PTH increases renal 25(OH)D3-1alpha -hydroxylase (CYP1alpha ) mRNA but not renal 1,25(OH)2D3 production in adult rats

H. J. Armbrecht1,2,3, M. A. Boltz1,2, and T. L. Hodam1,2

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


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 CYP1alpha , the cytochrome P-450 component of the 25(OH)D3-1alpha -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 CYP1alpha 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 CYP1alpha 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 CYP1alpha 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 CYP1alpha mRNA.

parathyroid hormone; cytochrome P-450; calcitriol


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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)D3-1alpha -hydroxylase enzyme complex (CYP1alpha ). PTH has been shown to significantly increase the mRNA levels of CYP1alpha 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 CYP1alpha mRNA. Decreased induction of CYP1alpha mRNA by PTH could also account for the decreased CYP1alpha mRNA levels seen in adult rats on a low-calcium diet (4). To that end, we also compared the response of CYP1alpha mRNA levels to a low-calcium diet with the response to PTH in both age groups.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 CYP1alpha 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.

CYP1alpha 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 beta -actin antisense control template from Ambion. Bands were quantitated by scanning densitometry, and CYP1alpha 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.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 CYP1alpha 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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Fig. 1.   Basal parameters in young and adult nonparathyroidectomized (PTX) rats. Plasma and renal parameters related to 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] production (1,25D Prod.) were measured in non-PTX rats aged 2-3 mo (young) and 10-12 mo (adult). Plasma 1,25(OH)2D3 and parathyroid hormone (PTH) were measured using commercial kits. Renal 1,25(OH)2D3 production was measured using isolated renal slices. Renal 25(OH)D3-1alpha -hydroxylase enzyme complex P-450 (CYP1alpha ) mRNA levels were quantitated by ribonuclease protection assay (RPA) relative to actin. Values are expressed relative to young rats (100%). Bars are means ± SE of 4-6 animals except for CYPalpha mRNA (9 animals). * Statistically different from young (P < 0.05, t-test).

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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Fig. 2.   Effect of PTH in young and adult PTX rats. Young (unshaded bars) and adult rats (shaded bars) were PTX and then given PTH or vehicle only (control) subcutaneously. A: plasma 1,25(OH)2D3 was measured by radioligand binding assay, and renal 1,25(OH)2D3 production was measured using renal slices. B: renal CYP1alpha mRNA levels were measured by RPA (C) and are expressed as percentage of maximum. Bars are means ± SE of 3-6 rats, and statistical significance is P < 0.05 (t-test). aSignificantly different from control of same age group. bSignificantly different from young of same treatment group.

To determine whether the differences in the action of PTH were due to differences in CYP1alpha expression, CYP1alpha mRNA levels were measured (Fig. 2B). PTH markedly increased CYP1alpha 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 CYP1alpha 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 CYP1alpha mRNA levels in response to PTH (Fig. 2B) with the response to a low-calcium diet (4). RPA was used to compare CYP1alpha mRNA levels in RNA pools from both sets of experiments (Fig. 3). In young rats, the CYP1alpha 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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Fig. 3.   Comparison of the effect of low dietary calcium and PTH in young and adult rats. Pools were made of renal RNA from the PTH-treated animals (Fig. 2B) and from the low-calcium-diet animals (4). The CYP1alpha mRNA levels in the pools were quantitated by RPA and are expressed as percent of maximum. Bars are means ± SE of 2 RPAs, and shaded bars indicate adult rats. aSignificantly different from young of same treatment group (P < 0.05, t-test).

Finally, the effect of PTH and forskolin on CYP1alpha 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 CYP1alpha 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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Fig. 4.   Effect of PTH on CYP1alpha mRNA levels in renal slices. Slices from PTX young (unshaded bars) and adult rats (shaded bars) were incubated with PTH (100 nM) and forskolin (Fsk; 10 µM) or vehicle only (control) for 8 h. CYP1alpha mRNA levels were quantitated by RPA and are expressed as percent of maximum response. A: bars are means ± SE of 8 slice incubations. aSignificantly different from control of same age group (P < 0.05, t-test). There were no significant differences between age groups. B: representative RPA of 8 individual slices.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 CYP1alpha mRNA. In the intact animal, the CYP1alpha 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 CYP1alpha 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 CYP1alpha 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 CYP1alpha mRNA levels in adult rats. First, it may be that there is decreased translation of CYP1alpha mRNA into CYP1alpha 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 CYP1alpha protein in the adult animal such that its activity is diminished. The CYP1alpha 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 CYP1alpha 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 CYP1alpha mRNA levels does not change with age.

In previous studies, we showed that the capacity of a low-calcium diet to increase renal CYP1alpha 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 CYP1alpha 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 CYP1alpha 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 CYP1alpha mRNA levels by PTH and other regulatory factors.

In summary, PTH increases renal CYP1alpha 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 CYP1alpha mRNA levels. The decreased adaptation of adult rats to a low-calcium diet in terms of CYP1alpha is not due to decreased responsiveness to short-term administration of PTH. It may reflect age differences in the regulation of CYP1alpha expression by other factors.


    ACKNOWLEDGEMENTS

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.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Armbrecht, HJ, Boltz MA, Christakos S, and Bruns ME. Capacity of 1,25-dihydroxyvitamin D to stimulate expression of calbindin D changes with age in the rat. Arch Biochem Biophys 352: 159-164, 1998[ISI][Medline].

2.   Armbrecht, HJ, Boltz MA, and Forte LR. Effect of age on parathyroid hormone and forskolin stimulated adenylate cyclase and protein kinase activity in the renal cortex. Exp Gerontol 21: 515-522, 1986[ISI][Medline].

3.   Armbrecht, HJ, Forte LR, Wongsurawat N, Zenser TV, and Davis BB. Forskolin increases 1,25-dihydroxyvitamin D3 production by rat renal slices in vitro. Endocrinology 114: 644-649, 1984[Abstract].

4.   Armbrecht, HJ, Hodam TL, Boltz MA, and Kumar VB. Capacity of a low calcium diet to induce the renal vitamin D 1alpha -hydroxylase is decreased in adult rats. Biochem Biophys Res Commun 255: 731-734, 1999[ISI][Medline].

5.   Armbrecht, HJ, Nemani RK, and Wongsurawat N. Regulation of calcium metabolism by the vitamin D hydroxylases. Advances Mol Cell Biol 14: 245-267, 1996.

6.   Armbrecht, HJ, Wongsurawat N, and Paschal RE. Effect of age on renal responsiveness to parathyroid hormone and calcitonin in rats. J Endocrinol 114: 173-178, 1987[Abstract].

7.   Armbrecht, HJ, Wongsurawat N, Zenser TV, and Davis BB. Differential effects of parathyroid hormone on the renal 1,25- dihydroxyvitamin D3 and 24,25-dihydroxyvitamin D3 production of young and adult rats. Endocrinology 111: 1339-1344, 1982[Abstract].

8.   Brenza, HL, and DeLuca HF. Regulation of 25-hydroxyvitamin D3 1alpha -hydroxylase gene expression by parathyroid hormone and 1,25-dihydroxyvitamin D3. Arch Biochem Biophys 381: 143-152, 2000[ISI][Medline].

9.   Friedlander, J, Janulis M, Tembe VRHK, Wong MS, and Favus MJ. Loss of parathyroid hormone-stimulated 1,25-dihydroxyvitamin D3 production in aging does not involve protein kinase A or C pathways. J Bone Miner Res 9: 339-345, 1994[ISI][Medline].

10.   Jones, G, Strugnell SA, and DeLuca HF. Current understanding of the molecular actions of vitamin D. Physiol Rev 78: 1193-1231, 1998[Abstract/Free Full Text].

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12.   Liao, C, Reaven E, and Azhar S. Age-related decline in the steroidogenic capacity of isolated Leydig cells: a defect in cholesterol mobilization and processing. J Steroid Biochem Mol Biol 46: 39-47, 1993[ISI][Medline].

13.   Murayama, A, Takeyama KI, Kitanaka S, Kodera Y, Kawaguchi Y, Hosoya T, and Kato S. Positive and negative regulations of the renal 25-hydroxyvitamin D3 1alpha -hydroxylase gene by parathyroid hormone, calcitonin, and 1alpha ,25(OH)2D3 in intact animals. Endocrinology 140: 2224-2231, 1999[Abstract/Free Full Text].

14.   Popplewell, PY, and Azhar S. Effects of aging on cholesterol content and cholesterol-metabolizing enzymes in the rat adrenal gland. Endocrinology 121: 64-73, 1987[Abstract].

15.   Sohal, RS, and Sohal BH. Hydrogen peroxide release by mitochondria increases during aging. Mech Ageing Dev 57: 187-202, 1991[ISI][Medline].

16.   Tsai, KS, Heath H, Kumar R, and Riggs BL. Impaired vitamin D metabolism with aging in women. Possible role in pathogenesis of senile osteoporosis. J Clin Invest 73: 1668-1672, 1984[ISI][Medline].

17.   Yan, LJ, Levine RL, and Sohal RS. Oxidative damage during aging targets mitochondrial aconitase. Proc Natl Acad Sci USA 94: 11168-11172, 1997[Abstract/Free Full Text].


Am J Physiol Renal Fluid Electrolyte Physiol 284(5):F1032-F1036




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