The Vitamin D Analog ED-71 Is a Potent Regulator of Intestinal Phosphate Absorption and NaPi-IIb

Endocrinology, Nov 2012

The vitamin D analog ED-71 [1α,25-dihydroxy-2β-(3-hydroxypropyloxy)vitamin D3] has been approved for treatment of osteoporosis in Japan, but its effects on mineral metabolism have not been fully explored. We investigated the actions of ED-71 on phosphate (Pi) absorption and induction of the intestinal sodium/phosphate cotransporters. Oral treatment of vitamin D-deficient rats with ED-71 (20 pmol every other day for 8 d) produced a maximal 8-fold increase in duodenal Pi absorption, measured by the in situ loop method, whereas 1,25-dihyroxyvitamin D3 [1,25(OH)2D3], at doses up to 150 pmol, had no effect. This action of ED-71 was attributable to a dramatic 24-fold induction of sodium-dependent Pi transporter type IIb (NaPi-IIb) mRNA in the duodenum; Pit-1 and Pit-2 mRNA levels were not increased. In vitamin D-replete rats, ED-71 treatment (50 pmol) at 72 and 24 h before death increased NaPi-IIb mRNA in the duodenum and jejunum, but not the ileum, whereas 1,25(OH)2D3 at 1000 pmol was ineffective in all segments. Single oral doses of ED-71 increased mouse intestinal NaPi-IIb mRNA and protein between 6 and 24 h. Surprisingly, rat lung NaPi-IIb was not increased by ED-71, despite its coexpression with the vitamin D receptor in alveolar type II cells. However, ED-71 did not induce intestinal NaPi-IIb in vitamin D receptor-ablated mice. The greater potency of ED-71 than 1,25(OH)2D3 on NaPi-IIb appears to be due to much higher and more prolonged levels of ED-71 in the circulation. In summary, ED-71, due to its disparate pharmacokinetics, is a much more potent inducer of intestinal Pi absorption and NaPi-IIb than 1,25(OH)2D3, suggesting a role for this analog in the treatment of Pi-wasting disorders.

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The Vitamin D Analog ED-71 Is a Potent Regulator of Intestinal Phosphate Absorption and NaPi-IIb

Received May The Vitamin D Analog ED-71 Is a Potent Regulator of Intestinal Phosphate Absorption and NaPi-IIb Alex J. Brown 0 Fanjie Zhang 0 Cynthia S. Ritter 0 0 Renal Division, Washington University School of Medicine , St. Louis, Missouri 63110 , USA The vitamin D analog ED-71 [1 ,25-dihydroxy-2 -(3-hydroxypropyloxy)vitamin D3] has been approved for treatment of osteoporosis in Japan, but its effects on mineral metabolism have not been fully explored. We investigated the actions of ED-71 on phosphate (Pi) absorption and induction of the intestinal sodium/phosphate cotransporters. Oral treatment of vitamin D-deficient rats with ED-71 (20 pmol every other day for 8 d) produced a maximal 8-fold increase in duodenal Pi absorption, measured by the in situ loop method, whereas 1,25-dihyroxyvitamin D3 [1,25(OH)2D3], at doses up to 150 pmol, had no effect. This action of ED-71 was attributable to a dramatic 24-fold induction of sodium-dependent Pi transporter type IIb (NaPi-IIb) mRNA in the duodenum; Pit-1 and Pit-2 mRNA levels were not increased. In vitamin D-replete rats, ED-71 treatment (50 pmol) at 72 and 24 h before death increased NaPi-IIb mRNA in the duodenum and jejunum, but not the ileum, whereas 1,25(OH)2D3 at 1000 pmol was ineffective in all segments. Single oral doses of ED-71 increased mouse intestinal NaPi-IIb mRNA and protein between 6 and 24 h. Surprisingly, rat lung NaPi-IIb was not increased by ED-71, despite its coexpression with the vitamin D receptor in alveolar type II cells. However, ED-71 did not induce intestinal NaPi-IIb in vitamin D receptor-ablated mice. The greater potency of ED-71 than 1,25(OH)2D3 on NaPi-IIb appears to be due to much higher and more prolonged levels of ED-71 in the circulation. In summary, ED-71, due to its disparate pharmacokinetics, is a much more potent inducer of intestinal Pi absorption and NaPi-IIb than 1,25(OH)2D3, suggesting a role for this analog in the treatment of Pi-wasting disorders. (Endocrinology 153: 5150 -5156, 2012) - Thydroxypropyloxy)vitamin D3 or ED-71] has been demhe vitamin D analog ED-71 [1 ,25-dihydroxy-2 -(3onstrated to slow bone resorption while maintaining bone formation, properties that have led to its development for treatment of osteoporosis ( 1–5 ). Its effects are superior to those of alfacalcidol (1 -hydroxyvitamin D3), a precursor of the natural vitamin D hormone 1,25(OH)2D3 (1 ,25-dihydroxyvitamin D3). The mechanism(s) responsible for the greater beneficial effects of ED-71 on bone are not known. We have previously reported that ED-71 is more potent than 1,25(OH)2D3 in stimulating intestinal absorption of calcium and Pi in rats ( 6 ). The enhanced calcium absorption was attributable to greater potency of the analog to induce the calcium channel TRPV6. In the present study, we further investigated the mechanism for the greater effect of ED-71 on intestinal Pi absorption. It is well known that 1,25(OH)2D3 can increase intestinal Pi absorption, mainly in the jejunum, and that the effect is relatively modest at less than 2-fold (reviewed in Ref. 7). However, the mechanism remains unclear. 1,25(OH)2D3 has been shown to up-regulate intestinal sodium-dependent Pi transporter type IIb (NaPi-IIb) protein, but the mRNA levels were not affected ( 8, 9 ). Furthermore, a recent report found that 1,25(OH)2D3-stimulated intestinal Pi absorption was sodium independent, which would exclude a role for NaPi-IIb in the regulation (10). However, the current study with ED-71 found that the analog increases Pi transport in the duodenum, that the increase is much larger than that reported for 1,25(OH)2D3, and involves induction of NaPi-IIb at the Abbreviations: BBM, Brush border membrane; BBMV, BBM vesicle; CYP, cytochrome P 450; DBP, vitamin D binding protein; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; NaPi-IIb, sodium-dependent phosphate transporter type IIb; 1,25(OH)2D3 , 1,25dihyroxyvitamin D3; Pi, phosphate; P.O., by mouth. mRNA level. The potential mechanism(s) involved are discussed. Materials and Methods Effects of ED-71 and 1,25(OH)2D3 on duodenal Pi absorption in vitamin D-deficient rats Weanling Sprague Dawley male rats were raised for 8 wk on a vitamin D-deficient diet containing 2.0% Ca, 1.25% P, and 20% lactose (Teklad TD96348; Harlan, Madison, WI) This diet prevents the development of hypocalcemia, hypophosphatemia, and secondary hyperparathyroidism ( 11 ). The rats were gavaged with vehicle (0.5 ml fractionated coconut oil), 1,25(OH)2D3, or ED-71 (20, 50, and 150 pmol) every other day for 8 d (n 6). Forty eight hours after the last injection, intestinal P transport was measured in situ by the isolated duodenal loop method used by our group previously to determine Ca absorption ( 12–14 ). Briefly, the first 6-cm segment of the small intestine was flushed with 12 ml saline, tied off, and filled with 0.3 ml of transport buffer [16 mM HEPES (pH 7.4), 140 mM NaCl, 3.5 mM KCl, 0.1 mM KH2PO4, and 40 Ci [32P]phosphate (NEN Life Science Products, Boston, MA)]. Blood was collected after 10 min and a 0.5-ml aliquot was counted for 32P. Pilot studies confirmed that [32P]phosphate increased linearly for at least 20 min. The duodenums were harvested, and RNA was prepared to determine the changes in Pi transporter gene expression. Effects of ED-71 and 1,25(OH)2D3 on Pi transporter gene expression in normal rats and mice Sprague Dawley male rats (250 g) maintained on normal rodent chow were treated twice with 50 or 1000 pmol of 1,25(OH)2D3 or 50 pmol ED-71 by mouth (P.O.) at 72 and 24 h before death. RNA was isolated from the duodenum, jejunum, ileum, kidney, and lung for analysis of Pi transporter gene expression. C57/BL6 male mice (40 g) were treated P.O. with vehicle or 50 ng of ED-71 P.O. 16 h before death. RNA was isolated from duodenum, jejunum, and ileum. The time required for induction of NaPi-IIb by ED-71 was determined by treating C57/BL6 male mice (40 g) with 50 ng ED-71 at 1, 2, 4, 6, and 24 h before death. RNA was isolated from duodenal mucosa. The role of the vitamin D receptor (VDR) in the induction of NaPi-IIb was determined using VDR-knockout mice (B6.129S4Vdrtm1Mbd/J, The Jackson Laboratory, Bar Harbor, ME) developed by Li et al. ( 15 ). The VDR / mice were maintained from weaning on a vitamin D-replete rescue diet (Teklad TD.96348). The VDR / and VDR / mice were fed the rescue diet for 1 wk before treatment. VDR / , VDR / , and VDR / mice (40 g) were treated with 50 ng ED-71 or vehicle (n 4/group), and duodenal mucosa was collected after 24 h. Both male and female mice were used in this study, and no sex-dependent responses were detected. To confirm that ED-71 induction of NaPi-IIb mRNA leads to an increase in NaPi-IIb protein, male C57/BL6 mice (40 g) were treated with vehicle or 50 ng of ED-71 (n 3), and, 16 h later, brush border membrane (BBM) vesicles (BBMV) were isolated from the proximal and distal halves of the small intestine using the method of Marks et al. ( 8 ). Briefly, the intestinal segments were slit longitudinally, and the mucosa was scraped off with a glass slide, suspended in buffer containing 50 mM mannitol, 2 mM HEPES (pH 7.2) and 0.25 mM phenylmethylsulfonyl fluoride (PMSF), and homogenized three times at half-speed for 20 sec using a Polytron tissue disruptor (Fisher Scientific, Pittsburgh, PA). Magnesium chloride (1 M) was added to the homogenate to a final concentration of 10 mM, followed by mixing on a rotary wheel for 20 min. The homogenate was centrifuged at 3000 g for 20 min, and the supernatant was centrifuged at 27,000 g for 20 min. The pellet was suspended in 300 mM mannitol, 20 mM HEPES (pH 7.2), 0.1 mM MgSO4, and 0.25 mM phenylmethylsulfonyl fluoride using a 21-gauge needle. The suspension was then centrifuged for 15 min at 6000 g, and the resulting supernatant was centrifuged at 27,000 g for 30 min. The purified BBM pellet was resuspended in the same buffer using a gel-loading pipetman tip. All steps were carried out at 4 C. Protein concentration of the BBMV was determined by the method of Bradford ( 16 ) using a kit from Bio-Rad Laboratories, Inc. (Hercules, CA). The clearance rates of ED-71 and 1,25(OH)2D3 were determined by gavaging mice (40 g) with [26,27-3H]ED-71 (Chugai Pharmaceuticals, Ltd., Tokyo, Japan) or 1,25-dihydroxy[26,273H]vitamin D3 (PerkinElmer Corp., Wellesley, MA ) (50 ng, 0.1 Ci) and collecting blood after 1, 3, 7, or 24 h (n 4). Plasma (100 l) was mixed with 4 ml ScintiVerse (Fisher Scientific), and tritium was measured by scintillation counting (Taurus; Micromedic Systems, Inc., Huntsville, AL). Pooled samples (100 l from each plasma sample) were mixed with radioinert 1,25(OH)2D3 or ED-71 (1 nmol) as internal standard and extracted using C18 cartridges (Fisher Scientific) by the method of Reinhardt et al. ( 17 ). The final eluent was resolved by normalphase HPLC using a 5- m silica column (BakerBond, Fisher Scientific) with hexane-isopropanol-methanol (85:14:1) as mobile phase at 1.5 ml/min on a Beckman model 338 HPLC (Beckman Coulter, Inc., Brea, CA). Fractions (30 sec) were collected and counted. Radioactivity eluting with the internal standard was corrected for recovery and expressed as picomoles of gavaged radioactivity per ml of plasma. All animal protocols were approved by the Animal Studies Committee of Washington University School of Medicine. Analysis of gene expression RNA samples from the intestinal segments, kidney, and lung were analyzed by RT-quantitative PCR as previously described ( 18 ). Total RNA was isolated using RNAzol Bee (Tel-Test, Friendswood, TX) per manufacturer’s instructions. RT of the RNA was carried out using oligo-dT primer and SMART MMLV reverse transcriptase (CLONTECH Laboratories, Mountain View, CA). Real-time PCR (quantitative PCR) was performed using Fast SYBR Green Master Mix (Applied Biosystems; Foster City, CA) in an Applied Biosystems 7900HT Fast Real-Time PCR System. The primers used for quantifying rat NaPi-IIa, NaPi-IIb, NaPi-IIc, Pit-1, Pit-2, and cytochrome P 450 (CYP)24A1, and mouse NaPi-IIb, CYP24A1, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) are shown in Supplemental Table 1 (published on The Endocrine Society’s Journals Online web site at http://jcem.endojournals.org). The primers for rat GAPDH were obtained from QIAGEN (QuantiTech primer no. QT00199633, Valencia, CA). The data are expressed as the ratio of target mRNA to GAPDH mRNA ( CT method). Immunoblot analysis NaPi-IIb protein content in BBMV from the proximal and distal halves of the small intestine was determined by immunoblot analysis. BBMV samples were resolved on 12% SDS-PAGE gels and transferred to polyvinylidene difluoride membranes. The membranes were blocked with PBS containing 0.1% Tween 20 and 5% nonfat dry milk at room temperature, and then incubated overnight in blocking buffer containing a 1:2000 dilution of NaPi-IIb antibody (no. NOO35–26C; US Biological, Swampscott, MA). After washing, membranes were incubated for 1 h in goat antirabbit secondary antibody conjugated to horseradish peroxidase (Sigma-Aldrich, Saint Louis, MO) at a 1:20,000 dilution. Blots were stripped and reprobed with mouse actin antibody (1:1000; Sigma-Aldrich). Enhanced chemiluminescent reagent from GE HealthCare (Piscataway, NJ) was used for immunodetection. Statistics Significance values in dose-responses curves for 1,25(OH)2D3 and ED-71 were determined by Dunnett’s Multiple Comparison Test. Results Effects of ED-71 and 1,25(OH)2D3 on duodenal Pi absorption The majority of the vitamin D-regulated intestinal uptake of Pi occurs in the proximal segments of the small intestine, although some previous studies have indicated that Pi absorption is not regulated by 1,25(OH)2D3 in the duodenum. Absorption in this segment was examined more closely using the in situ duodenal loop method. Basal absorption was minimized by performing the study in vitamin D-deficient rats. The rats were treated with vehicle or with ED-71 or 1,25(OH)2D3 at doses of 20, 50, and 150 pmol every other day for 8 d. ED-71 was much more potent than 1,25(OH)2D3 in enhancing duodenal Pi absorption, producing an 8-fold increase in transport (Fig. 1). This was accompanied by a 24-fold increase in NaPi-IIb mRNA (Fig. 2). Other sodium-dependent Pi transporters were not involved in the increased Pi absorption. Pit-2 mRNA was unchanged and Pit-1 mRNA decreased 75% (Fig. 2). Effects of ED-71 and 1,25(OH)2D3 on Pi transporter expression in small intestinal segments Previous studies in rats found that 1,25(OH)2D3 enhanced Pi absorption primarily in the jejunum, but increased NaPi-IIb mRNA was not observed. Therefore, the effects of ED-71 and 1,25(OH)2D3 on Pi transporters were examined in the duodenum, jejunum, and ileum of rats fed FIG. 1. Effects of ED-71 and 1,25(OH)2D3 on duodenal Pi absorption. Vitamin D-deficient rats maintained on the rescue diet were treated orally every other day for 8 d with vehicle (V) or the specified dose in picomoles of 1,25(OH)2D3 (D) or ED-71 (E). Duodenal Pi absorption was measured by the in situ loop method as detailed in Materials and Methods. Data are presented as mean SEM (n 4 – 6). DPM, Disintegrations per minute. *, P 0.05 vs. V. The data are expressed as the ratio of CYP24A1 mRNA to GAPDH mRNA ( CT method), and presented as mean a, P 0.05 vs. vehicle. normal, vitamin D-replete rodent chow (Purina). The rats were gavaged with vehicle, 50 pmol of ED-71, or 50 or 1000 pmol of 1,25(OH)2D3 at 72 and then at 24 h before death. As shown in Fig. 3, ED-71 significantly increased NaPi-IIb in both the duodenum and jejunum, whereas the same dose and a 20-fold higher dose of 1,25(OH)2D3 had no effect. It should be noted that basal jejunal NaPi-IIb was 5 times higher than basal duodenal NaPi-IIb mRNA, so that the NaPi-IIb/GAPDH mRNA ratio was higher in the jejunum than in the duodenum of ED-71-treated rats. Basal ileal NaPi-IIb mRNA was intermediate to the duodenum and jejunum, but no regulation by either compound was observed. Under these conditions, neither ED-71 nor 1,25(OH)2D3 altered Pit-1 or Pit-2 mRNA in any of the segments (data not shown). Although the relative basal levels of NaPi-IIb mRNA in the intestinal segments and the lack of regulation by 1,25(OH)2D3 agree with previous findings, the high degree of up-regulation of NaPi-IIb mRNA by ED-71, especially in the duodenum, is a novel observation. The ability of ED-71 to enhance Pi uptake and induce NaPi-IIb, whereas the same and 20-fold higher doses of 1,25(OH)2D3 were ineffective, may be attributable to its general higher potency in the gut under the conditions used. This is supported by data in Table 1, showing that ED-71 produced tremendously high levels of CYP24A1 mRNA that were 4 orders of magnitude higher than those in the 1,25(OH)2D3-treated rats in all of the rat intestinal segments. Thus, ED-71 appears to act as a superagonist in these segments despite its lower affinity than 1,25(OH)2D3 for the VDR. In contrast, induction of ileal CYP24A1 by ED-71 was more modest, indicating that the analog is generally less active in this segment. A similar pattern for induction of NaPi-IIb mRNA by ED-71 was observed in mouse small intestine: duodenum jejunum ileum (Supplemental Fig. 1). This allowed the use of a commercial antibody to mouse NaPi-IIb to determine protein content. Immunoblot analysis of BBMV from the proximal and distal halves of the mouse small intestine showed induction of NaPi-IIb protein by ED-71 that was similar to that for increase in NaPi-IIb mRNA (Fig. 4). The time course for induction of NaPi-IIb differed from that of CYP24A1. As shown in Figure 5 (upper panel), mouse duodenal NaPi-IIb mRNA increased between 6 and 24 h after ED-71 administration, whereas CYP24A1 mRNA began to increase at 2 h after treatment (Fig. 5, lower panel) as reported previously in rats ( 19 ). ED-71 does not induce NaPi-IIb in rat lung or type II transporter in the kidney NaPi-IIb is also expressed in the lung, specifically in alveolar type II cells ( 20 ), which also express the VDR ( 21 ). Surprisingly, ED-71 did not regulate NaPi-IIb in rat lung, despite a 1000-fold increase in 24-hydroxylase mRNA (Supplemental Fig. 2). The kidney expresses distinct type II sodium-phosphate cotransporters that regulate renal Pi reabsorption. ED-71 did not regulate rat kidney NaPi-IIa mRNA and slightly decreased expression of NaPi-IIc mRNA (Supplemental Fig. 3). Induction of NaPi-IIb by ED-71 is VDR dependent The lack of induction of NaPi-IIb by ED-71 in the lung and the inability of 1,25(OH)2D3 to induce intestinal NaPi-IIb suggested the possibility that the activation of NaPi-IIb expression in the intestine may be VDR independent. This hypothesis was tested in VDR-ablated mice. As shown in Fig. 6, no induction was observed in VDR / mice treated with ED-71, with an intermediate induction in VDR / mice. Similar results were obtained with CYP24A. Of note, the NaPi-IIb/GAPDH mRNA ratio was not decreased by VDR ablation, suggesting that endogenous 1,25(OH)2D3 levels have little effect on NaPi-IIb expression. Clearances of orally administered ED-71 and 1,25(OH)2D3 The reason for the differential effects of ED-71 and 1,25(OH)2D3 on intestinal NaPi-IIb induction is not clear. As mentioned above, ED-71 has a lower affinity than 1,25(OH)2D3 for the VDR. However, pharmacokinetic differences may provide the explanation. Previous studies by Okano et al. ( 2 ) found that ED-71 has a higher affinity for the serum vitamin D binding protein (DBP), which could prolong its circulating half-life. In addition, we recently reported that ED-71 is not a substrate for the vitamin D 24-hydroxylase (CYP24A1), and therefore is not subject to the inducible attenuation that limits the activities of other vitamin D compounds ( 22 ). We compared the bioavailabilities of ED-71 and 1,25(OH)2D3 in mice after oral administration of tritiated forms of the compounds at the dose used in the mouse studies above. Figure 7 demonstrates that the plasma levels of ED-71 remained constant over the 24-h time course, whereas peak levels of the exogenous 1,25(OH)2D3 were 5 times lower than those of ED-71 at 1 h and decreased by 90% by 7 h. These findings suggest that the higher activity of ED-71compared with 1,25(OH)2D3 on NaPi-IIb may be attributable to much greater bioavailability. Discussion The vitamin D analog ED-71 has been shown to be effective in increasing bone mass in postmenopausal women and in ovariectomized rats ( 1–5 ). However, its effects on mineral metabolism have not been fully investigated. In the current study, we demonstrated that with chronic oral administration, ED-71 is a very potent inducer of intestinal Pi absorption. The increased Pi flux was attributable to induction of NaPi-IIb, because the mRNA for the type 3 Pi transporters, Pit-1 and Pit-2, were not increased in the intestine. 1,25(OH)2D3, even at 20-fold higher doses, had no effect on NaPi-IIb mRNA, in agreement with the findings of Marks et al. ( 8 ), who reported that 1,25(OH)2D3 did not regulate Pi absorption in the rat duodenum. In addition, they found that 1,25(OH)2D3 did not induce NaPi-IIb mRNA in any of the small intestinal segments of the rat, suggesting that their observed enhancement of jejunal Pi absorption by 1,25(OH)2D3 may be independent of NaPi-IIb induction ( 8 ). In contrast, our findings with ED-71 demonstrate that the analog is a potent inducer of duodenal Pi absorption and duodenal NaPi-IIb mRNA. Although the degree of induction was less in the jejunum and still less in the ileum, the basal NaPi-IIb expression increases along the small intestine. Therefore, the relatively smaller fold increases in NaPi-IIb mRNA and protein in the distal segments still produce a substantial overall increase in NaPi-Iib, as evidenced by the immunoblot in Fig. 5. Considering the greater length of the more distal segments, their contribution to total Pi absorption would be greater than that of the duodenum. Therefore, the dramatic increases in NaPi-IIb and Pi absorption in the duodenum and the lesser increase in the jejunum would likely produce would have a less dramatic effect on overall Pi absorption along the entire small intestine, as was noted in our previous study ( 6 ). ED-71 was also much more potent than 1,25(OH)2D3 in inducing duodenal CYP24A1 mRNA, suggesting that this analog acts as a superagonist of the VDR in the gut with the oral dosing used in these experiments. This cannot be attributed to greater affinity for the VDR, because ED-71 has 8 times lower affinity than 1,25(OH)2D3 ( 23 ), but the possibility that ED-71 induces a distinct and more active VDR conformation cannot be excluded. It is more likely that the higher potency of ED-71 is attributable to its very different pharmacokinetics. The analog binds more tightly to the serum DBP, leading to a longer circulating half-life, as is illustrated in Fig, 7. In addition, we recently reported that ED-71 is very resistant to metabolism by CYP24A1 (24-hydroxylase), the major catabolic enzyme for vitamin D compounds ( 22 ). The slow catabolism would also contribute to the persistently high levels of ED-71 in the circulation, providing a much higher reserve of active compound for target gene activation. Our findings strongly point to pharmacokinetics as the explanation for the much higher potency of ED-71. Other factors such as altered VDR stability in response to ED-71 binding may also contribute. Surprisingly, ED-71 did not induce NaPi-IIb in the lung, despite the expression of both NaPi-IIb and the VDR by alveolar type II cells ( 20, 21 ), and a large induction of lung CYP24A1 mRNA. The explanation for the cell specificity of NaPi-IIb induction by ED-71 is under investigation. The mechanism for the induction of NaPi-IIb by ED-71 is unclear but clearly requires the VDR. Total ablation of the VDR eliminated the effect, and loss of one allele led to a dramatic decrease in induction. This finding, along with the apparent requirement for high levels of an active vitamin D compound and no decrease in NaPi-IIb mRNA with VDR ablation, indicate that NaPi-IIb is weakly regulated by vitamin D, and that the effects of endogenous levels of 1,25(OH)2D3 may be negligible. Similar findings for wild-type vs. VDR-knockout mice on the rescue diet were recently reported by Kaneko et al. ( 9 ). Further studies are needed to establish whether the enhancement of NaPiIIb mRNA by ED-71 is through activation of transcription or transcript stabilization. Although most studies have been unable to demonstrate induction of NaPi-IIb mRNA by calcitriol, Xu et al. ( 24 ) reported a 2-fold increase by calcitriol in the intestine of young, but not mature, rats. In rat intestinal cells, calcitriol treatment produced a 1.6-fold increase in the activity of the proximal promoter (1.1 kb), but the vitamin D-responsive elements were not identified. Future studies are needed to identify ED-71-stimulated VDR binding sites in or near the NaPi-IIb gene, using chromatin immunoprecipitation directed to the proximal promoter region ( 24 ), and chromatin immunoprecipitation-Seq to identify potential sites in more distal and intergenic regions ( 25, 26 ). The current findings indicate that the induction by ED-71 of duodenal Pi absorption (8-fold) and NaPi-IIb mRNA (24-fold) is greater than that of duodenal calcium absorption (2-fold) and TRPV6 (2-fold), as we reported recently ( 6 ). This preferential stimulation of Pi uptake suggests a role for ED-71 in the treatment of Pi-wasting disorders. In summary, we demonstrated that ED-71 is a very potent stimulator of Pi absorption and NaPi-IIb expression in the intestine. 1,25(OH)2D3, at 20-fold higher doses, had no effect. This could be attributable to the different pharmacokinetics, because ED-71 has a higher DBP affinity than 1,25(OH)2D3 and is not metabolized by the inducible CYP24A1. The prolonged higher levels of ED-71 may allow the enhanced NaPi-IIb gene expression. However, the molecular mechanism for induction of intestinal NaPi-IIb by ED-71 and the apparent tissue specificity for the activation require further investigation. Acknowledgments We thank Jane Boudreaux for assistance with the in vivo studies. Brown et al. Address all correspondence and requests for reprints to: Alex J. Brown, Ph.D., Box 8126, 660 South Euclid, St. Louis, Missouri 63110. E-mail: . This work was supported by a research grant from Chugai Pharmaceutical, Ltd. Disclosure Summary: The work was funded by a research grant (to A.J.B.) from Chugai Pharmaceuticals, maker of ED-71. 1. Matsumoto T , Kubodera N 2007 ED -71, a new active vitamin D3, increases bone mineral density regardless of serum 25(OH)D levels in osteoporotic subjects . J Steroid Biochem Mol Biol 103 : 584 - 586 2. Okano T , Tsugawa N , Masuda S , Takeuchi A , Kobayashi T , Nishii Y 1991 A novel synthetic vitamin D3 analogue, 2- -(3-hydroxypropoxy)-calcitriol (ED-71) : its biological activities and pharmacological effects on calcium metabolism . Contrib Nephrol 91 : 116 - 122 3. 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Brown, Alex J., Zhang, Fanjie, Ritter, Cynthia S.. The Vitamin D Analog ED-71 Is a Potent Regulator of Intestinal Phosphate Absorption and NaPi-IIb, Endocrinology, 2012, 5150-5156, DOI: 10.1210/en.2012-1587