Failure of high doses of calcitriol and hypercalcaemia to induce apoptosis in hyperplastic parathyroid glands of azotaemic rats

Aquiles Jara1,, Sergio González2, Arnold J. Felsenfeld3, Cecilia Chacón1, Andrés Valdivieso1, Roberto Jalil1 and Benedicto Chuaqui2

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



   Abstract
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Background. Whether calcitriol administration, which is used to treat secondary hyperparathyroidism in dialysis patients, induces regression of parathyroid-gland hyperplasia remains a subject of interest and debate. If regression of the parathyroid gland were to occur, the presumed mechanism would be apoptosis. However, information on whether high doses of calcitriol can induce apoptosis of parathyroid cells in hyperplastic parathyroid glands is lacking. Consequently, high doses of calcitriol were given to azotaemic rats and the parathyroid glands were evaluated for apoptosis.

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



   Introduction
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Secondary hyperparathyroidism (2nd HPT) continues to be a cause of morbidity in patients on chronic dialysis [1]. It is often observed early in renal failure and progresses as renal failure worsens. Factors responsible for the development of 2nd HPT include hypocalcaemia, phosphorus retention, a deficiency of calcitriol, and skeletal resistance to parathyroid hormone (PTH). These factors lead to increased PTH synthesis and secretion and result in parathyroid gland hyperplasia and perhaps some hypertrophy of individual cells [2,3]. The size of parathyroid glands increases over time in dialysis patients and the magnitude of hyperparathyroidism has been shown to correlate with parathyroid gland size or mass [46].

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.



   Subjects and methods
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Subjects
The study was performed in male Sprague–Dawley rats weighing 140–160 g at the start of the study. All rats underwent ligation of two of the three main renal branch arteries in the hilum of the left kidney, or sham operation. One week later a right nephrectomy or sham operation was performed. During surgical procedures, rats were anaesthetized with intraperitoneally administered ketamine 7.5 mg/100 g (Ketaset, Fort Dodge Laboratories, Fort Dodge, Iowa, USA) and xylazine 0.5 mg/100 g (AnaSed, Lloyd Laboratories, Shenandoah, Iowa, USA). Rats were housed in individual cages, given 15 g of food daily, and allowed free access to water. Rats ingesting less than 12 g per day were removed from the study.

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 4–6 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).



   Results
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 Subjects and methods
 Results
 Discussion
 References
 
The biochemical data at sacrifice are shown in Table 1Go. In rats not given calcitriol (groups 1 and 3), the PTH level was greater in the azotaemic than in the non-azotaemic group (178±42 vs 72±7 pg/ml, P<0.05). Hypercalcaemia developed in all groups in which calcitriol was given (groups 2, 4, and 5) and marked PTH suppression was observed during hypercalcaemia in both azotaemic and non-azotaemic groups. Despite the severe hyperphosphataemia in groups 4 and 5, calcitriol administration and the resultant hypercalcaemia markedly suppressed PTH levels.


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Table 1. Biochemical data

 
In the azotaemic rats, parathyroid glands were enlarged by inspection and histological examination showed the presence of hyperplasia (Figure 1Go). There did not appear to be any difference in the degree of parathyroid hyperplasia among the azotaemic rats whether or not calcitriol was given.



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Fig. 1. Light microscopy of a parathyroid gland from a sham-operated and from a 5/6 nephrectomized rat both without calcitriol. Rats received a high-phosphorus (1.2%) diet for 4 weeks after which the rat received a normal phosphorus (0.6%) diet for 2 weeks. Light microscopy shows (a) a representative parathyroid gland from a sham-operated rat in group 1 (not given calcitriol), and (b) a representative hyperplastic parathyroid gland from an azoaemic rat in group 3 (not given calcitriol); (original magnification x200).

 
DNA fragmentation in 2% agarose gel electrophoresis as DNA ladders (as indicative of internucleosomal endonucleolysis) was not observed in parathyroid tissue of the rats whether or not calcitriol was administered. The percentage of apoptotic cells detected by TUNEL stain (Figure 2Go and Table 2Go), was low in parathyroid tissue from sham-operated and from azotaemic rats whether or not calcitriol was given (Figures 2BGo and 2CGo). In placental tissue, which served as control, a high number of apoptotic cells were observed (Figure 2AGo). The percentage of apoptotic cells was low (<=0.25%) in all five groups (Table 2Go) and was not different among the five groups (ANOVA, P=0.92) nor among the three azotaemic groups (groups 3–5, ANOVA, P=0.69).



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Fig. 2. In situ end-labelling technique (TUNEL) for detection of apoptosis in parathyroid cells. Immunostaining was positive in (a) placental tissue, which served as the control, (b) a very low number of apoptotic cells in a parathyroid gland from an azotaemic rat which did not receive calcitriol, and (c) a very low number of apoptotic cells in a parathyroid gland from an azotaemic rat which did receive calcitriol (original magnification x200).

 

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Table 2. The percentage of apoptotic cells by TUNEL stain in the parathyroid glands

 



   Discussion
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
The present study shows that high doses of calcitriol together with the associated hypercalcaemia markedly suppress PTH levels in normal rats and even in azotaemic rats with hyperphosphataemia. While it has been previously suggested that the administration of high doses of calcitriol to azotaemic rats with secondary hyperparathyroidism could induce apoptosis of parathyroid cells [25], we found no evidence that high doses of calcitriol and hypercalcemia increased the rate of apoptosis in parathyroid tissue from normal or azotaemic rats.

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 5–10-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 0.2–0.3% [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.



   Acknowledgments
 
This work was supported by a grant from Fondo Nacional de Ciencias y Tecnología de Chile (FONDECYT No. 1960785). We are grateful to Mr Leonardo Andrade for assistance in immunohistochemical analysis and Ms Isabel Díaz for her expert secretarial assistance. Part of these data were presented at the 31st Annual Meeting of the American Society of Nephrology, 25–28 October, Philadelphia, USA, and published as an abstract (J Am Soc Nephrol 1998; 9: 566).



   Notes
 
Correspondence and offprint requests to: Dr Aquiles Jara, Department of Nephrology, Pontificia Universidad Católica de Chile, Marcoleta 345, Santiago, Chile. Back



   References
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 

  1. Felsenfeld AJ. Considerations for the treatment of secondary hyperparathyroidism in renal failure. J Am Soc Nephrol1997; 8: 993–1004[ISI][Medline]
  2. Parfitt AM. The hyperparathyroidism of chronic renal failure: A disorder of growth. Kidney Int1997; 52: 3–9[ISI][Medline]
  3. Drueke TB. Cell biology of parathyroid gland hyperplasia in chronic renal failure. J Am Soc Nephrol2000; 11: 1141–1152[Free Full Text]
  4. Ellis HA, Peart KM. Azotaemic renal osteodystrophy: a quantitative study on iliac bone. J Clin Pathol1973; 26: 83–101[ISI][Medline]
  5. Johnson WJ, McCarthy JT, van Heerden JA, Sterioff S, Grant CS, Kao PC. Results of subtotal parathyroidectomy in hemodialysis patients. Am J Med1988; 84: 23–32
  6. Malberti F, Farina M, Imbasciati E. The PTH–calcium curve and the set point of calcium in primary and secondary hyperparathyroidism. Nephrol Dial Transplant1999; 14: 2398–2406[Abstract/Free Full Text]
  7. Bover J, Rodriguez M, Trinidad P et al. Factors in the development of secondary hyperparathyroidism during graded renal failure in the rat. Kidney Int1994; 45: 953–961[ISI][Medline]
  8. Denda M, Finch J, Slatopolsky E. Phosphorus accelerates the development of parathyroid hyperplasia and secondary hyperparathyroidism in rats with renal failure. Am J Kidney Dis1996; 28: 596–602[ISI][Medline]
  9. Yi H, Fukagawa M, Yamato H, Kumagai M, Watanab T, Kurokawa K. Prevention of enhanced parathyroid hormone secretion, synthesis and hyperplasia by mild dietary phosphorus restriction in early chronic renal failure in rats: Possible direct role of phosphorus. Nephron1995; 70: 242–248[ISI][Medline]
  10. Miller MA, Chin J, Miller SC, Fox J. Disparate effects of mild, moderate and severe secondary hyperparathyroidism on cancellous and cortical bone in rats with chronic renal insufficiency. Bone1998; 23: 257–266[ISI][Medline]
  11. Silver J, Naveh-Many T, Mayer H, Schmelzer HJ, Popovtzer MM. Regulation by vitamin D metabolites of parathyroid hormone gene transcription in vivo in the rat. J Clin Invest1986; 78: 1296–1301[ISI][Medline]
  12. Szabo A, Merke J, Beier E, Mall G, Ritz E. 1,25(OH)2 Vitamin D3 inhibits parathyroid cell proliferation in experimental uremia. Kidney Int1989; 35: 1049–1056[ISI][Medline]
  13. Fukagawa M, Kaname S, Igarashi T, Ogata E, Kurokawa K. Regulation of parathyroid hormone synthesis in chronic renal failure in rats. Kidney Int1991; 39: 874–881[ISI][Medline]
  14. Hamdy NAT, Kanis JA, Beneton MNC et al. Effect of alfacalcidol on natural course of bone disease in mild to moderate renal failure. Br Med J1995; 310: 358–363[Abstract/Free Full Text]
  15. Ritz E, Küster S, Schmidt-Gayk H et al. Low-dose calcitriol prevents the rise in 1,84 iPTH without affecting serum calcium and phosphate in patients with moderate renal failure (prospective placebo-controlled multicentre trial). Nephrol Dial Transplant1995; 10: 2228–2234[Abstract]
  16. Slatopolsky E, Weerts C, Thielan J, Horst R, Harter H, Martin KJ. Marked suppression of secondary hyperparathyroidism by intravenous administration of 1,25-dihydroxy-cholecalciferol in uremic patients. J Clin Invest1984; 74: 2136–2143[ISI][Medline]
  17. Dunlay R, Rodriguez M, Felsenfeld A, Llach F. Direct inhibitory effect of calcitriol on parathyroid function (sigmoidal curve) in dialysis patients. Kidney Int1989; 36: 1093–1098[ISI][Medline]
  18. Rodriguez M, Caravaca F, Fernandez E et al. Parathyroid function as a determinant of the response to calcitriol treatment in the hemodialysis patient. Kidney Int1999; 56: 306–317[ISI][Medline]
  19. Silver J, Russell J, Sherwood LM. Regulation by vitamin D metabolites of messenger ribonucleic acid for preproparathyroid hormone in isolated bovine parathyroid cells. Proc Natl Acad Sci USA1985; 82: 4270–4273[Abstract]
  20. Kremer R, Bolivar I, Goltzman D, Hendy GN. Influence of calcium and 1,25-dihydroxycholecalciferol on proliferation and proto-oncogene expression in primary cultures of bovine parathyroid cells. Endocrinology1989; 125: 935–941[Abstract]
  21. Canalejo A, Almaden Y, Torregrosa V et al. The in vitro effect of calcitriol on parathyroid cell proliferation and apoptosis. J Am Soc Nephrol2000; 11: 1865–1872[Abstract/Free Full Text]
  22. Fukagawa M, Okazaki R, Takano K et al. Regression of parathyroid hyperplasia by calcitriol-pulse therapy in patients on long-term dialysis. N Engl J Med1990; 323: 421–422[ISI][Medline]
  23. Fukagawa M, Kitaoka M, Yi H et al. Serial evaluation of parathyroid size by ultrasonography is another useful marker for the long-term prognosis of calcitriol pulse therapy in chronic dialysis patients. Nephron1994; 68: 221–228[ISI][Medline]
  24. Drueke TB, Zhang P, Gogusev J. Apoptosis: background and possible role in secondary hyperparathyroidism. Nephrol Dial Transplant1997; 12: 2228–2233[Free Full Text]
  25. Fukagawa M, Yi H, Kurokawa K. Calcitriol induces apoptosis of hyperplastic parathyroid cells in uremic rats. J Am Soc Nephrol1991; 2: 635 (Abstract)
  26. Naveh-Many T, Rahamimov R, Livni N, Silver J. Parathyroid cell proliferation in normal and chronic renal failure rats: The effects of calcium, phosphate, and vitamin D. Clin Invest1995; 96: 1786–1793[ISI][Medline]
  27. Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, New York, 1989
  28. Jara A, Bover J, Lavigne J, Felsenfeld AJ. A comparison of two parathyroid hormone assays for the rat: The new immunoradiometric with the competitive-binding assay. J Bone Miner Res1994; 9: 1629–1633[ISI][Medline]
  29. Thompson EB. Apoptosis. Annu Rev Physiol1998; 60: 525–532[ISI][Medline]
  30. Wada M, Furuya Y, Sakiyama J et al. The calcimimetic compound NPS R-568 suppresses parathyroid cell proliferation in rats with renal insufficiency. J Clin Invest1997; 100: 2977–2983[Abstract/Free Full Text]
  31. Sugenoya Y, Yoshimura A, Uda K et al. Evidence of apoptosis-preventing Bcl-2 and its counteractive Bax is associated with cell proliferation in secondary hyperparathyroidism. J Am Soc Nephrol1996; 7: 1499 (Abstract)
  32. Zhang P, Duchambon P, Gogusev J et al. Apoptosis in parathyroid hyperlasia of patients with primary or secondary uremic hyperparathyroidism. Kidney Int2000; 57: 437–445[ISI][Medline]
  33. Wang W, Johansson H, Kvasnicka T, Farnebo L, Grimelius L. Detection of apoptotic cells and expression of Ki-67 antigen, Bcl-2, p53 oncoproteins in human parathyroid adenoma. APMIS1996; 104: 789–796[ISI][Medline]
  34. Zhang P, Gogusev J, Duchambon P, Sarfati E, Drueke T. Apoptosis in patients with primary and secondary hyperparathyroidism. J Am Soc Nephrol1996; 7: 1504 (Abstract)
  35. Wada M, Nagano N, Furuya Y, Chin J, Nemeth EF, Fox J. Calcimimetic NPS R-568 prevents parathyroid hyperplasia in rats with severe secondary hyperparathyroidism. Kidney Int2000; 57: 50–58[ISI][Medline]
  36. Chin J, Miller SC, Wada M, Nagano N, Nemeth EF, Fox J. Activation of the calcium receptor by a calcimimetic compound halts the progression of secondary hyperparathyroidism in uremic rats. J Am Soc Nephrol2000; 11: 903–911[Abstract/Free Full Text]
  37. Wang Q, Palnitkar S, Parfitt AM. Parathyroid cell proliferation in the rat: Effect of age and of phosphate administration and recovery. Endocrinology1996; 137: 4558–4562[Abstract]
  38. Quarles LD, Yohay DA, Carroll BA, Spritzer CE, Minda SA, Lobaugh BL. Prospective trial of pulse oral versus intravenous calcitriol treatment of hyperparathyroidism in ESRD. Kidney Int1994; 45: 1710–1721[ISI][Medline]
  39. Cannella G, Bonucci E, Rolla D et al. Evidence of healing of secondary hyperparathyroidism in chronically hemodialyzed uremic patients treated with long-term intravenous calcitriol. Kidney Int1994; 46: 1124–1132[ISI][Medline]
  40. Chudek J, Kokot F, Witkowicz J et al. No marked apoptosis of parathyroid cells after intraparathyroid injections of Calcijex—observation in a patient with tertiary hyperparathyroidism after successful renal transplantation. Nephrol Dial Transplant2000; 15: 424–425[Free Full Text]
  41. Henry HL, Taylor AN, Norman AW. Response of chick parathyroid glands to the vitamin D metabolites, 1,25-dihydroxycholecalciferol and 24,25-dihydroxycholecalciferol. J Nutr1977; 107: 1918–1926[ISI][Medline]
  42. Fukuda N, Tanaka H, Tominaga Y, Fukagawa M, Kurokawa K, Seino Y. Decreased 1,25-dihydroxyvitamin D3 receptor density is associated with a more severe form of parathyroid hyperplasia in chronic uremic patients. J Clin Invest1993; 92: 1436–1443[ISI][Medline]
  43. Brown AJ, Dusso A, Slatopolsky E. Vitamin D. Am J Physiol1999; 277: F157–175[Abstract/Free Full Text]
Received for publication: 2.12.99
Revision received 4. 9.00.