DataSheet_1_Vitamin D regulates transepithelial acid secretion in zebrafish (Danio rerio) larvae.pdf

Maintenance of an acid-base balance is essential for normal physiological processes in vertebrates. Freshwater fishes live in an aquatic environment with variable pH, and their buffering capacity for acid-base balance in body fluids is weak. Thus, after acid exposure, fishes secrete excess acid to prevent internal acidosis. Acid-secreting ionocytes present in the adult gills and embryonic skin are primarily responsible for acid secretion, and H+-ATPase and Na+/H+ exchanger 3 (NHE3) are the two main transporters responsible for apical acid secretion. Vitamin D is a well-known hormone involved in the maintenance of Ca2+ homeostasis and is suggested to be involved in acid-base regulation by modulating the activity and/or mRNA expression of NHE3 in mammalian models. It remains unclear whether vitamin D is involved in acid secretion in fishes. The aim of the present study was to use zebrafish as a model to determine whether vitamin D and its receptors influence acid secretion. Our results indicated that the levels of 1α, 25-dihydroxyvitamin D3 (1α,25(OH)2D3), the bioactive vitamin D, were significantly increased in 3 days post-fertilization zebrafish larvae after exposure to acidic freshwater (AFW, pH 4.0). Exogenous 1α,25(OH)2D3 (20 μg/L) incubation substantially enhanced the mRNA expression of acid-secreting transporters and acid secretion at the skin of the entire body and each H+-ATPase-rich cell (HRC), a type of acid-secreting ionocyte. Furthermore, the expression of vitamin D receptors (VDRs) was identified in HRCs of zebrafish. When both VDRa and VDRb were knocked down, acid secretion and the mRNA expression of acid-secreting transporters were significantly decreased. Moreover, double knockdown of VDRa/b prevented the increase in acid secretion induced by AFW and 1α,25(OH)2D3 treatment. This study is the first to indicate that vitamin D is involved in acid secretion in fish.

酸碱稳态的维持对于脊椎动物的正常生理过程至关重要。淡水鱼类栖息于pH值可变的水生环境中，其体液酸碱平衡的缓冲能力较弱。因此，鱼类在暴露于酸性环境后，会分泌过量酸类以避免体内酸中毒。存在于成鱼鳃及胚胎皮肤中的泌酸离子细胞（acid-secreting ionocytes）是介导酸分泌的主要细胞，而氢质子ATP酶（H+-ATPase）与钠氢交换体3（NHE3）是负责顶膜泌酸的两类主要转运蛋白。

维生素D是一类已知的参与维持钙离子稳态的激素，在哺乳动物模型中，其被认为可通过调控NHE3的活性及/或mRNA表达参与酸碱平衡调节。但目前尚不清楚维生素D是否参与鱼类的酸分泌过程。

本研究以斑马鱼为模型，旨在探究维生素D及其受体是否会影响酸分泌过程。本研究结果显示，在暴露于酸性淡水（AFW, pH 4.0）后，受精后3天的斑马鱼幼体内具有生物活性的维生素D——1α,25-二羟基维生素D3（1α,25(OH)2D3）的水平显著升高。外源施加1α,25(OH)2D3（20 μg/L）孵育可显著提升泌酸转运蛋白的mRNA表达水平，并增强整个幼体皮肤及每一类泌酸离子细胞——富氢质子ATP酶细胞（HRC）的酸分泌能力。此外，本研究在斑马鱼的HRC中检测到了维生素D受体（VDRs）的表达。当同时敲低VDRa与VDRb时，酸分泌能力及泌酸转运蛋白的mRNA表达水平均显著下降。此外，同时敲低VDRa/b可阻断AFW及1α,25(OH)2D3处理所诱导的酸分泌升高现象。本研究首次证实维生素D参与鱼类的酸分泌过程。