The important reactions that occur to the vitamin D molecule and the important reactions involved in the expression of the final active form of vitamin D are reviewed in a critical manner. After an overview of the metabolism of vitamin D to its active form and to its metabolic degradation products, the molecular understanding of the 1alpha-hydroxylation reaction and the 24-hydroxylation reaction of the vitamin D hormone is presented. Furthermore, the role of vitamin D in maintenance of serum calcium is reviewed at the physiological level and at the molecular level whenever possible. Of particular importance is the regulation of the parathyroid gland by the vitamin D hormone. A third section describes the known molecular events involved in the action of 1alpha,25-dihydroxyvitamin D3 on its target cells. This includes reviewing what is now known concerning the overall mechanism of transcriptional regulation by vitamin D. It describes the vitamin D receptors that have been cloned and identified and describes the coactivators and retinoid X receptors required for the function of vitamin D in its genomic actions. The presence of receptor in previously uncharted target organs of vitamin D action has led to a study of the possible function of vitamin D in these organs. A good example of a new function described for 1alpha,25-dihydroxyvitamin D3 is that found in the parathyroid gland. This is also true for the role of vitamin D hormone in skin, the immune system, a possible role in the pancreas, i.e., in the islet cells, and a possible role in female reproduction. This review also raises the intriguing question of whether vitamin D plays an important role in embryonic development, since vitamin D deficiency does not prohibit development, nor does vitamin D receptor knockout. The final section reviews some interesting analogs of the vitamin D hormone and their possible uses. The review ends with possible ideas with regard to future directions of vitamin D drug design.

Background/Aims: Vitamin D insufficiency and secondary hyperparathyroidism (SHPT) are associated with increased morbidity and mortality in chronic kidney disease (CKD) and are poorly addressed by current treatments. The present clinical studies evaluated extended-release (ER) calcifediol, a novel vitamin D prohormone repletion therapy designed to gradually correct low serum total 25-hydroxyvitamin D, improve SHPT control and minimize the induction of CYP24A1 and FGF23. Methods: Two identical multicenter, randomized, double-blind, placebo-controlled studies enrolled subjects from 89 US sites. A total of 429 subjects, balanced between studies, with stage 3 or 4 CKD, SHPT and vitamin D insufficiency were randomized 2:1 to receive oral ER calcifediol (30 or 60 µg) or placebo once daily at bedtime for 26 weeks. Most subjects (354 or 83%) completed dosing, and 298 (69%) entered a subsequent open-label extension study wherein ER calcifediol was administered without interruption for another 26 weeks. Results: ER calcifediol normalized serum total 25-hydroxyvitamin D concentrations (>30 ng/ml) in >95% of per-protocol subjects and reduced plasma intact parathyroid hormone (iPTH) by at least 10% in 72%. The proportion of subjects receiving ER calcifediol who achieved iPTH reductions of ≥30% increased progressively with treatment duration, reaching 22, 40 and 50% at 12, 26 and 52 weeks, respectively. iPTH lowering with ER calcifediol was independent of CKD stage and significantly greater than with placebo. ER calcifediol had inconsequential impact on serum calcium, phosphorus, FGF23 and adverse events. Conclusion: Oral ER calcifediol is safe and effective in treating SHPT and vitamin D insufficiency in CKD.

The intrarenal renin-angiotensin system (RAS) plays a key role in the development of diabetic nephropathy. Recently, we showed that combination therapy with an AT 1 receptor blocker (ARB) and an activated vitamin D analog produced excellent synergistic effects against diabetic nephropathy, as a result of blockade of the ARB-induced compensatory renin increase. Given the diversity of vitamin D analogs, here we used a pro-drug vitamin D analog, doxercalciferol (1␣-hydroxyvitamin D 2), to further test the efficacy of the combination strategy in long-term treatment. Streptozotocin-induced diabetic DBA/2J mice were treated with vehicle, losartan, doxercalciferol (0.4 and 0.6 g/kg), or losartan and doxercalciferol combinations for 20 wk. Vehicle-treated diabetic mice developed progressive albuminuria and glomerulosclerosis. Losartan alone moderately ameliorated kidney injury, with renin being drastically upregulated. A similar therapeutic effect was seen with doxercalciferol alone, which markedly suppressed renin and angiotensinogen expression. The losartan and doxercalciferol combination most effectively prevented albuminuria, restored glomerular filtration barrier structure, and dramatically reduced glomerulosclerosis in a dose-dependent manner. These effects were accompanied by blockade of intrarenal renin upregulation and ANG II accumulation. These data demonstrate an excellent therapeutic potential for doxercalciferol in diabetic renal disease and confirm the concept that blockade of the compensatory renin increase enhances the efficacy of RAS inhibition and produces synergistic therapeutic effects in combination therapy.renin-angiotensin system; compensatory renin increase; albuminuria; glomerulosclerosis DIABETIC NEPHROPATHY (DN) is the most common renal complication of diabetes mellitus and a leading cause of end-stage renal disease, accounting for 44% of new cases in 2005 (9). It is well established that the renin-angiotensin system (RAS) is a major mediator of progressive renal injury. Since renal interstitial angiotensin (ANG) II levels are much higher than in the plasma (28), the local RAS in the kidney is believed to play the major damaging role in diabetic nephropathy. Kidney cells, including mesangial cells and podocytes, are able to synthesize all components of the RAS, including renin, the (pro)renin receptor, angiotensinogen (AGT), and ANG II receptors independently of the systemic RAS, making the kidney capable of maintaining a high level of local ANG II. Intrarenal renin and AGT levels are induced in diabetic animals (4, 48). In vitro studies showed that when exposed to high glucose levels, mesangial cells and podocytes increase renin and ANG II production (13,38,42). Intrarenal ANG II promotes the progression of renal injury via multiple pathways that increase glomerular permeability, induce oxidative stress, and promote the synthesis of profibrotic and proinflammatory factors and extracellular matrix (8,15). The consequence of the progression of diabetic renal injury is the development of protein...

A full-length cDNA for the human liver mitochondrial cytochrome P-450 CYP27 was cloned from a human hepatoma HepG2 cDNA library and then subcloned into the mammalian expression vector pSG5. When CYP27 cDNA was trnsfected into COS-1 tansformed monkey kidney ceRs along with adrenodoin cDNA, transected cells revealed a 10-to 20-fold higher vitamin D3-25-hydroxylase activity than nontransfected cells. Transfected cells were capable of 25-hydroxylation of vitamin D3, la-hydroxyvitamin D3 and lahydroxydihydrotachysterol3. In each case they also showed the ability to 26(27)-hydroxylate the cholesterol-like (D3) side chain. The relative rates of 25-and 26(27)-hydroxylation of la-hydroxyvitamin D3 approximately mimicked the ratio of products observed in HepG2 cells. Vitamin D2 and lahydroxyvitamin D2, both with the ergosterol-like side chain, were 24-and 26(27)-hydroxylated by CYP27. The rate of side-chain 24-, 25-, or 26(27)-hydroxylation was greater for la-hydroxylated vitamin D analogs than for their nonhydroxylated counterparts. We conclude that CYP27 is capable of24-, 25-, and 26(27)-hydroxylation of vitamin D analogs and that the nature of products Is partially dictated by the side chain of the substrate. This work has revealed that the cytochrome P-450 CYP27 may be important in the metabolism of vitamin D analogs used as drugs.The cytochrome P45S-containing enzyme vitamin D3 25-hydroxylase has been extensively studied over the past 20 years or so since the first broken-cell systems were described (1). Subsequently, many studies involving purification and characterization ofthe enzyme(s) have been performed using the liver of different experimental animals, in particular that of the rat and rabbit (2, 3). In the rat, both microsomal and mitochondrial versions of the vitamin D3 25-hydroxylase have been reported (3, 4). The human liver counterpart was believed to exist exclusively in the mitochondrial inner membrane (5). This conclusion was subsequently challenged by the observation that the microsomal fraction from human liver might contain an inhibitor(s) of the enzyme (6).The cloning of the liver mitochondrial vitamin D3 25-hydroxylase cDNA has been reported for rat (7) and rabbit (8, 9). The cloning of the presumed human homolog of the liver mitochondrial vitamin D3 25-hydroxylase has also been reported (10, 11) and this cytochrome P-450 has been named CYP27. Though Cali and coworkers (10,12) (Kingston, ON).Cell Culture and Incubation with Vitamin D Analogs. HepG2 cells were cultured as described (14). COS-1 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and antibiotics (penicillin G, 100 pg/ml; gentamicin, 5 pg/ml; and Fungizone, 300 ng/ml).Tissue culture plates (100-or 150-mm diameter) were washed twice with phosphate-buffered saline and once with medium containing antibiotics plus 1% (wt/vol) bovine serum albumin. Serum-free medium containing 1% albumin (4 ml per 100-mm plate; 10 ml per 150-mm plate) was added to each plate, followed by vitamin D anal...