Kato, Akira

Most vertebrates, including cartilaginous fishes, maintain their plasma SO42−concentration ([SO42−]) within a narrow range of 0.2–1 mM. As seawater has a [SO42−] about 40 times higher than that of the plasma, SO42−excretion is the major role of kidneys in marine teleost fishes. It has been suggested that cartilaginous fishes also excrete excess SO42−via the kidney. However, little is known about the underlying mechanisms for SO42−transport in cartilaginous fish, largely due to the extraordinarily elaborate four-loop configuration of the nephron, which consists of at least 10 morphologically distinguishable segments. In the present study, we determined cDNA sequences from the kidney of holocephalan elephant fish ( Callorhinchus milii) that encoded solute carrier family 26 member 1 (Slc26a1) and member 6 (Slc26a6), which are SO42−transporters that are expressed in mammalian and teleost kidneys. Elephant fish Slc26a1 (cmSlc26a1) and cmSlc26a6 mRNAs were coexpressed in the proximal II (PII) segment of the nephron, which comprises the second loop in the sinus zone. Functional analyses using Xenopus oocytes and the results of immunohistochemistry revealed that cmSlc26a1 is a basolaterally located electroneutral SO42−transporter, while cmSlc26a6 is an apically located, electrogenic Cl−/SO42−exchanger. In addition, we found that both cmSlc26a1 and cmSlc26a6 were abundantly expressed in the kidney of embryos; SO42−was concentrated in a bladder-like structure of elephant fish embryos. Our results demonstrated that the PII segment of the nephron contributes to the secretion of excess SO42−by the kidney of elephant fish. Possible mechanisms for SO42−secretion in the PII segment are discussed.

Significance Cation/proton antiporters (CPAs) are essential to life. The sodium/proton exchanger (NHE) branch of the CPA family has been studied exhaustively and is an important drug target; however, much less is known about the recently discovered NHA branch, represented by two genes in both humans and flies. Here we show that sodium/hydrogen antiporter (NHA) function is essential to life, and that both NHAs protect against salt stress. Their transport mechanisms are radically different, however, suggesting that function cannot be inferred from structural similarity. Although NHA2 was found to be a Na + /H + exchanger as expected, NHA1 was seen to act as an electroneutral H + /Cl - cotransporter. This is an important finding for future studies of these transporters.

The kidney of marine teleosts is the major site of Mg2+excretion and produces urine with a high Mg2+concentration. However, the transporters involved in Mg2+excretion are poorly understood. The cyclin M (Cnnm; also known as ancient conserved domain protein) family comprises membrane proteins homologous to the bacterial Mg2+and Co2+efflux protein, CorC. To understand the molecular mechanism of Mg2+homeostasis in marine teleosts, we analyzed the expression of the C nnm family genes in the seawater (SW) pufferfish, torafugu ( Takifugu rubripes), and the closely related euryhaline species, mefugu ( Takifugu obscurus). Database mining and phylogenetic analysis indicated that the Takifugu genome contains six members of the Cnnm family: two orthologs of Cnnm1, one of Cnnm2, one of Cnnm3, and two of Cnnm4. RT-PCR analyses indicated that Cnnm2, Cnnm3, and Cnnm4a are expressed in the kidney, whereas other members are mainly expressed in the brain. Renal expression of Cnnm3 was upregulated in SW mefugu, whereas renal expression of Cnnm2 was upregulated in freshwater (FW) mefugu. No significant difference was observed in renal expression of Cnnm4a between SW and FW mefugu. In situ hybridization and immunohistochemical analyses of the SW mefugu kidney revealed that Cnnm3 is expressed in the proximal tubule, and its product localizes to the lateral membrane. When Cnnm3 was expressed in Xenopus laevis oocytes, whole cellular Mg2+content and free intracellular Mg2+activity significantly decreased. These results suggest that Cnnm3 is involved in body fluid Mg2+homeostasis in marine teleosts.

Zebrafish Na+/H+exchanger 3b (zNHE3b) is highly expressed in the apical membrane of ionocytes where Na+is absorbed from ion-poor fresh water against a concentration gradient. Much in vivo data indicated that zNHE3b is involved in Na+absorption but not leakage. However, zNHE3b-mediated Na+absorption has not been thermodynamically explained, and zNHE3b activity has not been measured. To address this issue, we overexpressed zNHE3b in Xenopus oocytes and characterized its activity by electrophysiology. Exposure of zNHE3b oocytes to Na+-free media resulted in significant decrease in intracellular pH (pHi) and intracellular Na+activity ( aNai). aNaiincreased significantly when the cytoplasm was acidified by media containing CO2-HCO3−or butyrate. Activity of zNHE3b was inhibited by amiloride or 5-ethylisopropyl amiloride (EIPA). Although the activity was accompanied by a large hyperpolarization of ∼50 mV, voltage-clamp experiments showed that Na+/H+exchange activity of zNHE3b is electroneutral. Exposure of zNHE3b oocytes to medium containing NH3/NH4+resulted in significant decreases in pHiand aNaiand significant increase in intracellular NH4+activity, indicating that zNHE3b mediates the Na+/NH4+exchange. In low-Na+(0.5 mM) media, zNHE3b oocytes maintained aNaiof 1.3 mM, and Na+-influx was observed when pHiwas decreased by media containing CO2-HCO3−or butyrate. These results provide thermodynamic evidence that zNHE3b mediates Na+absorption from ion-poor fresh water by its Na+/H+and Na+/NH4+exchange activities.

The second most abundant cation in seawater (SW), Mg2+, is present at concentrations of ∼53 mM. Marine teleosts maintain plasma Mg2+concentration at 1–2 mM by excreting Mg2+into the urine. Urine Mg2+concentrations of SW teleosts exceed 70 mM, most of which is secreted by the renal tubular epithelial cells. However, molecular mechanisms of the Mg2+secretion have yet to be clarified. To identify transporters involved in Mg2+secretion, we analyzed the expression of fish homologs of the Slc41 Mg2+transporter family in various tissues of SW pufferfish torafugu ( Takifugu rubripes) and its closely related euryhaline species mefugu ( Takifugu obscurus). Takifugu genome contained five members of Slc41 genes, and only Slc41a1 was highly expressed in the kidney. Renal expression of Slc41a1 was markedly elevated when mefugu were transferred from fresh water (FW) to SW. In situ hybridization analysis and immunohistochemistry at the light and electron microscopic levels revealed that Slc41a1 is localized to vacuoles in the apical cytoplasm of the proximal tubules. These results suggest that pufferfish Slc41a1 is a Mg2+transporter involved in renal tubular transepithelial Mg2+secretion by mediating Mg2+transport from the cytosol to the vacuolar lumen, and support the hypothesis that Mg2+secretion is mediated by exocytosis of Mg2+-rich vacuoles to the lumen.

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.