Raymond W. M. Kwong

The tight junction protein claudin-b regulates epithelial permeability and sodium handling in larval zebrafish, Danio rerio

The functional role of the tight junction protein claudin-b in larval zebrafish (Danio rerio) was investigated. We showed that claudin-b protein is expressed at epithelial cell-cell contacts on the skin. Translational gene knockdown of claudin-b protein expression caused developmental defects, including edema in the pericardial cavity and yolk sac. Claudin-b morphants exhibited an increase in epithelial permeability to the paracellular marker polyethylene glycol (PEG-4000) and fluorescein isothiocyanate-dextran (FD-4). Accumulation of FD-4 was confined mainly to the yolk sac and pericardial cavity in the claudin-b morphants, suggesting these regions became particularly leaky in the absence of claudin-b expression. Additionally, Na(+) efflux was substantially increased in the claudin-b morphants, which contributed to a significant reduction in whole-body Na(+) levels. These results indicate that claudin-b normally acts as a paracellular barrier to Na(+). Nevertheless, the elevated loss of Na(+) in the morphants was compensated by an increase in Na(+) uptake. Notably, we observed that the increased Na(+) uptake in the morphants was attenuated in the presence of the selective Na(+)/Cl(-)-cotransporter (NCC) inhibitor metolazone, or during exposure to Cl(-)-free water. These results suggested that the increased Na(+) uptake in the morphants was, at least in part, mediated by NCC. Furthermore, treatment with an H(+)-ATPase inhibitor bafilomycin A1 was found to reduce Na(+) uptake in the morphants, suggesting that H(+)-ATPase activity was essential to provide a driving force for Na(+) uptake. Overall, the results suggest that claudin-b plays an important role in regulating epithelial permeability and Na(+) handling in zebrafish.

The physiology of fish at low pH: the zebrafish as a model system

Ionic regulation and acid–base balance are fundamental to the physiology of vertebrates including fish. Acidification of freshwater ecosystems is recognized as a global environmental problem, and the physiological responses to acid exposure in a few fish species are well characterized. However, the underlying mechanisms promoting ionic and acid–base balance for most fish species that have been investigated remain unclear. Zebrafish (Danio rerio) has emerged as a powerful model system to elucidate the molecular basis of ionic and acid–base regulation. The utility of zebrafish is related to the ease with which it can be genetically manipulated, its suitability for state-of-the-art molecular and cellular approaches, and its tolerance to diverse environmental conditions. Recent studies have identified several key regulatory mechanisms enabling acclimation of zebrafish to acidic environments, including activation of the sodium/hydrogen exchanger (NHE) and H+-ATPase for acid secretion and Na+ uptake, cortisol-mediated regulation of transcellular and paracellular Na+ movements, and ionocyte proliferation controlled by specific cell-fate transcription factors. These integrated physiological responses ultimately contribute to ionic and acid–base homeostasis in zebrafish exposed to acidic water. In the present review, we provide an overview of the general effects of acid exposure on freshwater fish, the adaptive mechanisms promoting extreme acid tolerance in fishes native to acidic environments, and the mechanisms regulating ionic and acid–base balance during acid exposure in zebrafish.

The interactions of iron with other divalent metals in the intestinal tract of a freshwater teleost, rainbow trout (Oncorhynchus mykiss).

This study examined the concentration-dependent interactive effects of four essential (Cu(2+), Zn(2+), Ni(2+), Co(2+)) and two non-essential (Pb(2+) and Cd(2+)) divalent metals on intestinal iron (Fe(2+)) absorption in freshwater rainbow trout (Oncorhynchusmykiss) using an invitro gut sac technique. All of the divalent metals except cobalt inhibited the intestinal Fe(2+) absorption in fish, and the magnitude of inhibition followed the order of: Ni(2+)~Pb(2+)>Cd(2+)~Cu(2+)>Zn(2+). The mucosal epithelium of the intestine was found to be the most sensitive to inhibition relative to the mucus or blood compartment, suggesting that these interactions likely occur via the divalent metal transporter-1 (DMT1). In addition, the reciprocal effects of Fe(2+) on intestinal accumulation of lead and cadmium were investigated. Elevated Fe(2+) did not affect lead accumulation in the intestine, indicating a greater affinity of Pb(2+) to the Fe(2+) transport pathway and/or the existence of additional pathways for lead absorption. In contrast, the accumulation of cadmium in the intestine decreased considerably in the presence of excess Fe(2+), indicating the importance of the Fe(2+) absorption pathway in dietary cadmium accumulation in fish. Overall, our study provides important insights into the mechanisms of dietary uptake of several divalent metals in freshwater fish.

Cortisol regulates epithelial permeability and sodium losses in zebrafish exposed to acidic water.

The effects of cortisol on epithelial permeability and sodium (Na(+)) handling during acid exposure were investigated in larval zebrafish (Danio rerio). The results demonstrated that the whole-body absorption of the paracellular permeability marker polyethylene glycol-4000 (PEG-4000) decreased with increasing levels of exogenous cortisol. Western blot analysis revealed that the abundance of the epithelial tight junction proteins occludin-a and claudin-b was increased after cortisol treatment. Furthermore, translational gene knockdown of claudin-b using an antisense morpholino oligonucleotide caused an increase in the permeability to PEG-4000, which was mitigated by cortisol treatment, further suggesting a role for cortisol in reducing paracellular permeability. Exposure to acidic water (pH 4.0 vs 7.6) caused an expected increase in the diffusive loss of Na(+) and a decrease in whole-body Na(+) levels. These disruptive effects of acute acid exposure on Na(+) balance were reduced by treatment of larvae with exogenous cortisol. Translational knockdown of the glucocorticoid receptor (GR) abolished the effects of cortisol on epithelial PEG permeability, suggesting that activation of GR was probably the major signaling pathway for reducing epithelial permeability. During acid exposure, the epithelial PEG permeability in the GR morphants was significantly higher than in the control fish. Additionally, GR morphants exhibited a more pronounced diffusive loss of Na(+) than the control fish during acid exposure. These findings suggest that cortisol may help to minimize the negative consequences of acid exposure on Na(+) homoeostasis via GR-mediated reductions in epithelial permeability and paracellular Na(+) loss.

Effects of dietary cadmium exposure on tissue-specific cadmium accumulation, iron status and expression of iron-handling and stress-inducible genes in rainbow trout: Influence of elevated dietary iron

Recent evidences suggest that dietary cadmium (Cd) uptake likely occurs via the dietary iron (Fe) uptake pathway in freshwater fish, at least in part. The present study investigated the interactive effects of dietary Cd and Fe in juvenile rainbow trout (Oncorhynchus mykiss). Fish were treated for four weeks with four different diets: normal Fe, high Fe, normal Fe plus Cd, and high Fe plus Cd. Physiological parameters, tissue-specific Fe and Cd level, plasma Fe status, and tissue-specific mRNA expression of transferrin, metallothioneins (MT-A and MT-B) and heat shock proteins 70 (HSP70a and HSP70b) were analyzed. Exposure to dietary Cd increased Cd burden in the following order: intestine>kidney>stomach>liver>gill>carcass. Interestingly, high dietary Fe reduced Cd accumulation in the stomach and intestine as well as in the wholebody of fish. Dietary Cd increased hepatic transferrin mRNA expression and total Fe binding capacity in the plasma, indicating the effect of Cd on Fe handling in fish. The mRNA expression of MTs and HSP70s was also increased in various tissues following dietary Cd exposure, however the response profile of different MT and HSP70 genes was not consistent among different tissues. In general, MT-A was more responsive to Cd exposure in the intestine and liver, whereas MT-B was more responsive in the kidney. Similarly, HSP70a expression was more sensitive to Cd exposure than HSP70b, particularly in the intestine. Interestingly, high Fe diet suppressed Cd-induced induction of transferrin, MT and HSP70 genes in various tissues. Overall, our study suggests that elevated dietary Fe can reduce Cd accumulation and ameliorate Cd-induced stress responses in freshwater fish.

The role of hydrogen sulphide in the control of breathing in hypoxic zebrafish (Danio rerio)

Hydrogen sulphide (H2S), a gaseous neurotransmitter, is involved in oxygen sensing in glomus cells, which are oxygen‐sensing cells found in the mammalian carotid body. Neuroepithelial cells (NECs) are oxygen‐sensing cells of fish and are thought to be phylogenetic precursors of mammalian glomus cells; however, the oxygen‐sensing mechanisms of these cells remain largely unknown. Both adult and larval zebrafish responded to exogenous H2S by increasing ventilation in a dose‐dependent manner; H2S increased intracellular [Ca2+] in NECs. Inhibiting endogenous H2S production decreased or abolished the ventilatory response to hypoxia in both adult and larval zebrafish. The results demonstrate an important role for H2S in oxygen sensing in zebrafish.

Molecular evidence and physiological characterization of iron absorption in isolated enterocytes of rainbow trout (Oncorhynchus mykiss): Implications for dietary cadmium and lead absorption

Recent studies suggested the probable involvement of an apical iron (Fe(2+)) transporter, the divalent metal transporter-1 (DMT1), in the uptake of several divalent metals in fish. The present study examined the gastrointestinal expression of the DMT1 gene, and investigated the kinetics of Fe(2+) uptake and its interactions with cadmium and lead in isolated enterocytes of freshwater rainbow trout (Oncorhynchus mykiss). The expressions of two DMT1 isoforms (Nramp-beta and -gamma) were recorded along the entire gastrointestinal tract of fish as well as in the enterocytes. Fe(2+) uptake in isolated enterocytes was saturable and sensitive to the proton gradient and membrane potential, suggesting DMT1-mediated transport. Both cadmium and lead inhibited Fe(2+) uptake in isolated enterocytes in a concentration-dependent manner, and lead appeared to be a stronger inhibitor than cadmium. The kinetic characterization of Fe(2+) uptake revealed that the apparent affinity of uptake was significantly decreased (increased K(m)) in the presence of either cadmium or lead, whereas the maximum uptake rate (J(max)) remained unchanged-indicating that the interaction between Fe(2+) and cadmium or lead is competitive in nature. Overall, our study suggests that the uptake of dietary cadmium and lead may occur via the iron-transporting pathway in fish.

A role for nitric oxide in the control of breathing in zebrafish (Danio rerio).

ABSTRACT Nitric oxide (NO) is a gaseous neurotransmitter, which, in adult mammals, modulates the acute hypoxic ventilatory response; its role in the control of breathing in fish during development is unknown. We addressed the interactive effects of developmental age and NO in the control of piscine breathing by measuring the ventilatory response of zebrafish (Danio rerio) adults and larvae to NO donors and by inhibiting endogenous production of NO. In adults, sodium nitroprusside (SNP), a NO donor, inhibited ventilation; the extent of the ventilatory inhibition was related to the pre-existing ventilatory drive, with the greatest inhibition exhibited during exposure to hypoxia (PO2=5.6 kPa). Inhibition of endogenous NO production using l-NAME suppressed the hypoventilatory response to hyperoxia, supporting an inhibitory role of NO in adult zebrafish. Neuroepithelial cells (NECs), the putative oxygen chemoreceptors of fish, contain neuronal nitric oxide synthase (nNOS). In zebrafish larvae at 4 days post-fertilization, SNP increased ventilation in a concentration-dependent manner. Inhibition of NOS activity with l-NAME or knockdown of nNOS inhibited the hypoxic (PO2=3.5 kPa) ventilatory response. Immunohistochemistry revealed the presence of nNOS in the NECs of larvae. Taken together, these data suggest that NO plays an inhibitory role in the control of ventilation in adult zebrafish, but an excitatory role in larvae. Summary: Nitric oxide, a gaseous neurotransmitter, plays a modulatory role in controlling breathing in zebrafish during acute changes in environmental oxygen levels, and its role changes throughout development.

Evidence for a role of tight junctions in regulating sodium permeability in zebrafish ( Danio rerio ) acclimated to ion-poor water

Freshwater teleosts are challenged by diffusive ion loss across permeable epithelia including gills and skin. Although the mechanisms regulating ion loss are poorly understood, a significant component is thought to involve paracellular efflux through pathways formed via tight junction proteins. The mammalian orthologue (claudin-4) of zebrafish (Danio rerio) tight junction protein, claudin-b, has been proposed to form a cation-selective barrier regulating the paracellular loss of Na+. The present study investigated the cellular localization and regulation of claudin-b, as well as its potential contribution to Na+ homeostasis in adult zebrafish acclimated to ion-poor water. Using a green fluorescent protein-expressing line of transgenic zebrafish, we found that claudin-b was expressed along the lamellar epithelium as well as on the filament in the inter-lamellar regions. Co-localization of claudin-b and Na+/K+-ATPase was observed, suggesting its interaction with mitochondrion-rich cells. Claudin-b also appeared to be associated with other cell types, including the pavement cells. In the kidney, claudin-b was expressed predominantly in the collecting tubules. In addition, exposure to ion-poor water caused a significant increase in claudin-b abundance as well as a decrease in Na+ efflux, suggesting a possible role for claudin-b in regulating paracellular Na+ loss. Interestingly, the whole-body uptake of a paracellular permeability marker, polyethylene glycol-400, increased significantly after prolonged exposure to ion-poor water, indicating that an increase in epithelial permeability is not necessarily coupled with an increase in passive Na+ loss. Overall, our study suggests that in ion-poor conditions, claudin-b may contribute to a selective reduction in passive Na+ loss in zebrafish.

Involvement of the calcium-sensing receptor in calcium homeostasis in larval zebrafish exposed to low environmental calcium

The involvement of the calcium-sensing receptor (CaSR) in Ca(2+) homeostasis was investigated in larval zebrafish, Danio rerio. The expression of CaSR mRNA was first observed at 3 h posfertilization (hpf) and increased with development until plateauing at ∼48 hpf. At 4 dpf, CaSR mRNA was increased in fish acclimated to low Ca(2+) water (25 μM vs. 250 μM in normal water). Using immunohistochemistry and confocal microscopy, we demonstrated that the CaSR is expressed in the olfactory epithelium, neuromasts, ionocytes on the yolk sac epithelium, and corpuscles of Stannius. Results of double immunohistochemistry and/or in situ hybridization indicated that the CaSR is localized to a subset of mitochondrion-rich ionocytes enriched with Na(+)/K(+)-ATPase and epithelial Ca(2+) channel (ecac). Translational knockdown of the CaSR prevented 4 dpf larvae from regulating whole body Ca(2+) levels when exposed to a low Ca(2+) environment. Further, the increases in ecac mRNA expression and Ca(2+) influx, normally associated with exposure to low-Ca(2+) water, were prevented by CaSR knockdown. These findings demonstrate that larval zebrafish lacking the CaSR lose their ability to regulate Ca(2+) when confronted with a low-Ca(2+) environment. Results from real-time PCR suggested that the mRNA expression of the hypocalcemic hormone stanniocalcin (stc-1) remained elevated in the CaSR morphants following acclimation to low-Ca(2+) water. Overall, the results suggest that the CaSR is critical for Ca(2+) homeostasis in larval zebrafish exposed to low environmental Ca(2+) levels, possibly owing to its modulation of stanniocalcin mRNA expression.