Background

Vitamin D compounds are used clinically to control secondary hyperparathyroidism (SHPT) due to renal failure. Newer vitamin D compounds retain the suppressive action of 1,25(OH)₂D₃ on the parathyroid glands and may have less Ca²⁺-mobilizing activity, offering potentially safer therapies.

Methods

This study investigated the effect of a single dose of compound (1,25(OH)₂D₃, 1,24(OH)₂D₂, or 1(OH)D₂) on renal and intestinal Ca²⁺ transport proteins, including TRPV5 and TRPV6, and serum Ca²⁺, in a novel SHPT model, the 25-OH-D₃-1-hydroxylase knockout mouse, which lacks endogenous 1,25(OH)₂D₃ and is severely hypocalcemic. Animals were injected intraperitoneally with compound (100 ng/mouse).

Results

Serum levels of 1,25(OH)₂D₃ and 1,24(OH)₂D₂ peaked at four hours post-injection (pi), then declined rapidly. 1,25(OH)₂D₂ generated from 1(OH)D₂ peaked at 12 hours pi and then remained stable. Serum Ca²⁺ was increased to near-normal within four hours by 1,25(OH)₂D₃ and 1,24(OH)₂D₂, and within 12 hours by 1(OH)D₂. 1,25(OH)₂D₃ and 1,24(OH)₂D₂ up-regulated duodenal TRPV5 and TRPV6 mRNA to a similar degree within four hours; mRNA levels decreased by 12 hours after 1,24(OH)₂D₂ treatment, and by 24 hours after 1,25(OH)₂D₃ treatment. 1,25(OH)₂D₃ increased kidney levels of TRPV5, calbindin-D_28K, and calbindin-D_9K mRNA within four hours; 1,24(OH)₂D₂ did not change kidney TRPV5 levels and modestly increased calbindin D_9K by 48 hours. 1(OH)D₂ produced later-onset effects, increasing duodenal TRPV6 and calbindin-D_9K mRNA levels by 12 hours and TRPV5 by 48 hours.

Conclusion

In kidney, 1(OH)D₂ increased TRPV5, calbindin-D_28K, and calbindin-D_9K mRNA levels by 12 hours. This study indicates that Ca²⁺ transport proteins, including TRPV5 and TRPV6, are differentially up-regulated by vitamin D compounds.

METHODS

Animal protocol

25-hydroxyvitamin D₃-1-hydroxylase (1-OHase^-/-) knockout mice were recently generated by targeted ablation of exon 8 encoding the heme-binding domain of the enzyme²⁵. 1-OHase knockout mice were genotyped by Southern blot in combination with polymerase chain reaction (PCR) analysis at an age of 3 weeks directly after the weaning period, as described previously^17,25. Homozygous 1-OHase (1-OHase^-/-) knockout mice were fed a regular diet containing 1.1% (w/w) Ca²⁺, 0.8% (w/w) phosphorus, 0% (w/w) lactose (Harlan Teklad; Madison, WI, USA) from week three to eight. At week eight, 1-OHase^-/- mice were injected intraperitoneally (ip) with 1,25(OH)₂D₃, 1,24(OH)₂D₂, or 1(OH)D₂ (100 ng/mouse). At 4, 12, 24, 48, and 96 hours' post-injection (pi), mice (N = 3 to 5 in each group) were sacrificed and blood, kidney, and duodenum samples were taken, which were subsequently analyzed for Ca²⁺ and 1,25(OH)₂D serum concentrations and expression of the Ca²⁺ transporters. The animal ethics board of the University Medical Center Nijmegen (Nijmegen) approved all animal experimental procedures.

Serum biochemistry

Serum Ca²⁺ concentrations were analyzed using a colorimetric assay kit as described previously²⁶. Circulating levels of active metabolites were determined by total dihydroxyvitamin D analysis (Nichols Institute Diagnostics Kit, cat no. 40–6090; San Juan Capistrano, CA, USA) as follows: samples of mouse serum (0.1 mL) were delipidated, and the dihydroxyvitamin D fraction was separated from potential cross-reactants by incubation with a highly specific solid-phase monoclonal antibody. The immunoextraction gel was then washed, and the purified dihydroxyvitamin D fraction was eluted directly into glass assay tubes. Calibrators and reconstituted eluates were incubated overnight with a highly specific polyclonal sheep antibody. ¹²⁵I-1,25-dihydroxyvitamin-D was added and incubation was continued for two more hours. Separation of bound and free analytes was achieved during incubation with antisheep precipitant, followed by centrifugation, decanting, and counting. Bound radioactivity is inversely proportional to the concentration of dihydroxyvitamin-D. Metabolite concentrations were quantified using a standard curve.

Immunohistochemistry

The renal localization of TRPV5 was assessed by immunohistochemistry as described previously²⁷. In short, staining of renal cortex sections for TRPV5 was performed on 7-m sections of fixated frozen kidney samples. TRPV5 staining involved immersion of the kidney sections in boiled citrate target retrieval buffer (0.01 mol/L sodium citrate and 0.01 mol/L citric acid, pH 6.0), which was then left to cool for 30 minutes, and subsequent incubation in 0.3% (v/v) H₂O₂ in buffer (0.15 mol/L NaCl, 0.1 mol/L Tris-HCl, pH 7.6) for 30 minutes. Sections were incubated for 16 hours at 4°C with the affinity-purified guinea pig TRPV5 antibody (1:1000) and TRPV5 protein was detected as described previously^17,27. Images were made using a Nikon Diaphot confocal laser scanning microscope (Tokyo, Japan; MRC-1000; Bio-Rad, Richmond, CA, USA) and a Zeiss fluorescence microscope equipped with a digital photo camera (Nikon DMX1200). For semiquantitative determination of protein levels, digital images were analyzed with the Image Pro Plus 4.1 image analysis software (Media Cybernetics; Silver Spring, MD, USA), resulting in quantification of the protein levels as the mean of integrated optical density (IOD).

Real-time PCR analysis

Total RNA was extracted from kidney and duodenum using TriZol Total RNA Isolation Reagent (Gibco BRL; Breda, The Netherlands). The obtained RNA was subjected to DNAse treatment to prevent genomic DNA contamination. Thereafter, 2 g RNA was reverse transcribed by Moloney-Murine Leukemia Virus-Reverse Transcriptase (M-MLV-RT; Gibco BRL), as described previously¹⁷. The obtained renal cDNA was used to determine TRPV5, calbindin-D_9K, calbindin-D_28K, Na⁺-Ca²⁺ exchanger (NCX1), and plasma membrane ATPase (PMCA1b) mRNA expression levels, as well as mRNA levels of the housekeeping gene hypoxanthine-guanine phosphoribosyl transferase (HPRT) as an endogenous control. The cDNA transcribed from duodenum RNA was assayed for TRPV5, TRPV6, calbindin-D_9K, PMCA1b, and HPRT mRNA expression levels. Polymerase chain reaction (PCR) primers and fluorescent probes were designed using the computer program Primer Express (Applied Biosystems, Foster City, CA, USA) and purchased from Biolegio (Malden, The Netherlands). The primer and probe sequences for TRPV5, TRPV6, calbindin-D_9K, calbindin-D_28K, NCX1, PMCA1b, and HPRT were described previously¹⁷. Expression levels were quantified by real-time quantitative PCR on an ABI Prism 7700 Sequence Detection System (PE Biosystems, Rotkreuz, Switzerland).

Statistical analysis

The serum Ca²⁺ and vitamin D metabolite concentrations were expressed as the mean SEM. Overall statistical significance was determined by analysis of variance (ANOVA). In the case of significance (P < 0.05), individual groups were compared by contrast analysis according to Fisher.

RESULTS

Serum Ca²⁺ and vitamin D metabolites

Initially, a dose response analysis was carried out in 8-week-old 1-OHase^-/- mice using 50, 100, and 150 ng/animal of either 1,25(OH)₂D₃, 1,24(OH)₂D₂, or 1(OH)D₂. Maximal responses on the expression of the Ca²⁺ transport genes were observed at 100 ng (data not shown), and this dose was chosen for further investigation. Subsequently, the effects of 1,25(OH)₂D₃, 1(OH)D₂, and 1,24(OH)₂D₂ administration on serum Ca²⁺ and vitamin D metabolite levels in 1-OHase^-/- mice at time points from four to 96 hours were determined Figure 1. Control 1-OHase^-/- mice treated with vehicle exhibited severe hypocalcemia with serum Ca²⁺ concentrations around 1.5 mmol/L Figure 1a. Intraperitoneal administration of a single 100 ng dose of 1,25(OH)₂D₃ or 1,24(OH)₂D₂ significantly increased serum Ca²⁺ concentration to near-normal values of around 2 mmol/L within four hours Figure 1a. The serum Ca²⁺ concentrations remained increased up to 96 hours pi, declining slightly in the case of 1,24(OH)₂D₂. In contrast, serum Ca²⁺ was not increased by 1(OH)D₂ until 12 hours. This compound, a prodrug, requires systemic metabolism to active metabolites to exert effects. Serum Ca²⁺ levels were increased by 1(OH)D₂ over the remaining time points examined Figure 1a, but never exceeded the serum Ca²⁺ levels of normal littermates (2.5 mmol/L).

Figure 1.

Serum Ca²⁺ (A) and 1,25(OH)₂D (B) concentrations in 1-OHase^-/- mice supplemented with 1,25(OH)₂D₃ (black), 1(OH)D₂ (diagonal stripes) or 1,24(S)(OH)₂D₂ (white) at different time points in hour. Data are mean SEM (N = 3 to 5). *P < 0.05, significantly different from 1-OHase^-/- treated with vehicle only.

Full figure and legend (38K)

Analysis of vitamin D metabolite levels following dose administration showed that 1,25(OH)₂D₃ produced a significant increase in serum 1,25(OH)₂D concentrations that peaked at four hours pi and subsequently declined gradually Figure 1b. Serum levels of 1,24(OH)₂D₂ followed a time course similar to 1,25(OH)₂D_3, but were consistently about 25% of 1,25(OH)₂D₃ levels. Administration of 1(OH)D₂ produced 1,25(OH)₂D serum levels that reached approximately 600 pg/mL at 12 hours pi, and then remained relatively constant for the remaining time points Figure 1b.

Expression of Ca²⁺ transport genes in kidney

The effect of 1,25(OH)₂D₃, 1(OH)D₂, and 1,24(OH)₂D₂ on mRNA levels of Ca²⁺ transport genes (TRPV5, calbindin-D_9K, calbindin-D_28K, NCX1, and PMCA1b) in the kidney was investigated using total RNA isolated from kidney cortex and real-time quantitative PCR Figure 2. 1,25(OH)₂D₃ supplementation produced a 2.5-fold increase in TRPV5 mRNA expression at four hours pi compared to vehicle-treated mice Figure 2a. Expression of TRPV5 was significantly increased from four hours' pi onwards. Significant up-regulation of calbindin-D_28K (1.5- to 2-fold) and calbindin-D_9K (2- to 12-fold) mRNA was also produced by 1,25(OH)₂D₃ Figure 2b and c. NCX1 mRNA expression was not significantly affected by 1,25(OH)₂D₃ Figure 2d, whereas a slight reduction was observed for PMCA1b Figure 2e.

Figure 2.

The effect of 1,25(OH)₂D₃ (black), 1(OH)D₂ (diagonal stripes), and 1,24(S)(OH)₂D₂ (white) on the expression of the renal Ca²⁺ transport genes, including TRPV5 (A), calbindin-D_28K (B), calbindin-D_9K (C), NCX1 (D), and PMCA1b (E) in 1-OHase^-/- mice assessed by real-time polymerase chain reaction (PCR) analysis. Values are calculated as a ratio to the hypoxanthine-guanine phosphoribosyl transferase (HPRT) RNA level and expressed relative to levels of 1-OHase^-/- mice treated with vehicle. Values are presented as mean SEM (N = 3 to 5). *P < 0.05, significant different from 1-OHase^-/- treated with vehicle only (control).

Full figure and legend (48K)

Administration of 1,24(OH)₂D₂ did not significantly affect mRNA expression levels of TRPV5 and calbindin-D_28K Figure 2a and b. Remarkably, calbindin-D_9K and NCX1 mRNA were up-regulated at the late time points, 48 and 96 hours pi, whereas PMCA1b mRNA was slightly decreased Figure 2c to e.

Administration of 1(OH)D₂ increased mRNA expression of TRPV5, calbindin-D_28K, and calbindin-D_9K in the kidney. Although the degree of mRNA up-regulation induced by 1(OH)D₂ was comparable to those of 1,25(OH)₂D₃, there was a slight delay in the response to 1(OH)D₂ Figure 2a to c. Expression of the basolateral extrusion genes (NCX1 and PMCA1b) was not robustly affected by 1(OH)D₂ Figure 2d and e.

Analysis of TRPV5 protein levels in kidney

TRPV5 protein expression in the kidney was examined using immunohistochemistry. Figure 3a shows representative immunofluorescence labeling of distal convoluted (DCT) and connecting tubules (CNT) in sections of kidney cortex from 1-OHase knockout mice supplemented with 1,25(OH)₂D₃, 1(OH)D₂, or 1,24(OH)₂D₂. More TRPV5 protein was detected at 96 hours pi in the 1,25(OH)₂D₃- and 1(OH)D₂-supplemented mice, indicated by the increased staining in the kidney cortex. In these immunopositive tubules, TRPV5 was localized to the apical membrane of DCT and CNT. To semiquantify TRPV5 protein expression, the IOD was measured with computer analysis using the Image-Pro Plus software version 3.0 Figure 3b. In line with the observed TRPV5 expression at the mRNA level, the response on protein expression for 1(OH)D₂ was delayed (48 hours) compared to 1,25(OH)₂D₃ (12 hours). For 1,24(OH)₂D₂, a significant increase in TRPV5 expression was observed only at 96 hours pi Figure 3b.

Figure 3.

The effect of 1,25(OH)₂D₃, 1(OH)D₂, or 1,24(S)(OH)₂D₂ on renal TRPV5 protein expression in 1-OHase^-/- mice. Representative immunohistochemistry of TRPV5 in the kidney cortex at 4 and 96 hours post-administration (A). Averaged TRPV5 protein expression levels were calculated by measuring the integral optical density (IOD) using the Image-Pro Plus version 3.0 software (B). Values are presented as mean SEM (N = 3 to 5). *P < 0.05, significantly different from 1-OHase^-/- treated with vehicle only (control).

Full figure and legend (137K)

Expression of Ca²⁺ transport genes in duodenum

Subsequently, we investigated whether treatment with these vitamin D compounds altered the expression of genes encoding Ca²⁺ transport proteins involved in duodenal transcellular Ca²⁺ absorption. Administration of 1,25(OH)₂D₃ produced a >30-fold increase in TRPV6 mRNA levels by four hours' pi Figure 4b. In line with these findings, TRPV5 and calbindin-D_9K were also significantly up-regulated Figure 4a, c. Levels of TRPV5 and TRPV6 mRNA remained elevated at 12 hours pi, returning to baseline by 24 hours pi; calbindin-D_9K mRNA remained elevated out to 96 hours pi. PMCA1b mRNA expression was slightly, but significantly, increased at 48 to 96 hours post-administration of 1,25(OH)₂D₃ Figure 4d.

Figure 4.

The effect of 1,25(OH)₂D₃ (black), 1(OH)D₂ (diagonal stripes), and 1,24(S)(OH)₂D₂ (white) on the expression of the duodenal Ca²⁺ transport genes, including TRPV5 (A), TRPV6 (B), calbindin-D_9K (C), and PMCA1b (D) in 1-OHase^-/- mice assessed by real-time polymerase chain reaction (PCR) analysis. Values are calculated as a ratio to the hypoxanthine-guanine phosphoribosyl transferase (HPRT) RNA level and expressed relative to levels of 1-OHase^-/- mice treated with vehicle. Values are presented as mean SEM (N = 3 to 5). *P < 0.05, significantly different from 1-OHase^-/- treated with vehicle only (control).

Full figure and legend (40K)

1,24(OH)₂D₂ -treatment resulted in a rapid 24-fold up-regulation of TRPV6 mRNA in duodenum by four hours pi Figure 4b. TRPV5 and calbindin D_9K mRNA were also significantly increased Figure 4a and c. This rapid increase in TRPV5 and TRPV6 mRNA expression was transient and returned quickly to control values at 12 to 96 hours pi. Calbindin-D_9K mRNA expression remained elevated during the entire studied time interval. No significant change was observed in the level of mRNA encoding PMCA1b Figure 4d.

Supplementation with 1(OH)D₂ resulted in a delayed, but significant up-regulation of TRPV6 (160-fold) and TRPV5 (8-fold) mRNA that was maximal at 96 hours post-administration Figure 4a and b. In addition, up-regulation of both Ca²⁺ channels was accompanied by an increase in expression of calbindin-D_9K (3- to 15-fold) Figure 4c. In contrast, PMCA1b expression was elevated only at 96 hours pi Figure 4d.

DISCUSSION

Vitamin D compounds including 1,25(OH)₂D₃ are widely used in the treatment of SHPT because of their efficacy in reducing elevated PTH levels. High PTH levels are deleterious to the skeleton, particularly to cortical bone²⁸, and may have pathologic effects on other tissues as well⁶. Normalization of PTH levels and bone histology were observed in patients with renal failure who were treated with vitamin D compounds including 1(OH)D₃ and 1,25(OH)₂D₃^29,30. However, hypercalcemia develops frequently in patients treated with older compounds, such as 1,25(OH)₂D₃, limiting their clinical application³¹. Thus, novel vitamin D compounds, with efficacy in PTH reduction and reduced effects on systemic Ca²⁺, have been developed with the goal of minimizing this unwanted side effect^{7,8,9,10,11,12,14,15,16}.

Several experimental animal models have been utilized to study Ca²⁺ transport proteins and their disturbances linked to parathyroid activity. Recently, 1-OHase knockout mice strains have been generated that represent a valuable animal model for pseudovitamin D deficiency rickets (PDDR) and SHPT^25,32. Untreated 1-OHase^-/- mice presented the same clinical phenotype as patients with PDDR, including markedly elevated serum PTH levels, retarded growth, failure to thrive, severe hypocalcemia, bone abnormalities (including rickets and osteomalacia), and undetectably low serum levels of 1,25(OH)₂D₃. The last feature was particularly important because our goal was to quantitate the effects of vitamin D compounds on Ca²⁺ transport proteins. Because the transgenic mice completely lacked background levels of 1,25(OH)₂D₃, the immediate effects of exogenously administered vitamin D compounds could be assigned unambiguously. In the absence of renal 1-OHase activity, no feedback inhibition of endogenous 1,25(OH)₂D₃ synthesis by exogenous compounds was possible, simplifying analysis at later time points. Furthermore, the transgenic animals are a robust model, reliably developing the PDDR phenotype. Models employed in the past to produce low serum levels of 1,25(OH)₂D₃, such as the vitamin D–deficient rat, require subjecting experimental animals to a vitamin D–deficient diet under ultraviolet (UV)-free conditions for months, and in our experience, often meet with limited success in producing the desired phenotype. For the above reasons, these transgenic mice offer an ideal model in which to study the activity of vitamin D compounds on serum Ca²⁺ and the expression of Ca²⁺ transport genes. Recent work from our laboratory indicates that the renal Ca²⁺ transport proteins, including TRPV5, calbindin-D_9K, calbindin-D_28K, and NCX1 are down-regulated in kidneys of 1-OHase^-/- mice^17,19. Furthermore, mRNA encoding TRPV6, calbindin-D_9K, and PMCA1b is decreased in duodenum¹⁹. Treatment with 1,25(OH)₂D₃ normalizes serum Ca²⁺ levels and the expression of the Ca²⁺ transport proteins in these knockout animals¹⁷.

In the present study, we examined the effect of three vitamin D compounds, namely 1,25(OH)₂D₃, 1(OH)D₂, and 1,24(OH)₂D₂ on serum Ca²⁺ and vitamin D levels, and on the expression of Ca²⁺ transport genes in the kidney and duodenum in 1-OHase knockout mice. 1,25(OH)₂D₃ has been used previously in many studies of Ca²⁺ transport proteins and was included as the prototypic active vitamin D compound. 1(OH)D₂ is a vitamin D prodrug, less calcemic than 1,25(OH)₂D₃ in animal studies⁶, which must be metabolized to become active, resulting in altered pharmacokinetics relative to active vitamin D compounds. 1(OH)D₂ has recently been approved in the United States for the treatment of SHPT of renal failure. 1,24(OH)₂D₂ is an active metabolite of 1(OH)D₂ with greatly reduced calcemic activity relative to 1,25(OH)₂D₃^33,35.

All three compounds, administered as a single intraperitoneal dose of 100 ng, were able to increase serum Ca²⁺ levels toward the normal range in knockout mice. 1,25(OH)₂D₃ and 1,24(OH)₂D₂ increased serum Ca²⁺ from 1.5 to 2 mmol/L within four hours pi Figure 1a, while 1(OH)D₂ increased serum Ca²⁺ to 2 mmol/L by 12 hours pi. These increases in serum Ca²⁺ correlated well with the pharmacokinetics of the vitamin D compounds. Serum levels of both 1,24(OH)₂D₂ and 1,25(OH)₂D₃ increased rapidly after dose administration, reaching a peak at four hours pi, and then decreasing rapidly. This spike in serum levels of 1,24(OH)₂D₂ and 1,25(OH)₂D₃ resembled that seen in wild-type animals³³. In contrast, serum levels of 1,25(OH)₂D₂ produced from 1(OH)D₂ reached a peak at 12 hours pi, which reflects the delayed hepatic activation of this prodrug.

The additional time required to increase serum 1,25(OH)₂D₂ levels following 1(OH)D₂ administration, relative to active compounds, correlated well with the additional time required by 1(OH)D₂ to increase serum Ca²⁺. Unexpectedly, however, serum levels of 1,25(OH)₂D₂ produced from 1(OH)D₂ remained elevated out to 96 hours postdose, a significant departure from the pharmacokinetics observed previously for 1(OH)D₂, in which serum levels of 1,25(OH)₂D₂ returned to baseline by 48 hours postdose³⁴. The continued elevation of serum 1,25(OH)₂D₂ in 1-OHase^-/- animals 96 hours after a single dose of 1(OH)D₂ may result from low initial levels of CYP24²⁵, the major catabolic enzyme for vitamin D compounds, combined with the very large dose of 1(OH)D₂ used in this study.

Interestingly, like serum Ca²⁺ levels, TRPV5 and TRPV6 mRNA levels in duodenum increased in parallel with serum levels of vitamin D compounds. Both 1,25(OH)₂D₃ and 1,24(OH)₂D₂ induced a rapid increase in duodenal TRPV5 and TRPV6 levels (35-fold and 20-fold, respectively, for TRPV6) within four hours, while 1(OH)D₂ did not up-regulate TRPV6 levels until 12 hours pi. The effect of 1(OH)D₂ on TRPV5 mRNA in duodenum was also later onset, with no increase at early time points, and then pronounced up-regulation.

Effects of vitamin D compounds on Ca²⁺ regulatory genes in kidney were more diverse. Interestingly, 1,24(OH)₂D₂ did not up-regulate TRPV5 or calbindin mRNA in kidney at any time point. In contrast, 1,25(OH)₂D₃ significantly increased both TRPV5 and calbindin mRNA levels by four hours pi. 1(OH)D₂ significantly increased mRNA levels of TRPV5 and calbindin at 12 hours pi and later. Both 1,25(OH)₂D₃ and 1(OH)D₂ up-regulated mRNA levels of the Ca²⁺ transport genes in the kidney to a similar degree. TRPV5 protein expression was up-regulated by 1(OH)D₂ exclusively along the apical side of tubules in the distal part of the nephron, in agreement with previous studies¹⁸. In general, increases in expression of Ca²⁺ transport genes by vitamin D compounds were smaller in kidney than in duodenum, but still significant.

An intriguing aspect of the present study was that 1,25(OH)₂D₃, and to a lesser extent 1,24(OH)₂D₂, appeared to have a biphasic effect on mRNA levels of TRPV5 levels in kidney and duodenum, and TRPV6 levels in duodenum; mRNA levels were highly increased by four to 12 hours, declined at 24 hours, then increased again by 96 hours Figures 2a, 3b, and 4a. Levels of TRPV5 and TRPV6 mRNA in duodenum, and TRPV5 protein in kidney, were also highly induced at later time points (48 and 96 hours) by 1(OH)D₂. The reason for this biphasic or later onset effect is unclear, and further studies will be necessary to determine the underlying mechanism.

The effects of 1(OH)D₂ on mRNA levels of Ca²⁺ transport proteins were more pronounced at later time points in both duodenum and kidney. The sustained supraphysiologic levels of 1,25(OH)₂D₂ (approximately 600 pg/mL) at 12 to 96 hours pi following the large dose of 1(OH)D₂ (approximately 5 g/kg) produced strong induction of Ca²⁺ transport proteins at later time points in both duodenum and kidney (Figures 3b, 4a to d), which may account for the continued gradual increase in serum Ca²⁺ produced by 1(OH)D₂. Of note, even at the large dose used, the levels of serum Ca²⁺ in transgenic mice treated with 1(OH)D₂ never exceeded those of normal littermates (2.5 mmol/L).

The data in the present study may shed light on the mechanism underlying the reduced calcemic activity of 1,24(OH)₂D₂. As noted, 1,24(OH)₂D₂ is a metabolite of vitamin D₂ and 1(OH)D₂, which is 10- to 30-fold less calcemic than 1,25(OH)₂D₃³³. As in normal animals³³, 1,24(OH)₂D₂ serum levels in knockout animals were consistently about 25% of 1,25(OH)₂D₃ levels. Despite this, 1,24(OH)₂D₂ increased serum Ca²⁺ to the same extent as 1,25(OH)₂D₃ at four hours pi, and increased duodenal levels of TRPV5 and TRPV6 levels to the same degree Figure 4a and b. The equivalent ability of 1,24(OH)₂D₂ and 1,25(OH)₂D₃ to increase serum Ca²⁺ levels in this study may have resulted from the large dose of compound used, which was deliberately chosen to maximize the responses of Ca²⁺ transporter genes in the knockout animals. However, even at this large dose, animals treated with 1,24(OH)₂D₂ showed a rapid decline in TRPV5 and TRPV6 mRNA levels in duodenum after initial induction, returning to near baseline levels at 12 hours pi; in animals treated with 1,25(OH)₂D₃, TRPV5 and TRPV6 levels remained elevated or even increased further at 12 hours pi Figure 4a and b. As noted, 1,24(OH)₂D₂ did not increase kidney levels of TRPV5 or calbindin-D_28K at any time point. Taken together, the data suggest that the reduced calcemic effects of 1,24(OH)₂D₂ may result from a reduced effect on Ca²⁺ channel proteins in target organs, through either reduced magnitude of induction or reduced duration of induction. Further studies will be necessary to confirm this hypothesis.

CONCLUSION

This study provides evidence that TRPV5 and TRPV6 are differentially up-regulated by vitamin D analogs. The time course of up-regulation of these epithelial Ca²⁺ transporters appears to correlate well with serum levels of active vitamin D metabolites. 1(OH)D₂ provided normalization of serum Ca²⁺ and up-regulation of the epithelial Ca²⁺ transporters that was maintained for an extended period, whereas at the same dose 1,24(OH)₂D₂ and 1,25(OH)₂D₃ produced a more rapid increase in serum Ca²⁺ concentration. Further studies are under way to define the roles of TRPV5 and TRPV6 in vitamin D–regulated Ca²⁺ transport and to determine the relative contribution of these epithelial Ca²⁺ transporters to vitamin D–mediated Ca²⁺ homeostasis.

References

References

Acknowledgments

This work was supported by the Dutch Organization of Scientific Research (Zon-Mw 016.006.001, Zon-Mw 902.18.298), the Dutch Kidney Foundation (C10.1881 and C03.6017), and by Bone Care International. We thank R. St-Arnaud for kindly providing the 1-OHase knockout mice.