NKA Α-subunit Research Articles

Paralog switching facilitates diadromy: ontogenetic, microevolutionary and macroevolutionary evidence.

Identifying how the demands of migration are met at the level of gene expression is critical for understanding migratory physiology and can potentially reveal how migratory forms evolve from nonmigratory forms and vice versa. Among fishes, migration between freshwater and seawater (diadromy) requires considerable osmoregulatory adjustments, powered by the ion pump Na+, K+-ATPase (NKA) in the gills. Paralogs of the catalytic α-subunit of the pump (NKA α1a and α1b) are reciprocally upregulated in fresh- and seawater, a response known as paralog-switching, in gills of some diadromous species. We tested ontogenetic changes in NKA α-subunit paralog expression patterns, comparing pre-migrant and migrant alewife (Alosa pseudoharengus) sampled in their natal freshwater environment and after 24h in seawater. In comparison to pre-migrants, juvenile out-migrants exhibited stronger paralog switching via greater downregulation of NKA α1a in seawater. We also tested microevolutionary changes in the response, exposing juvenile diadromous and landlocked alewife to freshwater (0 ppt) and seawater (30 ppt) for 2, 5, and 15days. Diadromous and landlocked alewife exhibited salinity-dependent paralog switching, but levels of NKA α1b transcription were higher and the decrease in NKA α1a was greater after seawater exposure in diadromous alewife. Finally, we placed alewife α-subunit NKA paralogs in a macroevolutionary context. Molecular phylogenies show alewife paralogs originated independently of paralogs in salmonids and other teleosts. This study demonstrated that NKA paralog switching is tied to halohabitat profile and that duplications of the NKA gene provided the substrate for multiple, independent molecular solutions that support a diadromous life history.

Genome-wide identification and quantification of salinity-responsive Na+/K+-ATPase α-subunits in three salmonids

Na+/K+-ATPase (NKA) is a ubiquitous ion-transporting pump that functions to maintain intracellular homeostasis. We performed a comprehensive assessment of salinity response in terms of genome-wide identification, evolutionary footprint, structural characterization, expression patterns of NKA α-subunit genes and NKA activity in three salmonids, including rainbow trout (Oncorhynchus mykiss), steelhead trout (O. mykiss irideus), and Atlantic salmon (Salmo salar). From the O. mykiss and S. salar genomes, a total of 48 NKAα genes were identified and classified into nine isoforms: NKAα1 (a/b/b2/c), NKAα2 (a/b), and NKAα3 (a/b/b2). The intergenomic homologous genes across O. mykiss and S. salar exhibited multicollinearity relationships, with two tandem duplicate events occurring mainly in NKAα1b/b2 and α1c. The predicted motifs and domains of NKAα genes were highly conserved, accompanied by unequal transmembrane domains (TMDs) that contained specific functional sites (Na+-K+/ATP/phosphorylation). The expression profiles of the nine NKAα isoforms in three salmonids were tissue-specific, and the expression levels of NKAα1 except α1c were the highest in the osmoregulatory organs (e.g. gill and mesonephros), while those of NKAα2/α3 were mainly in the excitable tissues (e.g. muscle and brain). Exploration of potential indicators for three salmonids as follows: In the rainbow trout, NKAα2a and NKAα1b/b2 expression were extremely sensitive to salinity change in the gill following transfer to seawater. In the steelhead trout, NKAα3 (a/b/b2) responded more significantly in the heart. In the Atlantic salmon, NKAα1b/b2 isoforms were more prominent in the muscle exposed to seawater. These changes in expression of NKAα were associated with increased NKA activity in the gill and muscle and exhibited salinity-dependent variation and differences between the osmoregulatory capacity of parr and smolt. These results provide insights into the molecular mechanisms of NKAα-mediated salinity sensitivity as well as deliver effective indicators for elucidating the osmoregulation strategy of salmonids.

Effects of hypo-osmotic shock on osmoregulatory responses and expression levels of selected ion transport-related genes in the sesarmid crab Episesarma mederi (H. Milne Edwards, 1853)

This study examined the osmoregulatory responses to hypo-osmotic shock in the commercially and ecologically important crab Episesarma mederi (H. Milne Edwards, 1853). After the acclimation for one week at a salinity of 25 PSU, Adult males E. mederi were immediately exposed to salinities of 5 PSU and 25 PSU (the control group). The time course of changes in haemolymph osmolality, gill Na+/K+ ATPase (NKA) activity, oxygen uptake rates, and mRNA expression levels of ion-transport related genes, including the NKA-α subunit, V-type H+ATPase (VT) and Na+/K+/2Cl−(NKCC), were determined. The results showed that E. mederi was a strong hyperosmoregulator after exposure to 5 PSU, achieved by modulations of NKA activity in their posterior gills rather than the anterior gills. The crabs acclimated to 5 PSU increased oxygen uptake, especially during the initial exposure, reflecting increased energetic costs for osmotic stress responses. In the posterior gills, the NKA activities of the crabs acclimated to 5 PSU at 3, 72 and 168 h were significantly higher than those in the control group. Elevated NKA-α subunit expression levels were detected at 6 h and 12 h. Increased expression levels of VT and NKCC were identified at 6 h and 12 h, respectively. Our results indicate that elevated gill NKA activity at 3 h could result from enzyme activity and kinetic alterations. On the other hand, the gill NKA activity at 72 and 168 h was sustained by elevated NKA-α subunit expression. Hence, these adaptive responses in osmoregulation enable the crabs to withstand hypo-osmotic challenges and thrive in areas of fluctuating salinity in mangroves and estuaries.

DR region of NKAα1 is a target to ameliorate hepatic lipid metabolism disturbance in obese mice

BackgroundNa+/K+-ATPase (NKA), an ion pumping enzyme ubiquitously expressed in various cells, is critically involved in cellular ion homeostasis and signal transduction. However, the role of NKA in hepatic lipid homeostasis has yet to be fully characterized. MethodsThe activity of NKA and NKAα1 expression were determined in steatotic cells, mice and patients. The roles of NKAα1 in hepatosteatosis were detected using hepatocyte knockout or specific overexpression of NKAα1 in mice. ResultsHerein, we demonstrated that the expression and activity of α1 subunit of NKA (NKAα1) were lowered in the livers of nonalcoholic fatty liver disease (NAFLD) patients, high-fat diet (HFD)-induced obese mice, and genetically obese (ob/ob, db/db) mice, as well as oleic acid-induced hepatocytes. Hepatic deficiency of NKAα1 exacerbated, while adeno-associated virus-mediated liver specific overexpression of NKAα1 alleviated hepatic steatosis through regulation of fatty acid oxidation (FAO) and lipogenesis. Mechanistically, we revealed that NKAα1 upregulated sirtuin 1 (SIRT1) via interacting with ubiquitin specific peptidase 22 (USP22), a deubiquitinating enzyme for the stabilization and deubiquitination of SIRT1, thus activating the downstream autophagy signaling. Blockade of the SIRT1/autophagy signaling pathway eliminated the protective effects of NKAα1 against lipid deposition in hepatocytes. Importantly, we found that an antibody against the DR region (897DVEDSYGQQWTYEQR911) of NKAα1 subunit (DR-Ab) ameliorated hepatic steatosis through maintaining the membrane density of NKAα1 and inducing its activation. ConclusionsCollectively, this study renews the functions of NKAα1 in liver lipid metabolism and provides a new clue for gene therapy or antibody treatment of hepatic lipid metabolism disturbance by targeting NKAα1.

Anti–Na+/K+-ATPase DR antibody attenuates UUO-induced renal fibrosis through inhibition of Na+/K+-ATPase α1–dependent HMGB1 release

Reduced Na+/K+-ATPase (NKA) activity and NKAα1 expression are engaged in the pathologies of renal diseases. NKA-mediated Src activation is not the only reason for NKA-related renal fibrosis. In this study, we found that genetic reduction of NKAα1 exhibited exacerbated tubulointerstitial lesions and fibrosis in the UUO mice model. Activation of NKAα1 with an antibody against the extracellular DR region of the NKAα1 subunit (DRm217) prevented UUO-induced tubulointerstitial lesions, preserved kidney function, and decrease renal fibrosis. Further studies revealed that NKAα1 deficiency mice exhibited high inflammation factors expression when they suffered UUO surgery, compared with NKAα1+/+ (WT) mice. DRm217 alleviated inflammatory cell infiltration, suppress NF-κB phosphorylation, and decreased inflammatory factors expression in the UUO mice model. Released HMGB1 can trigger the inflammatory response and contribute to renal fibrosis. Knockdown of NKA in renal tubular cells or in NKAα1+/- mice was associated with more susceptibility to HMGB1 release in the UUO mice model. DRm217 exerted its antifibrotic effect via inhibiting HMGB1 release. Furthermore, AMPK activation participates in the effect of DRm217 on inhibiting HMGB1 release. Our findings suggest that NKAα1 is a regulator of renal fibrosis and its DR-region is a novel target on it.

Na+/K+-ATPase as a Target of Cardiac Glycosides for the Treatment of SARS-CoV-2 Infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), identified for the first time in Wuhan, China, causes coronavirus disease 2019 (COVID-19), which moved from epidemic status to becoming a pandemic. Since its discovery in December 2019, there have been countless cases of mortality and morbidity due to this virus. Several compounds such as chloroquine, hydroxychloroquine, lopinavir-ritonavir, and remdesivir have been tested as potential therapies; however, no effective treatment is currently recommended by regulatory agencies. Some studies on respiratory non-enveloped viruses such as adenoviruses and rhinovirus and some respiratory enveloped viruses including human respiratory syncytial viruses, influenza A, parainfluenza, SARS-CoV, and SARS-CoV-2 have shown the antiviral activity of cardiac glycosides, correlating their effect with Na+/K+-ATPase (NKA) modulation. Cardiac glycosides are secondary metabolites used to treat patients with cardiac insufficiency because they are the most potent inotropic agents. The effects of cardiac glycosides on NKA are dependent on cell type, exposure time, and drug concentration. They may also cause blockage of Na+ and K+ ionic transport or trigger signaling pathways. The antiviral activity of cardiac glycosides is related to cell signaling activation through NKA inhibition. Nuclear factor kappa B (NFκB) seems to be an essential transcription factor for SARS-CoV-2 infection. NFκB inhibition by cardiac glycosides interferes directly with SARS-CoV-2 yield and inflammatory cytokine production. Interestingly, the antiviral effect of cardiac glycosides is associated with tyrosine kinase (Src) activation, and NFκB appears to be regulated by Src. Src is one of the main signaling targets of the NKA α-subunit, modulating other signaling factors that may also impair viral infection. These data suggest that Src-NFκB signaling modulated by NKA plays a crucial role in the inhibition of SARS-CoV-2 infection. Herein, we discuss the antiviral effects of cardiac glycosides on different respiratory viruses, SARS-CoV-2 pathology, cell signaling pathways, and NKA as a possible molecular target for the treatment of COVID-19.

Anti-Na+/K+-ATPase immunotherapy ameliorates α-synuclein pathology through activation of Na+/K+-ATPase α1-dependent autophagy.

Na+/K+-ATPase (NKA) plays important roles in maintaining cellular homeostasis. Conversely, reduced NKA activity has been reported in aging and neurodegenerative diseases. However, little is known about the function of NKA in the pathogenesis of Parkinson's disease (PD). Here, we report that reduction of NKA activity in NKAα1+/- mice aggravates α-synuclein-induced pathology, including a reduction in tyrosine hydroxylase (TH) and deficits in behavioral tests for memory, learning, and motor function. To reverse this effect, we generated an NKA-stabilizing monoclonal antibody, DR5-12D, against the DR region (897DVEDSYGQQWTYEQR911) of the NKAα1 subunit. We demonstrate that DR5-12D can ameliorate α-synuclein-induced TH loss and behavioral deficits by accelerating α-synuclein degradation in neurons. The underlying mechanism for the beneficial effects of DR5-12D involves activation of NKAα1-dependent autophagy via increased AMPK/mTOR/ULK1 pathway signaling. Cumulatively, this work demonstrates that NKA activity is neuroprotective and that pharmacological activation of this pathway represents a new therapeutic strategy for PD.

DR-region of Na+/K+-ATPase is a target to ameliorate hepatic insulin resistance in obese diabetic mice.

Reduced hepatic Na+/K+-ATPase (NKA) activity and NKAα1 expression are engaged in the pathologies of metabolism diseases. The present study was designed to investigate the potential roles of NKAα1 in hepatic gluconeogenesis and glycogenesis in both hepatocytes and obese diabetic mice.Methods: Insulin resistance was mimicked by glucosamine (GlcN) in either human hepatocellular carcinoma (HepG2) cells or primary mouse primary hepatocytes. Obese diabetic mice were induced by high-fat diet (HFD) feeding for 12 weeks.Results: We found that both NKA activity and NKAα1 protein level were downregulated in GlcN-treated hepatocytes and in the livers of obese diabetic mice. Pharmacological inhibition of NKA with ouabain worsened, while activation of NKAα1 with an antibody against an extracellular DR region of NKAα1 subunit (DR-Ab) prevented GlcN-induced increase in gluconeogenesis and decrease in glycogenesis. Likewise, the above results were also corroborated by the opposite effects of genetic knockout/overexpression of NKAα1 on both gluconeogenesis and glycogenesis. In obese diabetic mice, hepatic activation or overexpression of NKAα1 stimulated the PI3K/Akt pathway to suppress hyperglycemia and improve insulin resistance. More importantly, loss of NKA activities in NKAα1+/- mice was associated with more susceptibility to insulin resistance following HFD feeding.Conclusions: Our findings suggest that NKAα1 is a physiological regulator of glucose homoeostasis and its DR-region is a novel target to treat hepatic insulin resistance.

AMPK alleviates high uric acid-induced Na+-K+-ATPase signaling impairment and cell injury in renal tubules

One of the mechanisms in hyperuricemia (HUA)-induced renal tubular injury is the impairment of Na+-K+-ATPase (NKA) signaling, which further triggers inflammation, autophagy, and mitochondrial dysfunction and leads to cell injury. Here, we used RNA sequencing to screen the most likely regulators of NKA signaling and found that the liver kinase B1(LKB1)/adenosine monophosphate (AMP)-activated protein kinase (AMPK)/ mammalian target of rapamycin (mTOR) pathway was the most abundantly enriched pathway in HUA. AMPK is a key regulator of cell energy metabolism; hence, we examined the effect of AMPK on HUA-induced dysregulation of NKA signaling and cell injury. We first detected AMPK activation in high uric acid (UA)-stimulated proximal tubular epithelial cells (PTECs). We further found that sustained treatment with the AMPK activator 5-aminoimidazole-4-carboxamide 1-β-d-ribofuranoside (AICAR), but not the AMPK inhibitor Compound C, significantly alleviated UA-induced reductions in NKA activity and NKA α1 subunit expression on the cell membrane by reducing NKA degradation in lysosomes; sustained AICAR treatment also significantly alleviated activation of the NKA downstream molecules Src and interleukin-1β (IL-1β) in PTECs. AICAR further alleviated high UA-induced apoptosis, autophagy, and mitochondrial dysfunction. Although AMPK activation by metformin did not reduce serum UA levels in hyperuricemic rats, it significantly alleviated HUA-induced renal tubular injury and NKA signaling impairment in vivo with effects similar to those of febuxostat. Our study suggests that AMPK activation may temporarily compensate for HUA-induced renal injury. Sustained AMPK activation could reduce lysosomal NKA degradation and maintain NKA function, thus alleviating NKA downstream inflammation and protecting tubular cells from high UA-induced renal tubular injury.

Effects of different photoperiod regimes on the smoltification and seawater adaptation of seawater-farmed rainbow trout (Oncorhynchus mykiss): Insights from Na+, K+–ATPase activity and transcription of osmoregulation and growth regulation genes

Photoperiod is thought to be the main zeitgeber that induces smoltification in salmonids. However, its effects on the smoltification of rainbow trout (Oncorhynchus mykiss) are not fully understood and no published data documents the effects of the photoperiod regime currently used commercially, continuous light (LL). The present study compared the effect of four different photoperiod regimes (i.e. advanced phase photoperiod (APP), delayed phase photoperiod (DPP), LL and simulated natural photoperiod (SNP)) on the smoltification and growth of juvenile rainbow trout during their freshwater phase of winter-spring and the following summer post smolt phase. Smoltification was evaluated by monitoring gill Na+,K+–ATPase (NKA) activity and transcription of NKA α-subunit isoforms 1a and 1b, and Na+,K+,2Cl‐cotransporter 1a. Growth was measured as specific growth rate of both length and weight, and through molecular growth proxies such as the levels of circulating insulin-like growth factor 1 (IGF-I) in plasma and transcription of igf-I, igf binding protein 1b (igfbp1b), growth hormone receptor 1 (ghr1) and cathepsin L (ctsl) in the liver. Results indicate that APP induces a longer smolt window and higher levels of plasma IGF-I in both freshwater and seawater (two months post transfer), while DPP led to a shorter smolt window, lower plasma IGF-I levels in freshwater and seawater, an earlier decrease in liver igf-I and ctsl transcription in freshwater (as seen by modelling over time) and lower specific growth rate in freshwater. The transcription analysis of osmoregulatory genes complemented NKA activity and allowed for the detection of a transient response to light and of differences between the osmoregulatory capacity of parr and desmolted fish. Furthermore, an upregulation of the liver transcription of igf-I, ghr1 and ctsl was found in all treatments during the smolt window, which corresponded to the periods with highest growth. Finally, both plasma IGF-I and liver igf-I in seawater were found to be significantly correlated to fish growth in seawater. However, our data did not show that plasma IGF-I prior to seawater transfer could be used as a reliable predictor of growth in seawater. Overall, and especially when compared with other salmonid species, photoperiod seems to be a weaker inducer of smoltification in rainbow trout, according to the parameters that were tested, suggesting that other environmental cues might be more important drivers of this process.

DR-region of Na+/K+ ATPase is a target to treat excitotoxicity and stroke

Na+/K+ ATPase (NKA) is important in maintaining cellular functions. We found that loss of NKA activities in NKAα1+/− mice is associated with increased susceptibility to ischemic injuries following transient middle cerebral artery occlusion (tMCAO). This is corroborated by the neuroprotective effects of an antibody raised against an extracellular DR region (897DVEDSYGQQWTYEQR911, sequence number as in rat) of NKAα subunit (DR-Ab) in both preventive and therapeutic settings. DR-Ab protects cortical neurons against glutamate-induced toxicity by stimulating activities of NKA and Na+/Ca2+ exchanger (NCX), which resulted in accelerated Ca2+ extrusion. DR-Ab also enhanced the association between NKA and GluR2 and therefore reduced the internalization of both proteins from membrane induced by glutamate toxicity. The mechanism appears to involve suppression of GluR2 phosphorylation through PKCα/PICK pathway. Our data indicate that DR-region of NKA may be a novel therapeutic target for drug development for the treatment of ischemic stroke.

Intestinal FXYD12 and sodium-potassium ATPase: A comparative study on two euryhaline medakas in response to salinity changes.

FXYD proteins are the regulators of sodium-potassium ATPase (Na+/K+-ATPase, NKA). In teleosts, NKA is a primary driving force for the operation of many ion transport systems in the osmoregulatory organs (e.g. intestines). Hence, the purpose of this study was to determine the expression of FXYD proteins and NKA α-subunit in the intestines of two closely related medakas (Oryzias dancena and O. latipes), which came from different salinity habitats and have diverse osmoregulatory capabilities, to illustrate the association between NKA and FXYD proteins of two medaka species in response to salinity changes. The results showed that the fxyd12 mRNA was the most predominant in the intestines of both medakas. The association of FXYD12 and NKA in the intestines of the two medaka species was demonstrated via double immunofluorescent staining and co-immunoprecipitation. Upon salinity challenge, the localization of FXYD12 and NKA was similar in the intestines of the two medaka species. However, the expression profiles of intestinal FXYD12 and NKA (mRNA and protein levels), as well as NKA activity differed between the medakas. These results showed that FXYD12 may play a role in modulating NKA activity in the intestines of the two medakas following salinity changes in the maintenance of internal homeostasis. These findings contributed to knowledge of the expression and potential role of vertebrate FXYD12, the regulators of NKA, upon salinity challenge.

Osmoregulation in the Plotosidae Catfish: Role of the Salt Secreting Dendritic Organ.

Unlike other marine teleosts, the Plotosidae catfishes reportedly have an extra-branchial salt secreting dendritic organ (DO). Salinity acclimation [brackishwater (BW) 3aaa, seawater (SWcontrol) 34aaa, and hypersaline water (HSW) 60aaa] for 14 days was used to investigate the osmoregulatory abilities of Plotosus lineatus through measurements of blood chemistry, muscle water content (MWC), Na+/K+-ATPase (NKA) specific activity and ion transporter expression in gills, DO, kidney and intestine. Ion transporter expression was determined using immunoblotting, immunohistochemistry (IHC) and quantitative polymerase chain reaction (qPCR). HSW elevated mortality, plasma osmolality and ions, and hematocrit, and decreased MWC indicating an osmoregulatory challenge. NKA specific activity and protein levels were significantly higher in DO compared to gill, kidney and intestine at all salinities. NKA specific activity increased in kidney and posterior intestine with HSW but only kidney showed correspondingly higher NKA α-subunit protein levels. Since DO mass was greater in HSW, the total amount of DO NKA activity expressed per gram fish was greater indicating higher overall capacity. Gill NKA and V-ATPase protein levels were greater with HSW acclimation but this was not reflected in NKA activity, mRNA or ionocyte abundance. BW acclimation resulted in lower NKA activity in gill, kidney and DO. Cl- levels were better regulated and the resulting strong ion ratio in BW suggests a metabolic acidosis. Elevated DO heat shock protein 70 levels in HSW fish indicate a cellular stress. Strong NKA and NKCC1 (Na+:K+:2Cl- cotransporter1) co-localization was observed in DO parenchymal cells, which was rare in gill ionocytes. NKCC1 immunoblot expression was only detected in DO, which was highest at HSW. Cystic fibrosis transmembrane regulator Cl- channel (CFTR) localize apically to DO NKA immunoreactive cells. Taken together, the demonstration of high NKA activity in DO coexpressed with NKCC1 and CFTR indicates the presence of the conserved secondary active Cl- secretion mechanism found in other ion transporting epithelia suggesting a convergent evolution with other vertebrate salt secreting organs. However, the significant osmoregulatory challenge of HSW indicates that the DO may be of limited use under more extreme salinity conditions in contrast to the gill based ionoregulatory strategy of marine teleosts.

The inner mantle of the giant clam, Tridacna squamosa, expresses a basolateral Na+/K+-ATPase α-subunit, which displays light-dependent gene and protein expression along the shell-facing epithelium.

Na+/K+-ATPase (NKA) is essential for maintaining the Na+ and K+ gradients, and supporting the secondary active transport of certain ions/molecules, across the plasma membrane of animal cells. This study aimed to clone the NKA α-subunit (NKAα) from the inner mantle adjacent to the extrapallial fluid of Tridacna squamosa, to determine its subcellular localization, and to examine the effects of light exposure on its transcript level and protein abundance. The cDNA coding sequence of NKAα from T. squamosa comprised 3105 bp, encoding 1034 amino acids with an estimated molecular mass of 114 kDa. NKAα had a basolateral localization along the shell-facing epithelium of the inner mantle. Exposure to 12 h of light led to a significantly stronger basolateral NKAα-immunofluorescence at the shell-facing epithelium, indicating that NKA might play a role in light-enhanced calcification in T. squamosa. After 3 h of light exposure, the transcript level of NKAα decreased transiently in the inner mantle, but returned to the control level thereafter. In comparison, the protein abundance of NKAα remained unchanged at hour 3, but became significantly higher than the control after 12 h of light exposure. Hence, the expression of NKAα in the inner mantle of T. squamosa was light-dependent. It is probable that a higher expression level of NKA was needed in the shell-facing epithelial cells of the inner mantle to cope with a rise in Na+ influx, possibly caused by increases in activities of some Na+-dependent ion transporters/channels involved in light-enhanced calcification.

Immunolocalization of Na+ /K+ -ATPase, Na+ /K+ /2Cl− co-transporter (NKCC) and mRNA expression of Na+ /K+ -ATPase α-subunit during short-term salinity transfer in the gills of Persian sturgeon (Acipenser persicus , Borodin, 1897) juveniles

The study tests the physiological responses of Persian sturgeon, Acipenser persicus, during the abrupt release of juveniles from freshwater (FW) into brackish waters (BW = 11‰) of the Caspian Sea. Fish weight at release was 2-3 g (2.55 ± 0.41 g; 8.8 ± 0.58 cm TL). Totals of 160 individuals were randomly distributed into four fiber-glass aerated tanks (volume 60-L). Two tanks served as controls (FW groups), and two as exposure tanks for BW (Caspian Sea water = CSW). Fish were sampled at 0, 3, 6, 12, 24, 48 and 96 hr after abrupt transfer to CSW. Plasma osmolality, immunolocalization of Na+, K+ -ATPase (NKA) and Na+/K+/2Cl– (NKCC) Co-transporter, NKA activity and the NKA α-subunit mRNA expression were analyzed. Blood osmolality of fish transferred from FW to CSW increased significantly within hours post-transfer (p < .05) and remained at a high level for up to 96 hr. Immunolocalization of NKCC indicated co-localization with NKA in the chloride cells in the gill epithelium. A partial sequence of the NKA α-subunit (632 bp) is described. Its expression levels were up-regulated at 12 and 48 hr following salinity transfer (p < .05). However, NKA activity sharply increased in CSW specimens by almost 2.8-fold (p < .05) between 48 and 96 hr after transfer. Gill NKCC co-transporter abundance increased, coinciding with increased gill NKA activity. The increased activity of NKCC during salt excretion in CSW may lead to an influx of Na+ into the chloride cells. Consequently, NKA activity increases to maintain intracellular Na+ homeostasis.

Abstract P268: The Effects of a Slow Pressor Dose of Angiotensin II on Sodium Transporters Expression in the Kidney Cortex are Independent of PPAR-alpha

A slow pressor dose of Angiotensin II (Ang II) has been shown to increase the expression of sodium transporters in the proximal tubules (NHE3), TALH (NKCC2) and distal nephrons (NCC) of Sprague Dawley rats before an increase in blood pressure. Peroxisome Proliferator Activated Receptor - alpha (PPAR-alpha) has been shown to be involved in pressure natriuresis through changes in sodium transport via ameloride and thiazide-dependent mechanisms. We hypothesized that the changes in expression of the sodium transporters during Ang II hypertension were dependent upon PPAR-alpha expression. To address this hypothesis, we treated wild-type (WT) and PPAR-alpha knockout (KO) mice with a slow pressor dose of Ang II (400 ng/kg/min) for 12 days. Mean arterial pressure (MAP) was measured by radiotelemetry. Control MAP was not different between WT (110 ± 8 mmHg) and PPAR-alpha KO mice (112 ± 12 mmHg). On day 12 of Ang II, MAP for PPAR-alpha KO (156 ± 16) mice was significantly higher than WT (138 ± 11 mmHg) mice. The expression of NHE3, NHERF1, NKA-α1 subunit, NKCC2, and NCC was detected in kidney cortical homogenates by western blotting. Kidneys were homogenized and 25 μg of supernatant proteins were separated by 10% SDS-PAGE, transferred to nitrocellulose paper, and blotted against antibodies to NHE3, NHERF1, NKA α1 subunit, NKCC2, and NCC. The slow pressor dose of Ang II decreased the expression of NHE3 in WT + Ang II (0.14 ± 0.02 ODU) and PPAR-alpha KO + Ang II (0.10 ± 0.02 ODU), when compared to WT (2.61 ± 0.93 ODU) and PPAR-alpha KO (2.20 ± 0.58 ODU) controls. Ang II-treatment also decreased NKCC2 in both WT (0.31 ± 0.10 ODU) and PPAR-alpha KO (0.22 ± 0.03 ODU). Ang II hypertension caused similar decreases in NCC and NHERF1 expression in WT and PPAR-α KO mice. NKA alpha1 subunit expression was increased during Ang II hypertension in both WT (1.06 ± 0.26 ODU) and PPAR-α KO (1.64 ± 0.26 ODU) mice. Our results suggest that the effects of a slow pressor dose of Ang II on expression of sodium transporters are independent of PPAR-alpha expression. Future studies are needed to determine the effects of decreasing NHE3, NKCC2, NCC and NHERF1 expression in the kidney on urinary salt excretion during a slow pressor dose of Ang II.

Flexible selection of diversified Na(+)/K(+)-ATPase α-subunit isoforms for osmoregulation in teleosts.

Background and methodsMultiple Na+/K+-ATPase (NKA) α-subunit isoforms express differentially in response to salinity transfer in teleosts but we observed that the isoform nomenclature is inconsistent with the phylogenetic relationship of NKA α-genes. We cloned the catalytic NKA α-subunit isoforms in eels and medaka, analyzed the time course of their expressions in osmoregulatory tissues after transfer from freshwater (FW) to seawater (SW), and performed phylogenetic analyses to deduce an evolutionary scenario that illustrates how various duplication events have led to the current genomic arrangement of NKA α-genes in teleosts.Results and discussionFive and six α-subunits were cloned in eels and medaka respectively. In eels, the commonly-reported α1a and α1b isoforms were absent while the α1c isoform was diversified instead (α1c-1, α1c-2, α1c-3, α2, and α3 in eels). Phylogenetic estimation indicated that the α1a and α1b isoforms from salmon, tilapia, and medaka were generated by independent duplication events and thus they are paralogous isoforms. Re-examination of expression changes of known isoforms after salinity challenge revealed that the isoforms selected as predominant SW-types varied among teleost lineages. Diversification of α1 isoforms occurred by various types of gene duplication, or by alternative transcription among tandem genes to form chimeric transcripts, but there is no trend for more α1 copies in euryhaline species. Our data suggest that the isoform switching between FW (α1a predominates) and SW (α1b predominates) that occurs in salmonids is not universal in teleosts. Instead, in eels, α1c-1 was the major α-subunit upregulated gill, intestine, and kidney in SW. Localization of both NKA mRNA and protein showed consistent upregulation in gill and intestine in SW eels, but not in renal distal and collecting tubules, where low transcript expression levels were accompanied by high protein levels, suggesting a tissue-specific translational regulation that determines and fine-tunes the NKA expression. In medaka, α1b was upregulated in SW in anterior intestine while most other α-subunit isoforms were less responsive to salinity changes.ConclusionBy integrating gene expression and phylogenetic results, we propose that the major NKA α-subunits for SW acclimation were not ancestrally selected, but rather were flexibly determined in lineage-specific fashion in teleosts.

Different expression patterns of renal Na+/K+-ATPase α-isoform-like proteins between tilapia and milkfish following salinity challenges.

Euryhaline teleosts can survive in a broad range of salinity via alteration of the molecular mechanisms in certain osmoregulatory organs, including in the gill and kidney. Among these mechanisms, Na+/K+-ATPase (NKA) plays a crucial role in triggering ion-transporting systems. The switch of NKA isoforms in euryhaline fish gills substantially contributes to salinity adaptation. However, there is little information about switches in the kidneys of euryhaline teleosts. Therefore, the responses of the renal NKA α-isoform protein switch to salinity challenge in euryhaline tilapia (Oreochromis mossambicus) and milkfish (Chanos chanos) with different salinity preferences were examined and compared in this study. Immunohistochemical staining in tilapia kidneys revealed the localization of NKA in renal tubules rather than in the glomeruli, similar to our previous findings in milkfish kidneys. Protein abundance in the renal NKA pan α-subunit-like, α1-, and α3-isoform-like proteins in seawater-acclimated tilapia was significantly higher than in the freshwater group, whereas the α2-isoform-like protein exhibited the opposite pattern of expression. In the milkfish, higher protein abundance in the renal NKA pan α-subunit-like and α1-isoform-like proteins was found in freshwater-acclimated fish, whereas no difference was found in the protein abundance of α2- and α3-isoform-like proteins between groups. These findings suggested that switches for renal NKA α-isoforms, especially the α1-isoform, were involved in renal osmoregulatory mechanisms of euryhaline teleosts. Moreover, differences in regulatory responses of the renal NKA α-subunit to salinity acclimation between tilapia and milkfish revealed that divergent mechanisms for maintaining osmotic balance might be employed by euryhaline teleosts with different salinity preferences.

Osmoregulation in larvae and juveniles of two recently separated Macrobrachium species: Expression patterns of ion transporter genes

In this comparative study, osmoregulatory mechanisms were analyzed in two closely related species of palaemonid shrimp from Brazil, Macrobrachium pantanalense and Macrobrachium amazonicum. A previous investigation showed that all postembryonic stages of M. pantanalense from inland waters of the Pantanal are able to hyper-osmoregulate in fresh water, while this species was not able to hypo-osmoregulate at high salinities. In M. amazonicum originating from the Amazon estuary, in contrast, all stages are able to hypo-osmoregulate, but only first-stage larvae, late juveniles and adults are able to hyper-osmoregulate in fresh water. The underlying molecular mechanisms of these physiological differences have not been known. We therefore investigated the expression patterns of three ion transporters (NKA α-subunit, VHA B-subunit and NHE3) following differential salinity acclimation in different ontogenetic stages (stage-V larvae, juveniles) of both species. Larval NKAα expression was at both salinities significantly higher in M. pantanalense than in M. amazonicum, whereas no difference was noted in juveniles. VHA was also more expressed in larvae of M. pantanalense than in those of M. amazonicum. When NHE3 expression is compared between the larvae of the two species, further salinity-related differences were observed, with generally higher expression in the inland species. Overall, a high expression of ion pumps in M. pantanalense suggests an evolutionary key role of these transporters in freshwater invasion.

FXYD11 mediated modulation of Na+/K+-ATPase activity in gills of the brackish medaka (Oryzias dancena) when transferred to hypoosmotic or hyperosmotic environments

FXYD proteins regulate Na+/K+-ATPase (NKA), which is a primary active pump that provides the driving force that triggers osmoregulatory systems in teleosts. To explore the regulatory mechanisms between FXYD and NKA in euryhaline teleosts, the expression of NKA (mRNA, protein, and activity) and FXYD11 and their interaction were examined in the gills of brackish medaka (Oryzias dancena) when transferred from brackish water (BW; 15‰) to fresh water (FW) or seawater (SW; 35‰). The mRNA expression of Odfxyd11 and Odnka-α was elevated 48h post-hypoosmotic transfer. Moreover, FXYD11 protein and NKA activity were upregulated 12h after transfer to FW. When transferred to SW, the protein abundance of FXYD11 and the NKA α-subunit did not show apparent changes, while Odfxyd11 and Odnka-α mRNA expression and NKA activity increased significantly 12h and 1h post-transfer, respectively. To clarify the FXYD11 mechanisms involved in modulating NKA activity via their interaction, co-immunoprecipitation was further applied to O. dancena gills. The results revealed that the levels of protein–protein interaction between branchial NKA and FXYD11 increased acutely 12h after the transfer from BW to FW. However, immediate upregulation of NKA activity 1h following post-exposure to SW, without the elevation of protein–protein interaction levels, was found. Hence, branchial NKA activity of O. dancena was suggested to be rapidly regulated by FXYD11 interaction with NKA when acclimated to hypoosmotic environments. To the best of our knowledge, this is the first study that focuses on the efficacy of interactions between FXYD11 and NKA in the gills of euryhaline teleosts.