1 Geriatric Research, Education, and Clinical Center, St. Louis Veterans Administration Medical Center, St. Louis 63125; and 2 Division of Geriatric Medicine and 3 Department of Biochemistry and Molecular Biology, St. Louis University Health Sciences Center, St. Louis, Missouri 63104
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ABSTRACT |
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The capacity of
parathyroid hormone (PTH) to stimulate renal 1,25-dihydroxyvitamin
D3 [1,25(OH)2D3] production
declines with age in the rat. The purpose of these studies was to
determine whether this decline is due to a decreased capacity of PTH to increase the mRNA levels of CYP1, the cytochrome P-450
component of the 25(OH)D3-1
-hydroxylase. Young (2 mo)
and adult (12 mo) male Fischer 344 rats were parathyroidectomized
(PTX). After 72 h, PTX rats were injected with PTH or vehicle at
24, 6, and 3 h before death, and renal CYP1
mRNA levels were
measured by ribonuclease protection assay. In young rats, PTH markedly
increased plasma 1,25(OH)2D3 and renal
1,25(OH)2D3 production. However, in adult rats,
the response to PTH was less than 30% of that seen in young rats.
Renal CYP1
mRNA levels, on the other hand, were increased over
fivefold by PTH in both young and adult rats. In in vitro studies,
PTH/forskolin increased CYP1
mRNA levels over twofold in renal
slices from both young and adult PTX rats. These studies demonstrate
that the decreased capacity of PTH to increase
1,25(OH)2D3 production in adult rats is not due
to decreased induction of CYP1
mRNA.
parathyroid hormone; cytochrome P-450; calcitriol
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INTRODUCTION |
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PARATHYROID HORMONE
(PTH) is one of the major regulators of the conversion of
25-hydroxyvitamin D3 [25(OH)D3] to
1,25-dihydroxyvitamin D3
[1,25(OH)2D3], the major biologically active
form of vitamin D, in the kidney. In young animals, a variety of
experiments have shown that PTH markedly stimulates
1,25(OH)2D3 production (5, 10).
PTH may accomplish this, in part, by increasing the expression of the
cytochrome P-450 component of the
25(OH)D3-1-hydroxylase enzyme complex (CYP1
). PTH has
been shown to significantly increase the mRNA levels of CYP1
in
intact animals (13) and renal cell lines (8).
With maturation and aging, the capacity of PTH to stimulate renal 1,25(OH)2D3 production declines. In the rat, the capacity of the kidney to make 1,25(OH)2D3 and increase plasma 1,25(OH)2D3 in response to PTH declines with age (6, 9). This has also been seen in human clinical studies where the capacity of PTH to increase plasma 1,25(OH)2D3 decreases with age (11, 16).
The decreased capacity of PTH to increase renal 1,25(OH)2D3 production in adults is also seen indirectly with regard to dietary calcium adaptation. Adult rats do not adapt to feeding on a low-calcium diet by increasing renal 1,25(OH)2D3 production and plasma 1,25(OH)2D3 levels as do young rats (4). This lack of adaptation occurs despite the fact that plasma PTH levels are markedly elevated by the low-calcium diet in both age groups.
The purpose of these studies was to determine whether the age-related
decline in PTH-stimulated 1,25(OH)2D3
production is due to decreased induction of renal CYP1 mRNA.
Decreased induction of CYP1
mRNA by PTH could also account for the
decreased CYP1
mRNA levels seen in adult rats on a low-calcium diet
(4). To that end, we also compared the response of CYP1
mRNA levels to a low-calcium diet with the response to PTH in both age groups.
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MATERIALS AND METHODS |
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Experiments were performed using male Fischer 344 rats that were 2-3 mo (young) and 10-12 mo (adult) of age. Rats were obtained from Harlan Industries (Indianapolis, IN) and were fed a semisynthetic diet containing 1.2% calcium, 0.8% phosphorus, and 3.3 IU/g of vitamin D3 (Purina Rodent Chow, Ralston-Purina, St. Louis, MO). Rats were parathyroidectomized (PTX) under pentobarbital sodium anesthesia. Parathyroid glands were identified and removed and/or cauterized under a dissecting microscope. On the third day after surgery, PTH was administered to half the animals and the other half received vehicle only. rPTH(1-34) (3 µg/100 g body wt) or vehicle (1 mM acetic acid, pH 4.0, 1.6% glycerol, 0.25% phenol) was injected subcutaneously at 24, 6, and 3 h before death. At death, kidneys were removed for isolation of RNA or for preparation of renal slices. Blood was collected for the measurement of plasma 1,25(OH)2D3 and PTH.
Renal slices for in vitro incubation were prepared as previously
described (7). Briefly, thin cortical slices were prepared using a Stadie-Riggs microtome and incubated in plastic vials containing Krebs-Ringer bicarbonate buffer (pH 7.4) at 37°C. The stoppered vials were gassed at 1-h intervals with 95%
O2-5% CO2. Slices were incubated with PTH and
forskolin or vehicle for the indicated length of time, and total RNA
was then isolated from the slices for determination of CYP1 mRNA levels.
In some experiments, renal slices were used to measure renal 1,25(OH)2D3 production, as described previously (6). Briefly, slices were incubated with 5 µM 3H-labeled 25(OH)D3. After 1 h, the 1,25(OH)2D3 product produced was quantitated by radioligand assay following partial purification by Sep-Paks. 1,25(OH)2D3 production was expressed as picomoles per minute per gram of slice weight.
CYP1 mRNA levels were measured by ribonuclease protection assay
(RPA) as previously described (4). Total RNA was isolated by RNAgents (Promega, Madison, WI). The RPA was performed using the
RPAII kit from Ambion (Austin, TX). The actin probe was the
-actin
antisense control template from Ambion. Bands were quantitated by
scanning densitometry, and CYP1
mRNA levels were normalized to
either actin mRNA or total RNA.
Plasma 1,25(OH)2D3 and PTH were measured using commercial kits (Nichols Institute Diagnostics, San Juan Capistrano, CA).
Data are reported as means ± SE for each treatment group. Statistical significance was determined by Student's t-test, and a confidence level greater than 95% was considered significant.
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RESULTS |
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In initial studies, basal parameters related to
1,25(OH)2D3 production were measured in non-PTX
young and adult rats (Fig. 1). Plasma
1,25(OH)2D3 levels and renal
1,25(OH)2D3 production were decreased by more
than 50% in adult rats compared with young rats. However, there was no
difference in renal CYP1 mRNA levels between young and adult rats.
In addition, there was no difference in plasma PTH levels. These
results suggested that the adult rats did not respond to plasma PTH to
the same degree as the young animals in terms of
1,25(OH)2D3 production.
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The effect of PTH on 1,25(OH)2D3 production was
studied directly using young and adult PTX rats (Fig.
2A). In
young animals, PTH markedly increased plasma
1,25(OH)2D3 levels over threefold. In adult
rats, PTH increased plasma 1,25(OH)2D3 levels
only slightly, and the levels attained were much less than those seen
in the young. A parallel pattern was seen with regard to renal
1,25(OH)2D3 production. PTH markedly increased
renal 1,25(OH)2D3 production in young rats, but
it only marginally increased it in adult rats.
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To determine whether the differences in the action of PTH were due to
differences in CYP1 expression, CYP1
mRNA levels were measured
(Fig. 2B). PTH markedly increased CYP1
mRNA levels in both young and adult rats. There was no difference in the magnitude of
the stimulation. In three experiments, the average stimulation by PTH
in the adult kidney was 94.8 ± 6.4% that in the young kidney (100%). This was not statistically different than 100%
(P > 0.46, t-test).
We showed previously that feeding a low-calcium diet also markedly
increases CYP1 mRNA levels in young animals (4). This presumably happens in response to the high levels of plasma PTH that
occur in response to the calcium deprivation. Therefore, it was of
interest to compare the magnitude of the CYP1
mRNA levels in
response to PTH (Fig. 2B) with the response to a low-calcium diet (4). RPA was used to compare CYP1
mRNA levels in
RNA pools from both sets of experiments (Fig.
3). In young rats, the CYP1
mRNA
levels induced by the low-calcium diet were much higher than those
induced by PTH. This was also seen in the adult animals, although to a
lesser degree.
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Finally, the effect of PTH and forskolin on CYP1 mRNA levels was
studied in vitro. Renal slices from young and adult PTX animals were
incubated in the presence and absence of PTH/forskolin for 8 h.
Forskolin was used along with PTH since this combination has been shown
to give the greatest sustained increase in renal slice
1,25(OH)2D3 production (3).
PTH/forskolin significantly increased CYP1
mRNA levels to the same
levels in slices from both young and adult rats (Fig.
4). This is of interest in light of
previous studies of 1,25(OH)2D3 production in
renal slices. In similar experiments, PTH/forskolin has been shown to
increase renal 1,25(OH)2D3 production in slices
from young rats but not in slices from adult rats (7).
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DISCUSSION |
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These studies provide evidence that the decreased renal
1,25(OH)2D3 production in the adult rat in
response to PTH is not due to decreased levels of CYP1 mRNA. In the
intact animal, the CYP1
mRNA levels in the adult kidney are the same
as those in the young (Fig. 1). However, plasma
1,25(OH)2D3 and renal
1,25(OH)2D3 production are decreased in the
adult animals (Fig. 1). In the direct studies of PTH action in PTX
animals, PTH increases CYP1
mRNA to the same levels in both young
and adult animals (Fig. 2B). However, there is a decreased
response to PTH in the adult animal in terms of plasma
1,25(OH)2D3 and renal
1,25(OH)2D3 production (Fig. 2A).
Finally, in isolated renal slices, PTH/forskolin significantly increases CYP1
mRNA to the same level in slices from both young and
adult animals (Fig. 4). However, in similar studies reported previously, PTH/forskolin significantly increased
1,25(OH)2D3 production only in renal slices
from young animals (7).
There are a number of possible explanations for decreased renal
1,25(OH)2D3 production despite normal CYP1
mRNA levels in adult rats. First, it may be that there is decreased
translation of CYP1
mRNA into CYP1
protein in adult animals. We
previously saw this in studying the effect of
1,25(OH)2D3 on the expression of calbindin in
the intestine of young and adult rats (1). We found that
1,25(OH)2D3 increases calbindin mRNA to similar levels in both young and adult rats. However, levels of calbindin protein are significantly lower in the adult rat intestine in response
to 1,25(OH)2D3.
Second, it may be that there is oxidative damage to the CYP1 protein
in the adult animal such that its activity is diminished. The CYP1
protein is located on the inner mitochondrial membrane and is,
therefore, particularly vulnerable to oxidative damage. Mitochondria
are a major source of free radicals, and mitochondrial free radical
production increases with age (15). Mitochondrial aconitase has been shown to accumulate oxidative damage with age (17).
A third possibility is that decreased renal production of 1,25(OH)2D3 may be due to decreased availability of the 25(OH)D3 substrate. Decreased substrate has been shown to contribute to the age-related decline seen in other steroidogenic systems. These include decreased cholesterol availability for the production of adrenal steroids by adrenal cells (14) and for the production of testosterone by Leydig cells (12). In our studies in the kidney, decreased renal 1,25(OH)2D3 production is seen even when renal slices are incubated in high concentrations of 25(OH)D3 (Fig. 4). Thus any decreased availability of 25(OH)D3 in older rats would be due to decreased transport of 25(OH)D3 into the kidney itself.
With regard to mechanisms, PTH and forskolin increase renal CYP1
mRNA levels via a cAMP-dependent mechanism. It has previously been
shown that the capacity of PTH to increase renal cAMP levels and
stimulate protein kinase A activity does not change with age (2,
9). Because the cAMP/protein kinase A signal transduction pathways are intact, it is not surprising that the effect of
PTH/forskolin on CYP1
mRNA levels does not change with age.
In previous studies, we showed that the capacity of a low-calcium diet
to increase renal CYP1 mRNA levels is markedly decreased in adult
rats (4). This occurs despite similar increases in plasma
PTH levels in response to the diet. One possible explanation for this
is that the adult kidney is refractory to the action of PTH in terms of
increasing renal CYP1
mRNA levels. However, the present studies show
that the adult kidney is not refractory to PTH in this regard (Fig.
2B).
When one compares the response of young animals to PTH and to a
low-calcium diet, the response to the diet is much greater (Fig. 3).
This is true despite the fact that injection of PTH increases plasma
PTH to levels higher than those seen with a low-calcium diet
(unpublished observations). Thus it would seem that it is the chronic
elevation of PTH over several weeks that markedly increases CYP1
mRNA levels in the young. In adult animals, although the acute effects
of PTH are similar to those seen in the young, the chronic effects of
PTH are different. It may be that there are age differences in the
chronic regulation of CYP1
mRNA levels by PTH and other regulatory factors.
In summary, PTH increases renal CYP1 mRNA levels to the same levels
in both young and adult animals. This is true whether PTH is injected
into the intact animal or PTH is incubated with isolated renal slices.
This suggests that the decreased renal 1,25(OH)2D3 production seen in the adult rat is
due to age-related changes distal to the elevation of CYP1
mRNA
levels. The decreased adaptation of adult rats to a low-calcium diet in
terms of CYP1
is not due to decreased responsiveness to short-term
administration of PTH. It may reflect age differences in the regulation
of CYP1
expression by other factors.
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ACKNOWLEDGEMENTS |
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This work was supported by the St. Louis Geriatric Research, Education, and Clinical Center and the Medical Research Service of the Department of Veterans Affairs.
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FOOTNOTES |
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Address for reprint requests and other correspondence: H. J. Armbrecht, Geriatric Center (11G-JB), St. Louis VA Medical Center, St. Louis, MO 63125 (E-mail: hjarmbrec{at}aol.com).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
10.1152/ajprenal.00306.2002
Received 27 August 2002; accepted in final form 17 January 2003.
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