Upon salinity challenge, the Na(+)-K(+)-ATPase (NKA) of fish kidney plays a crucial role in maintaining ion and water balance. Moreover, the FXYD protein family was found to be a regulator of NKA. Our preliminary results revealed that fxyd12 was highly expressed in the kidneys of the two closely related euryhaline medaka species (Oryzias dancena and O. latipes) from different natural habitats (brackish water and fresh water). In this study, we investigated the expression and association of renal FXYD12 and NKA α-subunit as well as potential functions of FXYD12 in the two medakas. These findings illustrated and compared the regulatory roles of FXYD12 for NKA in kidneys of the two medakas in response to salinity changes. In this study, at the mRNA and/or protein level, the expression patterns were similar for renal FXYD12 and NKA in the two medakas. However, different patterns of NKA activities and different interaction levels between FXYD12 and NKA were found in the kidneys of these two medakas. The results revealed that different strategies were used in the kidneys of the two medaka species upon salinity challenge. On the other hand, gene knockdown experiments demonstrated that the function of O. dancena FXYD12 allowed maintenance of a high level of NKA activity. The results of the present study indicated that the kidneys of the examined euryhaline medakas originating from brackish water and fresh water exhibited different modulatory mechanisms through which renal FXYD12 enhanced NKA activity to maintain internal homeostasis. Our findings broadened the knowledge of expression and functions of FXYD proteins, the modulators of NKA, in vertebrates.

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.

FXYD proteins are novel regulators of Na(+)-K(+)-ATPase (NKA). In fish subjected to salinity challenges, NKA activity in osmoregulatory organs (e.g., gills) is a primary driving force for the many ion transport systems that act in concert to maintain a stable internal environment. Although teleostean FXYD proteins have been identified and investigated, previous studies focused on only a limited group of species. The purposes of the present study were to establish the brackish medaka (Oryzias dancena) as a potential saltwater fish model for osmoregulatory studies and to investigate the diversity of teleostean FXYD expression profiles by comparing two closely related euryhaline model teleosts, brackish medaka and Japanese medaka (O. latipes), upon exposure to salinity changes. Seven members of the FXYD protein family were identified in each medaka species, and the expression of most branchial fxyd genes was salinity-dependent. Among the cloned genes, fxyd11 was expressed specifically in the gills and at a significantly higher level than the other fxyd genes. In the brackish medaka, branchial fxyd11 expression was localized to the NKA-immunoreactive cells in gill epithelia. Furthermore, the FXYD11 protein interacted with the NKA α-subunit and was expressed at a higher level in freshwater-acclimated individuals relative to fish in other salinity groups. The protein sequences and tissue distributions of the FXYD proteins were very similar between the two medaka species, but different expression profiles were observed upon salinity challenge for most branchial fxyd genes. Salinity changes produced different effects on the FXYD11 and NKA α-subunit expression patterns in the gills of the brackish medaka. To our knowledge, this report is the first to focus on FXYD expression in the gills of closely related euryhaline teleosts. Given the advantages conferred by the well-developed Japanese medaka system, we propose the brackish medaka as a saltwater fish model for osmoregulatory studies.