Identification of the TPO1 gene in yeast, and its human orthologue TETRAN, which cause resistance to NSAIDs

Non-steroidal anti-inflammatory drugs (NSAIDs), such as indomethacin, have serious gastrointestinal side effects. Since their direct cytotoxicity was suggested to be involved in this side effect, we here tried to identify NSAID-resistant genes. We screened for Saccharomyces cerevisiae genes whose overexpression causes indomethacin resistance and identified the TPO1 gene, which encodes a major facilitator superfamily transporter. Its overexpression or deletion made yeast cells resistant or sensitive, respectively, to some NSAIDs. A BLAST search identified the possible human orthologue of Tpo1p, tetracycline transporter-like protein (TETRAN), whose overexpression in cultured human cells caused resistance to some NSAIDs, suggesting that TETRAN is an efflux pump for some NSAIDs.     

Cloning and functional characterization of human SMCT2 (SLC5A12) and expression pattern of the transporter in kidney

Recently, we cloned two Na⁺-coupled lactate transporters from mouse kidney, a high-affinity transporter (SMCT1 or slc5a8) and a low-affinity transporter (SMCT2 or slc5a12). Here we report on the cloning and functional characterization of human SMCT2 (SLC5A12) and compare the immunolocalization patterns of slc5a12 and slc5a8 in mouse kidney. The human SMCT2 cDNA codes for a protein consisting of 618 amino acids. When expressed in mammalian cells or Xenopus oocytes, human SMCT2 mediates Na⁺-coupled transport of lactate, pyruvate and nicotinate. The affinities of the transporter for these substrates are lower than those reported for human SMCT1. Several non-steroidal anti-inflammatory drugs inhibit human SMCT2-mediated nicotinate transport, suggesting that NSAIDs interact with the transporter as they do with human SMCT1. Immunofluorescence microscopy of mouse kidney sections with an antibody specific for SMCT2 shows that the transporter is expressed predominantly in the cortex. Similar studies with an anti-SMCT1 antibody demonstrate that SMCT1 is also expressed mostly in the cortex. Dual-labeling of SMCT1 and SMCT2 with 4F2hc (CD98), a marker for basolateral membrane of proximal tubular cells in the S1 and S2 segments of the nephron, shows that both SMCT1 and SMCT2 are expressed in the apical membrane of the tubular cells. These studies also show that while SMCT2 is broadly expressed along the entire length of the proximal tubule (S1/S2/S3 segments), the expression of SMCT1 is mostly limited to the S3 segment. These studies suggest that the low-affinity transporter SMCT2 initiates lactate absorption in the early parts of the proximal tubule followed by the participation of the high-affinity transporter SMCT1 in the latter parts of the proximal tubule.     

Amino acid cotransporter SLC3A2 is selectively expressed in the early proximal segment of Xenopus pronephric kidney nephrons

The transport of amino acids across membranes is critical to all cells. As amino acids freely pass through the glomerular filtration barrier of the kidney, they must be efficiently resorbed to avoid depletion of circulating amino acid reserves. Not only do defects in amino acid resorption lead to costly wastage, they also cause congenital aminoacidurias. A clone encoding Xenopus SLC3A2 was identified and shown to be expressed at high levels in the early segment of the pronephric proximal tubules in developing tadpoles. The type II membrane glycoprotein encoded by this gene can associate with a wide variety of protein partners and participates in a broad spectrum of biological processes. In this report, the first whole-mount analysis of SLC3A2 during early embryonic development is presented. The expression pattern of SLC3A2 in the early proximal segment of the Xenopus pronephros is analogous to that of a previously described SLC7A8/XAA2 amino acid transporter. In mammals, SLC3A2 and SLC7A8/XAA2 associate to form a functional neutral amino acid transporter complex and coexpression of these two genes in a small domain within the pronephric tubules indicates that this is also the situation in the developing Xenopus kidney.     

Diclofenac-induced stimulation of SMCT1 (SLC5A8) in a heterologous expression system: A RPE specific phenomenon

SMCT1 is a Na⁺-coupled monocarboxylate transporter expressed in a variety of tissues including kidney, thyroid, small intestine, colon, brain, and retina. We found recently that several non-steroidal anti-inflammatory drugs (NSAIDs) inhibit the activity of SMCT1. Here we evaluated the effect of diclofenac, also a NSAID, on SMCT1. SMCT1 cDNA was expressed heterologously in the human retinal pigment epithelial cell lines HRPE and ARPE-19, the human mammary epithelial cell line MCF7, and in Xenopus laevis oocytes. Transport was monitored by substrate uptake and substrate-induced currents. Na⁺-dependent uptake/current was considered as SMCT1 activity. The effect of diclofenac was evaluated for specificity, dose-response, and influence on transport kinetics. To study the specificity of the diclofenac effect, we evaluated the influence of this NSAID on the activity of several other cloned transporters in mammalian cells under identical conditions. In contrast to several NSAIDs that inhibited SMCT1, diclofenac stimulated SMCT1 when expressed in HRPE and ARPE-19 cells. The stimulation was marked, ranging from 2- to 5-fold depending on the concentration of diclofenac. The stimulation was associated with an increase in the maximal velocity of the transport system as well as with an increase in substrate affinity. The observed effect on SMCT1 was selective because the activity of several other cloned transporters, when expressed in HRPE cells and studied under identical conditions, was not affected by diclofenac. Interestingly, the stimulatory effect on SMCT1 observed in HRPE and ARPE-19 cells was not evident in MCF7 cells nor in the X. laevis expression system, indicating that SMCT1 was not the direct target for diclofenac. The RPE-specific effect suggests that the target of diclofenac that mediates the stimulatory effect is expressed in RPE cells but not in MCF7 cells or in X. laevis oocytes. Since SMCT1 is a concentrative transporter for metabolically important compounds such as pyruvate, lactate, β-hydroxybutyrate, and nicotinate, the stimulation of its activity by diclofenac in RPE cells has biological and clinical significance.     

p-aminohippuric acid transport at renal apical membrane mediated by human inorganic phosphate transporter NPT1

Organic anions are secreted into urine via organic anion transporters across the renal basolateral and apical membranes. However, no apical membrane transporter for organic anions such as p-aminohippuric acid (PAH) has yet been identified. In the present study, we showed that human NPT1, which is present in renal apical membrane, mediates the transport of PAH. The K(m) value for PAH uptake was 2.66 mM and the uptake was chloride ion sensitive. These results are compatible with those reported for the classical organic anion transport system at the renal apical membrane. PAH transport was inhibited by various anionic compounds. Human NPT1 also accepted uric acid, benzylpenicillin, faropenem, and estradiol-17beta-glucuronide as substrates. Considering its chloride ion sensitivity, Npt1 is expected to function for secretion of PAH from renal proximal tubular cells. This is the first molecular demonstration of an organic anion transport function for PAH at the renal apical membrane.     

Expression and localization of aquaporins in the kidney of the musk shrew (Suncus murinus).

Expression and localization of members of the aquaporin (AQP) family (AQP1, 2, 3, 4, and 5) in the kidney of the musk shrew (Suncus murinus) was examined by immunohistochemistry. AQP1 was expressed in the proximal tubules and in the thin limb of the loops of Henle. AQP1 was the only water channel expressed in the proximal nephron examined, indicating that AQP1 may be an independent water transporter in the proximal nephron. AQP2 and AQP5 were localized to the apical cytoplasm of the cortical to medullary collecting duct (CD) cells and AQP3 and AQP4 were localized to the basal aspect of the cortical to medullary CD cells. AQP3 expression was weaker in the cortical cells compared with the medullary cells, whereas AQP4 was strongly positive throughout the CD. These indicate that the CD is the main water reabsorption segment of the nephron and is regulated by AQPs. Indeed, apical water transport of CD cells of the musk shrew may be controlled by both AQP2 and AQP5. The characteristic expression pattern of the AQPs in this animal provides a novel animal model for elucidating the regulation of water reabsorption by AQPs in the mammalian kidney.     

Luminal heterodimeric amino acid transporter defective in cystinuria

Mutations of the glycoprotein rBAT cause cystinuria type I, an autosomal recessive failure of dibasic amino acid transport (b(0,+) type) across luminal membranes of intestine and kidney cells. Here we identify the permease-like protein b(0,+)AT as the catalytic subunit that associates by a disulfide bond with rBAT to form a hetero-oligomeric b(0,+) amino acid transporter complex. We demonstrate its b(0,+)-type amino acid transport kinetics using a heterodimeric fusion construct and show its luminal brush border localization in kidney proximal tubule. These biochemical, transport, and localization characteristics as well as the chromosomal localization on 19q support the notion that the b(0,+)AT protein is the product of the gene defective in non-type I cystinuria.     

Regulation of renal proximal and distal tubule transport: sodium, chloride and organic anions

Renal tubular transport and its regulation are reviewed for Na(+) (and Cl(-)), and for fluid and organic anions (including urate). Filtered Na(+) (and Cl(-)) is reabsorbed along the tubules but only in mammals and birds does most reabsorption occur in the proximal tubules. Reabsorption involves active transport of Na(+) and passive reabsorption of Cl(-). The active Na(+) step always involves Na-K-ATPase at the basolateral membrane, but the entry step at luminal membrane varies among tubule segments and among vertebrate classes (except for Na(+)-2Cl(-)-K(+) cotransporter in diluting segment). Regulation can involve intrinsic, neural and endocrine factors. Proximal tubule fluid reabsorption is dependent on Na(+) reabsorption in all vertebrates studied, except ophidian reptiles. Fluid secretion occurs in glomerular and aglomerular fishes, reptiles and even mammals, but its significance is not always clear. A non-specific transport system for net secretion of organic anions (OAs) exists in the proximal renal tubules of almost all vertebrates. Net transepithelial secretion involves: (1) transport into the cells at the basolateral side against an electrochemical gradient by a tertiary active transport process, in which the final step involves OA/alpha-ketoglutarate exchange and (2) movement out of the cells across the luminal membrane down an electrochemical gradient by unknown carrier-mediated process(es). Regulation may involve protein kinase C and mitogen-activated protein kinase. Urate is net secreted in the proximal tubules of birds and reptiles. This process is urate-specific in reptiles but in birds, it may involve both a urate-specific system and the general OA system.     

Loss of ATP-dependent transport activity in pseudoxanthoma elasticum-associated mutants of human ABCC6 (MRP6)

Mutations in the ABCC6 (MRP6) gene cause pseudoxanthoma elasticum (PXE), a rare heritable disorder resulting in the calcification of elastic fibers. In the present study a cDNA encoding a full-length normal variant of ABCC6 was amplified from a human kidney cDNA library, and the protein was expressed in Sf9 insect cells. In isolated membranes ATP binding as well as ATP-dependent active transport by ABCC6 was demonstrated. We found that glutathione conjugates, including leukotriene C(4) and N-ethylmaleimide S-glutathione (NEM-GS), were actively transported by human ABCC6. Organic anions (probenecid, benzbromarone, indomethacin), known to interfere with glutathione conjugate transport of human ABCC1 and ABCC2, inhibited the ABCC6-mediated NEM-GS transport in a specific manner, indicating that ABCC6 has a unique substrate specificity. We have also expressed three missense mutant forms of ABCC6, which have recently been shown to cause PXE. MgATP binding was normal in these proteins; ATP-dependent NEM-GS or leukotriene C(4) transport, however, was abolished. Our data indicate that human ABCC6 is a primary active transporter for organic anions. In the three ABCC6 mutant forms examined, the loss of transport activity suggests that these mutations result in a PXE phenotype through a direct influence on the transport activity of this ABC transporter.     

Molecular characterization of the renal organic anion transporter 1

Organic anions of diverse chemical structures are secreted in renal proximal tubules. The first step in secretion, uptake of organic anions across the basolateral membrane of tubule cells, is mediated for the polyspecific organic anion transporter 1 (OAT1), which exchanges extracellular organic anions for intracellular α-ketoglutarate or glutarate. OAT1 orthologs cloned from various species show 12 putative transmembrane domains and possess several sites for potential post-translational modification. The gene for the human OAT1 is located on chromosome 11q13.1 and is composed of 10 exons. Alternative splicing within exon 9 gives rise to four variants, two of which (OAT1-1 and OAT1-2) are functional. Following heterologous expression in Xenopus laevis oocytes, flounder renal OAT1 transported p-aminohippurate, glutarate, several diuretics, and the nephrotoxic agent ochratoxin A. Two cationic amino acid residues, lysine 394 and arginine 478, were found to be important for interaction with glutarate. Anionic neurotransmitter metabolites and the heavy-metal chelator, 2,3-dimercaptopropane sulfonate, interacted with the rabbit renal OAT1, which is expressed in kidneys and the retina.     

Functional analysis of rat renal organic anion transporter OAT-K1: bidirectional methotrexate transport in apical membrane.

Renal organic anion transporter OAT-K1 was stably transfected in MDCK cells and examined for its transport characteristics and membrane localization. OAT-K1 mediated both uptake and efflux of methotrexate in the apical membranes. Immunoblotting showed that the apparent molecular mass of the expressed OAT-K1 was 50 kDa, which was comparable to that found in the rat renal brush-border membranes. The OAT-K1-mediated methotrexate transport was significantly inhibited in the presence of several organic anions such as folate and sulfobromophthalein. These findings suggest that OAT-K1 mediates bidirectional methotrexate transport across the apical membranes, and may be involved in the renal handling of methotrexate.     

ABCB4 exports phosphatidylcholine in a sphingomyelin-dependent manner

ABCB4, which is specifically expressed on the canalicular membrane of hepatocytes, exports phosphatidylcholine (PC) into bile. Because SM depletion increases cellular PC content and stimulates PC and cholesterol efflux by ABCA1, a key transporter involved in generation of HDL, we predicted that SM depletion also stimulates PC efflux through ABCB4. To test this prediction, we compared the lipid efflux activity of ABCB4 and ABCA1 under SM depletion induced by two different types of inhibitors for SM synthesis, myriocin and (1R,3S)-N-(3-hydroxy-1-hydroxymethyl-3-phenylpropyl)dodecanamide, in human embryonic kidney 293 and baby hamster kidney cells. Unexpectedly, SM depletion exerted opposite effects on ABCB4 and ABCA1, suppressing PC efflux through ABCB4 while stimulating efflux through ABCA1. Both ABCB4 and ABCA1 were recovered from Triton-X-100-soluble membranes, but ABCB4 was mainly recovered from CHAPS-insoluble SM-rich membranes, whereas ABCA1 was recovered from CHAPS-soluble membranes. These results suggest that a SM-rich membrane environment is required for ABCB4 to function. ABCB4 must have evolved to exert its maximum activity in the SM-rich membrane environment of the canalicular membrane, where it transports PC as the physiological substrate.     

Differential expression pattern of protein ARVCF in nephron segments of human and mouse kidney

The protein ARVCF is a member of the p120 subfamily of armadillo proteins whose members have been described to occur in junction-bound and non-junction-bound forms. Studies on ARVCF were constrained because the endogenous protein was difficult to detect with the available reagents. We have generated novel monoclonal and polyclonal antibodies usable for biochemical and localization studies. By systematic immunohistochemical analysis of various tissues protein ARVCF is prominently detected in mouse, bovine and human kidney. Using antibodies against specific markers of nephron segments protein ARVCF is localized in proximal tubules according to double label immunofluorescence. Besides its occurrence in proximal tubules of adult kidney and in renal cell carcinoma derived from proximal tubules ARVCF is also detected in maturing nephrons in early mouse developmental stages such as, for example, 15 days of gestation (E15). Immunoblotting of total extracts of cultured cells of renal origin showed that ARVCF is detected in all human and murine cultured cells analyzed. Upon immunolocalization ARVCF is mostly detected in the cytoplasm occurring in a fine granular form. This prominent cytoplasmic localization of ARVCF in cultured cells and its occurrence in proximal tubules implies an involvement of ARVCF in specific functional processes of proximal tubules of kidney. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Transport of a fluorescent cAMP analog in teleost proximal tubules

Previous studies have shown that killifish (Fundulus heteroclitus) renal proximal tubules express a luminal membrane transporter that is functionally and immunologically analogous to the mammalian multidrug resistance-associated protein isoform 2 (Mrp2, ABCC2). Here we used confocal microscopy to investigate in killifish tubules the transport of a fluorescent cAMP analog (fluo-cAMP), a putative substrate for Mrp2 and Mrp4 (ABCC4). Steady-state luminal accumulation of fluo-cAMP was concentrative, specific, and metabolism-dependent, but not reduced by high K+ medium or ouabain. Transport was not affected by p-aminohippurate (organic anion transporter inhibitor) or p-glycoprotein inhibitor (PSC833), but cell-to-lumen transport was reduced in a concentration-dependent manner by Mrp inhibitor MK571, leukotriene C4 (LTC4), azidothymidine (AZT), cAMP, and adefovir; the latter two compounds are Mrp4 substrates. Although MK571 and LTC4 reduced transport of the Mrp2 substrate fluorescein-methotrexate (FL-MTX), neither cAMP, adefovir, nor AZT affected FL-MTX transport. Fluo-cAMP transport was not reduced when tubules were exposed to endothelin-1, Na nitroprusside (an nitric oxide generator) or phorbol ester (PKC activator), all of which signal substantial reductions in cell-to-lumen FL-MTX transport. Fluo-cAMP transport was reduced by forskolin, and this reduction was blocked by the PKA inhibitor H-89. Finally, in membrane vesicles from Spodoptera frugiperda (Sf9) cells containing human MRP4, ATP-dependent and specific uptake of fluo-cAMP could be demonstrated. Thus, based on inhibitor specificity and regulatory signaling, cell-to-lumen transport of fluo-cAMP in killifish renal tubules is mediated by a transporter distinct from Mrp2, presumably a teleost form of Mrp4.     

Roles of ZIP8, ZIP14, and DMT1 in transport of cadmium and manganese in mouse kidney proximal tubule cells

Chronic exposure to cadmium causes preferential accumulation of cadmium in the kidney, leading to nephrotoxicity. In the process of renal cadmium accumulation, the cadmium bound to a low-molecular-weight metal-binding protein, metallothionein, has been considered to play an important role in reabsorption by epithelial cells of proximal tubules in the kidney. However, the role and mechanism of the transport of Cd(2+) ions in proximal tubule cells remain unclear. Zinc transporters such as Zrt, Irt-related protein 8 (ZIP8) and ZIP14, and divalent metal transporter 1 (DMT1) have been reported to have affinities for Cd(2+) and Mn(2+). To examine the roles of these metal transporters in the absorption of luminal Cd(2+) and Mn(2+) into proximal tubule cells, we utilized a cell culture system, in which apical and basolateral transport of metals can be separately examined. The uptake of Cd(2+) and Mn(2+) from the apical side of proximal tubule cells was inhibited by simultaneous addition of Mn(2+) and Cd(2+), respectively. The knockdown of ZIP8, ZIP14 or DMT1 by siRNA transfection significantly reduced the uptake of Cd(2+) and Mn(2+) from the apical membrane. The excretion of Cd(2+) and Mn(2+) was detected predominantly in the apical side of the proximal tubule cells. In situ hybridization of these transporters revealed that ZIP8 and ZIP14 are highly expressed in the proximal tubules of the outer stripe of the outer medulla. These results suggest that ZIP8 and ZIP14 expressed in the S3 segment of proximal tubules play significant roles in the absorption of Cd(2+) and Mn(2+) in the kidney.     

Structure and active transport in the plasma membrane of the tubules of frog kidney

The active transport of organic anions through the plasma membrane of the proximal tubules of frog kidney was studied. For this purpose a marker anion, fluorescein, was used, its flow into the tubules registered by the increase of fluorescense. The kinetics of transport was measured as function of time, concentration of substrate, concentration of a competing acid (p-aminohippuric acid) and temperature. The process is inhibited by strophantin, a specific poison for (Na⁺ + K⁺)-dependent ATPase. These data show that fluorescein transport is effected with the participation of a charged carrier, probably by the downfield mechanism postulated by Mitchell. To confirm this mechanism, a passive flow of K⁺ was created inwards across the membrane of the proximal tubules by means of valinomycin. It led to the discharge of the membrane and to the inhibition of fluorescein transport. Anions are transported downfield across the membrane, probably in a state of complexes with two Na⁺ ions.A magnetic field of 10 000–28 000 oersted inhibits the fluorescein transport strongly. This can be regarded as a proof of the liquid-crystalline structure of biological membranes and demonstrates the importance of this structure for active transport.     

Cloning and functional characterization of human SMCT2 (SLC5A12) and expression pattern of the transporter in kidney

Recently, we cloned two Na(+)-coupled lactate transporters from mouse kidney, a high-affinity transporter (SMCT1 or slc5a8) and a low-affinity transporter (SMCT2 or slc5a12). Here we report on the cloning and functional characterization of human SMCT2 (SLC5A12) and compare the immunolocalization patterns of slc5a12 and slc5a8 in mouse kidney. The human SMCT2 cDNA codes for a protein consisting of 618 amino acids. When expressed in mammalian cells or Xenopus oocytes, human SMCT2 mediates Na(+) -coupled transport of lactate, pyruvate and nicotinate. The affinities of the transporter for these substrates are lower than those reported for human SMCT1. Several non-steroidal anti-inflammatory drugs inhibit human SMCT2-mediated nicotinate transport, suggesting that NSAIDs interact with the transporter as they do with human SMCT1. Immunofluorescence microscopy of mouse kidney sections with an antibody specific for SMCT2 shows that the transporter is expressed predominantly in the cortex. Similar studies with an anti-SMCT1 antibody demonstrate that SMCT1 is also expressed mostly in the cortex. Dual-labeling of SMCT1 and SMCT2 with 4F2hc (CD98), a marker for basolateral membrane of proximal tubular cells in the S1 and S2 segments of the nephron, shows that both SMCT1 and SMCT2 are expressed in the apical membrane of the tubular cells. These studies also show that while SMCT2 is broadly expressed along the entire length of the proximal tubule (S1/S2/S3 segments), the expression of SMCT1 is mostly limited to the S3 segment. These studies suggest that the low-affinity transporter SMCT2 initiates lactate absorption in the early parts of the proximal tubule followed by the participation of the high-affinity transporter SMCT1 in the latter parts of the proximal tubule.     

Renal organic anion transport: a comparative and cellular perspective

A major system for net transepithelial secretion of a wide range of hydrophobic organic anions (OAs) exists in the proximal renal tubules of almost all vertebrates. This process involves transport into the cells against an electrochemical gradient at the basolateral membrane and movement from the cells into the lumen down an electrochemical gradient. Transport into the cells at the basolateral membrane, which is the dominant, rate-limiting step, is a tertiary active transport process, the final step which involves countertransport of the OA into the cells against its electrochemical gradient in exchange for alpha-ketoglutarate moving out of the cells down its electrochemical gradient. The outwardly directed gradient for alpha-ketoglutarate is maintained by metabolism ( approximately 40%) and by transport into the cells across both the basolateral and luminal membranes by separate sodium-dicarboxylate cotransporters ( approximately 60%). The inwardly directed sodium gradient driving alpha-ketoglutarate uptake is maintained by the basolateral Na(+)-K(+)-ATPase, the primary energy-requiring transport step in the total tertiary process. The basolateral OA/alpha-ketoglutarate exchange process now appears to be physiologically regulated by several factors in mammalian tubules, including peptide hormones (e.g., bradykinin) and the autonomic nervous system acting via protein kinase C (PKC) pathways and epidermal growth factor (EGF) working via the mitogen-activated protein kinase (MAPK) pathway.     

Blood-to-retina transport of creatine via creatine transporter (CRT) at the rat inner blood-retinal barrier

The purpose of this study was to elucidate the mechanisms of blood-to-retina creatine transport across the blood-retinal barrier (BRB) in vivo and in vitro, and to identify the responsible transporter(s). The creatine transport across the BRB in vivo and creatine uptake in an in vitro model of the inner BRB (TR-iBRB2 cells) were examined using [(14)C]creatine. Identification and localization of the creatine transporter (CRT) were carried out by RT-PCR, western blot, and immunoperoxidase electron microscopic analyses. An in vivo intravenous administration study suggested that [(14)C]creatine is transported from the blood to the retina against the creatine concentration gradient that exists between the retina and blood. [(14)C]Creatine uptake by TR-iBRB2 cells was saturable, Na(+)- and Cl(-)-dependent and inhibited by CRT inhibitors, suggesting that CRT is involved in creatine transport at the inner BRB. RT-PCR and western blot analyses demonstrated that CRT is expressed in rat retina and TR-iBRB2 cells. Moreover, using an immunoperoxidase electron microscopic analysis, CRT immunoreactivity was found at both the luminal and abluminal membranes of the rat retinal capillary endothelial cells. In conclusion, CRT is expressed at the inner BRB and plays a role in blood-to-retina creatine transport across the inner BRB.