Deficiency of Vitamin 25-Hydroxyvitamin John
D Metabolites Directly Stimulates Renal D,-1-Hydroxylase Activity in Rats
Fox, Uwe Kollenkirchen,
and Marian
R. Walters
Renal 2bhydroxyvitamin D,-I-hydroxylase (I-hydroxylase) enzyme activity in rats is known to be increased by parathyroid hormone (PTH), hypophosphatemia, and hypocalcemia. Thus, enzyme activity is markedly increased in vitamin D-deficient states, but whether this stimulation is a direct response to the vitamin D deficiency or only occurs following the associated chan9es in plasma calcium, phosphate, or PTH is unclear. We tested whether vitamin D deficiency per se influences I-hydroxylase activity in renal cortical slices using a normocalcemic rat model of vitamin D deficiency. Weanling male rats were fed one of the following three diets: (A) 0.8% Ca, 0.5% P, 2.2 IU vitamin D,/g; or vitamin D-deficient diets containing, (B) 0.8% Ca, 0.5% P; and (C) 2.0% Ca, 1.25% P, 20% lactose. Vitamin D-deficient rats fed diet B were hypocalcemic with elevated PTH at both test periods, and I-hydroxylase activity was increased more than IOO-fold compared with rats fed diet A. Plasma calcium, phosphate, and PTH levels were the same in groups A and C, but I-hydroxylase activity was also substantially elevated in 9roup C versus group A rats (104- and 17-fold increases after IO and 19 diet weeks, respectively). These data lead to the important conclusion that severe deficiency of vitamin D metabolites per se provides a strong and independent stimulus to renal I-hydroxylase activity in rats, perhaps due to the absence of 1,25(0H),D,-mediated enzyme inhibition. Copyright 0 1997 by W.B. Saunders Compeny
T
HE RENAL 25hydroxyvitamin D,-1-hydroxylase (lhydroxylase) enzyme catalyzes the conversion of 25 hydroxyvitamin D, [25(OH)D,] to the hormonally active metabolite, 1,25_dihydroxyvitamin D, [1,25(OH),D,].‘” Parathyroid hormone (PTH) is the major stimulator of l-hydroxylase activity>’ although hypophosphatemia and hypocalcemia have also been shown to stimulate the enzyme directly.“9 1,25(OH),D, can inhibit the 1-hydroxylase by a mechanism thought to involve a receptor-mediated process and, in a pattern of coordinate regulation, the hormone also increases the activity of the alternate enzyme, the 25(OH)D,24-hydroxylase.1-3 Importantly, the most potent stimulation of I-hydroxylase activity occurs under conditions of vitamin D deficiency.“3,‘0 Establishing whether this stimulation is a direct response to the deficiency of vitamin D metabolites or only occurs following the usually associated changes in plasma calcium, phosphate, and PTH levels is an important step in understanding the factors that regulate l-hydroxylase activity in normal and abnormal states. The availability of a well-characterized normocalcemic model of vitamin D deficiency in rats” allowed us to test the hypothesis that deficiency of vitamin D per se stimulates the enzyme in vivo. MATERIALS
AND METHODS
Animals
Normal, weanling, 21-day-old, male CD rats were obtained from Charles Riva cabs (Wilmington, MA). Upon receipt, they were randomly divided into four groups and housed in hanging wire cages under
incandescent
light. They were fed one of three diets:
From the Depatiment of Physiology, Tulane University School of Medicine, New Orleans, LA. Supported by a grant-in-aid from the American Heart Association, by USPHS-NIH Grant No. DK31847 (MR. W), and by a grant-in-aid from the American Heart Association-Louisiana (J.F.). Address reprint requests to John Fox, PhD, Department of Physiology, Tulane University School of Medicine, 1430 Tulane Ave, New Orleans, LA 701 I2. Copyright 0 1991 by KB. Saunders Company 00260495191/4004-0017$03.00/0
438
(A) 0.8% Ca, 0.5% P, 2.2 IU vitamin D,/g; (B) a vitamin D-deficient diet containing 0.8% Ca, 0.5% P; and (C) a vitamin D-deficient diet containing 2.0% Ca, 1.25% P, and 20% lactose, substituted for sucrose (Teklad, Madison, WI). After 10 and 19 diet weeks, five rats from each dietary group were anesthetized with pentobarbital sodium (60 mg/kg, intraperitoneally) and exsanguinated by cardiac puncture. Plasma was collected for the analyses described below. Both kidneys were removed and placed in ice-coid isotonic saline. Measurement
of Renal I-Hydroxylase Enzyme Activity
The kidneys were decapsulated and cortical slices (0.5 mm) were prepared using a tissue slicer (Stoelting, Wood Dale, IL). The slices (- 100 mg) were gassed with OJCO, (95/5) and incubated as described elsewhere.* The reaction was initiated by adding 1 ug 25(OH)D, in 10 FL ethanol [initial 25(OH)D, concentration, 2.5 umol/L]. Each incubation was regassed after 30 minutes and was terminated after 1 hour by adding 1 mL acetonitrile. Then, 1,000 dpm 1,25(OH)#H]D, (160 Ciimmol; Amersham, Arlington Heights, IL) was added to monitor recovery through the extraction and purification procedures. The slices and medium were homogenized in acetonitrile:water (50:50) and centrifuged. The generated 1,25(OH),D, was extracted from the supernatant and purified and assayed as described for plasma 1,25(OH),D, below. Renal l-hydroxylase enzyme activity was expressed as picograms 1,25(OH),D, produced per milligram cortex per hour.”
Plasma Analyses Before assay, vitamin D, metabolites were extracted and purified using established methods with minor modifications.“~” 25(OH)[‘H]D, (90 Ciimmol) and 1,25(OH),[‘H]D, (800 dpm) were added to each sample to monitor recovery through the purification. Vitamin D, metabolites were extracted from 0.5 to 1.0 mL plasma with an equal volume of acetonitrile. Following centrifugation, the supernatant was combined with 0.5 ~010.4 mol/L K*HPO,, pH 10.6, and applied to a C,,-OH Bond-Elm column (Analytichem, Harbor City, CA). The column was washed with water, 5 mL methanolwater (70/30), and 5 mL hexane. 25(OH)D, was eluted with 5 mL hexane-dichloromethane (90/10). The C,,-OH column was then washed with 8 mL hexane-dichloromethane-isopropanol (89/10/l) to remove 24,25(OH),D, and followed by 2.4 mL hexaneisopropanol (95/5) to elute 1,25(OH),D,. Before assay, the 25(OH)D, and 1,25(OH),D, fractions were further purified on a
Metabolism,
Vol40, No 4 (April), 1991: pp 438-441
STIMULATED
l-HYDROXYLASE
ACTIVITY
BY VITAMIN
439
D DEFICIENCY
Statistical comparisons were performed on a microcomputer using a software package (Stata; Computing Resource Center, Los Angeles, CA).
silica Bond-Elut column. The 25(OH)D, fraction was applied to the silica column and washed with 8 mL hexane-dichloromethaneisopropanol (89/10/l). 25(0H)D, was eluted with 10 mL hexaneisopropanol (95/5). The 1,25(OH),D, fraction was applied to the silica column, washed with 5 mL hexane-isopropanol (94/6), and eluted with 9 mL hexane-isopropanol (86/14). The final eluants were evaporated to dryness in a vacuum centrifuge and redissolved in ethanol for assay. Recoveries of 25(OH)D, and 1,25(OH),D3 averaged 70% to 80%. 25(OH)D, was quantitated by radioimmunoassay’4.15using sheep antiserum 02282.16The detection limit was approximately 1.5 to 2.0 pg/tube (y 6 to 8 pg/mL for 1 mL plasma). 1,25(OH),D, levels were quantitated by radioreceptor assay using a calf thymus receptor prepared using an established method.” The assay detection limit was 0.4 pg/tube ( _ 1 to 2 pg/mL for 1 mL plasma). Plasma amino-terminal PTH levels were measured with a homologous rat PTH(l-34) radioimmunoassay with goat antiserum G813-PTH, described previously.‘4.‘5The assay detection limit was 6 pg rat PTH(l-34)lmL. Plasma calcium levels were measured using methylthymol blue,” and plasma phosphate by the method of Chen et al.”
RESULTS
1 -Hydroxylase Activity In these animals, both time- and diet-dependent changes in 1-hydroxylase enzyme activity occurred. 1-Hydroxylase activity decreased significantly (P < .05) between 10 and 19 diet weeks in all dietary groups (Fig 1). Nevertheless, the hypocalcemic vitamin D-depleted rats with high PTH levels (diet B, documented below) exhibited substantially higher 1-hydroxylase activity after 10 and 19 diet weeks (109- and 140-fold elevation versus vitamin D-replete controls, respectively) (Fig 1). Enzyme activity was similarly elevated (104-fold) in normocalcemic, normal PTH rats (diet C) after 10 diet weeks. Although the degree of stimulation was somewhat reduced by 19 diet weeks, 1-hydroxylase activity remained significantly (P < .Ol) higher in these rats than in the vitamin D-replete controls (Fig 1).
StatisticalAnalysis All data are presented as mean ? SEM. The significance of differences between groups were determined by ANOVA and Tukey’s test. In the case of unequal variances (determined by Bartlett’s test), the data was log-transformed before ANOVA.
A.
Plasma Parameters Rats fed the 0.8% Ca, 0.5% P, vitamin D-deficient diet (diet B) were markedly hypocalcemic compared with the
10 weeks **
6.
19 weeks
**
--I!-
** +
**
IO
F +
+l
1
:
c T
-
Diet A
Diet B
-
Diet C
o.v---
Diet A
-
Diet B
Diet C
1-Hydroxylase enzyme activity in renal cortical slices from rats fed varying calcium and phosphorus diets for 10 and 19 weeks. Diet A, 0.8% Ca, 0.5% P, 2.2 IU vitamin DJg. Diet 6, 0.8% Ca, 0.5% P, vitamin D-deficient. Diet C, 2.0% Ca, 1.25% P, 20% lactose, vitamin D-deficient. Values are expressed as mean t SEM, n = 4 to B/group. l*P i .Ol: significance of difference from diet A (ANOVA and Tukey’s test).
440
FOX, KOLLENKIRCHEN, AND WALTERS
Table 1. Plasma Levels in Rats Fed Varying Calcium and Phosphorus Diets for 10 and 19 Weeks Diet A
Diet B
Diet C
Week IO
Ca (mg/dL)
10.8 ? 0.2
6.3 t 0.2t
10.2 t 0.1
5.3 + 0.6
8.0 + 0.5
6.0 ? 1.2
12 t 2
85 + 12t
26 f 4
25(OH)O, (pg/mL)
13.093 f 1,163
73 + 21t
1,25(OH),D, (pg/mL) Week 19
38 ? 8
7.7 * 1.7*
P (mg/dL) PTH (pg/mL)
Ca (mg/dL) P (mg/dL) PTH (pg/mL) 25(OH)D, (pg/mL) 1,25(OH),D, (pQ/mL)
75?
11t
5.5 2 2.4t
10.2 2 0.1
6.0 + 0.4t
10.3 r. 0.1
5.6 f 0.2
6.9 + 0.7
5.6 f 0.3
13 + 4
107?
28t
16 f 3
11,431 !z 4,743
31 ‘- lot
29 + 2t
27 t 2
4.7 & 1.1t
2.6 2 0.7t
NOTE. Values are expressed
as mean -t SEM, n = 5 individual
rats/group. Diet A, 0.8% Ca, 0.5% P, 2.2 IU vitamin DJg. Diet 6, 0.8% Ca, 0.5% P, vitamin D-deficient. Diet C, 2.0% Ca, 1.25% P, 20% lactose, vitamin D-deficient. *P < .05; tP < .Ol: significance of difference from diet A (ANOVA and Tukey’s test).
vitamin D-replete controls after both 10 and 19 diet weeks (Table 1). In contrast, rats fed the 2.0% Ca, 1.25% P, vitamin D-deficient diet, containing 20% lactose (diet C) were normocalcemic after 10 and 19 diet weeks. While variable, plasma phosphate levels were not changed significantly by any diet (Table 1). The normocalcemia in the vitamin D-deficient rats fed diet C was not maintained by elevated PTH secretion, because PTH levels were also not significantly different from the vitamin D-replete controls after either 10 or 19 diet weeks (Table 1). In contrast, plasma PTH levels were significantly (P < .Ol) increased in hypocalcemic, vitamin D-deficient rats (diet B). PTH levels were 7.1-fold higher after 10 diet weeks and 8.2-fold higher after 19 diet weeks. There were no significant timedependent differences in plasma calcium, phosphate, or PTH within any dietary group. Both groups of rats fed vitamin D-deficient diets were markedly vitamin D-depleted. 25(OH)D, levels were reduced by 99.4% and 99.7% after 10 and 19 diet weeks, respectively, in rats fed vitamin D-deficient diets B and C (Table 1). In rats fed diet B, 1,25(OH),D, levels were reduced by 80% and 83% after 10 and 19 diet weeks, respectively, and by 85% and 90% in rats fed diet C for 10 and 19 weeks, respectively (Table 1). While there was a tendency in all groups for plasma 1,25(OH),D, levels to be lower after 19 than at 10 diet weeks, the changes were not statistically significant (Table 1). DlSCUSSlON These studies were designed to determine the role of severe deficiency of vitamin D metabolites per se on renal 1-hydroxylase enzyme activity. While the condition of vitamin D-deficiency is well recognized as the major stimulator of the I-hydroxylase enzyme,“’ the actual mechanism for this stimulation has not been directly investigated due to the complicating factors of concurrent hypocalcemia, secondary hyperparathyroidism, and hypophosphatemia. To
facilitate study of the regulation of the 1-hydroxylase by vitamin D metabolites, we used the normocalcemic, normophosphatemic rat model of vitamin D deficiency that we have developed and characterized.” The results of these studies have demonstrated that deficiency of vitamin D metabolites alone can be a strong stimulus of the l-hydroxylase enzyme. In vitamin D-deficient rats maintained normocalcemic by dietary means (diet C), 1-hydroxylase activity was also substantially elevated above the level seen in vitamin D-replete control rats (Fig 1). The normocalcemia in rats fed diet C was not maintained by elevated PTH secretion, since PTH levels were also normal (Table 1). Furthermore, since the normocalcemic, normal PTH, vitamin D-deficient rats were also normophosphatemic (Table l), decreases in plasma phosphate also cannot account for the increased 1-hydroxylase enzyme activity. Thus, despite the fact that the plasma concentrations of three of the most important stimulators of 1-hydroxylase enzyme activity, ie, calcium, phosphate, and PTH, were entirely normal in rats fed diet C, a marked increase in 1-hydroxylase activity was still apparent. Nevertheless, the importance of calcium and PTH on the stimulation of 1-hydroxylase activity, as usually seen in vitamin D-deficient states, was still clearly apparent, since the hypocalcemic, vitamin D-deficient rats with markedly elevated PTH levels exhibited a 1-hydroxylase activity that was eightfold higher than seen in the normocalcemic vitamin D-deficient rats after 19 diet weeks (Fig 1). There was a time-dependent decrease in 1-hydroqlase activity in all dietary groups. There are two likely (and potentially related) explanations for this phenomenon. Undoubtably, the well-known age-related decrease in enzyme activity,” whose mechanism remains unclear, contributes to this phenomenon, in particular since similar declines in activity occur in both diet groups A and B. The mechanism underlying the larger decline in diet group C is unclear. It is tempting to just cite the higher PTH levels in diet group C at 10 weeks as the explanation. However, the nearly twofold numerical difference in PTH levels was not significantly different. The mechanism by which this deficiency of vitamin D metabolites stimulates l-hydroxylase activity is unknown, although there are numerous regulatory elements of the 1-hydroxylase at the cellular and mitochondrial levels. In particular, a mechanism involving reduced levels of 1,25(OH),D, is a likely explanation for the stimulation of 1-hydroxylase activity. 1,25(OH),D,, by a receptor-mediated mechanism, is known to be a potent and important inhibitor of the 1-hydroxylase and stimulator of the alternate renal enzyme, the 24-hydroxylase.“” The low plasma 1,25(OH),D, levels in the vitamin D-deficient rats may, therefore, produce increased 1-hydroxylase activity by the absence of this receptor-mediated enzyme inhibition. Alternatively, the absence of vitamin D metabolites may in some manner enhance transduction of the PTH signal to the 1-hydroxylase enzyme, permitting increased activity. In conclusion, these studies have demonstrated that hypocalcemia and secondary hyperparathyroidism are not solely responsible for the increased renal 25(OH)D,-l-
STIMULATED
l-HYDROXYLASE
ACTIVITY
BY VITAMIN
D DEFICIENCY
hydroxylase enzyme activity seen in vitamin D deficiency. Importantly, deficiency of vitamin D metabolites per se provides a strong and independent stimulus to l-hydroxylase activity, perhaps by loss of feedback inhibition of enzyme activity by 1,25(OH),D,.
441
ACKNOWLEDGMENT
We thank Drs Milan R. Uskokovic and Hunter Heath III for providing vitamin D, metabolites and antiserum (381%PTH, respectively. M. Bijoy Mathew and Diana Woods also provided expert technical assistance.
REFERENCES
1. Omdahl JL, DeLuca HF: Regulation of vitamin D metabolism and function. Physiol Rev 53:327-372, 1973 2. Fraser DR: Regulation of the metabolism of vitamin D. Physiol Rev 60:551-613,198O 3. Norman AW, Roth J, Orci L: The vitamin D endocrine system: Steroid metabolism, hormone receptors, and biological response. Endocrinol Rev 3:331-366,1982 4. Garabedian M, Holick MF, DeLuca HF: Control of 25hydroxycholecalciferol metabolism by parathyroid hormone. Proc Nat1 Acad Sci USA 69:1673-1676.1972 5. Fraser DR, Kodicek E: Regulation of 25-hydroxycholecalciferol-1-hydroxylase activity in kidney by parathyroid hormone. Nature 241:163-166, 1973 6. Friedlander EJ, Henry HL, Norman AW: Studies on the mode of action of calciferol. Effects of dietary calcium and phosphorus on the relationship between the 25-hydroxyvitamin D,-1-hydroxylase and production of chick intestinal calcium binding protein. J Biol Chem 252:8677-8683,1977 7. Swaminathan R, Sommerville BA, Care AD: Metabolism in vitro of 25-hydroxycholecalciferol in chicks fed on phosphorusdeficient diets. Clin Sci 541197-200, 1978 8. Gray RW, Napoli JL: Dietary phosphate deprivation increases 1,25-dihydroxyvitamin D, synthesis in rat kidney in vitro. J Biol Chem 258:1152-l 155, 1983 9. Favus MJ, Langman CB: Evidence for calcium-dependent control of 1,25-dihydroxyvitamin D, production by rat kidney proximal tubules. J Biol Chem 261:11224-11229,1986 10. Henry HL, Midgett RJ, Norman AW: Regulation of 25hydroxyvitamin D,-1-hydroqlase in vivo. J Biol Chem 249:75847592.1974
11. Kollenkirchen U, Fox J, Walters MR: Normocalcemia without hyperparathyroidism in vitamin D-deficient rats. J Bone Miner Res 6:273-278, 1991 12. Hollis BW, Kilbo T: The assay of circulating 1,25(OH)?D using non-end-capped C,, silica (C,,-OH): Performance and validation, in Norman AW, Schaefer K, Grigoleit H-G, et al (eds): Vitamin D. Molecular, Cellular and Clinical Endocrinology. Berlin, Germany, de Gruyter, 1988, pp 710-719 13. Reinhardt TA, Horst RL: Simplified assays for the determination of 25-OHD, 24,25-(OH),D and 1,25-(OH),D, in Norman AW, Schaefer K, Grigoleit H-G, et al (eds): Vitamin D. Molecular, Cellular and Clinical Endocrinology. Berlin. Germany, de Gruyter, 1988, pp 720-726 14. Fox J: Verapamil induces PTH resistance but increases duodenal calcium absorption in rats. Am J Physiol255:E702-E707. 1988 15. Fox J, Della-Santina CP: Oral verapamil and Ca and vitamin D metabolism in rats: Effect of dietary Ca. Am J Physiol257:E632E638,1989 16. Clemens TL, Hendy GN, Papapoulos SE, et al: Measurement of 1,25-dihydroxycholecalciferol in man by radioimmunoassay. Clin Endocrinol 11:225-234,1979 17. Gindler EM, King JD: Rapid calorimetric determination of calcium in biologic fluids with methylthymol blue. Am J Clin Pathol 581376-382, 1972 18. Chen PS Jr, Toribara TY, Warner H: Microdetermination of phosphorus. Anal Chem 28:1756-1758,1956 19. Armbrecht HJ, Forte LR, Halloran BP: Effect of age and dietary calcium on renal 25(OH)D metabolism, serum 1,25(OH)?D, and PTH. Am J Physiol246:E266-E270,1984
D Metabolites Directly Stimulates Renal D,-1-Hydroxylase Activity in Rats
Fox, Uwe Kollenkirchen,
and Marian
R. Walters
Renal 2bhydroxyvitamin D,-I-hydroxylase (I-hydroxylase) enzyme activity in rats is known to be increased by parathyroid hormone (PTH), hypophosphatemia, and hypocalcemia. Thus, enzyme activity is markedly increased in vitamin D-deficient states, but whether this stimulation is a direct response to the vitamin D deficiency or only occurs following the associated chan9es in plasma calcium, phosphate, or PTH is unclear. We tested whether vitamin D deficiency per se influences I-hydroxylase activity in renal cortical slices using a normocalcemic rat model of vitamin D deficiency. Weanling male rats were fed one of the following three diets: (A) 0.8% Ca, 0.5% P, 2.2 IU vitamin D,/g; or vitamin D-deficient diets containing, (B) 0.8% Ca, 0.5% P; and (C) 2.0% Ca, 1.25% P, 20% lactose. Vitamin D-deficient rats fed diet B were hypocalcemic with elevated PTH at both test periods, and I-hydroxylase activity was increased more than IOO-fold compared with rats fed diet A. Plasma calcium, phosphate, and PTH levels were the same in groups A and C, but I-hydroxylase activity was also substantially elevated in 9roup C versus group A rats (104- and 17-fold increases after IO and 19 diet weeks, respectively). These data lead to the important conclusion that severe deficiency of vitamin D metabolites per se provides a strong and independent stimulus to renal I-hydroxylase activity in rats, perhaps due to the absence of 1,25(0H),D,-mediated enzyme inhibition. Copyright 0 1997 by W.B. Saunders Compeny
T
HE RENAL 25hydroxyvitamin D,-1-hydroxylase (lhydroxylase) enzyme catalyzes the conversion of 25 hydroxyvitamin D, [25(OH)D,] to the hormonally active metabolite, 1,25_dihydroxyvitamin D, [1,25(OH),D,].‘” Parathyroid hormone (PTH) is the major stimulator of l-hydroxylase activity>’ although hypophosphatemia and hypocalcemia have also been shown to stimulate the enzyme directly.“9 1,25(OH),D, can inhibit the 1-hydroxylase by a mechanism thought to involve a receptor-mediated process and, in a pattern of coordinate regulation, the hormone also increases the activity of the alternate enzyme, the 25(OH)D,24-hydroxylase.1-3 Importantly, the most potent stimulation of I-hydroxylase activity occurs under conditions of vitamin D deficiency.“3,‘0 Establishing whether this stimulation is a direct response to the deficiency of vitamin D metabolites or only occurs following the usually associated changes in plasma calcium, phosphate, and PTH levels is an important step in understanding the factors that regulate l-hydroxylase activity in normal and abnormal states. The availability of a well-characterized normocalcemic model of vitamin D deficiency in rats” allowed us to test the hypothesis that deficiency of vitamin D per se stimulates the enzyme in vivo. MATERIALS
AND METHODS
Animals
Normal, weanling, 21-day-old, male CD rats were obtained from Charles Riva cabs (Wilmington, MA). Upon receipt, they were randomly divided into four groups and housed in hanging wire cages under
incandescent
light. They were fed one of three diets:
From the Depatiment of Physiology, Tulane University School of Medicine, New Orleans, LA. Supported by a grant-in-aid from the American Heart Association, by USPHS-NIH Grant No. DK31847 (MR. W), and by a grant-in-aid from the American Heart Association-Louisiana (J.F.). Address reprint requests to John Fox, PhD, Department of Physiology, Tulane University School of Medicine, 1430 Tulane Ave, New Orleans, LA 701 I2. Copyright 0 1991 by KB. Saunders Company 00260495191/4004-0017$03.00/0
438
(A) 0.8% Ca, 0.5% P, 2.2 IU vitamin D,/g; (B) a vitamin D-deficient diet containing 0.8% Ca, 0.5% P; and (C) a vitamin D-deficient diet containing 2.0% Ca, 1.25% P, and 20% lactose, substituted for sucrose (Teklad, Madison, WI). After 10 and 19 diet weeks, five rats from each dietary group were anesthetized with pentobarbital sodium (60 mg/kg, intraperitoneally) and exsanguinated by cardiac puncture. Plasma was collected for the analyses described below. Both kidneys were removed and placed in ice-coid isotonic saline. Measurement
of Renal I-Hydroxylase Enzyme Activity
The kidneys were decapsulated and cortical slices (0.5 mm) were prepared using a tissue slicer (Stoelting, Wood Dale, IL). The slices (- 100 mg) were gassed with OJCO, (95/5) and incubated as described elsewhere.* The reaction was initiated by adding 1 ug 25(OH)D, in 10 FL ethanol [initial 25(OH)D, concentration, 2.5 umol/L]. Each incubation was regassed after 30 minutes and was terminated after 1 hour by adding 1 mL acetonitrile. Then, 1,000 dpm 1,25(OH)#H]D, (160 Ciimmol; Amersham, Arlington Heights, IL) was added to monitor recovery through the extraction and purification procedures. The slices and medium were homogenized in acetonitrile:water (50:50) and centrifuged. The generated 1,25(OH),D, was extracted from the supernatant and purified and assayed as described for plasma 1,25(OH),D, below. Renal l-hydroxylase enzyme activity was expressed as picograms 1,25(OH),D, produced per milligram cortex per hour.”
Plasma Analyses Before assay, vitamin D, metabolites were extracted and purified using established methods with minor modifications.“~” 25(OH)[‘H]D, (90 Ciimmol) and 1,25(OH),[‘H]D, (800 dpm) were added to each sample to monitor recovery through the purification. Vitamin D, metabolites were extracted from 0.5 to 1.0 mL plasma with an equal volume of acetonitrile. Following centrifugation, the supernatant was combined with 0.5 ~010.4 mol/L K*HPO,, pH 10.6, and applied to a C,,-OH Bond-Elm column (Analytichem, Harbor City, CA). The column was washed with water, 5 mL methanolwater (70/30), and 5 mL hexane. 25(OH)D, was eluted with 5 mL hexane-dichloromethane (90/10). The C,,-OH column was then washed with 8 mL hexane-dichloromethane-isopropanol (89/10/l) to remove 24,25(OH),D, and followed by 2.4 mL hexaneisopropanol (95/5) to elute 1,25(OH),D,. Before assay, the 25(OH)D, and 1,25(OH),D, fractions were further purified on a
Metabolism,
Vol40, No 4 (April), 1991: pp 438-441
STIMULATED
l-HYDROXYLASE
ACTIVITY
BY VITAMIN
439
D DEFICIENCY
Statistical comparisons were performed on a microcomputer using a software package (Stata; Computing Resource Center, Los Angeles, CA).
silica Bond-Elut column. The 25(OH)D, fraction was applied to the silica column and washed with 8 mL hexane-dichloromethaneisopropanol (89/10/l). 25(0H)D, was eluted with 10 mL hexaneisopropanol (95/5). The 1,25(OH),D, fraction was applied to the silica column, washed with 5 mL hexane-isopropanol (94/6), and eluted with 9 mL hexane-isopropanol (86/14). The final eluants were evaporated to dryness in a vacuum centrifuge and redissolved in ethanol for assay. Recoveries of 25(OH)D, and 1,25(OH),D3 averaged 70% to 80%. 25(OH)D, was quantitated by radioimmunoassay’4.15using sheep antiserum 02282.16The detection limit was approximately 1.5 to 2.0 pg/tube (y 6 to 8 pg/mL for 1 mL plasma). 1,25(OH),D, levels were quantitated by radioreceptor assay using a calf thymus receptor prepared using an established method.” The assay detection limit was 0.4 pg/tube ( _ 1 to 2 pg/mL for 1 mL plasma). Plasma amino-terminal PTH levels were measured with a homologous rat PTH(l-34) radioimmunoassay with goat antiserum G813-PTH, described previously.‘4.‘5The assay detection limit was 6 pg rat PTH(l-34)lmL. Plasma calcium levels were measured using methylthymol blue,” and plasma phosphate by the method of Chen et al.”
RESULTS
1 -Hydroxylase Activity In these animals, both time- and diet-dependent changes in 1-hydroxylase enzyme activity occurred. 1-Hydroxylase activity decreased significantly (P < .05) between 10 and 19 diet weeks in all dietary groups (Fig 1). Nevertheless, the hypocalcemic vitamin D-depleted rats with high PTH levels (diet B, documented below) exhibited substantially higher 1-hydroxylase activity after 10 and 19 diet weeks (109- and 140-fold elevation versus vitamin D-replete controls, respectively) (Fig 1). Enzyme activity was similarly elevated (104-fold) in normocalcemic, normal PTH rats (diet C) after 10 diet weeks. Although the degree of stimulation was somewhat reduced by 19 diet weeks, 1-hydroxylase activity remained significantly (P < .Ol) higher in these rats than in the vitamin D-replete controls (Fig 1).
StatisticalAnalysis All data are presented as mean ? SEM. The significance of differences between groups were determined by ANOVA and Tukey’s test. In the case of unequal variances (determined by Bartlett’s test), the data was log-transformed before ANOVA.
A.
Plasma Parameters Rats fed the 0.8% Ca, 0.5% P, vitamin D-deficient diet (diet B) were markedly hypocalcemic compared with the
10 weeks **
6.
19 weeks
**
--I!-
** +
**
IO
F +
+l
1
:
c T
-
Diet A
Diet B
-
Diet C
o.v---
Diet A
-
Diet B
Diet C
1-Hydroxylase enzyme activity in renal cortical slices from rats fed varying calcium and phosphorus diets for 10 and 19 weeks. Diet A, 0.8% Ca, 0.5% P, 2.2 IU vitamin DJg. Diet 6, 0.8% Ca, 0.5% P, vitamin D-deficient. Diet C, 2.0% Ca, 1.25% P, 20% lactose, vitamin D-deficient. Values are expressed as mean t SEM, n = 4 to B/group. l*P i .Ol: significance of difference from diet A (ANOVA and Tukey’s test).
440
FOX, KOLLENKIRCHEN, AND WALTERS
Table 1. Plasma Levels in Rats Fed Varying Calcium and Phosphorus Diets for 10 and 19 Weeks Diet A
Diet B
Diet C
Week IO
Ca (mg/dL)
10.8 ? 0.2
6.3 t 0.2t
10.2 t 0.1
5.3 + 0.6
8.0 + 0.5
6.0 ? 1.2
12 t 2
85 + 12t
26 f 4
25(OH)O, (pg/mL)
13.093 f 1,163
73 + 21t
1,25(OH),D, (pg/mL) Week 19
38 ? 8
7.7 * 1.7*
P (mg/dL) PTH (pg/mL)
Ca (mg/dL) P (mg/dL) PTH (pg/mL) 25(OH)D, (pg/mL) 1,25(OH),D, (pQ/mL)
75?
11t
5.5 2 2.4t
10.2 2 0.1
6.0 + 0.4t
10.3 r. 0.1
5.6 f 0.2
6.9 + 0.7
5.6 f 0.3
13 + 4
107?
28t
16 f 3
11,431 !z 4,743
31 ‘- lot
29 + 2t
27 t 2
4.7 & 1.1t
2.6 2 0.7t
NOTE. Values are expressed
as mean -t SEM, n = 5 individual
rats/group. Diet A, 0.8% Ca, 0.5% P, 2.2 IU vitamin DJg. Diet 6, 0.8% Ca, 0.5% P, vitamin D-deficient. Diet C, 2.0% Ca, 1.25% P, 20% lactose, vitamin D-deficient. *P < .05; tP < .Ol: significance of difference from diet A (ANOVA and Tukey’s test).
vitamin D-replete controls after both 10 and 19 diet weeks (Table 1). In contrast, rats fed the 2.0% Ca, 1.25% P, vitamin D-deficient diet, containing 20% lactose (diet C) were normocalcemic after 10 and 19 diet weeks. While variable, plasma phosphate levels were not changed significantly by any diet (Table 1). The normocalcemia in the vitamin D-deficient rats fed diet C was not maintained by elevated PTH secretion, because PTH levels were also not significantly different from the vitamin D-replete controls after either 10 or 19 diet weeks (Table 1). In contrast, plasma PTH levels were significantly (P < .Ol) increased in hypocalcemic, vitamin D-deficient rats (diet B). PTH levels were 7.1-fold higher after 10 diet weeks and 8.2-fold higher after 19 diet weeks. There were no significant timedependent differences in plasma calcium, phosphate, or PTH within any dietary group. Both groups of rats fed vitamin D-deficient diets were markedly vitamin D-depleted. 25(OH)D, levels were reduced by 99.4% and 99.7% after 10 and 19 diet weeks, respectively, in rats fed vitamin D-deficient diets B and C (Table 1). In rats fed diet B, 1,25(OH),D, levels were reduced by 80% and 83% after 10 and 19 diet weeks, respectively, and by 85% and 90% in rats fed diet C for 10 and 19 weeks, respectively (Table 1). While there was a tendency in all groups for plasma 1,25(OH),D, levels to be lower after 19 than at 10 diet weeks, the changes were not statistically significant (Table 1). DlSCUSSlON These studies were designed to determine the role of severe deficiency of vitamin D metabolites per se on renal 1-hydroxylase enzyme activity. While the condition of vitamin D-deficiency is well recognized as the major stimulator of the I-hydroxylase enzyme,“’ the actual mechanism for this stimulation has not been directly investigated due to the complicating factors of concurrent hypocalcemia, secondary hyperparathyroidism, and hypophosphatemia. To
facilitate study of the regulation of the 1-hydroxylase by vitamin D metabolites, we used the normocalcemic, normophosphatemic rat model of vitamin D deficiency that we have developed and characterized.” The results of these studies have demonstrated that deficiency of vitamin D metabolites alone can be a strong stimulus of the l-hydroxylase enzyme. In vitamin D-deficient rats maintained normocalcemic by dietary means (diet C), 1-hydroxylase activity was also substantially elevated above the level seen in vitamin D-replete control rats (Fig 1). The normocalcemia in rats fed diet C was not maintained by elevated PTH secretion, since PTH levels were also normal (Table 1). Furthermore, since the normocalcemic, normal PTH, vitamin D-deficient rats were also normophosphatemic (Table l), decreases in plasma phosphate also cannot account for the increased 1-hydroxylase enzyme activity. Thus, despite the fact that the plasma concentrations of three of the most important stimulators of 1-hydroxylase enzyme activity, ie, calcium, phosphate, and PTH, were entirely normal in rats fed diet C, a marked increase in 1-hydroxylase activity was still apparent. Nevertheless, the importance of calcium and PTH on the stimulation of 1-hydroxylase activity, as usually seen in vitamin D-deficient states, was still clearly apparent, since the hypocalcemic, vitamin D-deficient rats with markedly elevated PTH levels exhibited a 1-hydroxylase activity that was eightfold higher than seen in the normocalcemic vitamin D-deficient rats after 19 diet weeks (Fig 1). There was a time-dependent decrease in 1-hydroqlase activity in all dietary groups. There are two likely (and potentially related) explanations for this phenomenon. Undoubtably, the well-known age-related decrease in enzyme activity,” whose mechanism remains unclear, contributes to this phenomenon, in particular since similar declines in activity occur in both diet groups A and B. The mechanism underlying the larger decline in diet group C is unclear. It is tempting to just cite the higher PTH levels in diet group C at 10 weeks as the explanation. However, the nearly twofold numerical difference in PTH levels was not significantly different. The mechanism by which this deficiency of vitamin D metabolites stimulates l-hydroxylase activity is unknown, although there are numerous regulatory elements of the 1-hydroxylase at the cellular and mitochondrial levels. In particular, a mechanism involving reduced levels of 1,25(OH),D, is a likely explanation for the stimulation of 1-hydroxylase activity. 1,25(OH),D,, by a receptor-mediated mechanism, is known to be a potent and important inhibitor of the 1-hydroxylase and stimulator of the alternate renal enzyme, the 24-hydroxylase.“” The low plasma 1,25(OH),D, levels in the vitamin D-deficient rats may, therefore, produce increased 1-hydroxylase activity by the absence of this receptor-mediated enzyme inhibition. Alternatively, the absence of vitamin D metabolites may in some manner enhance transduction of the PTH signal to the 1-hydroxylase enzyme, permitting increased activity. In conclusion, these studies have demonstrated that hypocalcemia and secondary hyperparathyroidism are not solely responsible for the increased renal 25(OH)D,-l-
STIMULATED
l-HYDROXYLASE
ACTIVITY
BY VITAMIN
D DEFICIENCY
hydroxylase enzyme activity seen in vitamin D deficiency. Importantly, deficiency of vitamin D metabolites per se provides a strong and independent stimulus to l-hydroxylase activity, perhaps by loss of feedback inhibition of enzyme activity by 1,25(OH),D,.
441
ACKNOWLEDGMENT
We thank Drs Milan R. Uskokovic and Hunter Heath III for providing vitamin D, metabolites and antiserum (381%PTH, respectively. M. Bijoy Mathew and Diana Woods also provided expert technical assistance.
REFERENCES
1. Omdahl JL, DeLuca HF: Regulation of vitamin D metabolism and function. Physiol Rev 53:327-372, 1973 2. Fraser DR: Regulation of the metabolism of vitamin D. Physiol Rev 60:551-613,198O 3. Norman AW, Roth J, Orci L: The vitamin D endocrine system: Steroid metabolism, hormone receptors, and biological response. Endocrinol Rev 3:331-366,1982 4. Garabedian M, Holick MF, DeLuca HF: Control of 25hydroxycholecalciferol metabolism by parathyroid hormone. Proc Nat1 Acad Sci USA 69:1673-1676.1972 5. Fraser DR, Kodicek E: Regulation of 25-hydroxycholecalciferol-1-hydroxylase activity in kidney by parathyroid hormone. Nature 241:163-166, 1973 6. Friedlander EJ, Henry HL, Norman AW: Studies on the mode of action of calciferol. Effects of dietary calcium and phosphorus on the relationship between the 25-hydroxyvitamin D,-1-hydroxylase and production of chick intestinal calcium binding protein. J Biol Chem 252:8677-8683,1977 7. Swaminathan R, Sommerville BA, Care AD: Metabolism in vitro of 25-hydroxycholecalciferol in chicks fed on phosphorusdeficient diets. Clin Sci 541197-200, 1978 8. Gray RW, Napoli JL: Dietary phosphate deprivation increases 1,25-dihydroxyvitamin D, synthesis in rat kidney in vitro. J Biol Chem 258:1152-l 155, 1983 9. Favus MJ, Langman CB: Evidence for calcium-dependent control of 1,25-dihydroxyvitamin D, production by rat kidney proximal tubules. J Biol Chem 261:11224-11229,1986 10. Henry HL, Midgett RJ, Norman AW: Regulation of 25hydroxyvitamin D,-1-hydroqlase in vivo. J Biol Chem 249:75847592.1974
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