1 Departments of Nephrology and 2 Pathology, Pontificia Universidad Católica de Chile, Santiago, Chile, 3 Department of Medicine, West Los Angeles VA Medical Center and UCLA, Los Angeles, California, USA
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Abstract |
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Methods. Rats were either sham-operated (two groups) or underwent a two-stage 5/6 nephrectomy (three groups). For the first 4 weeks, all rats were given a high (1.2%) phosphorus (P) diet to stimulate parathyroid gland growth and then were changed to a normal (0.6%) P diet for 2 weeks. At week 7, three of the five groups were given high doses of calcitriol (500 pmol/100 g body weight) intraperitoneally every 24 h during 72 h before sacrifice. The five groups during week 7 were: (i) normal renal function (NRF)+0.6% P diet; (ii) NRF+0.6% P+calcitriol; (iii) renal failure (RF)+0.6% P; (iv) RF+1.2% P+calcitriol; and (v) RF+0.6% P+calcitriol. Parathyroid glands were removed at sacrifice and the TUNEL stain was performed to detect apoptosis.
Results. At sacrifice, the respective serum calcium values in calcitriol-treated groups (groups 2, 4, and 5) were 15.52±0.26, 13.41±0.39 and 15.12±0.32 mg/dl. In group 3, PTH was 178±42 pg/ml, but in calcitriol-treated groups, PTH values were suppressed, 8±1 (group 2), 12±2 (group 4), and 7±1 pg/ml (group 5). Despite, the severe hypercalcaemia and marked PTH suppression in calcitriol-treated groups, the percentage of apoptotic cells in the parathyroid glands was very low (range 0.08±0.04 to 0.25±0.20%) and not different among the five groups.
Conclusions. We found no evidence in hyperplastic parathyroid glands that apoptosis could be induced in azotaemic rats by the combination of high doses of calcitriol and severe hypercalcaemia despite the marked reduction in PTH levels that was observed.
Keywords: apoptosis; calcitriol; hypercalcaemia; parathyroid gland; parathyroid hormone; rat
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Introduction |
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Phosphorus retention and decreased calcitriol levels have been shown to play an important role in the pathogenesis of 2nd HPT [7]. Phosphorus loading increases PTH secretion and induces parathyroid-gland enlargement. Recent studies have shown that a high-phosphorus diet induces marked parathyroid gland enlargement due to a combination of parathyroid cell hyperplasia and hypertrophy [810]. Discontinuation of the high-phosphorus diet resulted in resolution of the hypertrophy but not hyperplasia. A calcitriol deficiency has also been shown to play an important role in the genesis of uraemic hyperparathyroidism [1113] and calcitriol has been used to treat 2nd HPT both in the patient with moderate renal failure [14,15] and in the dialysis patient [1618]. In the parathyroid gland, calcitriol directly inhibits PTH mRNA transcription [11,13,19]. Calcitriol has also been suggested to be an important regulator of parathyroid cell growth [2,3] and may also act to directly suppress proliferation of parathyroid cells [12,20,21].
Whether calcitriol administration induces regression of parathyroid hyperplasia remains a subject of considerable interest and debate. Szabo et al. [12] showed that calcitriol administration suppressed the development of parathyroid hyperplasia independent of changes in serum calcium, but once parathyroid hyperplasia was established, hyperplasia was not reversed by calcitriol administration. Fukagawa et al. reported that calcitriol pulse therapy decreased the size of parathyroid glands of haemodialysis patients [22,23]. A decrease in the size of the parathyroid glands could result from either a reduction in size of hypertrophied cells, an enhancement of programmed cell death (apoptosis), or a combination of both. Current reports in the literature about the rate of apoptosis in hyperplastic parathyroid glands are contradictory [3,24], as is the minimal information available on whether calcitriol treatment induces parathyroid-cell apoptosis in an in vivo model [25,26].
The goal of our study was to determine whether high doses of calcitriol induce apoptosis in the hyperplastic parathyroid glands of azotaemic rats.
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Subjects and methods |
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Methods
All sham-operated and 5/6-nephrectomized rats were placed for 4 weeks on a 0.6% calcium and 1.2% phosphorus diet which contained 100 IU of vitamin D per 100 g of diet (ICN, Cleveland, Ohio, USA). In previous studies, this high-phosphorus diet has been shown to markedly exacerbate 2nd HPT in azotaemic rats and to induce marked parathyroid gland enlargement [7,10]. After 4 weeks of the high-phosphorus diet, all sham-operated and 5/6-nephrectomized rats were changed to a diet containing 0.6% calcium and 0.6% phosphorus (ICN, Cleveland, Ohio, USA) for 2 weeks to normalize serum calcium values in azotaemic rats. At the beginning of week 7, rats were divided into five groups and either (i) received three intraperitoneal doses of calcitriol (500 pmol/100 g of body weight per dose) (Abbott Laboratories, Chicago, Illinois, USA) at 72, 48 and 24 h before sacrifice, or (ii) received calcitriol vehicle at the same times. The 500 pmol/100 g dose of calcitriol (CTR) is equal to 2.08 µg/kg and is equivalent to giving a 70 kg human, 145.6 µg of calcitriol; this large dose was used to maximize the effect of calcitriol on the parathyroid gland.
The five study groups were: (i) group 1, sham-operated rats with normal renal function (NRF) were given a 0.6% calcium, 0.6% phosphorus (P) diet and received vehicle (NRF+0.6% P); (ii) group 2, sham-operated rats with normal renal function were given a 0.6% calcium, 0.6% phosphorus diet and received intraperitoneal CTR (NRF+0.6% P+CTR); (iii) group 3, 5/6 nephrectomized rats were given a 0.6% calcium, 0.6% phosphorus diet and received vehicle (RF+0.6% P); (iv) group 4, 5/6 nephrectomized rats were given a 0.6% calcium, 1.2% phosphorus diet and received intraperitoneal CTR (RF+1.2% P+CTR); and (v) group 5, 5/6 nephrectomized rats were given a 0.6% calcium, 0.6% phosphorus diet and received intraperitoneal CTR (RF+0.6% P+CTR).
At sacrifice, the parathyroid glands were selectively removed. In each rat, one gland was immediately frozen in liquid nitrogen and stored at -20°C for DNA extraction, according to standard methods [27]. Electrophoresis of the precipitated DNA was performed on a 2% agarose gel and later visualized with ethidium bromide staining under ultraviolet light. The other parathyroid gland was fixed in 4% paraformaldehyde and paraffin blocks prepared for histological analysis. The number of parathyroid glands prepared for histological analysis was less than the number of rats in each group because the first parathyroid gland identified was fixed for molecular analysis (DNA fragmentation) and the second one, which was not always located, was used for histological analysis.
In situ end-labelling (TUNEL) techniques of parathyroid tissue sections
The TUNEL stain was performed as described previously [26]. Paraffin tissue blocks were cut in 46 mm sections, deparaffinized in xylene and alcohol, and placed in PBS (pH 7.6). Later, tissue sections were treated with proteinase K and washed three times. Placental tissue which served as the control, was handled the same as the parathyroid gland. The in situ Cell Death Detection Kit, POD® (Boehringer Mannheim, Mannheim, Germany) was used for labelling of the free 3'-OH terminus.
Two authors (SG and BC) independently performed the counts on the parathyroid glands. A total of 1000 cells in each parathyroid gland were counted by each observer and the number of apoptotic cells are expressed as a percentage of the total.
Biochemical determinations
Serum calcium was measured by autoanalyser, serum phosphorus with a specific kit (Sigma Chemical Co. St Louis, Missouri, USA), serum creatinine with a creatinine analyser (Beckman Instruments, USA), and PTH with a rat immunoradiometric assay (Nichols Institute, San Clemente, California, USA) previously validated [28].
Statistics
Comparisons of the groups were performed with one-way analysis of variance (ANOVA) to establish whether differences were present among the five groups. If the ANOVA was P<0.05, a post-hoc test, the Fisher LSD, was used to compare the individual groups. In group 4, only one rat was not hypercalcaemic and in that rat, the serum phosphate (26.6 mg/dl), calcium (7.78 mg/dl), and PTH (97 pg/ml) values were more than two standard deviations from the group mean. As a result, the rat was excluded from the analysis. A P value <0.05 was considered significant. Results are expressed as the mean±standard error (SE).
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Results |
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Discussion |
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Conceptually, for growth of parathyroid tissue to occur, the proliferation rate must be greater than the apoptotic rate. In hyperplastic tissue, both may be increased but the proliferation rate must still exceed the rate of apoptosis for sustained growth to occur. However, a direct comparison of the rates of proliferation and apoptosis may be confounded by the fact that the cell cycle for cell proliferation is longer than the time required for the appearance and the removal of an apoptotic cell.
To date, results in uraemia regarding apoptosis of parathyroid tissue have not been entirely consistent. This could be due not only to the extremely low rate of proliferation of parathyroid cells, but also to the adequacy of techniques used to evaluate apoptosis. The majority of techniques rely only on evidence of DNA fragmentation as definite proof of apoptosis. Recently, it was shown that cells could undergo apoptosis even when internucleosomal DNA lysis was blocked. Furthermore, some of the cytoplasmic and membrane changes seen in apoptosis could be brought about without the presence of nuclei [29]. Thus, it is possible that if internucleosomal DNA lysis is an essential event in some cells but not in others, there is more than one final common pathway for apoptosis.
Several animal studies have evaluated apoptosis in normal and proliferating parathyroid glands. Naveh-Many et al. [26] and Wada et al. [30] did not find apoptotic cells in the parathyroid glands of normal and azotaemic rats even after the administration of calcitriol [26]. In some studies of normal human parathyroid glands and parathyroid glands from patients with primary HPT and 2nd HPT, the value for apoptotic cells has been reported to be low, in the 1% range [31,32]. However, other studies have reported values for apoptotic cells which were approximately 510-fold greater [33,34]. But in one of these latter studies, a reassessment was performed and lower values for apoptotic cells in the 1% range were reported for primary HPT and 2nd HPT [32]. In parathyroid glands removed from patients with primary HPT and 2nd HPT and studied by flow cytometry, the rate of apoptosis was only 0.20.3% [21]. Thus, it would seem that in most studies in animals and humans, the percentage of apoptotic cells in normal, adenomatous, and hyperplastic glands is low.
In 5/6 nephrectomized rats with hyperparathyroidism, treatment with the calcimimetic NPS R-568 immediately after the 5/6 nephrectomy blocked the proliferation of parathyroid cells [30] and prevented parathyroid gland hyperplasia [35]. But when NPS R-568 was given to azotaemic rats 4 weeks after 5/6 nephrectomy, a regression of the previously established parathyroid gland hyperplasia was not observed despite a marked reduction in PTH levels [36]. In non-azotaemic rats, Wang et al. [37] used a very high-phosphorus diet (3.41 %) for several weeks to induce parathyroid gland hyperplasia. Discontinuation of the high-phosphorus diet resulted in a reduction in PTH levels and parathyroid cell hypertrophy, but did not induce regression of parathyroid gland hyperplasia nor was apoptosis detected. Thus in 2nd HPT it seems that a marked reduction in PTH values as a result of the use of calcimimetics [36] or a reduction in dietary phosphorus [8,37] does not reduce established parathyroid gland hyperplasia.
A number of studies have shown that treatment with calcitriol or its analogues generally reduces PTH levels in dialysis patients with moderate to severe hyperparathyroidism [1618], but only a limited number of studies have addressed the important issue of whether calcitriol treatment reduces parathyroid gland size [22,23,38]. In one study, calcitriol treatment reduced parathyroid gland size [22], but in another study, parathyroid gland size remained unchanged after treatment [38]. However, the pretreatment parathyroid gland size was considerably greater in the latter study and it has been shown that the size of the parathyroid gland may affect the response to calcitriol treatment [23]. Another study has reported that calcitriol treatment decreased the intensity of isotope uptake during parathyroid scintigraphy, but this result could have been due to a reduction in parathyroid function rather than parathyroid gland size [39]. Even after 1 µg of calcitriol was twice directly injected into a hyperplastic parathyroid gland of a renal transplant recipient, apoptosis of parathyroid cells was not seen on removal of the parathyroid gland [40].
To decrease parathyroid gland size, it must be assumed that apoptosis would be a key component. In 1977, Henry et al. [41] reported the regression of parathyroid gland hyperplasia after vitamin D or high dose calcitriol treatment in the vitamin D deficient chick. Whether the ability to induce involution of the parathyroid gland is different in vitamin D deficiency than in uraemia is an important issue which has not been sufficiently studied. Potential differences in the response could include the presence of resistance to calcitriol in uraemia because of the decrease in the number of vitamin D receptors [42] and the presence of persistent hyperphosphataemia in uraemia and hypophosphataemia in vitamin D deficiency.
We elected to sacrifice the rats after 3 days of calcitriol treatment at a time when severe hypercalcaemia and a marked reduction in PTH levels were present. Despite the marked reduction in PTH values and the severe hypercalcaemia, it is possible that a longer duration of calcitriol treatment may be needed to induce apoptosis. However, in the study in the vitamin D deficient chick, a regression in parathyroid gland size of approximately 50% was already observed by 4 days after cholecalciferol or high doses of calcitriol were given [41].
In the present study, high doses of calcitriol were used in normal and azotaemic rats to determine whether these high doses combined with calcitriol-induced hypercalcaemia would induce apoptosis in the parathyroid gland. In all five study groups, a high-phosphorus diet, which in previous studies has resulted in marked elevations in PTH levels and parathyroid gland hyperplasia [7,8,10], was used for the first 4 weeks. Subsequently, some groups received high doses of calcitriol which also induced severe hypercalcaemia, but calcitriol treatment did not increase the rate of apoptosis which remained low in all settings. Since in previous studies calcitriol has acted as an antiproliferative agent [12,20,43], it may not be realistic to expect that calcitriol will induce apoptosis. Canalejo et al. [21] showed that the dispersal for 24 h in conditioned medium of parathyroid cells from recently removed parathyroid glands of patients with primary HPT and renal 2nd HPT resulted in a stress-induced increase in the rate of cell proliferation and apoptosis [21]. The addition of calcitriol to the medium resulted in a reduction of both cell proliferation and apoptosis.
In conclusion, we found no evidence in hyperplastic parathyroid glands that apoptosis could be induced in azotaemic rats by the combination of high doses of calcitriol and severe hypercalcaemia, despite the marked reduction in PTH levels that was observed.
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Acknowledgments |
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Notes |
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References |
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