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Front Physiol
2017 Jan 01;8:576. doi: 10.3389/fphys.2017.00576.
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FXYD8, a Novel Regulator of Renal Na+/K+-ATPase in the Euryhaline Teleost, Tetraodon nigroviridis.
Wang PJ
,
Yang WK
,
Lin CH
,
Hwang HH
,
Lee TH
.
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FXYD proteins are important regulators of Na+/K+-ATPase (NKA) activity in mammals. As an inhabitant of estuaries, the pufferfish (Tetraodon nigroviridis) responds to ambient salinity changes with efficient osmoregulation, including alterations in branchial, and renal NKA activities. Previous studies on teleostean FXYDs have mainly focused on the expression and potential functions of FXYD proteins in gills. The goal of the present study was to elucidate the potential role of FXYD8, a member of the fish FXYD protein family, in the modulation of NKA activity in the kidneys of this euryhaline pufferfish by using molecular, biochemical, and physiological approaches. The results demonstrate that T. nigroviridis FXYD8 (TnFXYD8) interacts with NKA in renal tubules. Meanwhile, the protein expression of renal TnFXYD8 was found to be significantly upregulated in hyperosmotic seawater-acclimated pufferfish. Moreover, overexpression of TnFXYD8 in Xenopus oocytes decreased NKA activity. Our results suggest the FXYD8 is able to modulate NKA activity through inhibitory effects upon salinity challenge. The present study further extends our understanding of the functions of FXYD proteins, the regulators of NKA, in vertebrates.
Figure 1. Alignment of amino acid sequences of teleostean FXYD8 proteins. The frame, epitope for specific antibody preparation; black triangle, potential phosphorylation site for TnFXYD8; asterisk, conserved glycine residue (G40); gray background, identical amino acids. All accession numbers are listed in Table S2. Od, brackish medaka (Oryzias dancena); Ol, Japanese medaka (Oryzias latipes); Ss, Atlantic salmon (Salmo salar); Tn, pufferfish (Tetraodon nigroviridis).
Figure 2. Phylogenetic tree of TnFXYD8 with known FXYD members from various teleosts. Numbers represent bootstrap values for the percentage of 1,000 replicates. All accession numbers are listed in Table S2. Aj, Japanese eel (Anguilla japonica); Dr, zebrafish (Danio rerio); Od, brackish medaka (Oryzias dancena); Ol, Japanese medaka (Oryzias latipes); Sa, spotted scat (Scatophagus argus); Ss, Atlantic salmon (Salmo salar); Tn, pufferfish (Tetraodon nigroviridis).
Figure 3. Expression of TnFXYD8 in various tissues of fresh water (FW)- and seawater (SW)-acclimated pufferfish by RT-PCR analysis. The Tnfxyd8 gene was found in the gill, kidney, gut, heart, eye, muscle, brain, skin, and liver of the pufferfish acclimated to either FW or SW. β-actin was used as an internal control.
Figure 4. Co-immunoprecipitation (Co-IP) of TnFXYD8 and NKA α-subunit (NKA α) in the pufferfish kidneys. TnFXYD8 or NKA were immunoprecipitated from renal crude membrane fractions of freshwater pufferfish using primary antibodies, then the immune complexes were analyzed by immunoblotting for NKA α (A) or TnFXYD8 (B), respectively. In immunoprecipitated NKA α or TnFXYD8, the immunoblotting analyses for NKAα and TnFXYD8 revealed immunoreactive bands at 100 kDa (A) and 13 kDa (B), respectively. In (B), the 55-kDa band in lane 3 is the IgG heavy chain of TnFXYD8 antibody. M, marker (kDa); lane 1, immunoblot detection of the opposing antibody (experimental group); lane 2, negative control for no antibody incubation in IP; lane 3, positive control using the same antibody with IP.
Figure 5. Effects of salinity on mRNA levels (A,B) and protein abundance (C,D) of renal TnFXYD8 in the pufferfish. (A) RT-PCR analysis confirmed primer specificity. (B) Comparisons of renal mRNA abundance between freshwater (FW)- and seawater (SW)-pufferfish quantified by real-time PCR. (C) Renal protein lysates revealed immunoreactive bands at 13 kDa. (D) The relative abundance of TnFXYD8 protein expressed in kidneys of pufferfish acclimated to FW was significantly lower than that in kidneys of the SW group. β-actin and actin were used as internal controls for mRNA and protein analyses, respectively. Values are presented as means ± standard error (N = 6 or 5 for mRNA or protein, respectively). The asterisk indicates a significant difference (P < 0.05) via Mann-Whitney U test. L, ladder (bp); M, marker (kDa); P, PCR production.
Figure 6. Xenopus oocyte Na+/K+-ATPase (NKA) activity in a TnFXYD8 overexpression experiment. Xenopus oocytes from the medium control and pCS2+ control groups were injected with equal volumes of Barth medium and the equal abundance (8 ng) of empty pCS2+ vector, respectively. NKA activity of the TnFXYD8-injected group (Tnfxyd8 cRNA) was significantly lower than that of the other two control groups. Values are means ± standard error (N = 6). Dissimilar letters indicate significant differences among various groups (P < 0.05).
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