched-chainl-amino acids. Since branched-chainl-amino acids (l-isoleucine,l-leucine, andl-valine) are essential for cell growth, cells which lack BCT were unable to proliferate in media containing -keto acids in place of the correspondingl-amino acids. CHW-1102, a Chinese hamster cell line, lacks BCT and does not grow in -keto acid media. Somatic cell hybrids were made by the fusion of CHW-1102 (HPRT^–) with several human cell lines and isolated on HAT medium. Growth assays of hybrid clones on -keto acid selection media independent of the HAT selection medium indicated two cell hybrid phenotypes: either (1) the hybrid clone, like the parental CHW-1102, could not utilize -keto acid media, or (2) the hybrid could proliferate on all three -keto acid media. The ability of hybrid cells to proliferate on -keto acid media correlated with the presence of either of two human genes which independently complemented the Chinese hamster deficiency. Two human genes, BCT1 assigned to chromosome 12 and BCT2 assigned to chromosome 19, were demonstrated to code for the expression of two molecular forms of BCT.A preliminary abstract of this work has been presented at the Human Gene Mapping Workshop V [Naylor, S.L. and Shows, T.B. (1979).Cytogenet. Cell Genet. 25:191–192].     

Molecular properties of the SLC13 family of dicarboxylate and sulfate transporters

The SLC13 gene family consists of five members in humans, with corresponding orthologs from different vertebrate species. All five genes code for sodium-coupled transporters that are found on the plasma membrane. Two of the transporters, NaS1 and NaS2, carry substrates such as sulfate, selenate and thiosulfate. The other members of the family (NaDC1, NaDC3, and NaCT) are transporters for di- and tri-carboxylates including succinate, citrate and -ketoglutarate. The SLC13 transporters from vertebrates are electrogenic and they produce inward currents in the presence of sodium and substrate. Substrate-independent leak currents have also been described. Structure–function studies have identified the carboxy terminal half of these proteins as the most important for determining function. Transmembrane helices 9 and 10 may form part of the substrate permeation pathway and participate in conformational changes during the transport cycle. This review also discusses new members of the SLC13 superfamily that exhibit both sodium-dependent and sodium-independent transport mechanisms. The Indy protein from Drosophila, involved in determining lifespan, and the plant vacuolar malate transporter are both sodium-independent dicarboxylate transporters, possibly acting as exchangers. The purpose of this review is to provide an update on new advances in this gene family, particularly on structure–function studies and new members of the family.     

An α-mating-type-specific mutation causing specific defect in sexual agglutinability in the yeast Saccharomyces cerevisiae

We have identified a recessive -mating-type-specific gene agl causing agglutinability defect without significant effects on other sexual activities. a cells carrying agl showed sexual agglutination with cells but cells carrying agl showed sexual agglutination with neither cells nor a cells. cells carrying agl produced pheromone and responded to a pheromone just like wild cells. cells carrying agl showed a little decreased but significant mating ability when tested on solid media or membrane filter. The agl mutant is different from any -specific ste mutants found so far in many sexual activities. The agl gene is recessive, and unlinked to the mating type locus. Biological significance of the mating type agglutinability is discussed based on the results obtained with the mutant.     

The limitations of renal epithelial cell line HK-2 as a model of drug transporter expression and function in the proximal tubule

Acquiring a mechanistic understanding of the processes underlying the renal clearance of drug molecules in man has been hampered by a lack of robust in vitro models of human proximal tubules. Several human renal epithelial cell lines derived from the renal cortex are available, but few have been characterised in detail in terms of transporter expression. This includes the HK-2 proximal tubule cell line, which has been used extensively as a model of nephrotoxicity. The aim of this study was to investigate the expression and function of drug transporters in HK-2 cells and their suitability as an in vitro model of the human proximal tubule. qPCR showed no mRNA expression of the SLC22 transporter family (OAT1, OAT3, OCT2) in HK-2 cells compared to renal cortex samples. In contrast, SLC16A1 (MCT1), which is important in the uptake of monocarboxylates, and SLCO4C1 (OATP4C1) were expressed in HK-2 cells. The functional expression of these transporters was confirmed by uptake studies using radiolabelled prototypic substrates dl-lactate and digoxin, respectively. The mRNA expression of apical membrane efflux transporters ABCB1 (MDR1) and several members of the ABCC family (multidrug resistance proteins, MRPs) was shown by qPCR. ABCG1 (BCRP) was not detected. The efflux of Hoechst 33342, a substrate for MDR1, was blocked by MDR1 inhibitor cyclosporin A, suggesting the functional expression of this transporter. Similarly, the efflux of the MRP-specific fluorescent dye glutathione methylfluorescein was inhibited by the MRP inhibitor MK571. Taken together, the results of this study suggest that HK-2 cells are of limited value as an in vitro model of drug transporter expression in the human proximal tubule.     

Interactions of P 1507, a new antioxidant agent,with phagocyte functions

Toxic oxygen free radicals are believed to play a role in the pathogenesis of a number of respiratory diseases. In particular, pulmonary emphysema may occur because of the oxidative impairment of 1-proteinase inhibitor (1-PI). We reportin vitro data on a new thiol agent, P 1507 [N-5-(thioxo-l-prolyl)-l-cysteine], obtained in a series of experiments designed in view of its therapeutic potential in these clinical conditions. We found that P 1507 at the concentration of 5×10^–6 M was able to almost fully abolish the PMA-triggered PMN-induced oxidative impairment of 1-PI. Protection may be due to the radical scavenger ability P 1507, that markedly reduced superoxide anion production from PMNs. We also found that P 1507 did not significantly impair other defence mechanisms of PMNs (i.e. phagocytosis, chemotaxis and bactericidal activity). The release of cytokines (TNF-, IL-6 and IL-8) from monocytes was not altered in the presence of P 1507. We conclude that the compound P 1507 may be considered for treatment of clinical conditions characterized by overload of oxidants, on the basis of its ability in preventing the oxidative damage of 1-PI and of a lack of unwanted inhibitory effects towards defence mechanisms of phagocytes.     

Effects of propionyl-l-carnitine and insulin on the electroretinogram,nerve conduction and nerve blood flow in rats with streptozotocin-induced diabetes

The effect of an analogue ofl-carnitine, propionyl-l-carnitine, on the electroretinogram, motor nerve conduction velocity and nerve blood flow was determined in rats with streptozotocin-induced diabetes, and was compared with the effects of insulin alone or combined therapy. Oral administration of propionyl-l-carnitine (3 g/kg daily for 4 weeks) significantly increased caudal nerve motor conduction velocity and sciatic nerve blood flow in diabetic rats. There were no differences in the effects of insulin (8–10 U daily for 4 weeks), propionyl-l-carnitine and combined therapy. Although propionyl-l-carnitine significantly shortened the peak latency of the electroretinogram b-wave in diabetic rats, its effect was far weaker than that of insulin or combined therapy, with combined therapy producing the greatest improvement. These effects of propionyl-l-carnitine were accompanied by a decrease of serum lipid levels, an increase of the sciatic nerve carnitine content, and no changes of the tissue (nerve and retinal) sorbitol andmyo-inositol concentrations. In contrast, insulin significantly reduced the tissue sorbitol content and markedly increasedmyo-inositol. These findings suggest that propionyl-l-carnitine may improve diabetic neuropathy and retinopathy without influencing the polyol pathway, and that this beneficial effect may be mediated through the amelioration of microcirculation and tissue carnitine content, thus probably increasing fatty acid oxidation.     

Histamine formation in guinea-pig brain and other tissues: Effect of α-methyl dopa

Pretreatment of guinea pigs with -methyl-l-dopa, an inhibitor of a non-specific decarboxylase, followed by injection of¹⁴C-l-histidine, had little effect on¹⁴C-histamine tissue levels relative to controls. Since earlier longterm tests showed that comparable doses of inhibitor markedly reduced urinary¹⁴C-histamine, it is tentatively concluded that the non-specific enzyme decarboxylatesl-histidine primarily in kidney, at a site from which newly-formed histamine is excreted directly, without entering the blood stream.¹⁴C-histamine levels in brain were relatively high and provide the strongest available evidence that histamine is formed in brain under physiological conditions.Supported by NIH Grant AM-10155.     

Influence of histamine H3-antagonists on human leukocytes

To determine the role of the histamine H₃-receptor on basophils, different specific H₃-antagonists were investigated. Incubation of washed leukocytes with N-acylated histamine-derivatives (N-ahd) induced elevated histamine levels. This process turned out to be dependent on dose, time and temperature, but independent of Ca²⁺ and Mg²⁺ ions. IgE-mediated histamine release was not modulated. [³H]-l-histidine was not decarboxylated into [³H]-histamine in spite of the observed histamine increase. Highly purified basophils did not show any histamine elevation but purified neutrophils and eosinophils were found to have increased histamine levels even after disintegration and subsequent incubation with N-ahd. It seems that the increased histamine levels result from the cleavage of the applied histamine amides. Other potent H₃-antagonists (e.g. thioperamide) neither produced increased histamine levels nor influenced IgE-mediated release from basophil leukocytes. The existence of H₃-receptors on human basophils therefore seems unlikely.This work was supported by Grant No. KI 622/1-1 from the Deutsche Forschungsgemeinschaft, Bonn, FRG.     

Substrate utilization in the isolated perfused cortical thick ascending limb of rabbit nephron

Isolated segments of cortical thick ascending limbs (cTAL) of rabbit kidney were perfused in vitro and the equivalent short circuit current (Isc) was measured. In a first series all substrates were removed on either side. Isc fell rapidly to 50±12% after 3 min and to 27±6% (n=5) after 10 min. This indicates that in cTAL segments Isc is strictly dependent on the presence of substrates. In series two it was tested what substrates can be utilized by the cTAL segment, and from which epithelial side [bath (b) or lumen (l)] the substrates are taken up. From the l-side only butyrate (10 mmol · l^–1) sustained the Isc at 95±2% (n=7). All other tested substrates (10 mmol · l^–1): pyruvate, acetate, -OH-butyrate,d-glucose, andl-lactate lead to a marked decline in Isc. From the b-side several substrates (5–10 mmol · l^–1) sustained the Isc:d-glucose,d-mannose, butyrate, -OH-butyrate, acetoacetate,l-lactate, acetate and pyruvate. Other compounds (1–10 mmol · l^–1): citrate, -ketoglutarate, succinate, glutamate, glutamine, propionate, caprylate and oleate did not sustain Isc. In the third series the mechanism of substrate utilization from the basolateral cell side was studied. It was shown that the Isc is a saturable function of thed-glucose,l-lactate, acetate, pyruvate or -OH-butyrate concentration with apparentK _m's between 0.05–1.0 mmol · l^–1. Several known inhibitors of sugar and of anion transport were tested at the bath side: phlorrhizin was without effect. Phloretin (500 mol · l^–1) inhibited Isc by 96%, yet its effect was not dependent on the presence of substrates on the b-side since inhibition ocurred also if the b-perfusate contained no substrate and Isc was driven by luminal butyrate. Also SITS (5 mmol · l^–1) exerted only a small inhibitory effect which was not specific since it was also observed with luminal butyrate. -Cyano-m-OH-cinnamate (10 mmol · l^–1) inhibited the Isc specifically whenl-lactate was the bath substrate. Probenecid (1 mmol · l^–1) had a similar yet less marked inhibitory effect. Thed-glucose uptake from the b-side was specifically inhibited by cytochalasin B at 5 · 10^–6 mol · l^–1. We conclude that the cTAL segment of the rabbit utilizesd-glucose and/or small anions such as pyruvate orl-lactate or acetate to energize salt reabsorption. The link between substrate availability and salt reabsorption is extremely close in this nephron segment. Substrate uptake occurs from the blood side. Sugar uptake can be inhibited by cytochalasin B andl-lactate uptake by probenecid and -cyano-m-OH-cinnamat. These data suggest that substrate uptake at the basolateral cell side occurs probably via carrier systems.Parts of this study have been presented at the 57th and 58th Tagung Deutsche Physiologische Gesellschaft, 17th Meeting Europ. Soc. Clin. Investigation, and XXIX Int. Congress Physiol. Sciences. This study was supported by Deutsche Forschungsgemeinschaft Gr 480/5-7     

Organic Cation Transporter 2 Mediates Cisplatin-Induced Oto- and Nephrotoxicity and Is a Target for Protective Interventions

The use of the effective antineoplastic agent cisplatin is limited by its serious side effects, such as oto- and nephrotoxicity. Ototoxicity is a problem of special importance in children, because deafness hampers their language and psychosocial development. Recently, organic cation transporters (OCTs) were identified in vitro as cellular uptake mechanisms for cisplatin. In the present study, we investigated in an in vivo model the role of OCTs in the development of cisplatin oto- and nephrotoxicity. The functional effects of cisplatin treatment on kidney (24 hours excretion of glucose, water, and protein) and hearing (auditory brainstem response) were studied in wild-type and OCT1/2 double-knockout (KO) mice. No sign of ototoxicity and only mild nephrotoxicity were observed after cisplatin treatment of knockout mice. Comedication of wild-type mice with cisplatin and the organic cation cimetidine protected from ototoxicity and partly from nephrotoxicity. For the first time we showed that OCT2 is expressed in hair cells of the cochlea. Furthermore, cisplatin-sensitive cell lines from pediatric tumors showed no expression of mRNA for OCTs, indicating the feasibility of therapeutic approaches aimed to reduce cisplatin toxicities by competing OCT2-mediated cisplatin uptake in renal proximal tubular and cochlear hair cells. These findings are very important to establish chemotherapeutical protocols aimed to maximize the antineoplastic effect of cisplatin while reducing the risk of toxicities.Cisplatin, one of the most effective and potent anticancer drugs, is used in the treatment of a wide variety of both pediatric and adult malignancies.¹ When combined with bleomycin and etoposide, cisplatin is considered to be curative treatment for testicular cancer.² However, the chemotherapeutic use of cisplatin is limited by serious side effects, such as nephrotoxicity and ototoxicity, sometimes requiring a reduction in dose or discontinuation of treatment. Even though nephrotoxicity can be managed with some success by concomitant use of i.v. hydration, it is still a major factor that limits the administration and efficacy of cisplatin in cancer therapy.³ Ototoxicity remains an unresolved clinical problem, especially in infants and younger children, where it leads to a considerable risk of delayed language development because of impaired perception of higher frequency consonant sounds that is of great importance in the presence of background noise. This may have devastating consequences for a young child''s social and educational development.⁴The molecular mechanisms of cellular cisplatin toxicity have been investigated intensively.² The ototoxic effects of cisplatin include loss of outer hair cells, blebbing of outer and inner hair cells, degeneration of the stria vascularis, and a decrease in the number of spiral ganglion cells.⁵ In the kidney, cisplatin highly accumulates in cells of the terminal proximal tubule and of the distal nephron, where it causes either apoptosis or necrosis, depending on exposure time and concentration.² Recently, some attention has been paid to the role of specific cellular uptake processes in cisplatin toxicity. The copper transporter (Ctr)1 seems to be involved in cisplatin accumulation in tumor cells⁶ as well as in the renal epithelial cells.⁷ However, the cisplatin uptake into renal tubular cells is also mediated by organic cation transporter (OCT)2 and the function of OCT2 is critically involved in the development of toxicity.^8,9 In humans, OCT2 (hOCT2) is highly expressed at the basolateral side of all three segments of the proximal tubule.¹⁰ It represents a subtype of three OCTs that belong to the SLC22 transporter family. OCTs are highly expressed in intestine, liver, and/or kidney and play a pivotal role in drug absorption and excretion.¹¹ They are polyspecific, electrogenic uniporters that may operate in both directions. Because the membrane potential provides part of the driving force, they preferably mediate organic cation uptake into cells at normal membrane potential.¹² OCT2 has been proposed as target for protective therapeutic interventions accompanying cisplatin treatment. In vitro studies showed that administration of cisplatin together with a second substrate of organic cation transporters decreases cellular cisplatin accumulation protecting renal cells from cisplatin toxicity.^8,9In the present study, we demonstrated in an in vivo model that OCT2 plays a pivotal role in the development of cisplatin induced oto- and nephrotoxicity. For the first time we showed that cisplatin ototoxicity is linked to the expression of OCT2 in hair cells of the cochlea. Furthermore, the feasibility of a therapeutic approach aimed to reduce cisplatin toxicities has been demonstrated in vivo. These findings are very important to establish chemotherapeutic protocols aimed to maximize the antineoplastic effect of cisplatin while reducing the risk of toxicities.     

Molecular specificity of the tubular resorption of “acidic” amino acids

Single sections of superficial proximal convolutions of rat kidney were microperfused in vivo and in situ. The perfusion fluids contained radioactively labelledl- ord-aspartate,l-glutamate,l-pyroglutamate, or N-methyl-d-aspartate.l--Carboxyglutamate as well as the other amino acids were added in the unlabelled from. Results.l- andd-Aspartate (0.073 mmol·1^–1) are quickly resorbed at about the same rate.d-Aspartate resorption was blocked byl-aspartate (5 mmol·1^–1) but not by -alanine (5 mmol·1^–1).l-Aspartate resorption was inhibited byl-glutamate (2 mmol·1^–1) but not byd-glutamate,l-asparagine,l-phenylalanine or by succinate (2 mmol·1^–1, each). The fast resorption ofl-glutamate (0.073 mmol·1^–1) was blocked byd-aspartate,l-cysteate (2 mmol·1^–1), but not by 3-mercaptopicolinic acid (0.15 mmol·1^–1),l-glutamine, 2-oxoglutarate, taurine, N-methyl-l-glutamate or kainic acid (2 mmol·1^–1, each).l--Carboxyglutamate (0.66 mmol·1^–1) and N-methyl-d-aspartate (2mol·1^–1) were found to be resorbed only at an extremely small rate.l-pyroglutamate (0.076 mmol·1^–1) resorption was not influenced byl-glutamate (1 mmol·1^–1). Fractional excretion of -carboxyglutamate was 7–25% (l-from) or 45–70% (d-form) at an artificially elevated plasma level of 12mol·1^–1.It is concluded thatl- andd-aspartate,l-glutamate,l-cysteate and, to a much smaller extent,l--carboxyglutamate, are accepted by the tubular resorption mechanism highly specific for acidic amino acids. N-Substitution, the amidation of the - or -carboxyl group, or the removal of the -amino moiety almost completely abolish the ability of such compounds to be resorbed via this carrier; N-methylated or -carboxylated derivatives of acidic amino acids are not resorbed at all from the proximal tubule. The resorption of glutamate, but not of aspartate, is highly stereospecific.Parts of this work were presented at meetings of the German Physiological Society in 1978 [28] and of the Gesellschaft für Nephrologie in 1980 [29] as well as at the VIIIth International Congress of Nephrology in Athens in 1981 [26]with technical assistance of Angelika Ascher and Gertaud Vetter