Molecular Physiological Evidence for the Role of Na+-Cl- Co-Transporter in Branchial Na+ Uptake in Freshwater Teleosts.

The gills are the major organ for Na+ uptake in teleosts. It was proposed that freshwater (FW) teleosts adopt Na+/H+ exchanger 3 (Nhe3) as the primary transporter for Na+ uptake and Na+-Cl- co-transporter (Ncc) as the backup transporter. However, convincing molecular physiological evidence to support the role of Ncc in branchial Na+ uptake is still lacking due to the limitations of functional assays in the gills. Thus, this study aimed to reveal the role of branchial Ncc in Na+ uptake with an in vivo detection platform (scanning ion-selective electrode technique, SIET) that has been recently established in fish gills. First, we identified that Ncc2-expressing cells in zebrafish gills are a specific subtype of ionocyte (NCC ionocytes) by using single-cell transcriptome analysis and immunofluorescence. After a long-term low-Na+ FW exposure, zebrafish increased branchial Ncc2 expression and the number of NCC ionocytes and enhanced gill Na+ uptake capacity. Pharmacological treatments further suggested that Na+ is indeed taken up by Ncc, in addition to Nhe, in the gills. These findings reveal the uptake roles of both branchial Ncc and Nhe under FW and shed light on osmoregulatory physiology in adult fish.

Responses of medaka (Oryzias latipes) ammonia production and excretion to overcome acidified environments.

Anthropogenic acidification of water is an on-going environmental disaster for freshwater fishes. Fishes rely on ammonia excretion to eliminate the excess acid and mitigate the harmful effects; however, it remains largely unknown how ammoniagenesis occurs and is coordinated with ammonia excretion upon acidic stress. Medaka (Oryzias latipes) was used to examine the effects of acidic stress on ammonia production and excretion. We reveal an undiscovered ammonia-producing cell type that is rich in glutaminase (GLS) and located adjacent to the ammonia-excreting ionocytes, Na+/H+ exchanger (NHE) cells, in the gills. The gills, comparing with other ammoniagenetic organs, is the quickest to respond to the acidic stress by triggering GLS-dependent ammonia production. The unique division of labor between GLS and NHE cells in the gills allows medaka to simultaneously upregulate GLS activity and ammonia excretion shortly after exposure to acidic environments. Pharmacological experiment with a GLS inhibitor abolished the activated ammonia excretion, further suggesting the essential role of the unique feature in the responses to acidic stress. Our study shades light on a novel physiological mechanism to timely and efficiently mitigate adverse effects of acidification, providing a new way to assess the impact of on-going environmental acidification on fish.

Teleostean fishes may have developed an efficient Na+ uptake for adaptation to the freshwater system.

Understanding Na+ uptake mechanisms in vertebrates has been a research priority since vertebrate ancestors were thought to originate from hyperosmotic marine habitats to the hypoosmotic freshwater system. Given the evolutionary success of osmoregulator teleosts, these freshwater conquerors from the marine habitats are reasonably considered to develop the traits of absorbing Na+ from the Na+-poor circumstances for ionic homeostasis. However, in teleosts, the loss of epithelial Na+ channel (ENaC) has long been a mystery and an issue under debate in the evolution of vertebrates. In this study, we evaluate the idea that energetic efficiency in teleosts may have been improved by selection for ENaC loss and an evolved energy-saving alternative, the Na+/H+ exchangers (NHE3)-mediated Na+ uptake/NH4 + excretion machinery. The present study approaches this question from the lamprey, a pioneer invader of freshwater habitats, initially developed ENaC-mediated Na+ uptake driven by energy-consuming apical H+-ATPase (VHA) in the gills, similar to amphibian skin and external gills. Later, teleosts may have intensified ammonotelism to generate larger NH4 + outward gradients that facilitate NHE3-mediated Na+ uptake against an unfavorable Na+ gradient in freshwater without consuming additional ATP. Therefore, this study provides a fresh starting point for expanding our understanding of vertebrate ion regulation and environmental adaptation within the framework of the energy constraint concept.

In Vivo Functional Assay in Fish Gills: Exploring Branchial Acid-Excreting Mechanisms in Zebrafish.

Molecular and physiological analyses in ionoregulatory organs (e.g., adult gills and embryonic skin) are essential for studying fish ion regulation. Recent progress in the molecular physiology of fish ion regulation was mostly obtained in embryonic skin; however, studies of ion regulation in adult gills are still elusive and limited because there are no direct methods for in vivo functional assays in the gills. The present study applied the scanning ion-selective electrode technique (SIET) in adult gills to investigate branchial H+-excreting functions in vivo. We removed the opercula from zebrafish and then performed long-term acid acclimation experiments. The results of Western blot and immunofluorescence showed that the protein expression of H+-ATPase (HA) and the number of H+-ATPase-rich ionocytes were increased under acidic situations. The SIET results proved that the H+ excretion capacity is indeed enhanced in the gills acclimated to acidic water. In addition, both HA and Na+/H+ exchanger (Nhe) inhibitors suppressed the branchial H+ excretion capacity, suggesting that H+ is excreted in association with HA and Nhe in zebrafish gills. These results demonstrate that SIET is effective for in vivo detection in fish gills, representing a breakthrough approach for studying the molecular physiology of fish ion regulation.

Estrogen-related receptor γ2 controls NaCl uptake to maintain ionic homeostasis.

Estrogen-related receptors (ERRs) are known to function in mammalian kidney as key regulators of ion transport-related genes; however, a comprehensive understanding of the physiological functions of ERRs in vertebrate body fluid ionic homeostasis is still elusive. Here, we used medaka (Oryzias melastigma), a euryhaline teleost, to investigate how ERRs are involved in ion regulation. After transferring medaka from hypertonic seawater to hypotonic freshwater (FW), the mRNA expression levels of errγ2 were highly upregulated, suggesting that Errγ2 may play a crucial role in ion uptake. In situ hybridization showed that errγ2 was specifically expressed in ionocytes, the cells responsible for Na+/Cl- transport. In normal FW, ERRγ2 morpholino knockdown caused reductions in the mRNA expression of Na+/Cl- cotransporter (Ncc), the number of Ncc ionocytes, Na+/Cl- influxes of ionocytes, and whole-body Na+/Cl- contents. In FW with low Na+ and low Cl-, the expression levels of mRNA for Na+/H+ exchanger 3 (Nhe3) and Ncc were both decreased in Errγ2 morphants. Treating embryos with DY131, an agonist of Errγ, increased the whole-body Na+/Cl- contents and ncc mRNA expression in Errγ2 morphants. As such, medaka Errγ2 may control Na+/Cl- uptake by regulating ncc and/or nhe3 mRNA expression and ionocyte number, and these regulatory actions may be subtly adjusted depending on internal and external ion concentrations. These findings not only provide new insights into the underpinning mechanism of actions of ERRs, but also enhance our understanding of their roles in body fluid ionic homeostasis for adaptation to changing environments during vertebrate evolution.

Insulin-like growth factor 1 triggers salt secretion machinery in fish under acute salinity stress.

Timely adjustment of osmoregulation upon acute salinity stress is essential for the survival of euryhaline fish. This rapid response is thought to be tightly controlled by hormones; however, there are still questions unanswered. In this work, we tested the hypothesis that the endocrine hormone, insulin-like growth factor 1 (Igf1), a slow-acting hormone, is involved in the activation of salt secretion mechanisms in euryhaline medaka (Oryzias melastigma) during acclimation to acute salinity stress. In response to a 30-ppt seawater (SW) challenge, Na+/Cl- secretion was enhanced within 0.5 h, with concomitant organization of ionocyte multicellular complexes and without changes in expression of major transporters. Igf1 receptor inhibitors significantly impair the Na+/Cl- secretion and ionocyte multicellular complex responses without affecting transporter expression. Thus, Igf1 may activate salt secretion as part of the teleost response to acute salinity stress by exerting effects on transporter function and enhancing the formation of ionocyte multicellular complexes. These findings provide new insights into hormonal control of body fluid ionic/osmotic homeostasis during vertebrate evolution.

Alkaline guts contribute to immunity during exposure to acidified seawater in the sea urchin larva.

Larval stages of members of the Abulacraria superphylum including echinoderms and hemichordates have highly alkaline midguts. To date, the reason for the evolution of such extreme pH conditions in the gut of these organisms remains unknown. Here, we test the hypothesis that, analogous to the acidic stomachs of vertebrates, these alkaline conditions may represent a first defensive barrier to protect from environmental pathogens. pH-optimum curves for five different species of marine bacteria demonstrated a rapid decrease in proliferation rates by 50-60% between pH 8.5 and 9.5. Using the marine bacterium Vibrio diazotrophicus, which elicits a coordinated immune response in the larvae of the sea urchin Strongylocentrotus purpuratus, we studied the physiological responses of the midgut pH regulatory machinery to this pathogen. Gastroscopic microelectrode measurements demonstrate a stimulation of midgut alkalization upon infection with V. diazotrophicus accompanied by an upregulation of acid-base transporter transcripts of the midgut. Pharmacological inhibition of midgut alkalization resulted in an increased mortality rate of larvae during Vibrio infection. Reductions in seawater pH resembling ocean acidification conditions lead to moderate reductions in midgut alkalization. However, these reductions in midgut pH do not affect the immune response or resilience of sea urchin larvae to a Vibrio infection under ocean acidification conditions. Our study addressed the evolutionary benefits of the alkaline midgut of Ambulacraria larval stages. The data indicate that alkaline conditions in the gut may serve as a first defensive barrier against environmental pathogens and that this mechanism can compensate for changes in seawater pH.

Did Acidic Stress Resistance in Vertebrates Evolve as Na+ /H+ Exchanger-Mediated Ammonia Excretion in Fish?

How vertebrates evolved different traits for acid excretion to maintain body fluid pH homeostasis is largely unknown. The evolution of Na+ /H+  exchanger (NHE)-mediated NH4+ excretion in fishes is reported, and the coevolution with increased ammoniagenesis and accompanying gluconeogenesis is speculated to benefit vertebrates in terms of both internal homeostasis and energy metabolism response to acidic stress. The findings provide new insights into our understanding of the possible adaptation of fishes to progressing global environmental acidification. In human kidney, titratable H+ and NH4+ comprise the two main components of net acid excretion. V-type H+ -ATPase-mediated H+ excretion may have developed in stenohaline lampreys when they initially invaded freshwater from marine habitats, but this trait is lost in most fishes. Instead, increased reliance on NHE-mediated NH4+ excretion is gradually developed and intensified during fish evolution. Further investigations on more species will be needed to support the hypothesis. Also see the video abstract here https://youtu.be/vZuObtfm-34.

Tipping points of gastric pH regulation and energetics in the sea urchin larva exposed to CO2 -induced seawater acidification.

Sea urchin larvae reduce developmental rates accompanied by changes in their energy budget when exposed to acidified conditions. The necessity to maintain highly alkaline conditions in their digestive systems led to the hypothesis that gastric pH homeostasis is a key trait affecting larval energy budgets leading to distinct tipping points for growth and survival. To test this hypothesis, sea urchin (Strongylocentrotus purpuratus) larvae were reared for 10 days in different pH conditions ranging from pH 7.0 to pH 8.2. Survival, development and growth rates were determined demonstrating severe impacts < pH 7.2. To test the effects of pH on midgut alkalization we measured midgut pH and monitored the expression of acid-base transporters. While larvae were able to maintain their midgut pH at 8.9-9.1 up to an acidification level of pH 7.2, midgut pH was decreased in the lower pH treatments. The maintenance of midgut pH under low pH conditions was accompanied by dynamic changes in the expression level of midgut acid-base transporters. Metabolic rates of the larvae increased with decreasing pH and reached a threshold between pH 7.0 and pH 7.3 where metabolic rates decreased again. Methylation analyses on promoter CpG islands were performed for midgut acid-base transporter genes to test for possible epigenetic modifications after 10-day exposure to different pH conditions. This analysis demonstrated no correlation between methylation level and pH treatments suggesting low potential for epigenetic modification of acid-base transporters upon short-term exposure. Since a clear tipping point was identified at pH 7.2, which is much lower than near-future ocean acidification (OA) scenarios, this study suggests that the early development of the purple sea urchin larva has a comparatively high tolerance to seawater acidification with substantial acclimation capacity and plasticity in a key physiological trait under near-future OA conditions.

Novel discoveries in acid-base regulation and osmoregulation: A review of selected hormonal actions in zebrafish and medaka.

Maintenance of internal ionic and acid-base homeostasis is critical for survival in all biological systems. Similar to mammals, aquatic fishes have developed sophisticated homeostatic mechanisms to mitigate metabolic or environmental disruptions in ionic and acid-base status of systemic body fluids via hormone-controlled transport of ions or acid equivalents. The present review summarizes newly discovered actions of several hormones in zebrafish (Danio rerio) and medaka (Oryzias latipes) that have greatly contributed to our overall understanding of ionic/acid-base regulation. For example, isotocin and cortisol were reported to enhance transport of various ions by stimulating the proliferation and/or differentiation of ionocyte progenitors. Meanwhile, stanniocalcin-1, a well-documented hypocalcemic hormone, was found to suppress ionocyte differentiation and thus downregulate secretion of H+ and uptake of Na+ and Cl-. Estrogen-related receptor and calcitonin gene-related peptide also regulate the differentiation of certain types of ionocytes to either stimulate or suppress H+ secretion and Cl- uptake. On the other hand, endothelin and insulin-like growth factor 1 activate the respective secretion of H+ and Na+/Cl through fast actions. These new findings enhance our understanding of how hormones regulate fish ionic and acid-base regulation while further providing new insights into vertebrate evolution, mammalian endocrinology and human disease-related therapeutics.

A SLC4 family bicarbonate transporter is critical for intracellular pH regulation and biomineralization in sea urchin embryos.

Efficient pH regulation is a fundamental requisite of all calcifying systems in animals and plants but with the underlying pH regulatory mechanisms remaining largely unknown. Using the sea urchin larva, this work identified the SLC4 HCO3- transporter family member SpSlc4a10 to be critically involved in the formation of an elaborate calcitic endoskeleton. SpSlc4a10 is specifically expressed by calcifying primary mesenchyme cells with peak expression during de novo formation of the skeleton. Knock-down of SpSlc4a10 led to pH regulatory defects accompanied by decreased calcification rates and skeleton deformations. Reductions in seawater pH, resembling ocean acidification scenarios, led to an increase in SpSlc4a10 expression suggesting a compensatory mechanism in place to maintain calcification rates. We propose a first pH regulatory and HCO3- concentrating mechanism that is fundamentally linked to the biological precipitation of CaCO3. This knowledge will help understanding biomineralization strategies in animals and their interaction with a changing environment.

Molecular Physiology of an Extra-renal Cl(-) Uptake Mechanism for Body Fluid Cl(-) Homeostasis.

The development of an ion regulatory mechanism for body fluid homeostasis was an important trait for vertebrates during the evolution from aquatic to terrestrial life. The homeostatic mechanism of Cl(-) in aquatic fish appears to be similar to that of terrestrial vertebrates; however, the mechanism in non-mammalian vertebrates is poorly understood. Unlike in mammals, in which the kidney plays a central role, in most fish species, the gill is responsible for the maintenance of Cl(-) homeostasis via Cl(-) transport uptake mechanisms. Previous studies in zebrafish identified Na(+)-Cl(-) cotransporter (NCC) 2b-expressing cells in the gills and skin as the major ionocytes responsible for Cl(-) uptake, similar to distal convoluted tubular cells in mammalian kidney. However, the mechanism by which basolateral ions exit from NCC cells is still unclear. Of the in situ hybridization signals of twelve members of the clc Cl(-) channel family, only that of clc-2c exhibited an ionocyte pattern in the gill and embryonic skin. Double in situ hybridization/immunocytochemistry confirmed colocalization of apical NCC2b with basolateral CLC-2c. Acclimation to a low Cl(-) environment increased mRNA expression of both clc-2c and ncc2b, and also the protein expression of CLC-2c in embryos and adult gills. Loss-of-function of clc-2c resulted in a significant decrease in whole body Cl(-) content in zebrafish embryos, a phenotype similar to that of ncc2b mutants; this finding suggests a role for CLC-2c in Cl(-) uptake. Translational knockdown of clc-2c stimulated ncc2b mRNA expression and vice versa, revealing cooperation between these two transporters in the context of zebrafish Cl(-) homeostasis. Further comparative genomic and phylogenetic analyses revealed that zebrafish CLC-2c is a fish-specific isoform that diverged from a kidney-predominant homologue, in the same manner as NCC2b and its counterparts (NCCs). Several lines of molecular and cellular physiological evidences demonstrated the cofunctional role of apical NCC2b and basolateral CLC-2c in the gill/skin Cl(-) uptake pathway. Taking the phylogenetic evidence into consideration, fish-specific NCC2b and CLC-2c may have coevolved to perform extra-renal Cl(-) uptake during the evolution of vertebrates in an aquatic environment.

Simplet/Fam53b is required for Wnt signal transduction by regulating ß-catenin nuclear localization.

Canonical ß-catenin-dependent Wnt signal transduction is important for several biological phenomena, such as cell fate determination, cell proliferation, stem cell maintenance and anterior-posterior axis formation. The hallmark of canonical Wnt signaling is the translocation of ß-catenin into the nucleus where it activates gene transcription. However, the mechanisms regulating ß-catenin nuclear localization are poorly understood. We show that Simplet/Fam53B (Smp) is required for Wnt signaling by positively regulating ß-catenin nuclear localization. In the zebrafish embryo, the loss of smp blocks the activity of two ß-catenin-dependent reporters and the expression of Wnt target genes, and prevents nuclear accumulation of ß-catenin. Conversely, overexpression of smp increases ß-catenin nuclear localization and transcriptional activity in vitro and in vivo. Expression of mutant Smp proteins lacking either the nuclear localization signal or the ß-catenin interaction domain reveal that the translocation of Smp into the nucleus is essential for ß-catenin nuclear localization and Wnt signaling in vivo. We also provide evidence that mammalian Smp is involved in regulating ß-catenin nuclear localization: the protein colocalizes with ß-catenin-dependent gene expression in mouse intestinal crypts; siRNA knockdown of Smp reduces ß-catenin nuclear localization and transcriptional activity; human SMP mediates ß-catenin transcriptional activity in a dose-dependent manner; and the human SMP protein interacts with human ß-catenin primarily in the nucleus. Thus, our findings identify the evolutionary conserved SMP protein as a regulator of ß-catenin-dependent Wnt signal transduction.

Anion exchanger 1b, but not sodium-bicarbonate cotransporter 1b, plays a role in transport functions of zebrafish H+-ATPase-rich cells.

Similar to mammalian proximal tubular cells, H(+)-ATPase rich (HR) cells in zebrafish skin and gills are also responsible for Na(+) uptake and acid secretion functions. However, the basolateral transport pathways in HR cells are still unclear. In the present study, we tested the hypothesis if there are specific slc4 members involved in basolateral ion transport pathways in HR cells. Fourteen isoforms were identified in the zebrafish(z) slc4 family, and the full-length cDNAs of two novel isoforms, zslc4a1b (anion exchanger, zAE1b) and zslc4a4b (Na(+)/HCO(3)(-) cotransporter, zNBCe1b), were sequenced. mRNA signals of zslc4a1b and zslc4a4b were mainly detected in certain groups of ionocytes in zebrafish skin/gills. Further double immunocytochemistry or in situ hybridization demonstrated that zAE1b, but not zNBCe1b, was localized to basolateral membranes of HR cells. Acclimation to low-Na(+) or acidic environments stimulated the mRNA expression of zslc4a1b in zebrafish gills, and loss-of-function of zslc4a1b with specific morpholinos caused significant decreases in both the whole body Na(+) content and the skin H(+) activity in the morphants. On the basis of these results, it was concluded that zAE1b, but not zNBCe1b, is involved in the basolateral transport pathways in Na(+) uptake/acid secretion mechanisms in zebrafish HR cells.

Role of SLC12A10.2, a Na-Cl cotransporter-like protein, in a Cl uptake mechanism in zebrafish (Danio rerio).

The thiazide-sensitive Na(+)-Cl(-) cotransporter (NCC), a member of the SLC12 family, is mainly expressed in the apical membrane of the mammalian distal convoluted tubule (DCT) cells, is responsible for cotransporting Na(+) and Cl(-) from the lumen into DCT cells and plays a major role in the mammalian renal NaCl reabsorption. The NCC has also been reported in fish, but the functional role in fish ion regulation is yet unclear. The present study used zebrafish as an in vivo model to test the hypothesis of whether the NCC plays a role in Na(+) and/or Cl(-) uptake mechanisms. Four NCCs were cloned, and only one of them, zebrafish (z) slc12a10.2 was found to predominately and specifically be expressed in gills. Double in situ hybridization/immunocytochemistry in zebrafish skin/gills demonstrated that the specific expression of zslc12a10.2 mRNA in a novel group of ionocytes differed from those of the previously-reported H(+)-ATPase-rich (HR) cells and Na(+)-K(+)-ATPase-rich (NaR) cells. Gill mRNA expression of zslc12a10.2 was induced by a low-Cl environment that stimulated fish Cl(-) influx, while a low-Na environment suppressed this expression. Incubation with metolazone, a specific inhibitor of the NCC, impaired both Na(+) and Cl(-) influx in 5-day postfertilization (dpf) zebrafish embryos. Translational knockdown of zslc12a10.2 with a specific morpholino caused significant decreases in both Cl(-) influx and Cl(-) content of 5-dpf zebrafish embryos, suggesting that the operation of zNCC-like 2 results in a net uptake of Cl(-) in zebrafish. On the contrary, zslc12a10.2 morphants showed increased Na(+) influx and content that resulted from upregulation of mRNA expressions of Na(+)-H(+) exchanger 3b and carbonic anhydrase 15a in HR cells. These results for the first time provide in vivo molecular physiological evidence for the possible role of the NCC in the Cl(-) uptake mechanism in zebrafish skin/gills.

The transcription factor, glial cell missing 2, is involved in differentiation and functional regulation of H+-ATPase-rich cells in zebrafish (Danio rerio).

H(+)-ATPase-rich (HR) cells in zebrafish are known to be involved in acid secretion and Na(+) uptake mechanisms in zebrafish gills/skin; however, little is known about how HR cells are functionally regulated. In the present work, we studied the roles of Drosophila glial cell missing (gcm), a cell fate-related transcription factor, in the differentiation and functional regulation of zebrafish HR cells. Zebrafish gcm2 (zgcm2) was found to begin expression in zebrafish embryos at 10 h postfertilization (hpf), and to be extensively expressed in gills but only mildly so in eyes, heart, muscles, and testes. By whole mount in situ hybridization, zgcm2 mRNA signals were found in a group of cells on the zebrafish yolk sac surface initially in the tail bud stage (10 hpf); they had disappeared at 36 hpf and thereafter appeared again in the gill region from 48 hpf. Double fluorescence in situ hybridization further demonstrated specific colocalization of zgcm2 mRNA in HR cells in zebrafish embryos. Knockdown of zgcm2 with a specific morpholino oligonucleotide caused the complete disappearance of HR cells with a concomitant decrease in H(+) activity at the apical surface of HR cells, but it did not affect the occurrence of Na(+)-K(+)-ATPase-rich cells. A decrease in the H(+)-ATPase subunit A (zatp6v1a) expression and no change in zgcm2 expression in zebrafish gills were seen from 12 h to 3 days after transfer to acidic fresh water, but a compensatory stimulation in the expressions of both genes appeared 4 days post-transfer. In conclusion, functional regulation of HR cells is probably achieved by enhancing cell differentiation via zGCM2 activation.

Carbonic anhydrase 2-like a and 15a are involved in acid-base regulation and Na+ uptake in zebrafish H+-ATPase-rich cells.

H(+)-ATPase-rich (HR) cells in zebrafish gills/skin were found to carry out Na+ uptake and acid-base regulation through a mechanism similar to that which occurs in mammalian proximal tubular cells. However, the roles of carbonic anhydrases (CAs) in this mechanism in zebrafish HR cells are still unclear. The present study used a functional genomic approach to identify 20 CA isoforms in zebrafish. By screening with whole mount in situ hybridization, only zca2-like a and zca15a were found to be expressed in specific groups of cells in zebrafish gills/skin, and further analyses by triple in situ hybridization and immunocytochemistry demonstrated specific colocalizations of the two zca isoforms in HR cells. Knockdown of zca2-like a caused no change in and knockdown of zca15a caused an increase in H+ activity at the apical surface of HR cells at 24 h postfertilization (hpf). Later, at 96 hpf, both the zca2-like a and zca15a morphants showed decreased H+ activity and increased Na+ uptake, with concomitant upregulation of znhe3b and downregulation of zatp6v1a (H+-ATPase A-subunit) expressions. Acclimation to both acidic and low-Na+ fresh water caused upregulation of zca15a expression but did not change the zca2-like a mRNA level in zebrafish gills. These results provide molecular physiological evidence to support the roles of these two zCA isoforms in Na+ uptake and acid-base regulation mechanisms in zebrafish HR cells.

Gene expression of Na+/H+ exchanger in zebrafish H+ -ATPase-rich cells during acclimation to low-Na+ and acidic environments.

In mammalian nephrons, most of the Na(+) and HCO(3)(-) is reabsorbed by proximal tubular cells in which the Na(+)/H(+) exchanger 3 (NHE3) is the major player. The roles of NHEs in Na(+) uptake/acid-base regulation in freshwater (FW) fish gills are still being debated. In the present study, functional genomic approaches were used to clone and sequence the full-length cDNAs of the nhe family from zebrafish (Danio rerio). A phylogenetic tree analysis of the deduced amino acid sequences showed that zNHE1-8 are homologous to their mammalian counterparts. By RT-PCR analysis and double/triple in situ hybridization/immunocytochemistry, only zebrafish NHE3b was expressed in zebrafish gills and was colocalized with V-H(+)-ATPase but not with Na(+)-K(+)-ATPase, indicating that H(+)-ATPase-rich (HR) cells specifically express NHE3b. A subsequent quantitative RT-PCR analysis demonstrated that acclimation to low-Na(+) FW caused upregulation and downregulation of the expressions of znhe3b and zatp6v0c (H(+)-ATPase C-subunit), respectively, in gill HR cells, whereas acclimation to acidic FW showed reversed effects on the expressions of these two genes. In conclusion, both NHE3b and H(+)-ATPase are probably involved in Na(+) uptake/acid-base regulation in zebrafish gills, like mammalian kidneys, but the partitioning of these two transporters may be differentially regulated depending on the environmental situation in which fish are acclimatized.