Developmentally regulated expression of organic ion transporters NKT (OAT1), OCT1, NLT (OAT2), and Roct

Anna Pavlova1, Hiroyuki Sakurai1,3, Baudouin Leclercq1, David R. Beier2, Alan S. L. Yu1, and Sanjay K. Nigam1,3

1 Renal and 2 Genetics Divisions, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115; and 3 Department of Pediatrics and Medicine, University of California, San Diego, La Jolla, California 92093


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Several xenobiotic (organic cation and anion) transporters have recently been identified, although their endogenous substrates, if such exist, remain unknown. When we initially identified NKT, also known as OAT1, the first member of the organic anion transporter (OAT) family (Lopez-Nieto CE, You G, Bush KT, Barros EJ, Beier DR, and Nigam SK. J Biol Chem 272: 6471-6478, 1997), we noted its expression in the embryonic kidney. We have now demonstrated its transporter function and more fully examined the spatiotemporal expression patterns of representative organic ion transporters, [NKT (OAT1), Roct, OCT1, and NLT, also known as OAT2] during murine development. In the kidney, NKT (OAT1), OCT1, and Roct transcripts appeared at midgestation, coinciding with proximal tubule differentiation, and gradually increased during nephron maturation. A similar pattern was observed for NLT (OAT2) in the liver and kidney, although, in the kidney, NLT (OAT2) transcription did not increase as dramatically. The roughly cotemporal expression of these related transporters in the developing proximal tubule may indicate common transcriptional regulation. Expression during embryogenesis in extrarenal sites could suggest a role in the formation and maintenance of nonrenal tissues. Importantly, all four genes were expressed in unexpected places during nonrenal organogenesis: Roct in the fetal liver (temporally coinciding with the onset of hematopoiesis) and neural tissue; NKT (OAT1) in the fetal brain; OCT1 in the ascending aorta and atrium; and NLT (OAT2) in the fetal lung, intestine, skin, and developing bone. Because these gene products mediate the transport of a broad range of metabolites and toxins, it seems likely that, apart from their known functions, these transporters play a role in transport of organic molecules, perhaps including those with morphogenetic activity. These genes could also play important developmental roles independent of transport function.

proximal tubule; kidney development; organogenesis; hematopoiesis; brain; OAT1; OAT2


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INTRODUCTION
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ORGANIC ION TRANSPORT MECHANISMS for the elimination of endogenous and exogenous toxins are necessary for the survival of mammalian species. Polyspecific organic ion transporters are a unique group of proteins, mediating transport of diverse and structurally unrelated molecules. Many of these are expressed in the proximal nephron of the kidney, consistent with the known role of this nephron segment in the elimination of organic ions and drugs.

Using codon optimized differential display (9), we recently cloned and characterized a novel gene product from mammalian kidney, NKT (OAT1) (10), and postulated that NKT (OAT1) functions as an organic ion transporter in mammalian kidneys. Subsequently, it was shown that NKT (OAT1) transports a wide variety of organic anions, including endogenous substrates such as cyclic nucleotides, prostaglandins, and uric acid, as well as a variety of structurally unrelated drugs and antibiotics (17, 19). NKT (OAT1) cDNA was found to share ~30% amino acid homology with the organic cation transporter (OCT1) (6), 35% homology to the rat liver-specific transporter NLT (OAT2) (10, 18) and 47% homology with the newly identified gene product Roct (2) The OCT1 protein and its isoforms (OCT1A and OCT1B) are known to mediate renal and hepatic transport of small organic cations, classified as type 1 substrates (3, 11). NLT (OAT2) has been reported to mediate sodium-independent transport of multispecific organic anions (16). The substrate specificity for Roct is yet to be determined. In the adult kidney, these genes are largely expressed in the proximal tubule, and, together, not only constitute a set of structurally and functionally related genes involved in drug elimination but also represent useful markers for the study of proximal tubule maturation during kidney development. However, little is known about the nature of their endogenous substrates, if such exist. Nevertheless, significant levels of expression of these transporters, especially in nonrenal/nonhepatic tissues during embryogenesis, might suggest a key role in transport of an endogenous substance or a role in development independent of transport.

There are some circumstantial data hinting at such a role for the organic ion transporters. The cation-like transporter gene (Orct), corresponding to the mammalian OCT proteins, was recently cloned from the lemming locus of Drosophila melanogaster (21). Analysis of the deduced amino acid sequences revealed ~34% of homology between Orct and NKT (OAT1) protein. Roct shares 31% of homology with the Orct gene of Drosophila. Mutations at the lemming locus of Drosophila result in the "embryonic segmentation fault" phenotype due to apoptotic cell death in dividing imaginal cells (21).

We report here the developmental analysis of organic ion transporters related to the Orct gene during mouse development. Northern blot and in situ hybridization analysis of mouse kidneys indicate that NKT (OAT1), Roct, OCT1, and NLT (OAT2) transcripts appear in proximal tubules during late embryogenesis and remain at high levels throughout adulthood. In contrast, their temporal and spatial expression profiles in extrarenal sites during embryogenesis are very different and largely unexpected. A common feature of the expression pattern between NKT (OAT1), OCT1, NLT (OAT2), and Roct in prenatal life is that, while transcription levels in kidneys (and liver for NLT (OAT2) and OCT1) dramatically increase toward birth, mRNA expression levels in other extrarenal sites become dramatically decreased.

Taken together, these data raise an intriguing possibility that organic ion transporters, in addition to their known functioning as kidney and/or liver transporters, play distinct, yet completely unexplored, roles during development.


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Tissue preparation. All tissues were isolated from CD-1 mice (Taconic Laboratories). Whole embryos and individual neonatal tissues were isolated after timed breedings, with the morning of the vaginal plug counted as 0 days postcoitum (pc). For postnatal tissues, the day of birth was counted as day 0 postpartum (pp).

Histology. Individual embryos (e11.5-e16.5, where e is embryonic day) and organs from postnatal mice (day 13 and 8 wk) were fixed in 4% paraformaldehyde/PBS solution, then dehydrated through a graded series of ethanol concentrations, clarified in xylene and then paraffin embedded. After that, 5-µm sections were obtained for in situ hybridization. In addition, commercially available (Novogen) paraffin sections of the staged murine embryos were extensively used for in situ hybridization analysis.

Neonatal mice (day 5 pp) were snap-frozen in at -30°C and cut on a cryostat 10 µm thick in dorsal orientation. For histological examination, sections were stained with hematoxylin and eosin.

In situ hybridization. In situ hybridization was performed as described earlier in detail (13, 15). Briefly, sense and antisense 35S-labeled RNA probes were generated under identical conditions by using T7, SP6, or T3 polymerase, then hydrolyzed to an average length of 100 bp and purified through a G-50 Sephadex column. Paraffin histological sections were dewaxed, rehydrated through a graded series of ethanol concentrations, digested with 27 ng/ml of proteinase K for 7 min, rinsed, and acetylated in 0.1 M triethanolamine/2.5% acetic anhydride. Hybridization was performed overnight at 52°C in a hybridization buffer containing 35,000 cpm/µl of probe. After hybridization, RNAse treatment and high-stringency washes in 5× standard sodium citrate (SSC)/10 mM DTT at 50°C; then 2× SSC/ 0.1M DTT/ 50% formamide at 65°C and 2× and 0.1× SSC at 37°C, slides were dehydrated, air dried, and exposed to Kodak autoradiographic film. Slides were then immersed in Kodak NTB-2 emulsion and exposed for 1 wk at 4°C. Photographs of whole embryos were printed directly from the autoradiographic film. Dark-field photomicrographs were taken by using a Nikon microscope with a neutral, red, or green filter. In situ analysis was generally performed twice per tissue sample with each probe. Sense probes controls were generally used once for each probe to confirm signal specificity.

RNA preparation. Total RNA samples were isolated from various embryonic and postnatal tissues (Charles River Laboratories) of e11.5-e20, newborn, days 5-13 pp, and adult mice, using a modified acid guanidinium thiocyanate-phenol-chlorophorm extraction method according to manufacturer's instructions (TRI Reagent, Molecular Research Center, Cincinnati, OH).

Northern blot analysis. Northern blot assay was performed as described earlier elsewhere (10). Total RNA samples were isolated by phenol-chloroform extraction. Fifteen micrograms of total RNA per lane were electrophoresed on 1% formamide/formaldehyde-agarose gels, transferred to nylon membranes overnight, and hybridized with random-primed 32P-labeled cDNA fragments of NKT (OAT1) (10), Roct (2), OCT1 [kindly provided by Dr. R. Green (5)], or NLT (OAT2) (kindly provided by Dr. R. Green, human Est accession no. T73363). 32P- labeled dCTP (50 µCi/1 reaction) was used according to the instruction of Ready-To-Go DNA labeling kit (Amersham Pharmacia, Piscataway, NJ). Ethidium bromide staining of the membrane was used to demonstrate equal loading.

Functional expression of organic solute transport in Xenopus laevis oocytes. Xenopus oocyte expression was performed by a modification of previously described methods (10). Defolliculated oocytes were injected with 100 ng of poly-A+ RNA isolated from day 17 mouse embryos or adult mice, using 30 ng of NKT cRNA or 50 nl water as negative controls. Radiolabeled organic solute uptake assays were performed 3 days later. As uptake via the multispecific organic anion transporter is known to be enhanced by transstimulation with dicarboxylic acid anions (17), oocytes for the organic anion uptake assay were first preloaded by incubation for 2 h in ND96 [containing in (mM) 96 NaCl, 2 KCl, 1.8 CaCl2, 1 MgCl2, and 5 HEPES, pH 7.4] supplemented with 2 mM sodium glutarate. For unilateral flux assays, oocytes were incubated for 1 h at room temperature in ND96 containing either 100 µM (40 Ci/mol) [14C]p-aminohippurate (PAH) for organic anion uptake or 0.6 mM (1.6 Ci/mol) [14C]tetraethylammonium bromide (TEA) for organic cation uptake. Transport was stopped by five washes in ice-cold ND96, the oocytes were transferred in pairs to individual scintillation vials and lysed in 10% sodium dodecyl sulfate, and radioactivity was measured in a liquid scintillation counter. Differences in mean uptakes were compared for statistical significance by one-way analysis of variance, and post hoc pairwise comparisons were performed by Fisher's least significant difference method.


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Using the combined approach of Northern blot analysis and in situ hybridization, we have analyzed the developmental expression and localization of the four related organic ion transporters NKT (OAT1), Roct, OCT1, and NLT (OAT2).

Developmental profile of NKT (OAT1): choroid plexus, dura matter, and kidneys as the primary sites of NKT (OAT1) expression during mouse development. Our studies have shown that the NKT (OAT1) gene is expressed during fetal and adult life in kidneys and brain. The first transcripts in the kidney were detected by in situ hybridization in the developing proximal tubules at e14 of mouse development (Fig. 1, E and F). No signal was detected in metanephric kidneys at earlier stages (e12-e13, not shown). Levels of expression increased by e16 and further increased in proximal tubules on maturation, which was detected by in situ hybridization and Northern blot analysis (Fig. 1, C, D, and K). The size (~2.4 kb) of the NKT (OAT1) transcript is consistent with a functional transporter (10, 17, 19). Thus because differentiation of proximal tubules in mice occurs around e14, the spatial-temporal distribution of NKT (OAT1) coincides with the differentiation of the proximal tubules of the kidney.


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Fig. 1.   NKT (OAT1) expression in developing brain structures and fetal kidney. Developmental profile of NKT (OAT1) expression was determined by in situ hybridization (A-J) and Northern blot (K) analysis. A and C: dark-field X-ray film images of murine embryo histological sections, hybridized with NKT (OAT1)-specific riboprobe. B: schematic picture of sagittal sections through whole embryos, shown in A (e14) and C (e16). NKT (OAT1) labeling is observed only in dura matter and choroid plexus and kidneys. At stage e14 NKT (OAT1) expression in dura matter can be detected throughout anterior-posterior axis of embryo (spinal cord). First transcripts of NKT (OAT1) in kidney appear in developing proximal tubules at e14 [A and well seen at higher power (E, F)]. By e16, signal is restricted to anterior part of embryo (head). A stronger signal in kidneys can now be seen clearly (C). D: dark-field image of dorsal section through newborn [5 days postpartum (p.p.)] mouse. At this stage NKT (OAT1) transcripts are localized in dura matter of brain and cortex of kidneys. E: dark-field microphotograph of metanephric kidney at e14. Positive signals are observed in metanephric mesenchyme-derived proximal tubules (PT), whereas no signal is detected in glomerular (G) or ureteric bud derived collecting tubules (CT). F: phase-contrast view of E. G and J: control high- and low-power pictures of adult kidney. NKT (OAT1) transcripts are located in cortex tubules but not in medulla. H and I: dark-field microphotographs of NKT (OAT1) expression in fetal (e14) brain (dura matter and choroid plexus, respectively). Arrows indicate NKT (OAT1) labeling in kidneys and in dura matter of brain. K: Northern blot analysis of NKT (OAT1) expression during kidney development showing signal in developing kidneys gradually increasing toward adulthood. Transcripts are detected in cortex but not in medulla. We observe 2 bands on blot, major (~2.4 kb) and less intense (~4.4 kb). Ethidium bromide staining of total RNA was shown to document loading.

In contrast to the developing kidney, the levels of NKT (OAT1) transcripts in the brain were found to be significantly higher in the fetal and early postnatal stage compared with adult life. Between e12 (the earliest stage examined) and e16 of embryo development, we observed labeling in the epithelial lining of ventricles, choroid plexus, and dura matter (data are shown for e14 and e16: Fig. 1, A-D, H, and I). Up until e14, we also observed NKT (OAT1) transcripts extending to the lining of root ganglions and spinal cord (Fig. 1A). By e16, no NKT (OAT1) signal was detected in the spinal cord (Fig. 1C). In the newborn (days 1-5 pp) and in the adult brain (not shown), a weak NKT (OAT1) signal was detected only in the meninges (Fig. 1D).

Roct gene expression in the developing nervous system, liver, and kidneys: transient expression in the fetal liver coincident with hematopoiesis during embryogenesis. Faint signals of Roct expression were first detected in the liver and brain of the mouse embryo at e12 (not shown). At e14, a clear signal for Roct expression was observed in the liver (Fig. 2, A and E) and in the nervous system (Fig. 2A). Strong labeling was observed throughout the entire nervous system (the epithelial lining and the body of the brain, including dura matter, choroid plexus, olfactory lobe, pituitary gland, and spinal cord) starting at e14 (Fig. 2, A-C). Transcription intensity decreased after e16 (Fig. 2B), and only very low levels of Roct mRNA were detected in the adult brain (not shown).


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Fig. 2.   Roct expression is restricted to fetal liver, kidney, and brain. Developmental profile of Roct expression was determined by in situ hybridization (A-D) and Northern blot analysis (E-F). A: Roct transcripts are localized in liver and nervous tissues of e14 murine embryo. No signal is detected in kidney. B: strong Roct expression is detected in cortex of e16 kidney, and lower levels of transcripts are present in liver and nervous tissue. C: schematic picture of sagittal sections through e14 (A) and e16 (B) embryos. D: dark-field X-ray image of cross section through adult kidney, hybridized with Roct riboprobe. Roct-specific signal is observed in cortex. E: on e14, Roct is detected in fetal liver but not in other organs tested. F: during kidney development Roct transcription is initiated after e14. Higher levels are detected in adult kidney (Ad). NB, Newborn kidney.

Kidney transcription was not detected until e16, where distinctive labeling in the renal cortex was observed (Fig. 2B). Levels of Roct expression in kidneys further increased at e19 and after birth (Fig. 2F).

Levels of Roct expression in the developing liver peaked by e14 and started to decrease at e16 (Fig. 2, A and B). We observed a decline in liver expression after e16 and a more dramatic downregulation of Roct expression toward adulthood. Only low levels of Roct expression could be detected in the adult liver by in situ hybridization (not shown). Interestingly, temporal expression of Roct (and intensity of its transcription) in the fetal and adult liver coincides with liver's role in hematopoeisis. Fetal hematopoiesis occurs almost exclusively in the liver from e12 through e16; however, separate hematopoietic foci in the liver may be detected later, even after birth. During this time period, most active transcription of Roct was found in the fetal liver. Very low levels of Roct transcripts were detected in the adult liver (not shown). Hematopoiesis in spleen and bone marrow is reported to occur after days 15 and 16 pc (14); Roct mRNA was not detected in the fetal or adult bone marrow and spleen.

Thus Roct transcription is not uniformly regulated among all organs: although levels of expression in the liver and nervous system gradually decline in late fetal life; levels in the kidneys rise and continue to do so after birth (Fig. 2).

OCT1 expression in fetal ascending aorta, kidneys, and liver during embryogenesis. High-stringency Northern blots and in situ hybridization were performed to analyze the tissue distribution and expression levels of OCT1 mRNA. No transcripts were detected by Northern blots in the several fetal tissues isolated from e14 embryos: spleen, heart, lung, liver, and kidney (Fig. 3, K and L). Nevertheless, by radioactive in situ hybridization highly localized signals for OCT1 expression in the upper portion of the developing atrium and ascending aorta were detected starting at e14 (Fig. 3, A, B, C, E, and F). Interestingly, no labeling was detected in other parts of the circulatory system (veins or aortas) or other organs. This site-specific OCT1 expression is a transient phenomenon: levels of expression start rapidly declining after e16 (Fig. 3C), and no postnatal signal was detected (Fig. 3D).


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Fig. 3.   OCT1 expression during development: kidney, liver, atrium, and ascending aorta. Developmental profile of OCT1 expression was determined by in situ hybridization (A-J) and Northern blot (K-L) analysis. OCT1 is expressed in atrium and ascending aorta during midembryogenesis and in kidneys and liver from late embryogenesis to adulthood. A and C: dark-field X-ray film images of murine embryo histological sections, hybridized with OCT1-specific riboprobe. B: schematic picture of sagittal sections through whole embryos, shown in A (e14) and C (e16). E and F: high-power photograph of e14 (A) showing OCT1 expression in developing atrium and aorta. V, ventricle; A, aorta. In situ hybridization analysis shows that at stage e14 OCT1 transcription is detected only in ascending aorta (arrowhead). By e16 signal is detected in atrium of heart (arrowhead), ascending aorta (arrowhead), and kidneys (arrow). Faint signal in liver (long arrowhead) can now be observed. D: dark-field image of dorsal section through newborn (5 days pp) mouse [schema (I)]. At this stage OCT1 transcripts were detected in liver and in cortex of kidneys. G: dark-field image of sagittal section through 2-wk-old mouse kidney. H: dark-field image of sagittal section through 2-wk-old mouse liver. I: schematic picture of 5-day-old mouse section shown in D. J: adult kidney, control. OCT1-specific signal is located in outer medulla and cortex. Postnatally (days 5 and 13 and adult) OCT1 transcripts are restricted to kidney and liver. K: At e14 OCT1 is not detected in tested tissues. L: OCT1 transcription during kidney development is initiated after e14.

OCT1 transcripts in the embryonic kidney appeared between e15 and e16 (Fig. 3C). Between e16 and e17, faint labeling in the liver could be detected (Fig. 3C). Both kidney and liver expression dramatically increased during development (Fig. 3D, G-J).

NLT (OAT2) expression during development includes liver, lung, kidneys, and developing bone and cartilage structures. It was previously reported that NLT (OAT2) transcripts were found in the adult human liver and at lower levels in kidneys (18). High-stringency Northern and in situ hybridization assays were performed by using human cDNA encoding part of the NLT (OAT2) gene to analyze its expression pattern during mouse development. Consistent with the expression pattern in the adult human tissues, high levels of NLT (OAT2) transcripts were detected by in situ hybridization in the adult liver and lower levels in adult kidneys (Fig. 4, D and E). During embryogenesis, we detected NLT (OAT2) mRNA transcripts in a broad range of embryonic tissues including liver, kidney, epithelial lining of the stomach, developing bone/cartilage structures, intestine, and skin (Fig. 4). Transcripts of NLT (OAT2) in the kidney could be detected by in situ hybridization by e14. In the newborn (day 5 pp), transcripts were present in the liver, kidneys, hind-brain, stomach, and skin (not shown). Levels of NLT (OAT2) transcription in fetal and adult murine kidneys were lower than in the liver, consistent with the findings in the adult human liver and kidneys.


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Fig. 4.   Developmental profile of NLT (OAT2) expression: liver, lung, kidneys, and developing bone structures. Developmental profile of NLT (OAT2) expression was determined by in situ hybridization (A-I) and Northern blot (J-K) analysis. NLT (OAT2) was found to be expressed in liver, lung, kidneys, and in developing bone structures during embryogenesis. A: dark-field X-ray film images of e14 embryo section, hybridized with human NLT (OAT2)-specific riboprobe. B: bright-field view of same embryo shown in A. In contrast to other transporters analyzed in this study, NLT (OAT2) transcripts are already in abundance in kidneys already by e14 (see also K). High levels of NLT (OAT2) RNA are detected in embryonic liver, lung (see also J), cartilage, and developing bone structures. C: schematic picture of sagittal section through whole embryos, shown in A. D: adult kidney, control. E: adult liver, control. Adult kidney and liver controls are shown to confirm NLT (OAT2) probe specificity. In adulthood high levels of NLT (OAT2) are detected in liver and lower levels in kidneys. This finding is consistent with levels of NLT (OAT2) expression reported for human adult tissues (17). F and G: dark- and bright-field microphotograph of e14 liver. NLT (OAT2) signal is in lining of bile duct (arrowhead). H and I: dark- and bright-field microphotograph of e14 kidney. J: At e14 NLT (OAT2) is detected in lung and liver but not in heart or spleen. K: NLT (OAT2) transcription is detected from e14 during kidney development.

Heterologous expression of organic solute transport in Xenopus oocytes. To determine whether the organic solute transporter expression we observed in mice by in situ hybridization represents stable, translatable mRNA capable of producing protein with solute transport activity, we isolated mouse embryonic and adult kidney mRNA and heterologously expressed them by injection into Xenopus oocytes. These oocytes are known to efficiently translate and process proteins encoded by exogenously introduced RNA. The functional expression of organic solute transporters was assayed by measuring the rates of cellular uptake of the prototypic organic anion PAH and organic cation TEA in RNA-injected oocytes compared with water-injected oocytes, which served as negative controls. PAH uptake was significantly increased in oocytes injected with adult kidney mRNA (Fig. 5A). We were also able to demonstrate PAH uptake in positive control oocytes that were injected with cRNA for NKT (OAT1), in contrast to a previous report (10). This difference is due to the introduction of conditions that favored organic anion uptake, particularly the use of a glutarate preloading protocol. TEA uptake was also increased in oocytes injected with adult kidney mRNA (Fig. 5B). These results demonstrate that there is indeed expression in adult mouse kidneys of stable, translatable mRNA capable of producing proteins with functional organic anion and cation transport activity. Uptake of PAH and TEA in oocytes injected with embryonic kidney mRNA, however, was not significantly different from that of water-injected controls. This likely reflects the much lower abundance of NKT (OAT1) and OCT mRNA as a proportional of the total kidney poly-A+ RNA pool in embryonic as opposed to adult mice.


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Fig. 5.   Functional expression of organic solute transport, assayed in Xenopus oocyte system. Uptake of organic anion [14C]p-aminohippurate (PAH; A) and organic cation, [14C]tetraethylammonium bromide (TEA; B) was assayed in oocytes injected with poly-A+ RNA isolated from kidneys of e17 embryos (EmbK) or adult mice (AdK). Control oocytes were injected with water, or with NKT (OAT1) cRNA. Each column represents mean ± SE for 4-10 samples. * P < 0.05 compared with water-injected oocytes.


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ABSTRACT
INTRODUCTION
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DISCUSSION
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The vital importance of organic ion transport for the adult organism is clear. However, our knowledge about the involvement of these transporters in formation and maintenance of tissues during embryogenesis is minimal. A potential developmental role was recently suggested for an "orphan" NTT4 transporter in central nervous system maturation (8). Thus developmental analysis of classically "renal" or "liver" transporters could provide an important new perspective on the role of these genes.

We report the developmental profiles of four organic ion transporters, related to the Orct gene of Drosophila: NKT (OAT1), Roct, OCT1, and NLT (OAT2) during murine embryogenesis. All of these gene products share between 31 and 47% of amino acid homology. Two of them, NKT (OAT1) and NLT (OAT2), are multispecific organic anion transporters, whereas OCT1 is known to mediate transport of small organic cations (11, 12). After the discovery of the mouse NKT (OAT1) gene (10), the rat homolog, sharing ~99% identity, was characterized. It was demonstrated that the rat homologue of NKT (OAT1) transports a wide variety of organic anions via a dicarboxylate exchange mechanism (17, 19). These molecules include endogenous substrates such as cyclic nucleotides, prostaglandins, and uric acid as well as a variety of unrelated drugs: antibiotics, a nonsteroidal anti-inflammatory drug, diuretics, an antineoplastic drug, and a uricosuric drug. It was recently reported that NLT (OAT2) mediates multispecific organic anion transport predominantly in liver. Substrate molecules included salicylates, dicarboxylates, and PAH (16). Substrate molecules of Roct, the gene sharing the highest homology with NKT (OAT1), NLT (OAT2), and OCT1, have yet to be identified.

The fact that NKT (OAT1) mediates the transport of a very wide variety of substances leads to the hypothesis that the spectrum of NKT (OAT1) substrates in the embryonic brain (or kidney) may be quite different from substances known to be transported in the adult kidneys. As embryogenesis progresses, the time course of expression patterns of NKT (OAT1) is roughly the inverse in developing brain and kidney. By in situ hybridization analysis, we demonstrated that NKT (OAT1) first appeared in the developing brain (including choroid plexus) and spinal cord. With development, levels of NKT (OAT1) declined and, postnatally, transcripts were detected only in the dura matter. In contrast, renal NKT (OAT1) expression was low at e14, which coincides with the onset of proximal tubule differentiation, and gradually increased toward birth. A similar pattern of developmental regulation in kidney and brain has been reported for the sodium/myoinositol cotransporter (7).

It had been previously reported that rat and human OCT1 genes exhibit notable differences in kinetic characteristics and expression pattern in adult tissues. Human OCT1 (hOCT1) was found to be expressed in human liver, whereas mRNA transcripts of the rat homolog (rOCT1) were localized in adult rat kidney, liver, intestine, and colon (23). In our studies, high levels of murine OCT1 transcripts were detected in a very narrow site in the embryo: the ascending aorta and the atrium of the fetal heart between e14 and e16. Interestingly, at e14 this is the only site of significant OCT1 expression detected in the whole embryo. At later stages of development (starting at e16), kidney and then liver are organs with high levels of OCT1 mRNA. We did not detect obvious OCT1 transcripts in fetal and newborn murine small intestine or colon (day e14 pc-day 5 pp). Interestingly, it has been demonstrated that, besides transport of the type 1 organic cation substrates, OCT1 efficiently transports catecholamines such as epinephrine, norepinephrine, dopamine, and indoleamine 5-hydroxytryptamine (3). Coincidentally, kidney and liver are known to mediate epinephrine and norepinephrine extraction from the circulation via nonclassical mechanisms for catecholamine transport (3). On the basis of these findings, it was concluded that OCT1 is a key element in hepatic and renal inactivation of circulating catecholamines (3). Expression of OCT1 transcripts in a very narrow spatiotemporal manner in the fetal heart and ascending aorta suggests that OCT1 may be playing a critical role in a very specific developmental process, perhaps in fetal catecholamine metabolism.

Transient Roct expression in the fetal liver coincides with the period when liver plays the major role in hematopoeisis. If Roct is involved in hematopoiesis or liver development, it is possible that it plays a role independent of any membrane transport activity. Roct is a newly cloned gene, and its substrates are yet to be identified. A member of a proton/organic cation transporter, OCTN1 was reported to have a similar developmental profile, where kidney expression is dramatically increased postnatally, whereas OCTN1 levels in liver peaked during fetal life and declined to undetectable levels in adulthood (20).

NLT (OAT2) is a multispecific transporter of organic anions via a Na-independent mechanism. It has been demonstrated that NLT (OAT2) mediates transport of a diverse group of molecules, including salicylate, acetylsalicylate, PGE2, dicarboxylates, and PAH (16). The possibility of at least partial overlap between NKT (OAT1) and NLT (OAT2) substrates is yet to be determined. Expression of NLT (OAT2) mRNA in adult (but not embryonic) kidney is lowest among the four transporters. To the extent that NLT (OAT2)'s substrate specificities in the adult kidney overlap with the other transporters, it is conceivable that NLT (OAT2) plays a "backup" role in case of inactivation of another organic anion transporter (such as NKT (OAT1)), or when the substrate load is great. Our finding of NLT (OAT2) transcripts in practically all chondrogenic structures of the developing bone system suggests involvement of NLT (OAT2) in this process and deserves further investigation.

There have been few studies analyzing expression of kidney tubular transporters during embryonic development. By Northern blot analysis, expression of Na-coupled glucose transporters SGLT-1 and -2 begins, respectively, around e18 and e17 in the rat (22). The water channel protein aquaporin-1 mRNA is not detectable until e20 in rat embryo by in situ hybridization and markedly upregulated after birth (1). The developmental expression of aquaporin-1 and -2 is remarkably different between humans and rats (4), suggesting that caution should be exercised in comparing chronological expression patterns of transporters among distantly related mammalian species. Nevertheless, at least in mice, all four organic ion transporters were expressed at relatively early stages of proximal tubular development.

The roughly coordinate expression of these closely related transporters in the developing proximal tubule suggests a common mechanism for their transcriptional regulation. Thus they serve as good markers for the onset of proximal tubulogenesis and maturation and could conceivably reflect the activity of a master regulatory gene involved in this key developmental process in the kidney (24, 25). However, they could also be involved in proximal tubule differentiation, perhaps by each transporting various organic molecules necessary for this to occur. Given their potentially overlapping substrate specificities, they may transport a single key morphogenetic molecule early in embryogenesis but diverse substrates as the nephron matures. In addition, they may serve some sort of local detoxifying function similar to their function in mature renal tissue.

Thus developmental analysis of a related group of organic ion transporters suggests that NKT (OAT1), OCT1, NLT (OAT2), and Roct are involved in key developmental events during embryogenesis, where each of these transporters may play a distinct role in the formation and maintenance of various tissues. Although it seems their most likely role would be in the transport of organic molecules involved in aspects of morphogenesis, it is also conceivable that their role is independent of a transport function, as might be the case with the Drosophila Orct gene. These speculations lead to the prediction that homozygous null mutants of this class of transporters will, at least in some cases, reveal developmental abnormalities.


    ACKNOWLEDGEMENTS

Present address of H. Sakurai: Dept. of Medicine/Pediatrics, Univ. of California, San Diego, La Jolla, CA 92093-0693.


    FOOTNOTES

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: S. K. Nigam, Dept. of Medicine/Pediatrics, Univ. of California, San Diego, 9500 Gilman Dr., La Jolla, CA 92093-0693 (E-mail: snigam{at}ucsd.edu).

Received 18 June 1999; accepted in final form 24 November 1999.


    REFERENCES
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ABSTRACT
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