Apical voltage-driven urate efflux transporter NPT4 in renal proximal tubule

Uric acid (urate) is the end product of purine metabolism in humans. Human kidneys reabsorb a large proportion of filtered urate. This extensive renal reabsorption, together with the fact that humans do not possess uricase that catalyzes the biotransformation of urate into allantoin, results in a higher plasma urate concentration in humans compared to other mammals. A major determinant of plasma urate concentration is renal excretion as a function of the balance between reabsorption and secretion. We previously identified that renal urate absorption in proximal tubular epithelial cells occurs mainly via apical urate/anion exchanger, URAT1/SLC22A12, and by facilitated diffusion along the trans-membrane potential gradient by the basolateral voltage-driven urate efflux transporter, URATv1/SLC2A9/GLUT9. In contrast, the molecular mechanism by which renal urate secretion occurs remains elusive. Recently, we reported a newly characterized human voltage-driven drug efflux transporter, hNPT4/SLC17A3, which functions as a urate exit pathway located at the apical side of renal proximal tubules. This transporter protein has been hypothesized to play an important role with regard to net urate efflux. An in vivo role of hNPT4 is supported by the fact that missense mutations in SLC17A3 present in hyperuricemia patients with urate underexcretion abolished urate efflux capacity in vitro. Herein, we report data demonstrating that loop diuretics and thiazide diuretics substantially interact with hNPT4. These data provide molecular evidence for loop and thiazide-diuretics-induced hyperuricemia. Thus, we propose that hNPT4 is an important transepithelial proximal tubular transporter that transports diuretic drugs and operates functionally with basolateral organic anion transporters 1/3 (OAT1/OAT3).     

Liver X receptor activation downregulates organic anion transporter 1 (OAT1) in the renal proximal tubule

Liver X receptors (LXRs) play an important role in the regulation of cholesterol by regulating several transporters. In this study, we investigated the role of LXRs in the regulation of human organic anion transporter 1 (hOAT1), a major transporter localized in the basolateral membrane of the renal proximal tubule. Exposure of renal S2 cells expressing hOAT1 to LXR agonists (TO901317 and GW3965) and their endogenous ligand [22(R)-hydroxycholesterol] led to the inhibition of hOAT1-mediated [(14)C]PAH uptake. This inhibition was abolished by coincubation of the above agonists with 22(S)-hydroxycholesterol, an LXR antagonist. Moreover, it was found that the effect of LXR agonists was not mediated by changes in intracellular cholesterol levels. Interestingly, the inhibitory effect of LXRs was enhanced in the presence of 9-cis retinoic acid, a retinoic X receptor agonist. Kinetic analysis revealed that LXR activation decreased the maximum rate of PAH transport (J(max)) but had no effect on the affinity of the transporter (K(t)). This result correlated well with data from Western blot analysis, which showed the decrease in hOAT1 expression following LXR activation. Similarly, TO901317 inhibited [(14)C]PAH uptake by the renal cortical slices as well as decreasing mOAT1 protein expression in mouse kidney. Our findings indicated for the first time that hOAT1 was downregulated by LXR activation in the renal proximal tubule.     

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.     

Hydrogen peroxide downregulates human organic anion transporters in the basolateral membrane of the proximal tubule

Hydrogen peroxide (H2O2) is known to be involved in drug-induced and ischemic proximal tubular damage. The purpose of this study was to elucidate the effects of hydrogen peroxide on organic anion transport mediated by human organic anion transporters 1 and 3 (hOAT1 and hOAT3), which are localized at the basolateral side of the proximal tubule. For this purpose, we established and utilized the second segment of the proximal tubule cells from mice stably expressing hOAT1 or hOAT3 (S2 hOAT1 or S2hOAT3, respectively). H2O2 induced a dose- and a time-dependent decrease in organic anion transport mediated by hOAT1 and hOAT3. Kinetic analysis revealed that H2O2 decreased the Vmax, but not Km of organic anion transport both in S2hOAT1 and S2hOAT3. The effects of gentamicin, known to induce proximal tubular damage via the production of H2O2, on the organic anion transporters were also examined. Gentamicin induced a significant decrease in organic anion transport in S2hOAT1 but not S2hOAT3. H2O2-induced decrease in organic anion transport was significantly inhibited by pretreatment with pyruvate as well as catalase, whereas the gentamicin-induced decrease was significantly inhibited by pretreatment with pyruvate but not with catalase. In conclusion, these results suggest that H2O2, which is produced during tubular injuries, downregulates organic anion transport mediated by both hOAT1 and hOAT3, leading to further modulation of pathophysiology.     

Identification of a novel human organic anion transporting polypeptide as a high affinity thyroxine transporter

Transport of various amphipathic organic compounds is mediated by organic anion transporting polypeptides (OATPs in humans, Oatps in rodents), which belong to the solute carrier family 21A (SLC21A/Slc21a). Several of these transporters exhibit a broad and overlapping substrate specificity and are expressed in a variety of different tissues. We have isolated and functionally characterized OATP-F (SLC21A14), a novel member of the OATP family. The cDNA (3059 bp) contains an open reading frame of 2136 bp encoding a protein of 712 amino acids. Its gene containing 15 exons is located on chromosome 12p12. OATP-F exhibits 47-48% amino acid identity with OATP-A, OATP-C, and OATP8, the genes of which are clustered on chromosome 12p12. OATP-F is predominantly expressed in multiple brain regions and Leydig cells of the testis. OATP-F mediates high affinity transport of T(4) and reverse T(3) with apparent K(m) values of approximately 90 nM and 128 nM, respectively. Substrates less well transported by OATP-F include T(3), bromosulfophthalein, estrone-3-sulfate, and estradiol-17beta-glucuronide. Furthermore, OATP-F-mediated T(4) uptake could be cis-inhibited by L-T(4) and D-T(4), but not by 3,5-diiodothyronine, indicating that T(4) transport is not stereospecific, but that 3',5'-iodination is important for efficient transport by OATP-F. Thus, in contrast to most other family members, OATP-F has a more selective substrate preference and may play an important role in the disposition of thyroid hormones in brain and testis.     

Characterization of mouse organic anion transporter 5 as a renal steroid sulfate transporter

A family of organic anion transporters (OAT) recently identified has important roles for the excretion or reabsorption of endogenous and exogenous compounds, and several new isoforms have been reported in this decade. Although the transepithelial transport properties of organic anions are gradually being understood, many portions of their functional characteristics in functions remain to be elucidated. A recently reported new cDNA encoding a mouse OAT5 (mOAT5) was constructed, using 3'-RACE PCR, with the total RNA isolated from a mouse kidney. When mOAT5 was expressed in Xenopus oocytes, mOAT5 transported estrone sulfate, dehydroepiandrosterone sulfate and ochratoxin A. Estrone sulfate uptake by mOAT5 displayed a time-dependent and sodium-independent manner. The Km values of estrone sulfate and dehydroepiandrosterone sulfate were 2.2 and 3.8 microM, respectively. mOAT5 interacted with chemically heterogeneous steroid or organic sulfates, such as nitrophenyl sulfate, methylumbelliferyl sulfate and estradiol sulfates. In contrast to the sulfate conjugates, mOAT5-mediated estrone sulfate uptake was not inhibited by the steroid or organic glucuronides. The mOAT5 protein having about 85 kDa molecular weight was shown to be mainly localized in the apical membrane of the proximal tubules of the outer medulla. These results suggest an important role of mOAT5 for the excretion or reabsorption of steroid sulfates in the kidney.     

Stereoselective renal secretion of carbenicillin in rabbits: Role of the organic anion transporter at the renal brush border membrane

Stereoselectivity in the renal secretion of carbenicillin (CBPC) was studied in rabbits. Significant renal secretion of CBPC was observed in vivo, with the secretion of the S-epimer being greater than that of the R-epimer. Stereoselective transport of CBPC was further studied in vitro using basolateral and brush border membrane vesicles prepared from rabbit kidneys. The transport of CBPC by the organic anion transporter into the basolateral membrane vesicles (BLMV) was not stereoselective. In contrast, a distinct stereoselectivity was observed in the transport of CBPC by the organic anion transporter into the brush border membrane vesicles (BBMV), with the transport of the S-epimer being more favorable. Significant epimer-epimer interactions were also observed in the transport into BBMV. The stereoselectivity of the transport of CBPC was calculated from the kinetic parameters with consideration of epimer-epimer interactions and was similar to that observed in vivo. It was concluded that the observed stereoselectivity in the renal secretion of CBPC in vivo reflected that of transport via the organic anion transporter located at the brush border membrane. Chirality 10:349–357, 1998. © 1998 Wiley-Liss, Inc.     

Identification of a novel gene family encoding human liver-specific organic anion transporter LST-1.

We have isolated a novel liver-specific organic anion transporter, LST-1, that is expressed exclusively in the human, rat, and mouse liver. LST-1 is a new gene family located between the organic anion transporter family and prostaglandin transporter. LST-1 transports taurocholate (Km = 13.6 microM) in a sodium-independent manner. LST-1 also shows broad substrate specificity. It transports conjugated steroids (dehydroepiandrosterone sulfate, estradiol-17beta-glucuronide, and estrone-3-sulfate), eicosanoids (prostaglandin E2, thromboxane B2, leukotriene C4, leukotriene E4), and thyroid hormones (thyroxine, Km = 3.0 microM and triiodothyronine, Km = 2.7 microM), reflecting hepatic multispecificity. LST-1 is probably the most important transporter in human liver for clearance of bile acids and organic anions because hepatic levels of another organic anion transporter, OATP, is very low. This is also the first report of the human molecule that transports thyroid hormones.     

Identification of a sodium-dependent organic anion transporter from rat adrenal gland

In this study, a novel sodium-dependent organic anion transporter (Soat) was identified. Soat is expressed in rat brain, heart, kidney, lung, muscle, spleen, testis, adrenal gland, small intestine, and colon. The Soat protein consists of 370 amino acids and shows 42% and 31% overall amino acid sequence identity to the ileal sodium-dependent bile acid transporter (Isbt) and the Na(+)/taurocholate cotransporting polypeptide (Ntcp), respectively. Soat is predicted to have nine transmembrane domains, with an N-terminus outside the cell and an intracellular C-terminus. The Soat gene is localized on chromosome 14 and is coded by six exons mapped in region 14p22. When expressed in Xenopus laevis oocytes, Soat shows transport function for estrone-3-sulfate (Km = 31 microM, Vmax = 5557 fmol/oocyte/30 min) and dehydroepiandrosterone sulfate (Km = 30 microM, Vmax = 5682 fmol/oocyte/30 min). Soat does not transport taurocholate, estradiol-17beta-glucuronide, nor ouabain.     

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.     

Shank2E binds NaP(i) cotransporter at the apical membrane of proximal tubule cells

Proteins expressing postsynaptic density (PSD)-95/Drosophila disk large (Dlg)/zonula occludens-1 (ZO-1) (PDZ) domains are commonly involved in moderating receptor, channel, and transporter activities at the plasma membrane in a variety of cell types. At the apical membrane of renal proximal tubules (PT), the type IIa NaP_i cotransporter (NaP_i-IIa) binds specific PDZ domain proteins. Shank2E is a spliceoform of a family of PDZ proteins that is concentrated at the apical domain of liver and pancreatic epithelial cell types and is expressed in kidney. In the present study, immunoblotting of enriched plasma membrane fractions and immunohistology found Shank2E concentrated at the brush border membrane of rat PT cells. Confocal localization of Flag-Shank2E and enhanced green fluorescent protein-NaP_i-IIa in cotransfected OK cells showed these proteins colocalized in the apical microvilli of this PT cell model. Shank2E coimmunoprecipitated with NaP_i-IIa from rat renal cortex tissue and HA-NaP_i-IIa coprecipitated with Flag-Shank2E in cotransfected human embryonic kidney HEK cells. Domain analysis showed that the PDZ domain of Shank2E specifically bound NaP_i-IIa and truncation of the COOH-terminal TRL motif from NaP_i-IIa abolished this binding, and Far Western blotting showed that the Shank2E- NaP_i-IIa interaction occurred directly between the two proteins. NaP_i-IIa activity is regulated by moderating its abundance in the apical membrane. High-P_i conditions induce NaP_i-IIa internalization and degradation. In both rat kidney PT cells and OK cells, shifting to high-P_i conditions induced an acute internal redistribution of Shank2E and, in OK cells, a significant degree of degradation. In sum, Shank2E is concentrated in the apical domain of renal PT cells, specifically binds NaP_i-IIa via PDZ interactions, and undergoes P_i-induced internalization. PDZ domains; endocytosis; degradation; epithelia     

Urate transport across the apical membrane of renal proximal tubules

Since the molecular cloning of the renal apical urate/anion exchanger URAT1 (SLC22A12), several membrane proteins relevant to urate transport have been identified. In addition, the identification of PDZ (PSD-95, DglA, and ZO-1) domain protein PDZK1 as a binding partner of URAT1, and the emerging role of PDZ scaffold for renal apical transporters have led to a new concept of renal urate transport: urate-transporting multimolecular complex, or "urate transportsome," that may form an ultimate functional unit at the apical membrane of renal proximal tubules. Elucidation of urate transportsome will lead to the new drug development for hyperuricemia.     

Transport of organic anions across the basolateral membrane of proximal tubule cells

Renal proximal tubules secrete diverse organic anions (OA) including widely prescribed anionic drugs. Here, we review the molecular properties of cloned transporters involved in uptake of OA from blood into proximal tubule cells and provide extensive lists of substrates handled by these transport systems. Where tested, transporters have been immunolocalized to the basolateral cell membrane. The sulfate anion transporter 1 (sat-1) cloned from human, rat and mouse, transported oxalate and sulfate. Drugs found earlier to interact with sulfate transport in vivo have not yet been tested with sat-1. The Na⁺-dicarboxylate cotransporter 3 (NaDC-3) was cloned from human, rat, mouse and flounder, and transported three Na⁺ with one divalent di- or tricarboxylate, such as citric acid cycle intermediates and the heavy metal chelator 2,3-dimercaptosuccinate (succimer). The organic anion transporter 1 (OAT1) cloned from several species was shown to exchange extracellular OA against intracellular α-ketoglutarate. OAT1 translocated, e.g., anti-inflammatory drugs, antiviral drugs, β-lactam antibiotics, loop diuretics, ochratoxin A, and p-aminohippurate. Several OA, including probenecid, inhibited OAT1. Human, rat and mouse OAT2 transported selected anti-inflammatory and antiviral drugs, methotrexate, ochratoxin A, and, with high affinities, prostaglandins E₂ and F_2α. OAT3 cloned from human, rat and mouse showed a substrate specificity overlapping with that of OAT1. In addition, OAT3 interacted with sulfated steroid hormones such as estrone-3-sulfate. The driving forces for OAT2 and OAT3, the relative contributions of all OA transporters to, and the impact of transporter regulation by protein kinases on renal drug excretion in vivo must be determined in future experiments. Electronic Publication     

Identification of an apical Cl-/HCO-3 exchanger in rat kidney proximal tubule

SLC26A6 (or putative anion transporter 1, PAT1) is located on the apical membrane of mouse kidney proximal tubule and mediates exchange in in vitro expression systems. We hypothesized that PAT1 along with a exchange is present in apical membranes of rat kidney proximal tubules. Northern hybridizations indicated the exclusive expression of SLC26A6 (PAT1 or CFEX) in rat kidney cortex, and immunocytochemical staining localized SLC26A6 on the apical membrane of proximal tubules, with complete prevention of the labeling with the preadsorbed serum. To examine the functional presence of apical exchanger, proximal tubules were isolated, microperfused, loaded with the pH-sensitive dye BCPCF-AM, and examined by digital ratiometric imaging. The pH of the perfusate and bath was kept at 7.4. Buffering capacity was measured, and transport rates were calculated as equivalent base flux. The results showed that in the presence of basolateral DIDS (to inhibit cotransporter 1) and apical EIPA (to inhibit Na⁺/H⁺ exchanger 3), the magnitude of cell acidification in response to addition of luminal Cl^– was 5.0-fold higher in the presence than in the absence of . The Cl^–-dependent base transport was inhibited by 61% in the presence of 0.5 mM luminal DIDS. The presence of physiological concentrations of oxalate in the lumen (200 µM) did not affect the exchange activity. These results are consistent with the presence of SLC26A6 (PAT1) and exchanger activity in the apical membrane of rat kidney proximal tubule. We propose that SLC26A6 is likely responsible for the apical (and Cl^–/OH^–) exchanger activities in kidney proximal tubule. putative anion transporter 1; chloride/formate exchanger; SLC26A6     

Mechanism of organic anion transport across the apical membrane of choroid plexus

The mechanism and membrane localization of choroid plexus (CP) organic anion transport were determined in apical (or brush border) membrane vesicles isolated from bovine choroid plexus and in intact CP tissue from cow and rat. Brush border membrane vesicles were enriched in Na(+),K(+)-ATPase (20-fold; an apical marker in CP) and demonstrated specific, sodium-coupled transport of proline, glucose, and glutarate. Vesicular uptake of the anionic herbicide 2, 4-dichlorophenoxyacetic acid (2,4-D) was markedly stimulated by an inward sodium gradient but only in the presence of glutarate, indicating the presence of apical dicarboxylate/organic anion exchange. Consistent with this interpretation, an imposed outward glutarate gradient stimulated 2,4-D uptake in the absence of sodium. Under both conditions, uptake was dramatically slowed and overshoot was abolished by probenecid. Likewise, apical accumulation of 2,4-D by intact bovine choroid plexus tissue in vitro was stimulated by external glutarate in the presence of sodium. Glutarate stimulation was abolished by 5 mM LiCl. Identical findings were obtained using rat CP tissue, which showed both sodium/glutarate-stimulated 2,4-D (tissue/medium (T/M) approximately 8) and p-aminohippurate (T/M = 2) transport. Finally, since the renal exchanger (rROAT1) has been cloned in rat kidney, a rROAT1-green fluorescent protein construct was used to analyze exchanger distribution directly in transiently transfected rat CP. As predicted by the functional studies, the fluorescently tagged transporter was seen in apical but not basolateral membranes of the CP.