Skip Navigation



NDT Advance Access published online on June 13, 2008
Nephrology Dialysis Transplantation, doi:10.1093/ndt/gfn338
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
23/11/3479    most recent
gfn338v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Carrillo-López, N.
Right arrow Articles by Cannata-Andía, J. B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Carrillo-López, N.
Right arrow Articles by Cannata-Andía, J. B.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

© The Author [2008]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org


Simultaneous changes in the calcium-sensing receptor and the vitamin D receptor under the influence of calcium and calcitriol

Natalia Carrillo-López1, Daniel Álvarez-Hernández1, Ignacio González-Suárez1, Pablo Román-García1, Jose M. Valdivielso2, José Luis Fernández-Martín1 and Jorge B. Cannata-Andía1

1 Bone and Mineral Research Unit, Instituto Reina Sofía de Investigación, Hospital Universitario Central de Asturias, REDinREN del ISCIII, Universidad de Oviedo, Oviedo, Asturias 2 Experimental Nephrology Laboratory, Hospital Universitario Arnau de Vilanova, IRBLLEIDA, REDinREN del ISCIII, Lleida, Spain

Correspondence and offprint requests to: Jorge B. Cannata-Andía, Bone and Mineral Research Unit, Instituto Reina Sofía de Investigación, Hospital Universitario Central de Asturias, C/Julián Clavería s/n, 33006 Oviedo, Asturias, Spain. Tel: +34-985106137; Fax: +34-985106142; E-mail: metoseo{at}hca.es



   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Background. The regulatory mechanisms of parathyroid hormone (PTH) synthesis are complex, involving calcium, calcitriol, the calcium-sensing receptor (CaR) and the vitamin D receptor (VDR). In this study, the effects of calcium and calcitriol on the simultaneous expression of CaR and VDR mRNA and protein levels were assessed in parathyroid glands cultured in vitro.

Methods. Parathyroid glands (N = 424) were removed and cultured for 24 h to study the effect of calcium on the CaR, VDR and PTH. In addition, the effect of calcitriol at low calcium concentrations (0.6 mM) on CaR and VDR levels was studied after 48 h of incubation. CaR, VDR and PTH mRNAs were measured by quantitative real-time PCR (qRT-PCR), and CaR and VDR protein levels were measured by immunohistochemistry.

Results. PTH gene expression was reduced by high calcium concentration. No differences were found in the CaR mRNA levels among the different calcium concentrations tested (0.6 mM calcium: 100%; 1.2 mM calcium: 120%; 2.0 mM calcium: 112%; median values), but VDR gene expression rose when calcium increased (0.6 mM calcium: 100%; 1.2 mM calcium: 164%; 2.0 mM calcium: 195%; median values). Calcitriol increased both CaR (control: 100%; 10–8 M calcitriol: 196%; median values) and VDR genes expression (control: 100%; 10–8 M calcitriol: 176%; median values). The same findings were corroborated at protein levels for both CaR and VDR.

Conclusions. In parathyroid glands cultured in vitro, calcium up-regulates VDR but not CaR. Conversely, calcitriol up-regulates both VDR and CaR mRNAs and protein levels, even at low calcium concentrations.

Keywords: calcitriol; calcium; CaR; VDR



   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Calcium and vitamin D are two important factors involved in the regulation of parathyroid hormone (PTH) synthesis and secretion acting in the parathyroid cell through their specific receptors: calcium-sensing receptor (CaR) and vitamin D receptor (VDR). While the CaR is a cell-membrane receptor and a member of the G-protein-coupled receptor family, the VDR is a nuclear receptor that, when bound to vitamin D, acts as a transcription factor. The differences in the nature of the two ligands and their receptors lead to two different mechanisms of action with a complementary function on the parathyroid cells.

On one hand, small decreases in extracellular calcium concentrations are rapidly sensed by the CaR, triggering an increase of PTH release within seconds or minutes. Small increases in calcium are also sensed by the CaR yielding opposite results [1–3]. On the other hand, vitamin D acts through a slower mechanism. Vitamin D enters into the parathyroid cell and binds the VDR. Then, the activated VDR translocates to the nucleus where the complex represses PTH gene transcription and therefore PTH synthesis [4–6].

PTH regulation is more complex; if low calcium concentration persists for periods longer than seconds or minutes, PTH gene transcription is up-regulated and parathyroid cell proliferation increases [7]. In contrast, calcitriol is a well-known parathyroid cell proliferation inhibitor [8].

Finally, calcium and calcitriol may modulate parathyroid cell function by regulating both CaR and VDR expression. There could be indirect actions that may depend on the interrelationship of calcium, calcitriol, CaR and VDR contributing to the regulation of the gland that is still not completely understood. Several papers have concentrated on this likely regulation; however, most of them have been performed in vivo [9–12], where it is hard to ascertain the specific role of each modulator since it is difficult to keep all of the previous factors under control [11,12]. The effect of calcium on the regulation of the VDR has been already assessed both in vitro and in vivo [13]; nevertheless, the in vitro studies were carried out in very short term periods. In addition, the effect of calcium and calcitriol on their own receptors regulation has been studied only in vivo [9–13]. Furthermore, the different studies concerning the effect of calcitriol on CaR regulation have shown contradictory results [11,12].

In this study we aimed to assess the long-term effect of calcium and calcitriol on CaR and VDR simultaneous expression in intact rat parathyroid glands cultured in vitro.



   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Parathyroid tissue culture
Parathyroid glands were obtained from 3-month-old male Wistar rats (n = 212), with normal renal function, as previously described [14]. The parathyroid glands were randomly placed in groups of eight glands into a nylon basket containing a permeable membrane of 12 µm pore size, physically isolating the glands from the bottom of the well (Transwell Costar, Corning, NY, USA). The glands were constantly shaken (AOS-0 SBS Instruments SA, Badalona, Spain) at 37°C with 2 mL of a standard medium containing 125 mmol/L NaCl, 5.9 mmol/L KCl, 1.2 mmol/L MgCl2, 1 mmol/L sodium pyruvate, 4 mmol/L L-glutamine, 12 mmol/L D-glucose, 25 mmol/L sodium Hepes, 0.1 U/mL insulin, 0.1% bovine serum albumin (BSA), 100 U/mL penicillin, 100 µg/mL streptomycin sulphate, 1 mmol/L phosphate (added as NaH2PO4 and Na2HPO4 in 1:2 proportions) and 0.6 mM CaCl2, buffered at pH 7.4 (all reactives from Sigma-Aldrich Co., St Louis, MO, USA). The University of Oviedo Ethics Committee for the use of laboratory animals approved this study.

In the calcium experiments, three different calcium concentrations were used (0.6, 1.2 and 2.0 mM). Culture media with 1.2 mM and 2.0 mM calcium were prepared from the standard medium adding CaCl2, to obtain the desired concentration. In the calcitriol experiments, 10–8 M calcitriol (Sigma-Aldrich) or vehicle was added to the standard medium. The glands were washed for 8 h in the standard medium changing the medium every 2 h. Then the parathyroid glands were cultured for an additional 24 h for calcium and 48 h for calcitriol experiments, changing the culture medium every 12 h. After the culture period, the glands were gathered and submerged in an RNAse inhibitor solution (RNAlater® Tissue Solution, Ambion, Austin, TX, USA) and frozen at –70°C until their study.

To assess the effect of calcium, 11 independent experiments were performed, using a total amount of 264 parathyroid glands. Each experiment consisted of 24 glands that once removed from the rats were randomly divided into three different groups. Every group was cultured for 24 h after the washing period in different culture media containing different calcium concentrations (0.6, 1.2 and 2.0 mM). To assess the effect of calcitriol, 10 independent experiments were performed using 160 parathyroid glands. Each experiment consisted of 16 glands that once removed were divided into two groups and cultured in the standard medium for 48 h after the washing period, one group with calcitriol 10–8 M and the other with vehicle.

Cell viability was measured at the beginning and at the end of the culture period by flow cytometry (FACScan, Becton-Dickinson, Franklin Lakes, NJ, USA) as previously described [14], always showing values >85%.

Phosphorus, calcium and pH, measured in the culture medium at the beginning and at the end of all experiments, remained stable during both the 24- and 48-h periods in culture. Phosphorus was measured by routine colorimetric assay, and ionized calcium and pH were measured with a calcium-selective electrode (Ciba Corning®).

RNA extraction
Total RNA was extracted from every pool of parathyroid glands with a TRITM reagent (Sigma-Aldrich). Despite the fact that parathyroid glands in culture maintain their functionality up to 4 days [14], RNA degradation might occur, and thus we ensured that every pool of glands used had highly pure total RNA (A260/A280 > 1.9). For this reason, four calcium and three calcitriol experiments were discarded, since the RNA of at least one pool of glands of each experiment did not satisfy the purity criteria.

Reverse transcription
The cDNA was obtained from 1 µg of total RNA and reverse-transcribed using SuperScriptTM reverse transcriptase (InvitrogenTM, Carlbad, CA, USA) following the manufacturer's instructions.

Real-time PCR
Quantitative real-time PCR (qRT-PCR) was performed on an ABI Prism 7000 Sequence Detection System (PE Applied Biosystems, Foster City, CA, USA) using TaqMan® Universal PCR Master Mix (PE Applied Biosystems). PTH, CaR, VDR genes and endogenous control (18s) were analysed using TaqMan® pre-developed assay reagents (TaqMan® Gene Expression Assays-On-Demand Rn 00566882_m1, Rn 00566496_m1, Rn 00566976_m1 and Eukaryotic 18s rRNA Endogenous Control Reagent, respectively) (PE Applied Biosystems). All reactions (in triplicate) were performed by amplifying endogenous and target genes in the same plate. To check reaction sensitivity, serial dilutions of cDNA (1/2, 1/4 and 1/10) were used to amplify endogenous and target genes. The absolute value of the slope of log input amount versus {Delta}CT was <0.1 in all cases (PTH versus 18s: –0.0762, CaR versus 18s: 0.0112 and VDR versus 18s: –0.0915). Relative quantitative evaluation of target genes was performed by comparing threshold cycles using the {Delta}{Delta}CT method, as previously described [15,16].

Immunohistochemistry
The CaR and VDR were detected by immunohistochemistry in 5-µm-thick serial sections from paraffin-embedded parathyroid glands using specific antibodies and haematoxylin counterstaining (Dako RealTM EnVisionTM Detection System, Peroxidase/DAB+, Rabbit/Mouse, Dako, Carpinteria, CA, USA, and rat ABC Staining System, Santa Cruz Biotechnology, CA, USA). The CaR was detected using a rabbit polyclonal antibody against a 23-amino-acid peptide within the extracellular domain (residues 203–226) [17]. The VDR was detected using a rat monoclonal antibody against the C-terminal of the DNA binding zinc finger domain (residues 89–105) (Chemicon-Millipore, Billerica, MA, USA). All the samples were processed at the same time in order to keep all the possible variables of the immunostaining procedure constant.

Statistical analysis
Statistical analysis was performed using SPSS 12.0 (SPSS Inc., Chicago, IL, USA) and comparisons were performed using non-parametric tests (Wilcoxon). Significant differences were considered when P < 0.05.



   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Effect of calcium on PTH gene expression
Parathyroid glands cultured for 24 h in a medium containing 1.2 mM calcium showed a significantly reduced PTH gene expression. The presence of a higher calcium concentration (2.0 mM) did not show a further decrease in PTH mRNA levels (Figure 1).


Figure 1
View larger version (17K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 1 PTH mRNA levels measured by qRT-PCR in parathyroid glands cultured in vitro for 24 h with different calcium concentrations (0.6, 1.2 or 2.0 mM). Median and mean ± standard error of six independent experiments are shown. *P < 0.05, compared to the reference group (0.6 mM Ca).

 
Effect of calcium on CaR and VDR gene expression and protein levels
The parathyroid gland CaR gene expression was similar after 24 h in culture among the three different calcium concentrations tested (0.6, 1.2 and 2.0 mM) (Figure 2A). In contrast, VDR gene expression increased when the calcium concentration in the medium increased, achieving statistical signification with 2.0 mM of calcium (Figure 2B).


Figure 2
View larger version (14K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 2 Effect of calcium on CaR and VDR gene expression in parathyroid glands after 24 h in culture. (A) CaR mRNA levels measured by qRT-PCR in parathyroid glands cultured in 0.6, 1.2 or 2.0 mM calcium. (B) VDR mRNA levels in the same three groups. Median and mean ± standard error of six independent experiments are shown. *P < 0.05, compared to the reference group (0.6 mM Ca). ° represents outlier values.

 
The results of gene expression were corroborated at a protein level. No differences were observed in CaR levels detected by immunohistochemistry among the three different calcium concentrations (Figure 3A, B, C), whereas VDR protein levels increased when parathyroid glands were cultured in higher calcium concentrations (Figure 3E, F, G).


Figure 3
View larger version (74K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 3 Effect of calcium on CaR and VDR protein levels in parathyroid glands after 24 h in culture. (A), (B), (C) Photomicrographies of representative immunohistochemical staining of the CaR in parathyroid glands cultured with 0.6 mM, 1.2 mM or 2.0 mM calcium, respectively. (D) Negative control for the CaR protein in a representative parathyroid gland. (E), (F), (G) Representative immunohistochemical staining for the VDR in parathyroid glands cultured with 0.6 mM, 1.2 mM or 2.0 mM calcium, respectively. (H) Negative control for the VDR protein in a representative parathyroid gland. Haematoxylin counterstaining (magnification x20).

 
Effect of calcitriol on VDR and CaR genes expression and protein levels
Both VDR and CaR genes significantly increased their expression in parathyroid glands after 48 h in culture with calcitriol (10–8 M) (Figure 4). The same effect was found when VDR and CaR protein levels were analysed by immunohistochemistry in parathyroid glands cultured with calcitriol 10–8 M (Figure 5).


Figure 4
View larger version (11K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 4 Effect of calcitriol on CaR and VDR gene expression in parathyroid glands after 48 h in culture. (A) CaR mRNA levels measured by qRT-PCR in parathyroid glands cultured with or without calcitriol 10–8 M. (B) VDR mRNA levels in the same two groups. Median and mean ± standard error of six independent experiments are shown. *P < 0.05, compared to the reference group (control group without calcitriol).

 

Figure 5
View larger version (83K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 5 Effect of calcitriol on CaR and VDR protein levels in parathyroid glands after 48 h in culture. (A), (B) Photomicrographies of representative immunohistochemical staining of the CaR in parathyroid glands cultured with vehicle or 10–8 M calcitriol, respectively. (C) Negative control for the CaR protein in a representative parathyroid gland. (D), (E) Representative immunohistochemical staining for the VDR in parathyroid glands cultured with vehicle or 10–8 M calcitriol, respectively. (F) Negative control for the VDR protein in a representative parathyroid gland. Haematoxylin counterstaining (magnification x20).

 


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
The purpose of this study was to assess the effect of calcium and calcitriol on the simultaneous expression of CaR and VDR mRNAs and protein levels in parathyroid glands cultured in vitro. On one hand, the increase in calcium concentration in the culture medium up-regulated VDR expression but did not modify CaR expression. On the other hand, calcitriol up-regulated not only VDR expression but also CaR expression.

The study was carried out using a long-term culture model of parathyroid tissue previously set up in our laboratory [14]. We demonstrated that under certain conditions parathyroid tissue is maintained viable and functional up to 4 days in culture [14]. However, based on the previous published results, we considered that 24 h in culture for calcium and 48 h in culture for calcitriol experiments were the most adequate times for the study. We demonstrated that incubations of up to 24 h were necessary to reach a stable PTH secretion and to obtain an adequate response to calcium [14,18], although prolonged stimuli with high calcium concentrations could trigger a protective mechanism against the CaR long-term activation [19]. Additionally, we also demonstrated that incubations of up to 48 h in culture with calcitriol were necessary to observe a significant decrease of PTH synthesis and secretion [14,18].

In the present study we took a step forward, demonstrating that calcium concentrations of 1.2 mM decreased not only PTH secretion but also PTH synthesis measured by qRT-PCR after 24 h in culture, showing no further reduction if the calcium concentration was increased up to 2.0 mM (Figure 1). Similar results had been previously reported both in vitro, after shorter periods of culture (6 h) [20], and in vivo [21,22].

The reduction of PTH mRNA observed when calcium concentration in the culture media increased cannot be explained by changes in CaR gene expression, since CaR mRNA levels did not show significant differences after doubling and even triplicating the calcium concentration in the medium (Figure 2A). Thus, the PTH synthesis inhibition observed (Figure 1) must be due to the CaR activation but not to an increase in CaR gene expression. Similarly, previous in vivo studies had also found no changes in CaR mRNA in rats fed with a diet containing different contents of calcium, suggesting, like in our study, a weak or even inexistent regulatory effect of extracellular calcium on CaR expression [11,12]. However, in previous in vivo studies the other biochemical parameters (calcitriol, phosphorous) varied under different conditions, while in our study only calcium changed in the different experimental groups. This lack of the effect of calcium on CaR expression was also corroborated at a protein level (Figure 3A, B, C).

In contrast, our results demonstrate that, despite the fact that VDR is the specific receptor for calcitriol and other active vitamin D metabolites, parathyroid glands’ VDR gene expression is modulated by calcium, showing that the higher the calcium, the higher VDR mRNA (Figure 2B) and the protein levels (Figure 3E, F, G). This effect had been previously described both in vivo [10,13] and in vitro [13] but using very short incubation periods compared to those used in our study.

The second aim of this study was to investigate the effect that calcitriol exerts simultaneously on VDR and CaR expression. We have demonstrated for the first time in an in vitro model that calcitriol up-regulates not only mRNA (Figure 4B) but also protein levels of its own receptor (Figure 5D, E). This effect had been previously described in vivo in both clinical and experimental studies using calcitriol [9,10,23,24] or maxacalcitol [25,26]. The in vitro effect of calcitriol had been previously observed in parathyroid glands cultured only for 6 h and with high calcium concentrations [13], while our results showed for the first time that calcitriol increased VDR levels after a more prolonged period of 48 h of culture even with low calcium concentrations (0.6 mM). According to the previously discussed VDR results obtained with low calcium, this effect cannot be attributed to calcium but to calcitriol, since the control and the calcitriol groups were both cultured with low calcium concentration.

Furthermore, we found that calcitriol, besides its effect on VDR, increased CaR gene expression, showing higher mRNA (Figure 4A) and CaR protein levels (Figure 5A, B). These results are in agreement with previous in vivo findings in parathyroid and kidney tissues [11,27] and also with a recent study describing vitamin D responsive elements in the promoters of the CaR gene [28], and they support the hypothesis that calcitriol up-regulates CaR gene expression, even at low calcium concentrations (0.6 mM) (Figure 4A). This in vitro effect of calcitriol on CaR and VDR expression has not been studied previously at the same time as it has been carried out in our study. In addition, in our experiments, the simultaneous measurements of CaR and VDR mRNAs and the protein levels have been studied during longer periods of culture (24 and 48 h) compared with the previous very short term culture period (up to 6 h) carried out measuring only the VDR [13].

Parathyroid cell proliferation and/or hyperplasia have been reported to be associated with a decrease in CaR [17] and VDR [24] expression by several authors. Moreover, some authors had speculated that parathyroid cell hyperplasia precedes down-regulation of CaR expression in the uraemic rat model [29]. Although after 24 or 48 h of culture it is not expected to find changes in cell proliferation, the parathyroid glands were immunostained for proliferating cell nuclear antigen. We could not find any proliferating parathyroid cell at any of the different calcium and calcitriol concentrations used (data not shown). This lack of proliferation activity is not surprising since the non-hyperplasic parathyroid tissue has a very low proliferation rate [30–32].

In summary, calcium seems to up-regulate VDR but not CaR mRNA and protein levels in parathyroid glands cultured in vitro for longer periods than previous studies. In contrast, calcitriol up-regulates both VDR and CaR levels even in the presence of low calcium concentrations.



   Acknowledgments
 
The authors wish to thank Dr Alex J. Brown and Dr Eduardo Slatopolsky for providing the CaR antibody and Dr Primitiva Menéndez for her assistance in the immunohistochemical analyses. They also thank Dr Manuel Naves-Díaz for his critical review of this manuscript and Marino Santirso for the language review. This work was supported by Fondo de Investigaciones Sanitarias (FIS 02/0688), ISCIII-Retic-RD06, REDinREN (16/06) and Fundación Renal Íñigo Álvarez de Toledo. DAH was supported by FIS (BEFI 03/00099) and NCL was supported by FICYT (IB05-060, 01/3139) and ISCIII-Retic-RD06, REDinREN (16/06).

Conflict of interest statement. None of the authors declare any conflict of interests. This work has not been published before except in abstract form.



   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

  1. Brown EM, Gamba G, Riccardi D, et al. Cloning and characterization of an extracellular Ca(2+)-sensing receptor from bovine parathyroid. Nature (1993) 366:575–580.[CrossRef][Medline]
  2. Silver J, Levi R. Regulation of PTH synthesis and secretion relevant to the management of secondary hyperparathyroidism in chronic kidney disease. Kidney Int Suppl (2005) S8–S12.
  3. Slatopolsky E. The role of calcium, phosphorus and vitamin D metabolism in the development of secondary hyperparathyroidism. Nephrol Dial Transplant (1998) 13(Suppl_3):3–8.[Abstract/Free Full Text]
  4. Pike JW. Vitamin D: receptors and the mechanism of action of 1,25-dihydroxyvitamin D3. In: Vitamin Receptors: Vitamins as Ligands in Cell Communication—Dakshinamurti K, ed. (1994) New York: Cambridge University Press. 59–77.
  5. Goodman WG. The flavors of vitamin D: tasting the molecular mechanisms. Kidney Int (2004) 66:1286–1287.[CrossRef][Web of Science][Medline]
  6. Naveh-Many T, Silver J. Regulation of parathyroid hormone gene expression and secretion by vitamin D. In: Vitamin D: Physiology, Molecular Biology and Clinical Applications—Holick MF, ed. (1998) Totowa: Humana Press. 217–237.
  7. Naveh-Many T, Rahamimov R, Livni N, et al. Parathyroid cell proliferation in normal and chronic renal failure rats. The effects of calcium, phosphate, and vitamin D. J Clin Invest (1995) 96:1786–1793.[Web of Science][Medline]
  8. Canalejo A, Almaden Y, Torregrosa V, et al. The in vitro effect of calcitriol on parathyroid cell proliferation and apoptosis. J Am Soc Nephrol (2000) 11:1865–1872.[Abstract/Free Full Text]
  9. Brown AJ, Zhong M, Finch J, et al. The roles of calcium and 1,25-dihydroxyvitamin D3 in the regulation of vitamin D receptor expression by rat parathyroid glands. Endocrinology (1995) 136:1419–1425.[Abstract]
  10. Russell J, Bar A, Sherwood LM, et al. Interaction between calcium and 1,25-dihydroxyvitamin D3 in the regulation of preproparathyroid hormone and vitamin D receptor messenger ribonucleic acid in avian parathyroids. Endocrinology (1993) 132:2639–2644.[Abstract/Free Full Text]
  11. Brown AJ, Zhong M, Finch J, et al. Rat calcium-sensing receptor is regulated by vitamin D but not by calcium. Am J Physiol (1996) 270:F454–F460.[Web of Science][Medline]
  12. Rogers KV, Dunn CK, Conklin RL, et al. Calcium receptor messenger ribonucleic acid levels in the parathyroid glands and kidney of vitamin D-deficient rats are not regulated by plasma calcium or 1,25-dihydroxyvitamin D3. Endocrinology (1995) 136:499–504.[Abstract]
  13. Garfia B, Canadillas S, Canalejo A, et al. Regulation of parathyroid vitamin D receptor expression by extracellular calcium. J Am Soc Nephrol (2002) 13:2945–2952.[Abstract/Free Full Text]
  14. Alvarez-Hernandez D, Gonzalez-Suarez I, Naves M, et al. Long-term response of cultured rat parathyroid glands to calcium and calcitriol: the effect of cryopreservation. J Nephrol (2005) 18:141–147.[Web of Science][Medline]
  15. Ginzinger DG. Gene quantification using real-time quantitative PCR: an emerging technology hits the mainstream. Exp Hematol (2002) 30:503–512.[CrossRef][Web of Science][Medline]
  16. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods (2001) 25:402–408.[CrossRef][Web of Science][Medline]
  17. Brown AJ, Ritter CS, Finch JL, et al. Decreased calcium-sensing receptor expression in hyperplastic parathyroid glands of uremic rats: role of dietary phosphate. Kidney Int (1999) 55:1284–1292.[CrossRef][Web of Science][Medline]
  18. Gonzalez-Suarez I, Alvarez-Hernandez D, Carrillo-Lopez N, et al. Aluminum posttranscriptional regulation of parathyroid hormone synthesis: a role for the calcium-sensing receptor. Kidney Int (2005) 68:2484–2496.[CrossRef][Web of Science][Medline]
  19. Kifor O, Kifor I, Moore FD Jr. m-Calpain colocalizes with the calcium-sensing receptor (CaR) in caveolae in parathyroid cells and participates in degradation of the CaR. J Biol Chem (2003) 278:31167–31176.[Abstract/Free Full Text]
  20. Rodriguez M, Almaden Y, Canadillas S, et al. The calcimimetic R-568 increases vitamin D receptor expression in rat parathyroid glands. Am J Physiol Renal Physiol (2007) 292:F1390–1395.[Abstract/Free Full Text]
  21. Yamamoto M, Igarashi T, Muramatsu M, et al. Hypocalcemia increases and hypercalcemia decreases the steady-state level of parathyroid hormone messenger RNA in the rat. J Clin Invest (1989) 83:1053–1056.[Web of Science][Medline]
  22. Naveh-Many T, Silver J. Regulation of parathyroid hormone gene expression by hypocalcemia, hypercalcemia, and vitamin D in the rat. J Clin Invest (1990) 86:1313–1319.[Web of Science][Medline]
  23. Naveh-Many T, Marx R, Keshet E, et al. Regulation of 1,25-dihydroxyvitamin D3 receptor gene expression by 1,25-dihydroxyvitamin D3 in the parathyroid in vivo. J Clin Invest (1990) 86:1968–1975.[Web of Science][Medline]
  24. Fukuda N, Tanaka H, Tominaga Y, et al. Decreased 1,25-dihydroxyvitamin D3 receptor density is associated with a more severe form of parathyroid hyperplasia in chronic uremic patients. J Clin Invest (1993) 92:1436–1443.[Web of Science][Medline]
  25. Shiizaki K, Negi S, Hatamura I, et al. Biochemical and cellular effects of direct maxacalcitol injection into parathyroid gland in uremic rats. J Am Soc Nephrol (2005) 16:97–108.[Abstract/Free Full Text]
  26. Shiizaki K, Fukagawa M, Yuan Q, et al. Direct injection of calcitriol or its analog improves abnormal gene expression in the hyperplastic parathyroid gland in uremia. Am J Nephrol (2008) 28:59–66.[CrossRef][Web of Science][Medline]
  27. Yao JJ, Bai S, Karnauskas AJ, et al. Regulation of renal calcium receptor gene expression by 1,25-dihydroxyvitamin D3 in genetic hypercalciuric stone-forming rats. J Am Soc Nephrol (2005) 16:1300–1308.[Abstract/Free Full Text]
  28. Canaff L, Hendy GN. Human calcium-sensing receptor gene: vitamin D response elements in promoters P1 and P2 confer transcriptional responsiveness to 1,25-dihydroxyvitamin D. J Biol Chem (2002) 277:30337–30350.[Abstract/Free Full Text]
  29. Ritter CS, Finch JL, Slatopolsky EA, et al. Parathyroid hyperplasia in uremic rats precedes down-regulation of the calcium receptor. Kidney Int (2001) 60:1737–1744.[CrossRef][Web of Science][Medline]
  30. Naccarato AG, Marcocci C, Miccoli P, et al. Bcl-2, p53 and MIB-1 expression in normal and neoplastic parathyroid tissues. J Endocrinol Invest (1998) 21:136–141.[Web of Science][Medline]
  31. Loda M, Lipman J, Cukor B, et al. Nodular foci in parathyroid adenomas and hyperplasias: an immunohistochemical analysis of proliferative activity. Hum Pathol (1994) 25:1050–1056.[CrossRef][Web of Science][Medline]
  32. Wang Q, Palnitkar S, Parfitt AM. The basal rate of cell proliferation in normal human parathyroid tissue: implications for the pathogenesis of hyperparathyroidism. Clin Endocrinol (Oxf) (1997) 46:343–349.[CrossRef][Medline]
Received for publication: 5. 2.08
Accepted in revised form: 22. 5.08


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 Am. J. Physiol. Renal Physiol.Home page
T. Meir, R. Levi, L. Lieben, S. Libutti, G. Carmeliet, R. Bouillon, J. Silver, and T. Naveh-Many
Deletion of the vitamin D receptor specifically in the parathyroid demonstrates a limited role for the receptor in parathyroid physiology
Am J Physiol Renal Physiol, November 1, 2009; 297(5): F1192 - F1198.
[Abstract] [Full Text] [PDF]

Home page
 J. Am. Soc. Nephrol.Home page
N. Carrillo-Lopez, P. Roman-Garcia, A. Rodriguez-Rebollar, J. L. Fernandez-Martin, M. Naves-Diaz, and J. B. Cannata-Andia
Indirect Regulation of PTH by Estrogens May Require FGF23
J. Am. Soc. Nephrol., September 1, 2009; 20(9): 2009 - 2017.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
23/11/3479    most recent
gfn338v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Carrillo-López, N.
Right arrow Articles by Cannata-Andía, J. B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Carrillo-López, N.
Right arrow Articles by Cannata-Andía, J. B.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?