Hiroyasu Tsukaguchi

A family of mammalian Na+-dependent L-ascorbic acid transporters.

Vitamin C (L-ascorbic acid) is essential for many enzymatic reactions, in which it serves to maintain prosthetic metal ions in their reduced forms (for example, Fe2+, Cu+),, and for scavenging free radicals in order to protect tissues from oxidative damage. The facilitative sugar transporters of the GLUT type can transport the oxidized form of the vitamin, dehydroascorbic acid, but these transporters are unlikely to allow significant physiological amounts of vitamin C to be taken up in the presence of normal glucose concentrations, because the vitamin is present in plasma essentially only in its reduced form. Here we describe the isolation of two L-ascorbic acid transporters, SVCT1 and SVCT2, from rat complementary DNA libraries, as the first step in investigating the importance of L-ascorbic acid transport in regulating the supply and metabolism of vitamin C. We find that SVCT1 and SVCT2 each mediate concentrative, high-affinity L-ascorbic acid transport that is stereospecific and is driven by the Na+ electrochemical gradient. Despite their close sequence homology and similar functions, the two isoforms of the transporter are discretely distributed: SVCT1 is mainly confined to epithelial systems (intestine, kidney, liver), whereas SVCT2 serves a host of metabolically active cells and specialized tissues in the brain, eye and other organs.

Molecular Cloning and Characterization of a Channel-like Transporter Mediating Intestinal Calcium Absorption

Calcium is a major component of the mineral phase of bone and serves as a key intracellular second messenger. Postnatally, all bodily calcium must be absorbed from the diet through the intestine. Here we report the properties of a calcium transport protein (CaT1) cloned from rat duodenum using an expression cloning strategy in Xenopus laevis oocytes, which likely plays a key role in the intestinal uptake of calcium. CaT1 shows homology (75% amino acid sequence identity) to the apical calcium channel ECaC recently cloned from vitamin D-responsive cells of rabbit kidney and is structurally related to the capsaicin receptor and the TRP family of ion channels. Based on Northern analysis of rat tissues, a 3-kilobase CaT1 transcript is present in rat duodenum, proximal jejunum, cecum, and colon, and a 6.5-kilobase transcript is present in brain, thymus, and adrenal gland. In situ hybridization revealed strong CaT1 mRNA expression in enterocytes of duodenum, proximal jejunum, and cecum. No signals were detected in kidney, heart, liver, lung, spleen, and skeletal muscle. When expressed inXenopus oocytes, CaT1 mediates saturable Ca2+uptake with a Michaelis constant of 0.44 mm. Transport of Ca2+ by CaT1 is electrogenic, voltage-dependent, and exhibits a charge/Ca2+uptake ratio close to 2:1, indicating that CaT1-mediated Ca2+ influx is not coupled to other ions. CaT1 activity is pH-sensitive, exhibiting significant inhibition by low pH. CaT1 is also permeant to Sr2+ and Ba2+ (but not Mg2+), although the currents evoked by Sr2+ and Ba2+ are much smaller than those evoked by Ca2+. The trivalent cations Gd3+ and La3+ and the divalent cations Cu2+, Pb2+, Cd2+, Co2+, and Ni2+ (each at 100 μm) do not evoke currents themselves, but inhibit CaT1-mediated Ca2+ transport. Fe3+, Fe2+, Mn2+, and Zn2+ have no significant effects at 100 μm on CaT1-mediated Ca2+ transport. CaT1 mRNA levels are not responsive to 1,25-dihydroxyvitamin D3 administration or to calcium deficiency. Our studies strongly suggest that CaT1 provides the principal mechanism for Ca2+ entry into enterocytes as part of the transcellular pathway of calcium absorption in the intestine.

Mutations in the formin gene INF2 cause focal segmental glomerulosclerosis

Focal segmental glomerulosclerosis (FSGS) is a pattern of kidney injury observed either as an idiopathic finding or as a consequence of underlying systemic conditions. Several genes have been identified that, when mutated, lead to inherited FSGS and/or the related nephrotic syndrome. These findings have accelerated the understanding of glomerular podocyte function and disease, motivating our search for additional FSGS genes. Using linkage analysis, we identified a locus for autosomal-dominant FSGS susceptibility on a region of chromosome 14q. By sequencing multiple genes in this region, we detected nine independent nonconservative missense mutations in INF2, which encodes a member of the formin family of actin-regulating proteins. These mutations, all within the diaphanous inhibitory domain of INF2, segregate with FSGS in 11 unrelated families and alter highly conserved amino acid residues. The observation that alterations in this podocyte-expressed formin cause FSGS emphasizes the importance of fine regulation of actin polymerization in podocyte function.

Functional and molecular characterization of the human neutral solute channel aquaporin-9

In metabolically active cells, the coordinated transport of water and solutes is important for maintaining osmotic homeostasis. We recently identified a broad selective-neutral solute channel, AQP9, from rat liver that allows the passage of a wide variety of water and neutral solutes (H. Tsukaguchi, C. Shayakul, U. V. Berger, B. Mackenzie, S. Devidas, W. B. Guggino, A. N. van Hoek, and M. A. Hediger. J. Biol. Chem. 273: 24737-24743, 1998). A human homolog (hAQP9) with 76% amino acid sequence identity to rat AQP9 (rAQP9) was described, but its permeability was found to be restricted to water and urea (K. Ishibashi, M. Kuwahara, Y. Gu, Y. Tanaka, F. Marumo, and S. Sasaki. Biochem. Biophys. Res. Commun. 244: 268-274, 1998). Here we report a reevaluation of the functional characteristics of hAQP9, its tissue distribution, the structure of its gene, and its chromosomal localization. When expressed in Xenopus oocytes, hAQP9 allowed passage of a wide variety of noncharged solutes, including carbamides, polyols, purines, and pyrimidines in a phloretin- and mercurial-sensitive manner. These functional characteristics are similar to those of rAQP9. Based on Northern blot analysis, both rat and human AQP9 are abundantly expressed in liver, whereas, in contrast to rAQP9, hAQP9 is also expressed in peripheral leukocytes and in tissues that accumulate leukocytes, such as lung, spleen, and bone marrow. The human AQP9 gene is composed of 6 exons and 5 introns distributed over approximately approximately 25 kb. The gene organization is strikingly similar to that reported for human AQP3 and AQP7, suggesting their evolution from a common ancestral gene. The promoter region contains putative tonicity and glucocorticoid-responsive elements, suggesting that AQP9 may be regulated by osmolality and catabolism. Fluorescence in situ hybridization assigned its locus to chromosome 15 q22.1-22.2. Our data show that hAQP9 serves as a promiscuous solute channel expressed in both liver and peripheral leukocytes, where it is ideally suited to transport of metabolites and/or nutrients into and out of these cellsIn metabolically active cells, the coordinated transport of water and solutes is important for maintaining osmotic homeostasis. We recently identified a broad selective-neutral solute channel, AQP9, from rat liver that allows the passage of a wide variety of water and neutral solutes (H. Tsukaguchi, C. Shayakul, U. V. Berger, B. Mackenzie, S. Devidas, W. B. Guggino, A. N. van Hoek, and M. A. Hediger. J. Biol. Chem. 273: 24737-24743, 1998). A human homolog (hAQP9) with 76% amino acid sequence identity to rat AQP9 (rAQP9) was described, but its permeability was found to be restricted to water and urea (K. Ishibashi, M. Kuwahara, Y. Gu, Y. Tanaka, F. Marumo, and S. Sasaki. Biochem. Biophys. Res. Commun. 244: 268-274, 1998). Here we report a reevaluation of the functional characteristics of hAQP9, its tissue distribution, the structure of its gene, and its chromosomal localization. When expressed in Xenopus oocytes, hAQP9 allowed passage of a wide variety of noncharged solutes, including carbamides, polyols, purines, and pyrimidines in a phloretin- and mercurial-sensitive manner. These functional characteristics are similar to those of rAQP9. Based on Northern blot analysis, both rat and human AQP9 are abundantly expressed in liver, whereas, in contrast to rAQP9, hAQP9 is also expressed in peripheral leukocytes and in tissues that accumulate leukocytes, such as lung, spleen, and bone marrow. The human AQP9 gene is composed of 6 exons and 5 introns distributed over approximately ∼25 kb. The gene organization is strikingly similar to that reported for human AQP3 and AQP7, suggesting their evolution from a common ancestral gene. The promoter region contains putative tonicity and glucocorticoid-responsive elements, suggesting that AQP9 may be regulated by osmolality and catabolism. Fluorescence in situ hybridization assigned its locus to chromosome 15 q22.1-22.2. Our data show that hAQP9 serves as a promiscuous solute channel expressed in both liver and peripheral leukocytes, where it is ideally suited to transport of metabolites and/or nutrients into and out of these cells.

Cloning and characterization of the urea transporter UT3: localization in rat kidney and testis.

Urea transport in the kidney plays an important role in urinary concentration and nitrogen balance. Recently, three types of urea transporters have been cloned, UT1 and UT2 from rat and rabbit kidney and HUT11 from human bone marrow. To elucidate the physiological role of the latter urea transporter, we have isolated the rat homologue (UT3) of HUT11 and studied its distribution of expression and functional characteristics. UT3 cDNA encodes a 384 amino acid residue protein, which has 80% identity to the human HUT11 and 62% identity to rat UT2. Functional expression in Xenopus oocytes induced a large (approximately 50-fold) increase in the uptake of urea compared with water-injected oocytes. The uptake was inhibited by phloretin (0.75 mM) and pCMBS (0.5 mM) (55 and 32% inhibition, respectively). Northern analysis gave a single band of 3.8 kb in kidney inner and outer medulla, testis, brain, bone marrow, spleen, thymus, and lung. In situ hybridization of rat kidney revealed that UT3 mRNA is expressed in the inner stripe of the outer medulla, inner medulla, the papillary surface epithelium, and the transitional urinary epithelium of urinary tracts. Co-staining experiments using antibody against von Willebrand factor showed that UT3 mRNA in the inner stripe of the outer medulla is expressed in descending vasa recta. These data suggest that UT3 in kidney is involved in counter current exchange between ascending and descending vasa recta, to enhance the cortico-papillary osmolality gradient. In situ hybridization of testis revealed that UT3 is located in Sertoli cells of seminiferous tubules. The signal was only detected in Sertoli cells associated with the early stages of spermatocyte development, suggesting that urea may play a role in spermatogenesis.

Toll-Like Receptor 9 Affects Severity of IgA Nephropathy

Environmental pathogens are suspected to aggravate renal injury in IgA nephropathy (IgAN), but neither underlying mechanisms nor specific exogenous antigens have been identified. In this study, a genome-wide scan of ddY mice, which spontaneously develop IgAN, was performed, and myeloid differentiation factor 88 (MyD88) was identified as a candidate gene for progression of renal injury (chi(2) = 21.103, P = 0.00017). For evaluation of the potential influence of environmental pathogens on progression of renal injury, ddY mice were housed in either conventional or specific pathogen-free conditions. Expression of genes encoding toll-like receptors (TLR) and the signaling molecule MyD88 were quantified by real-time reverse transcription-PCR in splenocytes. Although the housing conditions did not affect the prevalence of IgAN, the severity of renal injuries was higher in the conventionally housed group. Mice that had IgAN and were housed in conventional conditions had higher levels of TLR9 and MyD88 transcripts than mice that had IgAN and were housed in specific pathogen-free conditions. Furthermore, nasal challenge with CpG-oligodeoxynucleotides, which are ligands for TLR9, aggravated renal injury, led to strong Th1 polarization, and increased serum and mesangial IgA. For investigation of whether these results may be generalizable to humans, single-nucleotide polymorphisms in the TLR9 and MyD88 genes were analyzed in two cohorts of patients with IgAN; an association was observed between TLR9 polymorphisms and disease progression. In summary, these findings suggest that activation of the TLR9/MyD88 pathway by common antigens may affect the severity of IgAN.

Molecular and functional analysis of SDCT2, a novel rat sodium-dependent dicarboxylate transporter

Kidney proximal tubule cells take up Krebs cycle intermediates for metabolic purposes and for secretion of organic anions through dicarboxylate/organic anion exchange. Alteration in reabsorption of citrate is closely related to renal stone formation. The presence of distinct types of sodium-coupled dicarboxylate transporters has been postulated on either side of the polarized epithelial membrane in the kidney proximal tubule. Using a PCR-based approach, we isolated a novel member of the sodium-dependent dicarboxylate/sulfate transporter called SDCT2. SDCT2 is a 600-amino acid residue protein that has 47-48% amino acid identity to SDCT1 and NaDC-1, previously identified in kidney and intestine. Northern analysis gave a single band of 3.3 kb for SDCT2 in kidney, liver, and brain. In situ hybridization revealed that SDCT2 is prominently expressed in kidney proximal tubule S3 segments and in perivenous hepatocytes, consistent with the sites of high-affinity dicarboxylate transport identified based on vesicle studies. A signal was also detected in the meningeal layers of the brain. SDCT2 expressed in Xenopus oocytes mediated sodium-dependent transport of di- and tricarboxylates with substrate preference for succinate rather than citrate, but excluding monocarboxylates. SDCT2, unlike SDCT1, displayed a unique pH dependence for succinate transport (optimal pH 7.5-8.5) and showed a high affinity for dimethylsuccinate, two features characteristic of basolateral transport. These data help to interpret the mechanisms of renal citrate transport, their alteration in pathophysiological conditions, and their role in the elimination of organic anions and therapeutic drugs.

Binding-, intracellular transport-, and biosynthesis-defective mutants of vasopressin type 2 receptor in patients with X-linked nephrogenic diabetes insipidus.

Nephrogenic diabetes insipidus (NDI) is most often an X-linked disorder in which urine is not concentrated due to renal resistance to arginine vasopressin. We recently identified four vasopressin type 2 receptor gene mutations in unrelated X-linked NDI families, including R143P, delta V278, R202C, and 804insG. All these mutations reduced ligand binding activity to < 10% of the normal without affecting mRNA accumulation. To elucidate whether the receptors are expressed on the cell surface, we analyzed biosynthesis and localization of tagged or untagged receptors stably expressed in Chinese hamster ovary (CHO) cells, using two antibodies directed against distinct termini. Whole-cell and surface labeling studies revealed that the R202C clone had both surface-localized (50-55 kD) and intracellular proteins (40 and 75 kD), similar to the wild-type AVPR2 clone, whereas the R143P and delta V278 clones lacked the surface receptors, despite relatively increased intracellular components. The 804insG mutant cell produced no proteins despite an adequate mRNA level. Immunofluorescence staining confirmed that the R202C mutant reaches the cell surface, whereas the R143P and delta V278 mutants are retained within the cytoplasmic compartment. Thus, R202C, R143P/delta V278, and 804insG result in three distinct phenotypes, that is, a simple binding impairment at the cell surface, blocked intracellular transport, and ineffective biosynthesis or/and accelerated degradation of the receptor, respectively, and therefore are responsible for NDI. This phenotypic classification will help understanding of molecular pathophysiology of this disorder.

Distribution of mRNA for the facilitated urea transporter UT3 in the rat nervous system

Abstract Recently, the cDNA encoding the rat urea transporter UT3 has been cloned from rat kidney. Here we describe the cellular localization of this transporter in the brain as detected by non-radioactive in situ hybridization. UT3 is expressed in astrocytes throughout the central nervous system as well as in Bergmann glia in the cerebellum. The expression in astrocytes was verified by double staining using the astrocytic marker GFAP. UT3 mRNA is also strongly expressed by the ependymal cells lining the cerebral ventricles and by Müller cells in the retina. Furthermore, UT3 expression was detected in subgroups of neurons in the inferior colliculus and dorsal root ganglia, as well as in cells in the anterior pituitary gland. Other types of brain cells, including oligodendrocytes, microglia, tanycytes, endothelial cells of blood vessels, and epithelial cells in the choroid plexus were devoid of UT3 mRNA. Northern blot analysis confirmed that the mRNA species in the brain and in dorsal root ganglia are identical, and that cultured astrocytes and C6 cells also express the UT3 mRNA. UT3 mRNA expression by astrocytes is markedly upregulated in quinolinic acid-induced gliosis, possibly as a result of increased urea levels during gliosis induced polyamine formation. We propose that UT3 in astrocytes represents a mechanism to control urea formed in the brain by equilibrating it throughout the astrocyte network and guiding it to blood vessels and the CSF for disposal.

Phosphorylation of Nephrin Triggers Its Internalization by Raft-Mediated Endocytosis

Proper localization of nephrin determines integrity of the glomerular slit diaphragm. Slit diaphragm proteins assemble into functional signaling complexes on a raft-based platform, but how the trafficking of these proteins coordinates with their signaling function is unknown. Here, we demonstrate that a raft-mediated endocytic (RME) pathway internalizes nephrin. Nephrin internalization was slower with raft-mediated endocytosis than with classic clathrin-mediated endocytosis. Ultrastructurally, the RME pathway consisted of noncoated invaginations and was dependent on cholesterol and dynamin. Nephrin constituted a stable, signaling-competent microdomain through interaction with Fyn, a Src kinase, and podocin, a scaffold protein. Tyrosine phosphorylation of nephrin triggered its own RME-mediated internalization. Protamine-induced hyperphosphorylation of nephrin led to noncoated invaginations predominating over coated pits. These results demonstrate that an RME pathway couples nephrin internalization to its own signaling, suggesting that RME promotes proper spatiotemporal assembly of slit diaphragms during podocyte development or injury.