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 vitamin D (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.