Endocrinology Vol. 142, No. 7 3223-3230
Copyright © 2001 by The Endocrine Society
Hydroxylases Involved in Vitamin D Metabolism Are Differentially Expressed in Murine Embryonic Kidney: Application of Whole Mount in Situ Hybridization1
Masayo Yamagata,
Akihito Kimoto,
Toshimi Michigami,
Masahiro Nakayama and
Keiichi Ozono
Departments of Environmental Medicine (M.Y., T.M., K.O.) and
Pathology (A.K., M.N.), Osaka Medical Center and Research Institute for
Maternal and Child Health, 840 Murodo-cho, Izumi, Osaka 594-1101,
Japan
Address all correspondence and requests for reprints to: Dr. Keiichi Ozono, Department of Environmental Medicine, Osaka Medical Center and Research Institute for Maternal and Child Health, 840 Murodo-cho, Izumi city, Osaka 594-1101, Japan. E-mail: j61642{at}center.osaka-u.ac.jp
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Abstract
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In this study we examined the expression of 25-hydroxyvitamin
D-1
-hydroxylase (1-hydroxylase) and 25-hydroxyvitamin
D-24-hydroxylase (24-hydroxylase) by RT-PCR and whole mount in
situ hybridization using organ culture of kidney taken from
mouse embryo. First, the kidneys of mouse embryo at 11.5–17.5 days
gestation were cultured in the presence or absence of forskolin and
1,25-dihydroxyvitamin D3
[1,25-(OH)2D3]. Forskolin and
1,25-(OH)2D3 induced the expression of
1-hydroxylase and 24-hydroxylase, respectively, in a dose- and
time-dependent manner. In the absence of stimulants, the expression of
1-hydroxylase and 24-hydroxylase was detected from days 13.5–17.5
gestation. The expression of vitamin D receptor and megalin was
detected from days 13.5 and 11.5, respectively. Next, signals for the
expression of either 1-hydroxylase or 24-hydroxylase were detected
by whole mount in situ hybridization in kidney explants
taken from embryo at 15.5 days gestation after the appropriate
stimulation. However, the localization of signals differed between the
two enzymes; 1-hydroxylase messenger RNA was expressed in the inner
area of the kidney explants, whereas 24-hydroxylase messenger RNA was
expressed in the surface area. The expression of both hydroxylases was
restricted to the epithelium of developing renal tubules. The pattern
of megalin expression was similar to that of 1-hydroxylase
expression. To confirm the difference in distribution of
1-hydroxylase and 24-hydroxylase transcripts, the explants were
hybridized with probes for both 1-hydroxylase and 24-hydroxylase
using double labeling techniques after simultaneous stimulation with
forskolin and 1,25-(OH)2D3, resulting in the
detection at different locations of positive signals for the two
enzymes. These results suggest that the expression of 1-hydroxylase
is induced in a distinct epithelium of renal tubules from that of
24-hydroxylase even at the early stage of kidney development before
glomerulogenesis.
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Introduction
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BIOACTIVATION of vitamin D consists of two
steps: 25-hydroxylation of vitamin D in liver and 1-hydroxylation of
25-hydroxyvitamin D (25OHD) in kidney. The latter hydroxylation is
catalyzed by 25-hydroxyvitamin D-1-hydroxylase (1-hydroxylase),
whose complementary DNA (cDNA) and gene have been recently cloned
(1, 2, 3, 4, 5). The product, 1,25-dihydroxyvitamin D
[1,25-(OH)2D], is an active form of vitamin
D and plays an important role in bone and mineral metabolism, and the
effect of 1,25-(OH)2D is mainly mediated by
the intracellular molecule known as the vitamin D receptor (VDR)
(6). However, human genetic diseases involving impaired
function of 1-hydroxylase or VDR and a mouse model generated by
targeting the VDR gene show no apparent bone defects in fetus and
neonates, although osteomalatic bone lesions appear after a weaning
period (7, 8, 9, 10). These facts raise questions about the role
of vitamin D in bone formation and the maintenance of calcium
metabolism in fetus.
On the other hand, the vitamin D deficiency is often encountered
clinically in breast-fed premature babies unless vitamin D, calcium and
phosphorus are supplemented (11, 12). These findings
indicate the importance of vitamin D in the perinatal period to prevent
hypocalcemia and bone deformity. Serum concentrations of vitamin D
metabolites have been investigated in premature infants, and it is
suggested that the synthesis of the active vitamin D in kidney starts
around the 28–30th week of gestation (11, 13). In
utero, the diffusion of 1,25-(OH)2D from
mother to fetus through the placenta is very limited, suggesting that
most of the 1,25-(OH)2D circulating in the
fetus is derived from fetal sources (14, 15). The fetal
kidney and placenta are supposed to be main sites of
1-hydroxylation. In chick embryo,
1,25-(OH)2D was shown to be produced by kidney
on day 17 (16). The synthesis of
1,25-(OH)2D by placenta was shown in an
in vitro study (17), although some studies
reported that the enzymes involved in the synthesis were different from
the 1-hydroxylase identified in kidney (1, 3). In
contrast, the sharp increase in the serum concentration of
1,25-(OH)2D in response to PTH in neonates
just after birth suggests a capability for synthesizing
1,25-(OH)2D in fetal kidney (18). It
is necessary to examine the expression of 1-hydroxylase in
developing kidney to understand the physiological control of vitamin D
metabolism.
25-Hydroxyvitamin D-24-hydroxylase (24-hydroxylase) is another enzyme
involved in the hydroxylation of 25OHD. The product,
24,25-dihydroxyvitamin D [24,25-(OH)2D], is
generally accepted to be an inactive metabolite in vivo,
although specific actions of 24,25-(OH)2D have
been reported (19, 20, 21). A recent study using
24-hydroxylase-ablated mice has shown the physiological role of
24-hydroxylase enzyme in the regulation of
1,25-(OH)2D, another substrate of the enzyme
(22). Although 24-hydroxylase has a broad tissue
distribution, the predominant site of 24-hydroxylase expression is the
renal proximal tubule, as it is for the expression of 1-hydroxylase
(23). However, recent studies showed that the renal distal
tubules also express both hydroxylases with different regulation in the
proximal tubule (24, 25, 26). The site of expression of
1-hydroxylase and 24-hydroxylase remains to be determined in fetal
kidney. As 1,25-(OH)2D is the most potent
inducer of 24-hydroxylase (27), the expression of
1-hydroxylase may be correlated with that of 24-hydroxylase,
especially in terms of the ontogeny of both hydroxylases.
Megalin is an endocytic receptor expressed on the luminal surface of
the renal tubule (28). A crucial role for megalin in
1-hydroxylation at renal tubules has been shown in megalin-knockout
mice (29). It was found that the substrate, 25OHD, is
supplied not from basal membrane, in other words, from blood vessels,
but from luminal membrane to mitochondria in renal tubular cells.
Therefore, it is important to examine the expression of megalin during
the development of kidney when investigating vitamin D metabolism in
the fetus, because dynamic changes in the blood supply and the
production of urine occur in the fetal kidney. However, no study has
been performed to address this issue to date.
In the present study the expression of 1-hydroxylase as well as
24-hydroxylase, VDR, and megalin in mouse developing kidney was
investigated by RT-PCR. In addition, the distribution of
1-hydroxylase transcripts was examined by whole mount in
situ hybridization using organ culture of metanephros taken from
embryos at various stages and compared with that of 24-hydroxylase
transcripts.
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Materials and Methods
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Organ culture of mouse embryonic kidney
Animal protocols were approved by the institutional animal care
and use committee at Osaka Medical Center and Research Institute for
Maternal and Child Health. Five-week-old mice (female ICR, male
BDF1) were supplied by Clea Japan (Tokyo, Japan),
and were kept under pathogen-free conditions. Timed pregnant mice were
killed by cervical dislocation at 11.5–17.5 days gestation, and
metanephric tissue was aseptically obtained from the embryos. The age
of the embryos was determined by the day when a vaginal plug was
detected in the mother, which was designated day 0. At least six
different embryos from different dams were studied at each embryonic
stage. Whole embryonic metanephros were placed on the culture well
inserts (Becton Dickinson and Co., Franklin Lakes, NJ) and
cultured with 500 µl culture medium at 37 C in an atmosphere of 5%
CO2 (30, 31). The culture medium
consisted of DMEM (Nissui Pharmaceutical Co., Tokyo, Japan)
supplemented with 15% FBS (ICN Biochemicals, Inc.,
Irvine, CA), 1% penicillin/streptomycin, pyruvic acid (final
concentration, 0.22 mg/ml), and glutamine (final concentration, 1.8
mg/ml). The medium was replaced every 48 h when the culture was
maintained for a long period.
RNA analysis
To examine the effects of agents on the expression of the
hydroxylases in the kidney explants,
1,25-(OH)2D3 (Wako Co.,
Osaka, Japan) or forskolin (Sigma, St. Louis, MO) was
added to the culture medium. Six hours after the incubation with
forskolin and
1,25-(OH)2D3, unless
otherwise indicated, total RNA was extracted from cultured kidney of
mouse embryo using TRIzol reagent (Life Technologies, Inc., Grand Island, NY). The RNA samples were subjected to
RT-PCR for the expression of 1-hydroxylase, 24-hydroxylase, VDR,
megalin, and ß-actin. Total RNA (2.5 µg) was reverse transcribed
using random hexamers and Superscript II reverse transcriptase
(Life Technologies, Inc.) according to the manufacturer’s
instructions. PCR was performed using the following sets of specific
primers: mouse 1-hydroxylase: sense, 5'-CAAGCAGCCGCGGGCTATGCTGG-3';
antisense, 5'-GGAATTCCCGTGTCCCAGACA-3'; mouse 24-hydroxylase:
sense, 5'-CCAAGCTTCGTGCGCCAAAAGAGGTGC-3'; antisense,
5'-CCCGTGGAGATCATGAAGCTGGA-3'; mouse VDR: sense,
5'-ACATGTGCTGCTCATGGCCATCTG-3'; antisense,
5'-TGTGAGCTTCATGCTGTTCTCC-3'; mouse megalin (28): sense,
5'-CCTTGCCAAACCCTCTGAAAAT-3'; antisense,
5'-CACAAGGTTTGCGGTGTCTTTA-3'; and ß-actin: sense,
5'-GTGGGGCGCCCCAGGCACCA-3'; antisense, 5'-CTCCTTAATGTCACGCACGATTTC-3'.
Each reaction mixture (25 µl) contained the following: 4.0 µl of
the RT reaction products, except for ß-actin (1.0 µl) and megalin
(2.0 µl), 10 mM Tris-HCl (pH 9.0), 50 mM KCl,
1.5 mM MgCl2, 0.1% Triton X-100,
deoxy-NTP mix (0.2 mM each), the pair of primers (0.5
µM each), and 0.5 U recombinant Taq DNA
polymerase (Promega Corp., Madison, WI). The thermocycling
protocol comprised 25, 30, and 35 cycles for ß-actin, megalin, and
the others, respectively, of a denaturation step at 94 C for 0.5 min,
followed by 55 C for 1 min for annealing and 72 C for 1 min for
extension. The PCR products were then separated on 1.5–2.5% agarose
gels containing ethidium bromide and visualized under an UV
illuminator. Amplification of the expected fragments was confirmed by
sequencing using an automated sequencer (model 377, Perkin-Elmer Corp., Norwalk, CT).
Preparation of RNA probes for 1-hydroxylase, 24-hydroxylase, and
megalin
Whole coding sequences of cDNA for mouse 1-hydroxylase and
24-hydroxylase (GenBank accession no. AB006034 and D49438,
respectively) were obtained by PCR amplification. The PCR reaction
using primers for megalin described above was performed to obtain the
partial coding sequence of cDNA for mouse megalin (GenBank accession
no. C80829). Total RNA extracted from the kidney of a vitamin
D-deficient mouse (provided by Dr. N. Tsugawa, Kobe Pharmaceutical
University, Kobe, Japan) served as a template for the synthesis
of cDNA of the mouse 1-hydroxylase gene. To obtain the cDNA of the
mouse 24-hydroxylase gene, total RNA extracted from a mouse
osteoblastic cell line, MC3T3-E1, 24 h after the addition of
1,25-(OH)2D3 was used as
a template. The amplified fragments were subcloned into pT7 vector
(Novagen, Madison, WI) and then recloned into pGEM3zf(+) vector
(Promega Corp.). The sequences of the fragments were
verified by sequencing using an automated sequencer 377. Digoxigenin
(DIG)-labeled sense and antisense riboprobes for in situ
hybridization were prepared with SP6 and T7 RNA polymerases according
to the manufacturer’s protocol after linearization by digestion with
the appropriate restriction enzymes (digoxigenin RNA labeling kit,
Roche Molecular Biochemicals, Indianapolis, IN).
Whole kidney mount in situ hybridization
Whole kidney mount in situ hybridization was
performed according to a previous report with some modifications
(32). Entire kidney explants taken from mouse embryo at
15.5 days gestation were fixed with 4% paraformaldehyde for 24 h
and then dehydrated in a series of increasing methanol concentrations
(25%, 50%, 75%, and 100%). Subsequently, the explants were
rehydrated in PBS and treated with 10 µg/ml proteinase K at 37 C for
30 min. The digestion was terminated by incubation in 2 mg/ml glycine
for 10 min. Then, the explants were fixed again with 4%
paraformaldehyde and 0.2% glutaraldehyde for 20 min and incubated in
prehybridization solution [50% formamide, 5 x SSC (standard
saline citrate; pH 4.5), 50 µg/ml transfer RNA, 50 µg/ml heparin,
and 1% SDS] at 70 C for 2 h. Finally, the explants were
hybridized with DIG-labeled antisense or sense RNA probes in
hybridization solution [50% formamide, 5 x SSC (pH 4.5), 50
µg/ml transfer RNA, 50 µg/ml heparin, 1% SDS, and 1 µg/ml
DIG-labeled RNA probe] at 70 C overnight. After the hybridization, the
explants were washed and incubated in the blocking buffer [10% sheep
serum, 0.1% Tween-20, and 2 mM levamizol in 20
mM Tris-buffered saline (pH 7.5)] at room
temperature for 2.5 h. Then, the explants were reacted with the
anti-DIG antibody-coupled alkaline phosphatase at 4 C overnight, and
the probed molecules were detected using NBT/BCIP for the anti-DIG
antibody-coupled alkaline phosphatase (Roche Molecular Biochemicals).
For the double labeling (33), probes for 1-hydroxylase
and 24-hydroxylase were labeled using the DIG RNA labeling kit and the
fluorescein RNA labeling kit, respectively, according to the
manufacturer’s protocol (Roche Molecular Biochemicals).
The fluorescein isothiocyanate (FITC)-labeled probes were purified
using a Sephadex G-50 column (Amersham Pharmacia Biotech,
Piscataway, NJ). After prehybridization as described above, the
explants were incubated in the prehybridization buffer for 2 h and
then hybridized with hybridization buffer containing 1 µg/ml of both
probes at 70 C overnight. After the hybridization, FITC-labeled
molecules were detected using Fast Red tablets (Roche Molecular Biochemicals) for staining of the anti-FITC antibody-coupled
alkaline phosphatase (Roche Molecular Biochemicals).
Subsequently, the explants were refixed with 4% paraformaldehyde at 4
C for 24 h and washed with PBS three times. Then, alkaline
phosphatase conjugated with FITC-labeled antibody was inactivated in
PBS containing 0.1% Tween-20 at 65 C for 1 h. The explants were
incubated in the blocking buffer at room temperature for 2.5 h,
and then treated with the anti-DIG antibody-coupled alkaline
phosphatase at 4 C overnight. Finally, DIG-labeled molecules were
detected using 5-bromo-4-chloro-3-indolyl-phosphate as substrate for
alkaline phosphatase.
Histological examination
Explants were fixed in 10% buffered formalin, embedded in
paraffin, and cut in 5-µm-thick sections. Slides were stained with
hematoxylin and eosin (HE) and periodic acid-Schiff for light
microscopic analysis (model BX50, Olympus Corp., Tokyo,
Japan). Some of the explants subjected to whole mount in
situ hybridization were also fixed in 10% buffered formalin and
processed as described above for HE staining.
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Results
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Analysis of 1-hydroxylase and 24-hydroxylase expression in mice
embryonic kidney by RT-PCR
The expression of 1-hydroxylase and 24-hydroxylase during the
development of mouse embryonic kidneys was investigated by RT-PCR
following the extraction of RNA from metanephric explants. Consistent
with other reports (30, 31), explants could be cultured
longer than 5 days in this system and differentiated to some extent, as
suggested by the finding of glomerulogenesis. Thus, the period of
incubation with the agent was unlikely to be a factor in the
deterioration of kidney in terms of the maintenance of phenotype.
Therefore, using this system, the dose response and time course of the
response to stimulatory agents were investigated in kidney explants at
15.5 days gestation (Fig. 1
). The
addition of forskolin and
1,25-(OH)2D3 markedly
increased the expression of 1-hydroxylase and 24-hydroxylase,
respectively, in a time- and dose-dependent manner (Fig. 1). Based on
these results, 10-4
M forskolin and
10-8 M
1,25-(OH)2D3, added at
6 h, were selected as agents to examine the expression of
1-hydroxylase and 24-hydroxylase, respectively, in subsequent
experiments.
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Figure 1. Time- and dose-dependent expression of
1-hydroxylase and 24-hydroxylase in mouse embryonic kidney explant
after the stimulation. The expression of 1-hydroxylase (A and B) and
24-hydroxylase (C and D) was analyzed by RT-PCR in the presence or
absence of forskolin and 1,25-(OH)2D, respectively. The
conditions for the RT-PCR were described in Materials and
Methods. A and C, Time-dependent expression of 1-hydroxylase
and 24-hydroxylase after stimulation, respectively. The number
described above each lane indicates the hours of
incubation with 10-4 M forskolin
and 10-8 M
1,25-(OH)2D3 in A and C, respectively. M,
DNA molecular marker. B and D, Dose-dependent induction of
1-hydroxylase and 24-hydroxylase expression by forskolin and
1,25-(OH)2D3, respectively. The number
(except 0) above each lane indicates the logalistic
number of concentration of forskolin and
1,25-(OH)2D3 in A and C, respectively. 0,
Vehicle.
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To determine at which embryonic stage the expression and the regulation
of these hydroxylase transcripts begin, the expression of
1-hydroxylase and 24-hydroxylase was also investigated by RT-PCR in
embryonic kidney harvested at 11.5–17.5 days gestation with and
without the addition of the stimulatory agents (Fig. 2). The expression of 1-hydroxylase
and 24-hydroxylase was detected from 13.5 days gestation and enhanced
apparently by the addition of forskolin and
1,25-(OH)2D3,
respectively (Fig. 2, A and B).
RT-PCR analysis of VDR and megalin expression in mouse embryonic
kidney
The expression of VDR and megalin was also investigated by RT-PCR
in embryonic kidney harvested at 11.5–17.5 days gestation (Fig. 2
).
The expression of VDR was observed from day 13.5, which is
consistent with a previous report (Fig. 2C) (34).
The megalin gene was expressed in the kidney as early as day 11.5, and
the expression increased along with gestational age, suggesting that it
started as the tubulogenesis began and increased because of the
increased number of renal tubular cells (Fig. 2D).
Distribution of 1-hydroxylase and 24-hydroxylase messenger RNA
(mRNA) in mouse embryonic kidney examined by whole mount in situ
hybridization
To investigate the distribution of 1-hydroxylase and
24-hydroxylase transcripts, we first tried to establish conditions
under which the addition of
10-4> M
forskolin and 10-8 M
1,25-(OH)2D3 would
induce the expression of both hydroxylases at the same time, because
1,25-(OH)2D3 is known to
suppress the expression of 1-hydroxylase. The simultaneous
expression of both enzymes was detected by RT-PCR in the explants
treated with the two agents for 6 h (Fig. 3). Therefore, these explants were
subjected to whole mount in situ hybridization for either
the 1-hydroxylase or the 24-hydroxylase gene. Positive signals for
1-hydroxylase were detected as a coiled string pattern, like a tail,
in the inner area of kidney explants (Fig. 4A). No signals were detected in
kidney when sense probe was used (Fig. 4B). In addition, no signals
were detected in kidney cultured in the absence of forskolin (data not
shown). Microscopic observation of sections made after the refixation
and paraffin embedding revealed signals in the epithelium of developing
tubules in explants obtained from fetus at 15.5 days gestation (Fig. 5). No signals were observed in immature
glomerulus, including S-shape types, ureteric bud branches, or
mesenchymal cells.
Positive signals for 24-hydroxylase were also detected by whole mount
in situ hybridization. However, the mRNA was expressed in
the outer area, although the signals were also detected as a coiled
string pattern (Fig. 6). The sections
revealed that the expression of 24-hydroxylase was also restricted to
the epithelium of renal tubules (data not shown).
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Figure 6. Whole mount in situ hybridization
for 24-hydroxylase mRNA. Kidney explants obtained from mouse embryo at
15.5 days gestation were incubated with 10-8
M 1,25-(OH)2D and
10-4 M forskolin for 6 h and
hybridized with the antisense (A) or sense (B) probes for
24-hydroxylase mRNA as described in Materials and
Methods. Signals were observed when the explants were
hybridized with the antisense probe, but not with the sense probe.
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Distribution of megalin mRNA similar to that of 1-hydroxylase
mRNA in mouse embryonic kidney examined by whole mount in situ
hybridization
The distribution of megalin transcripts was investigated in mouse
embryonic kidney by whole mount in situ hybridization.
Positive signals for megalin were detected in the kidney with no
addition of stimulatory agents as a coiled string pattern, like a tail,
in the inner area of kidney explants (Fig. 7). The distribution of megalin mRNA was
similar to that of 1-hydroxylase mRNA, as shown in Fig. 4. No
signals were detected in kidney when the sense probe was used (data not
shown).
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Figure 7. Whole mount in situ hybridization
for megalin mRNA. Kidney explants obtained from mouse embryo at 15.5
days gestation were hybridized with the antisense probe for megalin
mRNA. Signals were visualized as described in Materials and
Methods.
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Difference in distribution between 1-hydroxylase and
24-hydroxylase mRNA shown by whole mount in situ hybridization using
double labeling techniques
The difference in distribution of 1-hydroxylase and
24-hydroxylase transcripts was further examined by whole mount in
situ hybridization using double labeling techniques after
simultaneous stimulation with forskolin and
1,25-(OH)2D3. Consistent
with the data for whole mount in situ hybridization reported
above, in which a single probe was separately labeled and hybridized,
the localization of positive signals for both enzymes was distinct; the
signals for 24-hydroxylase were localized in the surface area compared
with those for
1,25-(OH)2D3 (Fig. 8).
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Discussion
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The identification of cDNA of the 1-hydroxylase gene has
allowed us to examine gene expression. In the present study we
performed developmental analysis of the expression of 1-hydroxylase
and compared the results with those for 24-hydroxylase, using organ
culture of mouse embryonic kidney. Tubulogenesis and glomerulogenesis
start at 11.5 and 13.5 days gestation, respectively, in mouse embryonic
kidney (35). This means that tubulogenesis begins before
glomerulogenesis and proceeds to urine production. In our study HE
staining of kidney explants demonstrated that tubulogenesis and
glomerulogenesis were under way on the expected days (data not shown),
indicating that the kidney explant taken from mouse embryo in the
present study is an appropriate model for developmental analysis. In
addition, organ culture of kidney allows investigation of the direct
effects of various factors on the development of fetal kidney.
With respect to the ontogeny of 1-hydroxylase, its expression was
detected by RT-PCR in the metanephros obtained from mouse embryos at
the age of 13.5 days and later (Fig. 2A). In the same period the
expression of 24-hydroxylase was also detected. The addition of
forskolin markedly increased the expression of 1-hydroxylase during
the embryonic stage, suggesting that cAMP is an important messenger
even in the fetal kidney, although the effects of natural inducers,
including PTH and calcitonin, were not examined. A previous study of
the serum concentration of 1,25-(OH)2D in
neonates suggested that the neonatal kidney has the ability to produce
1,25-(OH)2D via 1-hydroxylation of 25OHD as
early as the 28th gestational week, when a substantial number of
nephrons including glomeruli are developed (12). Our data
indicate that the kidney is at least one major site of 1-hydroxylase
expression in the fetus. In addition, the expression of
1-hydroxylase can be induced just after tubulogenesis begins, which
is earlier than previously expected.
The kidney is a unique organ in which transition of mesenchyme to
epithelium (of tubules and glomerulus) occurs via the interaction of
ureteral bud and metanephric mesenchyme (36). In this
sense, metanephric mesenchyme is potentially capable of expressing
genes characteristic to epithelium under certain conditions. To
elucidate the distribution of 1-hydroxylase mRNA, we performed whole
mount in situ hybridization of cultured kidney. This method
is suitable for investigating the expression of genes
three-dimensionally and adopted mainly in studies of fetal development,
because the permeabilization of labeled probe occurs easily in the
fetus under the age of 17.5 days gestation. Thus, we applied this
technique to examine the expression of 1-hydroxylase and
24-hydroxylase. As shown in Fig. 4, a tubular pattern of
1-hydroxylase expression was observed in the kidney explants treated
with forskolin. After fixation of the kidney labeled with probe, the
specimen was histologically examined by making conventional thin
sections. The expression of the hydroxylase was confined to the tubular
epithelium and was not observed in the glomerulus, collecting ducts, or
mesenchymal cells.
Consistent with previous reports, the expression of 24-hydroxylase was
also restricted to renal tubular cells in the present study (24, 37, 38). One of the factors that determines the cell type
expressing 24-hydroxylase mRNA is the existence of VDR in epithelial
cells of renal tubules (31). Using polyclonal antibody
against 24-hydroxylase, the proximal and distal tubules of the kidney
were found to express 24-hydroxylase, although the distal tubule
exhibited more intense signals than the proximal tubule
(24). Immunohistochemical analysis revealed that the
expression of VDR was first identified on day 15 of gestation in mouse
embryonic kidneys (38). Our PCR method is so sensitive
that the expression of VDR was detected on day 13.5 of gestation.
Concerning the expression of 1-hydroxylase, many studies suggested
that renal 1-hydroxylase activity is localized exclusively in the
cells of the proximal tubules. However, analysis of the expression of
1-hydroxylase using in situ hybridization and RT-PCR
revealed that the distal tubules also express 1-hydroxylase,
especially in the vitamin D-replete condition (39). We
found that 1-hydroxylase mRNA was distributed further inside the
kidney than 24-hydroxylase, suggesting that different cells in renal
tubules are responsible for the expression of these hydroxylases. The
difference in the expression sites of 1-hydroxylase and
24-hydroxylase was further examined by whole mount in situ
hybridization using double labeling techniques and was confirmed in
developing kidney after the appropriate stimulation. Unfortunately, we
could not tell whether the proximal or distal tubule is responsible for
the expression of 1-hydroxylase due to the immature nature of
developing kidney.
A recent study using megalin-knockout mice revealed that 25OHD is
reabsorbed from lumen together with vitamin D-binding protein by
tubular epithelium and is hydroxylated at the position of
1-hydroxylase (29). The results suggest that the
production of 1,25-(OH)2D requires urine
production and subsequent reabsorption of 25OHD with vitamin D-binding
protein mediated by megalin located in the luminal surface membrane.
Our finding that the expression of megalin started from 11.5 days
gestation is interesting, because the expression proceeded
glomerulogenesis, which is essential for urine production. In addition,
the expression of megalin preceded that of 1-hydroxylase and
24-hydroxylase in developing kidney. In megalin-knockout mice, a
decrease in the number and size of large endosomes in tubular
epithelial cells was demonstrated, although gross malformation of the
kidney was not observed (40). Moreover, the distribution
of megalin was similar to that of 1-hydroxylase in the kidney
obtained from mouse embryo at 15.5 days gestation, suggesting that
megalin may function to synthesize 1,25-(OH)2D
in cooperation with 1-hydroxylase even in embryonic kidney. The role
of megalin in the early stages of kidney development is an interesting
issue to be studied.
In conclusion, we investigated the expression of 1-hydroxylase and
24-hydroxylase in mouse embryonic kidney explants by whole mount
in situ hybridization using double labeling techniques and
found different distributions of mRNA of the two enzymes, although the
expression of both hydroxylases was restricted to tubular
epithelium.
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Acknowledgments
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We thank Dr. Y. Matsui, Osaka Medical Center and Research
Institute for Maternal and Child Health, for instruction on whole mount
in situ hybridization. We thank Drs. N. Tsugawa and T.
Okano, Kobe Pharmaceutical University, for providing vitamin
D-deficient mice. We acknowledge Ms. Tomoko Hayashi for secretarial
help.
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Footnotes
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1 This work was supported in part by a grant from Japanese Ministry of
Education (to K.O.). 
Received October 3, 2000.
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Abstract
Introduction
