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Endocrinology Vol. 143, No. 2 587-595
Copyright © 2002 by The Endocrine Society

PTH-CALCITONIN-VITAMIN D-BONE

Dietary Phosphorus Transcriptionally Regulates 25-Hydroxyvitamin D-1{alpha}-Hydroxylase Gene Expression in the Proximal Renal Tubule

Martin Y. H. Zhang, Xuemei Wang, Jonathan T. Wang, Nathalie A. Compagnone, Synthia H. Mellon, Jean L. Olson, Harriet S. Tenenhouse, Walter L. Miller and Anthony A. Portale

Departments of Pediatrics (M.Y.H.Z., X.W., J.T.W., W.L.M., A.A.P.), Medicine (A.A.P.), Obstetrics, Gynecology, and Reproductive Sciences (N.A.C., S.H.M.), and Pathology (J.L.O.), University of California, San Francisco, California 94143; and Departments of Pediatrics and Human Genetics, McGill University and Montreal Children’s Hospital Research Institute (H.S.T.), Montréal, Québec, Canada

Address all correspondence and requests for reprints to: Anthony A. Portale, M.D., Room U-585, University of California, 533 Parnassus Avenue, San Francisco, California 94143-0748. E-mail: aportale{at}peds.ucsf.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Synthesis of the hormone 1,25-dihydroxyvitamin D, the biologically active form of vitamin D, occurs in the kidney and is catalyzed by the mitochondrial cytochrome P450 enzyme, 25-hydroxyvitamin D-1{alpha}-hydroxylase (1{alpha}-hydroxylase). We sought to characterize the effects of changes in dietary phosphorus on the kinetics of renal mitochondrial 1{alpha}-hydroxylase activity and the renal expression of P450c1{alpha} and P450c24 mRNA, to localize the nephron segments involved in such regulation, and to determine whether transcriptional mechanisms are involved. In intact mice, restriction of dietary phosphorus induced rapid, sustained, approximately 6- to 8-fold increases in renal mitochondrial 1{alpha}-hydroxylase activity and renal P450c1{alpha} mRNA abundance. Immunohistochemical analysis of renal sections from mice fed the control diet revealed the expression of 1{alpha}-hydroxylase protein in the proximal convoluted and straight tubules, epithelial cells of Bowman’s capsule, thick ascending limb of Henle’s loop, distal tubule, and collecting duct. In mice fed a phosphorusrestricted diet, immunoreactivity was significantly increased in the proximal convoluted and proximal straight tubules and epithelial cells of Bowman’s capsule, but not in the distal nephron. Dietary phosphorus restriction induced a 2-fold increase in P450c1{alpha} gene transcription, as shown by nuclear run-on assays. Thus, the increase in renal synthesis of 1,25-dihydroxyvitamin D induced in normal mice by restricting dietary phosphorus can be attributed to an increase in the renal abundance of P450c1{alpha} mRNA and protein. The increase in P450c1{alpha} gene expression, which occurs exclusively in the proximal renal tubule, is due at least in part to increased transcription of the P450c1{alpha} gene.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE HORMONE 1,25-dihydroxyvitamin D [1,25-(OH)2D] plays a critical role in calcium and phosphorus metabolism, bone growth, and cellular differentiation. Renal synthesis of 1,25-(OH)2D from its endogenous precursor, 25-hydroxyvitamin D (25OHD), is catalyzed by the mitochondrial cytochrome P450 enzyme, 25OHD-1{alpha}-hydroxylase (1{alpha}-hydroxylase). Recently, we and others cloned the cDNA and gene for human (1, 2, 3), rat ( 4, 5), and mouse (6) 1{alpha}-hydroxylases, designated P450c1{alpha}. Human P450c1{alpha} cDNA is 2.4 kb in length and encodes a protein of 508 amino acids with a predicted molecular mass of 56 kDa (1). The single copy human gene for P450c1{alpha}, located on chromosome 12q13–14, is 5 kb in length and comprises nine exons and eight introns (2). Although it is substantially smaller than genes for the other mitochondrial P450 enzymes, its intron/exon organization is very similar (2), suggesting that these genes belong to a single evolutionary lineage. We and others have identified mutations in the P450c1{alpha} gene in patients with autosomal recessive 1{alpha}-hydroxylase deficiency, also known as vitamin D-dependent rickets type 1, thus establishing the molecular genetic basis of this disease (1, 7).

The activity of the renal 1{alpha}-hydroxylase enzyme is subject to complex regulation by PTH, calcium, phosphorus, and 1,25-(OH)2D itself (8). With the cloning of P450c1{alpha}, attention has focused on the molecular mechanisms involved in the regulation of 1{alpha}-hydroxylase activity. In intact animals, an increase in P450c1{alpha} mRNA expression is induced by the administration of PTH and calcitonin (9, 10), restriction of dietary calcium (5), and vitamin D deficiency ( 4, 5). Conversely, the administration of 1,25-(OH)2D3 induces a decrease in P450c1{alpha} mRNA expression (4, 6, 10) and prevents the increase induced by PTH and calcitonin (10). The effect of PTH and forskolin to induce, and of 1,25-(OH)2D to inhibit, P450c1{alpha} gene expression occurs at least in part at the transcriptional level, as shown using promoter-reporter constructs of the human (11, 12) and mouse (13) genes.

Phosphorus has long been known to be an important determinant of the renal production of 1,25-(OH)2D. Restriction of dietary phosphorus in animals increases the synthesis and serum concentration of 1,25-(OH)2D (14, 15, 16, 17, 18, 19, 20); the rate of 1,25-(OH)2D synthesis varies inversely with the serum concentration of phosphorus, and the effect is independent of PTH (14, 15). In healthy human subjects, restricting dietary phosphorus induces an increase (21, 22, 23, 24, 25) and supplementation induces a decrease (24, 25, 26) in the serum concentration of 1,25-(OH)2D; these changes entirely reflect changes in the production rate of 1,25-(OH)2D, as its MCR did not change (24). In mice in which hypophosphatemia is due to homozygous disruption of the Npt2 gene, renal P450c1{alpha} gene expression is appropriately increased (27). However, the cellular and molecular mechanisms by which phosphorus regulates the renal production of 1,25-(OH)2D are unknown.

The serum concentration of 1,25-(OH)2D is determined by both its rate of synthesis and its rate of degradation. Metabolic inactivation of vitamin D is accomplished by the 25OHD-24-hydroxylase, P450c24, which converts 1,25-(OH)2D to its final inactivation product, calcitroic acid. Although 24-hydroxylase activity is expressed primarily in kidney, it can be induced by 1,25-(OH)2D in vitamin D target tissues such as intestine, lymphocytes, fibroblasts, bone, skin, macrophages, and possibly other tissues (28). In the rat, dietary phosphorus restriction induced a substantial decrease in renal 24-hydroxylase activity and P450c24 mRNA abundance (29), although the mediating molecular mechanisms are unknown.

The objectives of the present study were to characterize in normal mice the effects of changes in dietary phosphorus on the kinetics of renal mitochondrial 1{alpha}-hydroxylase activity and the renal expression of P450c1{alpha} and P450c24 mRNA, to localize the nephron segments involved in such regulation, and to determine whether transcriptional mechanisms are involved. We demonstrate that dietary phosphorus regulates P450c1{alpha} mRNA and protein expression in the proximal renal tubule at least in part via a transcriptional mechanism.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
To determine the effect of dietary phosphorus intake on renal 1{alpha}-hydroxylase activity and on renal P450c1{alpha} and P450c24 mRNA expression, we studied male C57BL/6 mice, 90–150 d of age (Charles River Laboratories, Inc., Wilmington, MA). All animals first were fed a vitamin D-replete control diet containing 1.0% phosphorus and 1.0% calcium (test diet 86129, Harlan Teklad, Madison, WI) for 7 d. Then, some groups of animals continued to receive the control diet, and other groups received an otherwise identical diet containing lower amounts of phosphorus [0.02% (test diet 86128, Teklad), 0.2%, or 0.6% phosphorus content]; each control and test diet was given for 1–8 d, as indicated in Results. Animals were anesthetized with pentobarbital, and blood was drawn by cardiac puncture for determination of serum phosphorus concentration, using a kit from Stanbio Laboratories (San Antonio, TX). The kidneys were removed; one kidney was rapidly frozen in liquid nitrogen for subsequent extraction of RNA, and the other was placed in homogenizing medium at 4 C for isolation of renal mitochondria. All procedures were approved by the committee on animal research, University of California (San Francisco, CA).

Renal mitochondrial 1{alpha}-hydroxylase activity
Renal mitochondria were isolated by the method of Vieth and Fraser (30). The mitochondrial pellet was suspended in buffer containing 125 mM KCl, 20 mM HEPES, 10 mM L-malic acid, 2 mM MgSO3, 1 mM dithiothreitol, and 0.25 mM EDTA adjusted to pH 7.42. Duplicate 1-ml aliquots of mitochondrial protein (2.0–3.0 mg/ml) were incubated with 500 nM chromatographically purified 25OHD3 in 20 µl 95% ethanol in a shaking water bath at 24 C for 15 min. The reaction was stopped by the addition of 1.0 ml acetonitrile, and lipid extraction of the sample was performed. 1,25-(OH)2D was isolated from the lipid extract by sequential C18 and silica column chromatography and quantitated in duplicate by RRA, as previously described (1).

The specificity of 1,25-(OH)2D3 quantitation was confirmed by subjecting a portion of selected samples to sequential straight phase HPLC using a Zorbax Sil column (0.46 x 25 cm; DuPont Instruments, Wilmington, DE) at a flow rate of 2.0 ml/min and equilibrated first in isopropanol-hexane (11:89) and then in isopropanol-dichloromethane (5:95) before quantitation by RRA. HPLC using these procedures can distinguish 1,25-(OH)2D from other metabolites of vitamin D (31). Paired values of 1,25-(OH)2D (determined without or with HPLC) did not differ significantly from one another. The protein concentration of the mitochondrial suspension was measured by a modification of the method of Smith et al. (32) (BCA Protein Assay, Pierce Chemical Co., Rockford, IL). The activity of 1{alpha}-hydroxylase in each sample was expressed as picograms of 1,25-(OH)2D produced per mg mitochondrial protein, and for each kidney the values from duplicate incubations were averaged.

Renal 1{alpha}- and 24-hydroxylase mRNA expression
cDNA fragments corresponding to nucleotides 176–626 of mouse P450c1{alpha} cDNA (6) and nucleotides 1262–1637 of mouse P450c24 cDNA (33) were prepared by RT-PCR of mouse kidney RNA, subcloned, and sequenced. A linearized TRIPLEscript plasmid containing a 250-bp mouse ß-actin cDNA fragment was used as a control template. Antisense RNA probes for P450c1{alpha}, P450c24, and ß-actin were prepared by transcription of subcloned cDNA fragments using either T7 or T3 RNA polymerases (Maxiscript protocol, Ambion, Inc., Austin, TX) and {alpha}-32P-labeled UTP (800 Ci/mmol; NEN Life Science Products, Boston, MA). The predicted sizes of the ribonuclease-protected fragments were: P450c1{alpha}, 493 bp; P450c24, 376 bp; and ß-actin, 250 bp.

The ribonuclease protection assay was performed as we described previously (34) using the HybSpeed RPA assay kit (Ambion, Inc.). Total RNA (20 µg), isolated from kidneys using the TRIzol reagent (Life Technologies, Inc./BRL, Gaithersburg, MD), was hybridized with the appropriate riboprobes (5 x 105 cpm) at 68 C for 10 min, and treated with ribonuclease A (5 U/ml) and T1 (200 U/ml) at 37 C for 30 min. The remaining protected RNA fragments were precipitated, denatured, and resolved on a denaturing 5% acrylamide/8 M urea gel. The gel was dried and exposed to a PhosphorImager screen (Molecular Dynamics, Inc., Sunnyvale, CA) for quantitation. Results are expressed as the ratio of P450c1{alpha} or P450c24 mRNA to ß-actin mRNA.

1{alpha}-Hydroxylase antibody
A human P450c1{alpha} cDNA fragment suitable for cloning in a bacterial expression vector was generated by PCR amplification of our cloned full-length P450c1{alpha} cDNA (1), using the sense primer 5'-CGGATCCATGACCCAGACCCTCAAG-3' (start codon underlined) and the antisense primer 5'-GGAATTCTATCTGTCCAAAAACTGTAGGTT-3' (stop codon underlined), thus deleting the 5'- and 3'-untranslated regions. Truncated P450c1{alpha} cDNA fragments also were generated using the same antisense primer and either the sense primer N-13 (5'-TCGGATCCGCTGGGCGCCCGAGTTG-3'), which deletes the first 13 N-terminal amino acids of the expressed protein, or primer N-33 (5'-CCGGATCCTGCAGACATCCCAGGC-3'), which deletes the first 33 N-terminal amino acids. The sense primers include a BamHI site, and the antisense primer includes an EcoRI site for cloning. The cDNA fragments were cloned into the bacterial expression vector, pLW01, that uses the strong T7 promoter and is replicated in high copy number in Escherichia coli strains that carry the T7 polymerase gene (35). The plasmids were transformed into E. coli BL21 (DE3)-pLysS (Novagen, Madison, WI), which also carries the lysogen plasmid to repress leaky expression before induction of T7 polymerase. Single colonies of transformed E. coli were inoculated into 5 ml L-broth containing 100 µg/ml ampicillin and 34 µg/ml chloramphenicol, grown overnight, and used to inoculate 150 ml broth (36). Isopropyl-ß-D-thiogalactoside (1 mM) was added at OD 0.8–1.0 to induce expression, and the culture was harvested by centrifugation 4 h later. The bacteria were resuspended in 5 ml 10 mM Tris, 1 mM EDTA, and 50 mM KCl at pH 7.5, sonicated, and centrifuged at 10,000 x g for 20 min. The pellet was resuspended in SDS gel sample buffer, analyzed by electrophoresis on a SDS-10% polyacrylamide gel, and stained with Coomassie Blue. The stained protein band was excised from the gel, emulsified, and used to inject two rabbits (Lampire Biological Laboratories, Pipersville, PA).

Western immunoblotting
The 1{alpha}-hydroxylase antibody was characterized by immunoblotting, using mitochondrial protein from mouse kidney and human adrenocortical carcinoma NCI-H295A cells (37), and total cellular protein from primary cultures of human keratinocytes, which express abundant 1{alpha}-hydroxylase activity (1). Proteins (10 µg/lane) were separated using SDS-8% PAGE and electroblotted onto Immobilon-P membranes (Millipore Corp.) in buffer containing 25 mM Tris, 192 mM glycine (pH 8.3), and 20% methanol. Membranes were blocked in PBS containing 0.1% Tween 20 (Sigma, St. Louis, MO) and 5% nonfat dry milk at 4 C overnight, washed, incubated with rabbit antisera to human P450c1{alpha} at a 1:20,000 dilution at room temperature for 1 h in the same buffer, washed again, and then incubated with goat antirabbit IgG conjugated with horseradish peroxidase (Promega Corp.) at a 1:5,000 dilution at room temperature for 1 h. The antigen-antibody complexes were detected by enhanced chemiluminescence (SuperSignal West Dura Extended Duration Substrate, Pierce Chemical Co.). In control experiments, primary antibody was omitted.

Immunohistochemistry
Animals were anesthetized, and the kidneys were perfused via the abdominal aorta with 4% paraformaldehyde. Immunohistochemistry was performed using rabbit antisera to human P450c1{alpha} (described above). Paraffin-embedded sections of mouse kidney (4 µm thick) were dewaxed in xylene and decreasing concentrations of alcohol, incubated with blocking solution (Roche, Indianapolis, IN), and incubated with antiserum diluted 1:1000 in 1x blocking solution at room temperature overnight. After washing three times in PBS, slides were incubated with using a fluorescein isothiocyanate-labeled antirabbit IgG antibody (1:200; Sigma) at room temperature for 60 min. Visualization was performed by indirect immunofluorescence. Similar experiments were performed using sheep antisera directed against peptides 266–289 of mouse P450c1{alpha} (38) as the primary antibody, and phycoerythrin-labeled antigoat/sheep IgG as the secondary antibody (1:200). For control sections, the primary antibody was omitted. To facilitate localizing specific regions of the nephron, we also used a mouse antihuman antibody to Tamm-Horsfall protein (The Binding Site, Inc., San Diego, CA), which is specific for the thick ascending limb of Henle’s loop, and sheep antihuman 11ß-hydroxysteroid dehydrogenase type 2 antibody, which is specific for cortical and medullary collecting ducts (39).

Isolation of nuclei and nuclear run-on assay
Nuclei were isolated from kidneys as previously described (40). Mice were fed either a control or a low (0.02%) phosphorus diet for 12, 24, and 48 h, and the kidneys were removed and homogenized using a Teflon/glass homogenizer in buffer containing 20 mM tricine, 2 mM CaCl2, 1 mM MgCl2, 0.25 M sucrose, and 2 mM dithiothreitol. Nuclei were pelleted by centrifugation at 1500 rpm for 5 min at 4 C; rehomogenized in the same buffer and centrifuged; washed and centrifuged twice with the above buffer plus 0.1% Triton; resuspended in storage buffer of 50 mM Tris-HCl, 40% glycerol, 5 mM MgCl2, and 0.1 mM EDTA; and frozen at -70 C.

Run-on transcription assays were performed as previously described (41). Briefly, 20 x 106 nuclei were incubated in reaction buffer containing 5 mM Tris-HCl, 2.5 mM MgCl2, 150 mM KCl, 2.5 mM dithiothreitol, and 10 U ribonuclease inhibitor (Life Technologies, Inc.) with 0.5 mM ATP, 0.25 mM CTP, 0.25 mM GTP (Roche ), and 200 µCi [{alpha}-32P]UTP (NEN Life Science Products) at 37 C for 30 min. The nuclei were then successively digested with ribonuclease-free deoxyribonuclease I and proteinase K. RNA was extracted with phenol/chloroform/isoamyl, and unincorporated nucleotides were removed using a Centricon 100 concentrator (Amicon, Beverly, MA). Equal amounts of radioactive elongated RNA (1–3 x 106 dpm) were hybridized to 5 µg linearized denatured plasmids containing cDNA inserts for P450c1{alpha}, P450c24, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), or control plasmid without insert that had been spotted onto nylon filters at 42 C for 4 d. The filters were washed under high stringency conditions and exposed to a phosphorimager screen for 3 d.

Statistical analysis
Data are reported as the mean ± SEM. Statistical comparison of mean values between control and low phosphorus diets was performed using the unpaired t test; P < 0.05 was taken to be statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Renal 1{alpha}-hydroxylase activity
In mice fed a vitamin D-replete control diet, production of 1,25-(OH)2D by isolated renal mitochondria was time dependent and reached a maximum after approximately 30 min, in agreement with findings obtained in rat renal mitochondria (30). Subsequent determinations of enzyme activity were made after 15 min of incubation. In animals treated with 1,25-(OH)2D3 (1.5 ng/g) 18 h before death, 1{alpha}-hydroxylase activity was suppressed to 42 ± 8% of control values; enzyme activity was not detectable in mitochondria that had been boiled for 2 min before incubation.

To determine the time course of response of renal mitochondrial 1{alpha}-hydroxylase activity to dietary phosphorus restriction, groups of mice were fed either control (1.0%) or low (0.02%) phosphorus diet for 1, 2, 4, and 8 d. A separate control group was studied concurrently with each of the four low phosphorus groups. After 1 d of phosphorus restriction, 1{alpha}-hydroxylase activity was not significantly different from that in mice fed the control diet (Fig. 1AGo). After 2 d of phosphorus restriction, 1{alpha}-hydroxylase activity was significantly increased (P < 0.05), and increased further with 4 and 8 d of phosphorus restriction (Fig. 1AGo). Maximal activity was attained after 8 d, at which time 1,25-(OH)2D production was approximately 7-fold higher than that in mice fed the control diet (Fig. 1AGo). Enzyme activity in different groups of mice fed the control diet varied slightly but not significantly over the time interval examined.



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  		Figure 1. Effect of dietary phosphorus on renal mitochondrial 1{alpha}hydroxylase activity. Mice were anesthetized, the kidneys were removed, and renal mitochondria were prepared and incubated with 25OHD to measure 1{alpha}-hydroxylase activity. A, Time course of response of renal 1{alpha}-hydroxylase activity to dietary phosphorus restriction. Mice were fed the control (1.0% phosphorus) diet ({square}) or the low phosphorus (0.02%) diet () for 1, 2, 4, and 8 d. Data are the mean ± SEM from four mice per group; for each time point, values were compared using unpaired t test. *, P < 0.05; #, P < 0.01. B, Effect of degree of phosphorus restriction on renal 1{alpha}-hydroxylase activity. Mice were fed a control (1.0% phosphorus) diet ({square}) or diets containing 0.02%, 0.2%, or 0.6% phosphorus (), each for 4 d. Data are the mean ± SEM from four mice per group; for each pair of diets, values were compared using unpaired t test. #, P < 0.01.

 
To determine the degree of phosphorus restriction necessary to stimulate 1{alpha}-hydroxylase activity, groups of mice were fed a diet containing 0.02%, 0.2%, 0.6%, or 1.0% (control) phosphorus for 4 d. A separate control group was studied concurrently with each of the three low phosphorus groups. Both severe (0.02%) and moderate (0.2%) phosphorus restriction induced 6- and 8-fold increases, respectively, in 1{alpha}-hydroxylase activity, whereas the 0.6% phosphorus diet had no effect on enzyme activity when compared with the control diet (Fig. 1BGo). When expressed as a percentage of the mean value in animals fed the control diet, enzyme activity with the 0.02% and 0.2% phosphorus diets did not differ significantly from one another (567 ± 111% and 796 ± 91%, respectively).

Eadie-Hofstee analysis of the effect of substrate concentration on mitochondrial enzyme activity yielded an apparent Michaelis-Menten constant (Km) of 5.9 x 10-7 M (Fig. 2Go). This value is comparable to that reported in chick and pig kidney, human skin (42, 43, 44), and mouse Leydig tumor MA-10 cells transfected with the cloned full-length human 1{alpha}-hydroxylase cDNA (1). With dietary phosphorus restriction, the observed increase in 1{alpha}-hydroxylase activity could be attributed to a substantial increase in the maximum velocity (Vmax) of the enzyme, from 22 to 157 fmol/mg protein·15 min for control and low (0.02%) phosphorus, respectively (Fig. 2Go). No significant change in the Km of the enzyme was observed. As the 0.2% phosphorus diet induced an increase in 1{alpha}-hydroxylase activity comparable to that induced by the 0.02% diet, it seems likely that the effect on enzyme kinetics would be the same with either diet.



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  		Figure 2. Effect of 25OHD concentration on renal mitochondrial 1{alpha}-hydroxylase activity. Renal mitochondria were prepared and incubated with the appropriate concentration of 25OHD for 15 min. Shown is an Eadie-Hofstee plot of data from a typical experiment in normal mice fed the control (1.0% phosphorus) diet ({bullet}; Vmax = 22 fmol/mg·15 min; Km = 6.0 x 10-7 M) or the low phosphorus (0.02%) diet ({circ}; Vmax = 157 fmol/mg·15 min; Km = 9.2 x 10-7 M) for 8 d.

 
Phosphorus restriction induced a significant decrease in serum phosphorus concentration that was detectable after 1 d of restriction and was further decreased after 2–8 d of restriction (Table 1Go). Both severe (0.02%) and moderate (0.2%) degrees of phosphorus restriction induced a significant decrease in serum phosphorus concentration, but the 0.6% phosphorus diet did not, compared with the control diet (Table 2Go).


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  		Table 1. Temporal effect of dietary phosphorus on serum phosphorus concentration

 

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  		Table 2. Effect of degree of phosphorus restriction on serum phosphorus concentration

 
1{alpha}-Hydroxylase and 24-hydroxylase mRNA expression
Severe phosphorus restriction (0.02%) induced a substantial increase in renal P450c1{alpha} mRNA abundance. After 2 d on the low phosphorus diet, P450c1{alpha} mRNA abundance was approximately 4-fold higher than that in animals on the control diet, and after 4 and 8 d of restriction, it was approximately 8-fold higher (Fig. 3AGo); no increase was detected after 1 d of phosphorus restriction. Moderate phosphorus restriction (0.2%) for 4 d also induced a substantial (>8-fold) increase in renal P450c1{alpha} mRNA abundance (Fig. 3BGo). However, with the 0.6% phosphorus diet, P450c1{alpha} mRNA expression did not change significantly from that on the control diet.



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  		Figure 3. Effect of dietary phosphorus on renal abundance of P450c1{alpha} mRNA. Mice were anesthetized, the kidneys were removed, and renal total RNA was prepared to estimate the abundance of P450c1{alpha} mRNA relative to ß-actin mRNA. A, Time course of response of renal P450c1{alpha} mRNA to dietary phosphorus restriction. Mice were fed the control (1.0% phosphorus) diet ({square}) or the low phosphorus (0.02%) diet () for 1, 2, 4, or 8 d. Data are the mean ± SEM from four mice per group; for each time point, values were compared using unpaired t test and are expressed as a percentage of the mean value for mice fed the control diet. *, P < 0.05; #, P < 0.01. B, Effect of degree of phosphorus restriction on renal P450c1{alpha} mRNA. Mice were fed the control diet ({square}) or diets containing 0.02%, 0.2%, or 0.6% phosphorus (), each for 4 d. Data are the mean ± SEM from four mice per group; for each pair of diets, values were compared using unpaired t test and are expressed as a percentage of the mean value for mice fed the control diet. *, P < 0.05; #, P < 0.01.

 
We examined the relationship between renal 1{alpha}-hydroxylase activity and P450c1{alpha} mRNA expression in animals fed either the control or 0.02% phosphorus diet for 1, 2, 4, or 8 d. Values of mitochondrial 1{alpha}-hydroxylase activity varied directly and significantly with those of P450c1{alpha} mRNA abundance (r = 0.81; P < 0.001; Fig. 4AGo). Values of P450c1{alpha} mRNA abundance varied inversely and significantly with serum phosphorus concentrations (r = -0.59; P < 0.001; Fig. 4BGo), although at lower phosphorus concentrations, the variation observed in P450c1{alpha} mRNA abundance was considerable. Values of renal 1{alpha}-hydroxylase activity also varied inversely with serum phosphorus concentration (r = -0.64; P < 0.001). Similarly, when all data for animals fed each dietary intake of phosphorus (1.0%, 0.6%, 0.2%, and 0.02% phosphorus) for 4 d were analyzed as a single set, we found a direct relationship between 1{alpha}-hydroxylase activity and P450c1{alpha} mRNA abundance (r = 0.79; P < 0.001) and an inverse relationship between P450c1{alpha} mRNA abundance and serum phosphorus concentration (r = -0.72; P < 0.001; data not shown).



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  		Figure 4. A, Relationship between renal mitochondrial 1{alpha}-hydroxylase activity and P450c1{alpha} mRNA abundance in mice fed the control (1.0% phosphorus; {circ}) or the low (0.02%) phosphorus ({bullet}) diet for 1, 2, 4, or 8 d (r = 0.81; P < 0.001). B, Relationship between renal P450c1{alpha} mRNA abundance and serum phosphorus concentration in mice fed the control (1.0% phosphorus; {circ}) or the low (0.02%; {bullet}) phosphorus diet for 1, 2, 4, or 8 d (r = -0.59; P < 0.001). A and B, Each point represents data from a single animal; for each duration of the low phosphorus diet, values are expressed as a percentage of the mean value for mice concurrently fed the control diet.

 
To determine the effect of dietary phosphorus on renal expression of P450c24, the enzyme responsible for renal catabolism of 1,25-(OH)2D, we estimated P450c24 mRNA abundance in mice fed control and low phosphorus diets. As expected (29), phosphorus restriction induced a substantial decrease in renal P450c24 mRNA abundance, the values decreasing by more than 70% compared with those in mice fed the control diet, at 2, 4, and 8 d of restriction (Fig. 5AGo). Similarly, moderate phosphorus restriction (0.2%) for 4 d also induced a 70% decrease in P450c24 mRNA expression. With the 0.6% phosphorus diet, P450c24 mRNA abundance decreased by 35% compared with values on the control diet (Fig. 5BGo).



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  		Figure 5. Effect of dietary phosphorus on renal abundance of P450c24 mRNA. Mice were anesthetized, the kidneys were removed, and renal total RNA was prepared to estimate the abundance of P450c24 mRNA relative to ß-actin mRNA. A, Time course of response of renal P450c24 mRNA abundance to dietary phosphorus restriction. Mice were fed the control (1.0% phosphorus) diet ({square}) or the low phosphorus (0.02%) diet () for 1, 2, 4, or 8 d. Data are the mean ± SEM from four mice per group; for each time point, values were compared using unpaired t test and are expressed as a percentage of the mean value for mice fed the control diet. *, P < 0.05. B, Effect of degree of phosphorus restriction on renal P450c24 mRNA abundance. Mice were fed the control diet ({square}) or diets containing 0.02%, 0.2%, or 0.6% phosphorus (), each for 4 d. Data are the mean ± SEM from four mice per group; for each pair of diets, values were compared using unpaired t test and are expressed as a percentage of the mean value for mice fed the control diet. #, P < 0.01.

 
Validation of antiserum to P450c1{alpha}
Expression of full-length human P450c1{alpha} in E. coli failed to produce readily detectable quantities of protein. Because the N-terminal membrane-anchoring sequence can attenuate bacterial expression of P450 proteins (45), we constructed vectors for N-13 and N-33 P450c1{alpha}; only the N-33 form was abundantly expressed (Fig. 6AGo). The band of bacterially expressed N-33 P450c1{alpha} protein was injected into rabbits to raise an antiserum. To determine the specificity of the P450c1{alpha} antibody, immunoblotting was performed using mitochondrial protein prepared from mouse kidney and adrenal NCI-H295A cells and total cellular protein from primary cultures of human keratinocytes. With mouse renal mitochondria and human keratinocytes, a single P450c1{alpha} band was detected (Fig. 6BGo). P450c1{alpha} protein abundance was greatly suppressed in renal mitochondria from mice injected with 1,25-(OH)2D3 compared with that in mice fed the control diet. No bands were detected using mitochondrial protein prepared from adrenal NCI-H295A cells (Fig. 6BGo).



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  		Figure 6. Preparation of antiserum to P450c1{alpha}. A, Expression of N-33 P450c1{alpha} in E. coli, shown on a 12% polyacrylamide gel stained with Coomassie Blue. Relatively little P450c1{alpha} is produced without isopropyl-ß-D-thiogalactoside induction (lane 4); with induction for 1 h (lanes 2 and 5) and 4 h (lane 3), most of the N-33 P450c1{alpha} is found in cytosol (supernatant; lanes 2 and 3) rather than in inclusion bodies (pellet; lane 5). B, Western immunoblotting with the antiserum to human N-33 P450c1{alpha}. Protein (10 µg) samples are from kidneys of mice fed the control (1% phosphorus) diet (CP) or the low (0.02%) phosphorus (LP) diet for 4 d, from kidneys of mice treated with 1,25-(OH)2D3 (1.5 ng/g) for 18 h (1 25 ), from human keratinocytes (KC) grown in low calcium medium, and from human adrenal NCI-H293A cells (NCI).

 
Immunohistochemistry
Immunohistochemical analysis of 1{alpha}-hydroxylase protein expression in kidney from mice fed the control diet, revealed weak staining of the proximal convoluted tubule (PCT), proximal straight tubule (PST), epithelial cells of Bowman’s capsule, thick ascending limb of Henle’s loop, distal convoluted tubule, and cortical collecting duct (Fig. 7Go, A and C). We also observed staining in these nephron segments when we used a sheep antibody (38) raised against peptides 266–289 of the mouse 1{alpha}-hydroxylase sequence, confirming the findings of Zehnder et al. (38) (data not shown). In mice fed the low phosphorus diet for 8 d, strongly increased immunoreactivity was observed in the PCT, PST, and epithelial cells of Bowman’s capsule; however, no increase in immunoreactivity was observed in the distal tubule or the collecting duct (Fig. 7Go, B and D). No staining was observed when the primary antibody was omitted under either dietary condition (data not shown).



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  		Figure 7. Localization of P450c1{alpha} protein by immunohistochemistry in paraffin sections of renal cortex (upper panels) and outer medulla (lower panels) from mice fed either the control (1.0%) or low (0.02%) phosphorus diet for 7 d. On the control diet (A and C), P450c1{alpha} protein is detected (positive staining green) in the renal cortex, including the PCT, epithelial cells of Bowman’s capsule (BC), and distal tubule (DT). With the low phosphorus diet (B and D), much stronger P450c1{alpha} expression is observed in PCT, BC, and PST, whereas no upregulation is seen in the DT or collecting duct (CD). The glomeruli (G) and vessels are negative. No staining was observed when the primary antibody was omitted under either dietary condition (data not shown).

 
Nuclear run-on transcription assay
To determine whether the increase in P450c1{alpha} mRNA expression induced by low phosphorus diet was due to transcriptional or posttranscriptional events, we performed nuclear run-on assays using nuclei from mice fed either control or low phosphorus diets for 12, 24, and 48 h. With phosphorus restriction, the level of P450c1{alpha} gene transcription, relative to that of GAPDH transcription, was increased to about 200% of control values, as early as 12 h after phosphorus restriction and remained increased after 24 and 48 h (Fig. 8Go). By contrast, with phosphorus restriction the level of P450c24 gene transcription, relative to that of GAPDH, did not change significantly (85 ± 3% of control values). In mice injected with 1,25-(OH)2D3 2 h before they were killed, P450c24 transcription increased to 4000% of control values.



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  		Figure 8. Effect of phosphorus restriction on P450c1{alpha} gene transcription. Nuclear run-on transcription assay was performed using nuclei from mice fed the control (1.0% phosphorus) diet ({square}) or the low phosphorus (0.02%) diet () for 12, 24, or 48 h. The intensity of P450c1{alpha} gene transcription was related to that of GAPDH transcription and determined by phosphorimager analysis. Two or three independent experiments were performed at each time point.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Restriction of dietary phosphorus increases the renal production of 1,25-(OH)2D (14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26). In the present study we demonstrate that 1{alpha}-hydroxylase activity, measured in isolated renal mitochondria from mice fed control and low phosphorus diets, increased within 2 d of phosphorus restriction, and increased further at 4 and 8 d of restriction to values approximately 9-fold higher than those in mice fed the control diet. Both severe (0.02%) and moderate (0.2%) degrees of phosphorus restriction significantly stimulated 1{alpha}-hydroxylase activity, whereas the 0.6% phosphorus diet did not, compared with the control (1%) diet. The increase in mitochondrial 1{alpha}-hydroxylase activity could be attributed to a substantial increase in Vmax of the enzyme, as no change in its Km was detected.

We found that the increase in renal 1{alpha}-hydroxylase activity induced by phosphorus restriction was associated with a corresponding increase in the abundance of renal P450c1{alpha} mRNA. Indeed, both the time course and magnitude of increase in P450c1{alpha} mRNA were similar to those of 1{alpha}hydroxylase activity. In animals fed either control or low phosphorus diet for 4 d, we found that values of renal 1{alpha}-hydroxylase activity varied directly and significantly with those of P450c1{alpha} mRNA. In addition, values of both renal 1{alpha}-hydroxylase activity and P450c1{alpha} mRNA varied inversely and significantly with serum phosphorus concentrations, although at lower phosphorus concentrations, the variation observed in P450c1{alpha} mRNA abundance was considerable. Thus, the present findings demonstrate that the increase in renal production and serum concentration of 1,25-(OH)2D induced by restricting dietary phosphorus is due to an increase in renal P450c1{alpha} mRNA, as recently reported in the phosphorus-restricted rat (46).

In prior studies that employed microdissected nephron segments from fetal rabbits (47) or from vitamin D-deficient rats or chicks (48, 49), 1{alpha}-hydroxylase activity was observed only in the proximal nephron. In the present study, however, immunohistochemical analysis of mouse kidney, using two different 1{alpha}-hydroxylase antibodies, revealed staining not only of the PCT and PST, but also of epithelial cells of Bowman’s capsule, the distal tubule, and the cortical collecting duct, confirming the findings in human kidney (38). Furthermore, in mice fed the low phosphorus diet for 8 d, a strong increase in immunoreactivity was observed in the PCT, PST, and epithelial cells of Bowman’s capsule, with no increase apparent in the distal tubule or collecting duct. These findings in intact mice demonstrate that 1{alpha}-hydroxylase protein is expressed in both the proximal and distal nephron under conditions of a normal phosphorus (and calcium) diet, and that phosphorus restriction up-regulates 1{alpha}-hydroxylase protein expression only in the proximal convoluted and proximal straight portion of the nephron. Thus, the proximal tubule appears to be the nephron segment responsible for increased production and circulating levels of 1,25-(OH)2D observed when enzyme activity is stimulated, as by phosphorus restriction. Our findings are consistent with prior observations in which 1{alpha}-hydroxylase activity was exclusively localized to the proximal nephron in vitamin D-deficient or fetal animals (47, 48, 49), conditions in which enzyme is predicted to be greatly stimulated.

We found that restriction of dietary phosphorus induced a significant reduction in renal mRNA abundance of P450c24, the enzyme responsible for renal catabolism of 1,25-(OH)2D; the decrease was detectable after only 1 d phosphorus restriction, with maximal suppression observed at 4 and 8 d. These findings are in agreement with those of Brown et al. (29), who observed a decrease in renal P450c24 mRNA and a corresponding decrease in renal 24-hydroxylase activity with phosphorus restriction in the rat. Our data suggest that renal expression of P450c24 responds more quickly to phosphorus restriction than does P450c1{alpha}b; the reason for this, however, is unknown. Thus, the present data demonstrate that dietary and serum phosphorus regulate renal expression of the P450c1{alpha} and P450c24 genes in a reciprocal manner. These findings raise the question of whether the increase in serum concentration of 1,25-(OH)2D induced by phosphorus restriction reflects not only an increase in its renal production but also, possibly preceded by, a decrease in its renal catabolism. However, the relative contribution of renal catabolism of 1,25-(OH)2D to its MCR in the intact organism is unknown. It is possible that with phosphorus restriction, a decrease in 24-hydroxylase activity in the kidney is offset by an increase in its activity in extrarenal tissue, the latter induced by 1,25-(OH)2D (33, 50), which results in little net change in the metabolic clearance of 1,25-(OH)2D in the intact organism, as we observed in phosphorus-restricted normal men (24).

Using nuclear run-on assays, we demonstrate that dietary phosphorus restriction increases transcription of the P450c1{alpha} gene. Approximately a 2-fold increase in transcriptional activity was observed after 12, 24, and 48 h of phosphorus restriction. Thus, we conclude that transcriptional activation of the P450c1{alpha} gene accounts at least in part for the increase in P450c1{alpha} mRNA expression observed in phosphorusrestricted mice. Whatever the mediating cellular and molecular mechanisms, the stimulation of P450c1{alpha} gene expression by phosphorus restriction is abolished by hypophysectomy and restored by administration of GH or IGF-I, indicating that an intact GH/IGF-I axis is required for the effect (46, 51, 52). The extent to which posttranscriptional mechanisms contribute to regulation of P450c1{alpha} gene expression by dietary phosphorus remains to be determined. Dietary phosphorus can regulate other factors posttranscriptionally; the increase in renal expression of the sodium phosphate cotransporter gene, Npt2, induced by a low phosphorus diet appears to occur by both posttranscriptional (53) and transcriptional ( 54) mechanisms, and the decrease in PTH mRNA expression in parathyroid glands induced by low phosphorus diet occurs by decreased PTH mRNA stability that results from the binding of cytoplasmic proteins to the 3'-untranslated region of the PTH transcript (55). We found that transcription of the P450c24 gene was decreased slightly, but not significantly, by phosphorus restriction and, by contrast, was greatly stimulated by injection of 1,25-(OH)2D, confirming the findings of Roy et al. (56).

In summary, we demonstrate in normal mice that the increase in renal synthesis of 1,25-(OH)2D induced by restricting dietary phosphorus reflects an increase in renal P450c1{alpha} mRNA expression and protein abundance. Such increases in P450c1{alpha} gene expression are due at least in part to increased transcription of the P450c1{alpha} gene, which occurs exclusively in the proximal renal tubule.


   Acknowledgments
 
We thank Dr. Martin Hewison for gifts of the 11ß-hydroxysteroid dehydrogenase type 2 antibody and sheep antimouse 1{alpha}-hydroxylase antibody.


   Footnotes
 
This work was supported by NIH Grants DK-54433 (to A.A.P.) and DK-37922 and DK-42154 (to W.L.M.), the Medical Research Council of Canada (GR-13297, to H.S.T.), and gifts from the David Carmel Trust (to A.A.P.).

Abbreviations: 1,25-(OH)2D, 1,25-dihydroxyvitamin D; 25OHD, 25-hydroxyvitamin D; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Km, Michaelis-Menten constant; PCT, proximal convoluted tubule; PST, proximal straight tubule; Vmax, maximum velocity.

Received May 22, 2001.

Accepted for publication October 15, 2001.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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