Vitamin D is involved in the regulation of Cl− uptake in zebrafish (Danio rerio)

Cl⁻ is a major anion in the bodily fluids of vertebrates, and maintaining its homeostasis is essential for normal physiological functions. Fishes inhabiting freshwater (FW) passively lose body fluid ions, including Cl⁻, to the external environment because of the electrochemical gradient of ions across the body surface. Therefore, FW fishes have to actively absorb Cl⁻ from the surroundings to maintain ion homeostasis in their bodily fluids. Hormonal control is vital for modulating ion uptake in fish. Vitamin D is involved in the regulation of Ca²⁺ uptake and acid secretion in fish. In the present study, we found that the levels of bioactive vitamin D, 1α,25-dihydroxyvitamin D₃ (1α,25(OH)₂D₃), significantly increased in zebrafish embryos and adults after exposure to water containing low levels of Cl⁻. Moreover, the administration of 1α,25(OH)₂D₃ treatment (20 μg/L) in zebrafish embryos, and intraperitoneal (i.p.) injection of 1α,25(OH)₂D₃ (5 μg/kg body mass) in zebrafish adults, resulting the increased Cl⁻ content in bodily fluid in zebrafish. Na⁺-Cl⁻ cotransporter 2b (NCC2b) and Cl⁻ channel 2c (CLC2c) are specifically expressed during Cl⁻ uptake by ionocytes in zebrafish. Our results indicated that the mRNA and protein expression of NCC2b and CLC2c considerably increased in the zebrafish with exogenous 1α,25(OH)₂D₃ treatment. Additionally, exogenous 1α,25(OH)₂D₃ administration increased the number of NCC2b- and CLC2c-expressing cells in yolk skins of zebrafish embryos and the gill filaments of zebrafish adults. Transcript signals of vitamin D receptors (VDRs) were identified in NCC2b-expressing cells. Knockdown of VDRa and VDRb significantly reduced the expression of NCC2b and CLC2c and the number of NCC2b- and CLC2c-expressing cells. These results indicate that vitamin D can affect Cl⁻ uptake in zebrafish and extend our knowledge of the role of vitamin D in fish physiology.