Kato, Akira

Na+/H+exchanger 3 (NHE3) provides one of the major Na+absorptive pathways of the intestine and kidney in mammals, and recent studies of aquatic vertebrates (teleosts and elasmobranchs) have demonstrated that NHE3 is expressed in the gill and plays important roles in ion and acid-base regulation. To understand the role of NHE3 in elasmobranch osmoregulatory organs, we analyzed renal and intestinal expressions and localizations of NHE3 in a marine elasmobranch, Japanese banded houndshark ( Triakis scyllium). mRNA for Triakis NHE3 was most highly expressed in the gill, kidney, spiral intestine, and rectum. The kidney and intestine expressed a transcriptional isoform of NHE3 (NHE3k/i), which has a different amino terminus compared with that of NHE3 isolated from the gill (NHE3g), suggesting that NHE3k/i and NHE3g arise from a single gene by alternative promoter usage. Immunohistochemical analyses of the Triakis kidney demonstrated that NHE3k/i is expressed in the apical membrane of a part of the proximal and late distal tubules in the sinus zone. In the bundle zone of the kidney, NHE3k/i was expressed in the apical membrane of the early distal tubules known as the diluting segment. In the spiral intestine and rectum, NHE3k/i was localized toward the apical membrane of the epithelial cells. The transcriptional levels of NHE3k/i were increased in the kidney when Triakis was acclimated in 130% seawater, whereas those in the spiral intestine were increased in fish acclimated in diluted seawater. These results suggest that NHE3 is involved in renal Na+reabsorption, urine acidification, and intestinal Na+absorption in elasmobranchs.

Secretion of HCO3− at the apical side of the epithelial cells of the choroid plexus is an essential step in the formation of cerebrospinal fluid. Anion conductance with a high degree of HCO3− permeability has been observed and suggested to be the major pathway for HCO3− transport across the apical membrane. Recently, it was found that NBC (Na+/HCO3− co-transporter) 4, an electrogenic member of the NBC family, was expressed in the choroid plexus. We found that a novel variant of the NBC4 [NBC4g/Slc4a5 (solute carrier family 4, sodium bicarbonate co-transporter, member 5)] is almost exclusively expressed in the apical membrane of rat choroid plexus epithelium at exceptionally high levels. RNA interference-mediated knockdown allowed the functional demonstration that NBC4g is the major player in the HCO3− transport across the apical membrane of the choroid plexus epithelium. When combined with a recent observation that in choroid plexus epithelial cells electrogenic NBC operates with a stoichiometry of 3:1, the results of the present study suggest that NBC4g mediates the efflux of HCO3− and contributes to cerebrospinal fluid production.

Marine fish drink seawater and eliminate excess salt by active salt transport across gill and gut epithelia. Euryhaline pufferfish ( Takifugu obscurus , mefugu) forms a CaCO3 precipitate on the luminal gut surface after transitioning to seawater. NBCe1 (Slc4a4) at the basolateral membrane of intestinal epithelial cell plays a major role in transepithelial intestinal HCO3− secretion and is critical for mefugu acclimation to seawater. We assayed fugu-NBCe1 (fNBCe1) activity in the Xenopus oocyte expression system. Similar to NBCe1 found in other species, fNBCe1 is an electrogenic Na+/HCO3− cotransporter and sensitive to the stilbene inhibitor DIDS. However, our experiments revealed several unique and distinguishable fNBCe1 transport characteristics not found in mammalian or other teleost NBCe1-orthologs: electrogenic Li+/ nHCO3− cotransport; HCO3− independent, DIDS-insensitive transport; and increased basal intracellular Na+ accumulation. fNBCe1 is a voltage-dependent Na+/ nHCO3− cotransporter that rectifies, independently from the extracellular Na+ or HCO3− concentration, around −60 mV. Na+ removal (0Na+ prepulse) is necessary to produce the true HCO3−-elicited current. HCO3− addition results in huge outward currents with quick current decay. Kinetic analysis of HCO3− currents reveals that fNBCe1 has a much higher transport capacity (higher maximum current) and lower affinity (higher Km) than human kidney NBCe1 (hkNBCe1) does in the physiological range (membrane potential = −80 mV; [HCO3−] = 10 mM). In this state, fNBCe1 is in favor of operating as transepithelial HCO3− secretion, opposite of hkNBCe1, from blood to the luminal side. Thus, fugu-NBCe1 represents the first ortholog-based tool to study amino acid substitutions in NBCe1 and how those change ion and voltage dependence.

Seawater (SW) contains ∼10 mM Ca2+, yet marine fish must drink seawater as their major water source. Thus marine teleosts fish need to excrete Ca2+to maintain whole body Ca2+homeostasis. In the intestine, seawater Ca2+interreacts with epithelial-secreted HCO3−by the intestinal epithelium, and the resulting CaCO3precipitates, which is rectally excreted. Recently the transporters involved in intestinal HCO3−secretion were identified. Ca2+is also excreted by the kidney, but the protein(s) involved in renal Ca2+excretion have not been identified. Here we identified a candidate transporter by using SW pufferfish torafugu ( Takifugu rubripes ) and its closely related euryhaline species mefugu ( Takifugu obscurus ), which are becoming useful animal models for studying molecular mechanisms of seawater adaptation. RT-PCR analyses of Na+/Ca2+exchanger (NCX) family members in various torafugu tissues demonstrated that only NCX2a is highly expressed in the kidney. Renal expression of NCX2a was markedly elevated when mefugu were transferred from freshwater to seawater. In situ hybridization and immunohistochemical analyses indicated that NCX2a is expressed in the proximal tubule at the apical membrane. NCX2a, expressed in Xenopus oocytes, conferred [Ca2+]out- and Na+-dependent currents. These results suggest that NCX2a mediates renal Ca2+secretion at the apical membrane of renal proximal tubules and has an important role in whole body Ca2+homeostasis of marine teleosts.

Na+ and Cl− movement across the intestinal epithelium occurs by several interconnected mechanisms: (a) nutrient-coupled Na+ absorption, (b) electroneutral NaCl absorption, (c) electrogenic Cl− secretion by CFTR, and (d) electrogenic Na+ absorption by ENaC. All these transport modes require a favorable electrochemical gradient maintained by the basolateral Na+/K+-ATPase, a Cl− channel, and K+ channels. Electroneutral NaCl absorption is observed from the small intestine to the distal colon. This transport is mediated by apical Na+/H+ (NHE2/3) and Cl−/HCO3− (Slc26a3/a6 and others) exchangers that provide the major route of NaCl absorption. Electroneutral NaCl absorption and Cl− secretion by CFTR are oppositely regulated by the autonomic nerve system, the immune system, and the endocrine system via PKAα, PKCα, cGKII, and/or SGK1. This integrated regulation requires the formation of macromolecular complexes, which are mediated by the NHERF family of scaffold proteins and involve internalization of NHE3. Through use of knockout mice and human mutations, a more detailed understanding of the integrated as well as subtle regulation of electroneutral NaCl absorption by the mammalian intestine has emerged.

Sulfate (SO42−) is the second most abundant anion in seawater (SW), and excretion of excess SO42−from ingested SW is essential for marine fish to survive. Marine teleosts excrete SO42−via the urine produced in the kidney. The SO42−transporter that secretes and concentrates SO42−in the urine has not previously been identified. Here, we have identified and characterized candidates for the long-sought transporters. Using sequences from the fugu database, we have cloned cDNA fragments of all transporters belonging to the Slc13 and Slc26 families from mefugu ( Takifugu obscurus ). We compared Slc13 and Slc26 mRNA expression in the kidney between freshwater (FW) and SW mefugu. Among 14 clones examined, the expression of a Slc26a6 paralog (mfSlc26a6A) was the most upregulated (30-fold) in the kidney of SW mefugu. Electrophysiological analyses of Xenopus oocytes expressing mfSlc26a6A, mfSlc26a6B, and mouse Slc26a6 (mSlc26a6) demonstrated that all transporters mediate electrogenic Cl−/SO42−, Cl−/oxalate2−, and Cl−/ nHCO3−exchanges and electroneutral Cl−/formate−exchange. Two-electrode voltage-clamp experiments demonstrated that the SO42−-elicited currents of mfSlc26a6A is quite large (∼35 μA at +60 mV) and 50- to 200-fold higher than those of mfSlc26a6B and mSlc26a6. Conversely, the currents elicited by oxalate and HCO3−are almost identical among mfSlc26a6A, mfSlc26a6B, and mSlc26a6. Kinetic analysis revealed that mfSlc26a6A has the highest SO42−affinity as well as capacity. Immunohistochemical analyses demonstrated that mfSlc26a6A localizes to the apical (brush-border) region of the proximal tubules. Together, these findings suggest that mfSlc26a6A is the most likely candidate for the major apical SO42−transporter that mediates SO42−secretion in the kidney of marine teleosts.