Calcidiol insufficiency is highly prevalent in chronic kidney disease (CKD), but the reasons for this are incompletely understood. CKD associates with a decrease in liver cytochrome P450 (CYP450) enzymes, and specific CYP450 isoforms mediate vitamin D 3 C-25-hydroxylation, which forms calcidiol. Abnormal levels of parathyroid hormone (PTH), which also modulates liver CYP450, could also contribute to the decrease in liver CYP450 associated with CKD. Here, we evaluated the effects of PTH and uremia on liver CYP450 isoforms involved in calcidiol synthesis in rats. Uremic rats had 52% lower concentrations of serum calcidiol than control rats (P Ͻ 0.002). Compared with controls, uremic rats produced 71% less calcidiol and 48% less calcitriol after the administration of vitamin D 3 or 1␣-hydroxyvitamin D 3 , respectively, suggesting impaired C-25-hydroxylation of vitamin D 3 . Furthermore, uremia associated with a reduction of liver CYP2C11, 2J3, 3A2, and 27A1. Parathyroidectomy prevented the uremia-associated decreases in calcidiol and liver CYP450 isoforms. In conclusion, these data suggest that uremia decreases calcidiol synthesis secondary to a PTH-mediated reduction in liver CYP450 isoforms. ] deficiency has also been demonstrated in patients with stages 3 and 4 chronic kidney disease (CKD) and in patients who are on dialysis. [1][2][3][4][5][6][7][8] In fact, low serum 25(OH)D 3 is so intimately associated with CRF that in one study, only 29 and 17% of patients with stages 3 and 4 CKD, respectively, had sufficient levels [defined as a serum 25(OH)D 3 concentrations Ͼ75 nmol/L or 30 ng/ml]. 2 A more recent study showed a prevalence of calcidiol insufficiency and deficiency as high as 98% in predialysis patients with a mean GFR of 18.3 ml/min. 4 Prevalence of low serum 25(OH)D 3 was 78 and 89% in two large cohorts of hemodialysis patients 9,10 and 87% in a large cohort of peritoneal dialysis patients. 11 The metabolic consequences of calcidiol defi-

High sensitivity of the rat hepatic vitamin D3-25 hydroxylase CYP27A to 1,25-dihydroxyvitamin D 3 administration. Am J Physiol Endocrinol Metab 284: E138-E147, 2003. First published October 1, 2002 10.1152 10. /ajpendo.00303.2002 is considered the main vitamin D3 (D3)-25 hydroxylase in humans. Our purpose was to evaluate the effect of the D3 nutritional and hormonal status on hepatic CYP27A mRNA, cellular distribution, transcription rate, and enzyme activity. Studies were carried out in normal and in D-depleted rats supplemented with D3, 25OHD3, or 1,25(OH)2D3. CYP27A exhibited a significant gender difference and was observed throughout the hepatic acinus not only in hepatocytes but also in sinusoidal endothelial, stellate, and Kupffer cells. Neither D3 nor 25OHD3 influenced CYP27A mRNA levels. However, 1,25(OH)2D3 repletion led to a 60% decrease in CYP27A mRNA, which was accompanied by a 46% decrease in mitochondrial D3-25 hydroxylase activity. The effect of 1,25(OH)2D3 was mediated by a significant decrease in CYP27A transcription, whereas its mRNA half-life remained unchanged. Our data indicate that CYP27A is present in hepatic parenchymal and sinusoidal cells and that the gene transcript is not influenced by the D3 nutritional status but is transcriptionally regulated by 1,25(OH)2D3 exposure.bile acid biosynthesis; Kupffer cells; stellate cells; hepatocytes; sinusoidal endothelial cells THE SECOSTEROID VITAMIN D 3 (D 3 ) of endogenous or exogenous origin has, in its native form, no biological activity. Once in circulation, D 3 is efficiently taken up by the liver (26) and hydroxylated at C-25 by a mitochondrial mixed-function oxidase CYP27A [C 27 sterol hydroxylase (EC 1.14.13.15)] (15). In humans, the enzyme is presumed to be the only D 3 -25 hydroxylase (51). However, a microsomal D 3 -25 hydroxylase has also been reported in rodents (11), chickens (12), and pigs (35), but only the porcine enzyme (which has been termed CYP2D25) has been cloned to date (34,44).CYP27A is a cytochrome P-450 that catalyzes the first step in the oxidation of the cholesterol side chain in the secondary "acidic" bile acid biosynthesis pathway (13). CYP27A is also able to hydroxylate D 3 and D 3 metabolites at position C-25 (51) as well as at other positions on the secosteroid side chain (29, 54). It has also been reported to be able to catalyze the 1␣-hydroxylation of 25-hydroxyvitamin D 3 (25OHD 3 ), albeit at a much lower rate than the transformation of D 3 into 25OHD 3 (3). However, unlike the tight regulation by the D 3 endocrine system associated with the renal 25-hydroxyvitamin D 3 -1␣-hydroxylase, the sensitivity of the gene encoding CYP27A to D 3 or to D 3 metabolites has not been characterized. The presence of regulatory mechanisms related to the D 3 status as a modulator of the 25-hydroxylation of the vitamin is, however, a widely accepted notion, which rests on the studies of DeLuca's group in the early 1970s (Bhattacharyya and DeLuca, Refs. 10, 12). Several laboratories have attempted to evaluate the mechanism(s) inv...

Little attention has been given to the consequences of the in vivo calcium status on intracellular calcium homeostasis despite several pathological states induced by perturbations of the in vivo calcium balance. The aim of these studies was to probe the influence of an in vivo calcium deficiency on the resting cytoplasmic Ca2+ concentration and the inositol-1,4,5-trisphosphate-sensitive Ca2+ pools. Studies were conducted in hepatocytes (a cell type well characterized for its cellular Ca2+ response) isolated from normal and calcium-deficient rats secondary to vitamin D depletion. Both resting cytoplasmic Ca2+ concentration and Ca2+ mobilization from inositol-1,4,5-trisphosphate-sensitive cellular pools were significantly lowered by calcium depletion. In addition, Ca deficiency was shown to significantly reduce calreticulin messenger RNA and protein levels but calcium entry through store-operated calcium channels remained unaffected, indicating that the Ca2+ entry mechanisms are still fully operational in calcium deficiency. The effects of calcium deficiency on cellular calcium homeostasis were reversible by repletion with oral calcium feeding alone or by the administration of the calcium-regulating hormone 1,25-dihydroxyvitamin D3, further strengthening the tight link between extra- and intracellular calcium. These data, therefore, challenge the currently prevailing hypothesis that extracellular Ca2+ has no significant impact on cellular Ca2+ by demonstrating that despite the large Ca2+ gradient between extra- and intracellular Ca2+ concentrations, calcium deficiency in vivo significantly alters the hormone-sensitive cellular calcium homeostasis.