PTH increases renal 25(OH)D 3-1 -hydroxylase (CYP1 ) mrna but not renal 1,25(OH) 2 D 3 production in adult rats

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1 Am J Physiol Renal Physiol 284: F1032 F1036, 2003; /ajprenal PTH increases renal 25(OH)D 3-1 -hydroxylase (CYP1 ) mrna but not renal 1,25(OH) 2 D 3 production in adult rats H. J. Armbrecht, 1,2,3 M. A. Boltz, 1,2 and T. L. Hodam 1,2 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 Submitted 27 August 2002; accepted in final form 17 January 2003 Armbrecht, H. J., M. A. Boltz, and T. L. Hodam. PTH increases renal 25(OH)D 3-1 -hydroxylase (CYP1 ) mrna but not renal 1,25(OH) 2 D 3 production in adult rats. Am J Physiol Renal Physiol 284: F1032 F1036, 2003; / ajprenal The capacity of parathyroid hormone (PTH) to stimulate renal 1,25-dihydroxyvitamin D 3 [1,25- (OH) 2 D 3 ] 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)D 3-1 -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) 2 D 3 and renal 1,25(OH) 2 D 3 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) 2 D 3 production in adult rats is not due to decreased induction of CYP1 mrna. parathyroid hormone; cytochrome P-450; calcitriol PARATHYROID HORMONE (PTH) is one of the major regulators of the conversion of 25-hydroxyvitamin D 3 [25(OH)D 3 ] to 1,25-dihydroxyvitamin D 3 [1,25(OH) 2 D 3 ], 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) 2 D 3 production (5, 10). PTH may accomplish this, in part, by increasing the expression of the cytochrome P-450 component of the 25(OH)D 3-1 -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) 2 D 3 production declines. In the rat, the capacity of the kidney to make 1,25(OH) 2 D 3 and increase plasma 1,25(OH) 2 D 3 in response to PTH declines with age (6, 9). This has also been seen in Address for reprint requests and other correspondence: H. J. Armbrecht, Geriatric Center (11G-JB), St. Louis VA Medical Center, St. Louis, MO ( hjarmbrec@aol.com). F1032 human clinical studies where the capacity of PTH to increase plasma 1,25(OH) 2 D 3 decreases with age (11, 16). The decreased capacity of PTH to increase renal 1,25(OH) 2 D 3 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) 2 D 3 production and plasma 1,25(OH) 2 D 3 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) 2 D 3 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 lowcalcium 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. MATERIALS AND METHODS Experiments were performed using male Fischer 344 rats that were 2 3 mo (young) and 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 D 3 (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 g/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) 2 D 3 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 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.

2 F1033 Plasma 1,25(OH) 2D 3 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. Fig. 1. Basal parameters in young and adult nonparathyroidectomized (PTX) rats. Plasma and renal parameters related to 1,25- dihydroxyvitamin D 3 [1,25(OH) 2D 3] production (1,25D Prod.) were measured in non-ptx rats aged 2 3 mo (young) and mo (adult). Plasma 1,25(OH) 2D 3 and parathyroid hormone (PTH) were measured using commercial kits. Renal 1,25(OH) 2D 3 production was measured using isolated renal slices. Renal 25(OH)D 3-1 -hydroxylase enzyme complex P-450 (CYP1 ) 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 CYP mrna (9 animals). *Statistically different from young (P 0.05, t-test). with 95% O 2-5% CO 2. 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) 2D 3 production, as described previously (6). Briefly, slices were incubated with 5 M 3 H-labeled 25(OH)D 3. After 1 h, the 1,25(OH) 2D 3 product produced was quantitated by radioligand assay following partial purification by Sep-Paks. 1,25(OH) 2D 3 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. 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) 2D 3 was measured by radioligand binding assay, and renal 1,25(OH) 2D 3 production was measured using renal slices. B: renal CYP1 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). a Significantly different from control of same age group. b Significantly different from young of same treatment group.

3 F1034 RESULTS In initial studies, basal parameters related to 1,25(OH) 2 D 3 production were measured in non-ptx young and adult rats (Fig. 1). Plasma 1,25(OH) 2 D 3 levels and renal 1,25(OH) 2 D 3 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) 2 D 3 production. The effect of PTH on 1,25(OH) 2 D 3 production was studied directly using young and adult PTX rats (Fig. 2A). In young animals, PTH markedly increased plasma 1,25(OH) 2 D 3 levels over threefold. In adult rats, PTH increased plasma 1,25(OH) 2 D 3 levels only slightly, and the levels attained were much less than those seen in the young. A parallel pattern was seen Fig. 4. Effect of PTH on CYP1 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 M) or vehicle only (control) for 8 h. CYP1 mrna levels were quantitated by RPA and are expressed as percent of maximum response. A: bars are means SE of 8 slice incubations. a Significantly 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. 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 CYP1 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. a Significantly different from young of same treatment group (P 0.05, t-test). with regard to renal 1,25(OH) 2 D 3 production. PTH markedly increased renal 1,25(OH) 2 D 3 production in young rats, but it only marginally increased it in adult rats. 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 % 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. 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) 2 D 3 production (3). PTH/forsko-

4 F1035 lin 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) 2 D 3 production in renal slices. In similar experiments, PTH/forskolin has been shown to increase renal 1,25(OH) 2 D 3 production in slices from young rats but not in slices from adult rats (7). DISCUSSION These studies provide evidence that the decreased renal 1,25(OH) 2 D 3 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) 2 D 3 and renal 1,25(OH) 2 D 3 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) 2 D 3 and renal 1,25(OH) 2 D 3 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) 2 D 3 production only in renal slices from young animals (7). There are a number of possible explanations for decreased renal 1,25(OH) 2 D 3 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) 2 D 3 on the expression of calbindin in the intestine of young and adult rats (1). We found that 1,25(OH) 2 D 3 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) 2 D 3. 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) 2 D 3 may be due to decreased availability of the 25(OH)D 3 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) 2 D 3 production is seen even when renal slices are incubated in high concentrations of 25(OH)D 3 (Fig. 4). Thus any decreased availability of 25(OH)D 3 in older rats would be due to decreased transport of 25(OH)D 3 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) 2 D 3 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. 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. 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. 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