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Hydroxylases Involved in Vitamin D Metabolism Are Differentially Expressed in Murine Embryonic Kidney: Application of Whole Mount in Situ Hybridization¹

Departments of Environmental Medicine (M.Y., T.M., K.O.) and Pathology (A.K., M.N.), Osaka Medical Center and Research Institute for Maternal and Child Health, 840 Murodo-cho, Izumi, Osaka 594-1101, Japan

Address all correspondence and requests for reprints to: Dr. Keiichi Ozono, Department of Environmental Medicine, Osaka Medical Center and Research Institute for Maternal and Child Health, 840 Murodo-cho, Izumi city, Osaka 594-1101, Japan. E-mail: j61642{at}center.osaka-u.ac.jp

In this study we examined the expression of 25-hydroxyvitamin D-1-hydroxylase (1-hydroxylase) and 25-hydroxyvitamin D-24-hydroxylase (24-hydroxylase) by RT-PCR and whole mount in situ hybridization using organ culture of kidney taken from mouse embryo. First, the kidneys of mouse embryo at 11.5–17.5 days gestation were cultured in the presence or absence of forskolin and 1,25-dihydroxyvitamin D₃ [1,25-(OH)₂D₃]. Forskolin and 1,25-(OH)₂D₃ induced the expression of 1-hydroxylase and 24-hydroxylase, respectively, in a dose- and time-dependent manner. In the absence of stimulants, the expression of 1-hydroxylase and 24-hydroxylase was detected from days 13.5–17.5 gestation. The expression of vitamin D receptor and megalin was detected from days 13.5 and 11.5, respectively. Next, signals for the expression of either 1-hydroxylase or 24-hydroxylase were detected by whole mount in situ hybridization in kidney explants taken from embryo at 15.5 days gestation after the appropriate stimulation. However, the localization of signals differed between the two enzymes; 1-hydroxylase messenger RNA was expressed in the inner area of the kidney explants, whereas 24-hydroxylase messenger RNA was expressed in the surface area. The expression of both hydroxylases was restricted to the epithelium of developing renal tubules. The pattern of megalin expression was similar to that of 1-hydroxylase expression. To confirm the difference in distribution of 1-hydroxylase and 24-hydroxylase transcripts, the explants were hybridized with probes for both 1-hydroxylase and 24-hydroxylase using double labeling techniques after simultaneous stimulation with forskolin and 1,25-(OH)₂D₃, resulting in the detection at different locations of positive signals for the two enzymes. These results suggest that the expression of 1-hydroxylase is induced in a distinct epithelium of renal tubules from that of 24-hydroxylase even at the early stage of kidney development before glomerulogenesis.

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