Excess secretion of parathyroid hormone (PTH) and enlargement of the parathyroid glands (PTGs) are hallmarks of secondary hyperparathyroidism (2°HPT), a complication frequently associated with chronic renal failure. While much has been learned in the past decade about the control of PTH secretion, the importance of regulation of parathyroid gland size has recently gained more attention. The systemic factors involved in the pathogenesis of parathyroid hyperplasia include decreases in serum calcium and 1,25-dihydroxyvitamin D3, and an increase in serum phosphorus. Down-regulation of both the calcium-sensing receptor (CaR) and the vitamin D receptor (VDR) in hyperplastic PTGs of uremic patients have been reported, and also may be involved in the pathogenesis of parathyroid hyperplasia1,2,3,4,5,6.
We recently have examined the association between CaR expression and parathyroid cell proliferation in a uremic rat model7. We found that down-regulation of the CaR occurred in chronic renal failure, but only when the PTGs were hyperplastic. Restriction of dietary phosphate, which inhibits parathyroid cell hyperplasia, prevented down-regulation of the CaR. In addition, areas of decreased CaR protein expression were associated with areas of parathyroid cell proliferation. This association between CaR expression and parathyroid cell proliferation also has been demonstrated in vitro. We and others have shown that the rapid loss of calcium response in monolayer cultures of proliferating bovine parathyroid cells is associated with a dramatic decrease in the expression of CaR mRNA and protein8,9. The speculation that down-regulation of the CaR could be a function of the proliferative or dedifferentiated state of hyperplastic parathyroid cells is supported by Nygren et al, who found that proliferating bovine parathyroid monolayers exhibited a decreased suppressibility of PTH by calcium, whereas non-proliferating cells retained much of their ability to regulate secretion of PTH10.
Although an association between CaR down-regulation and parathyroid hyperplasia is apparent, the full nature of their temporal relationship is unclear. In essence, does the decline in CaR content occur as an early, perhaps initiating, event in the development of parathyroid hyperplasia, or does it occur subsequently to parathyroid cell proliferation or the conditions that promote it? The current study used a uremic rat model to examine the onset of parathyroid gland enlargement in relation to the time course of the fall in parathyroid CaR expression.
METHODS
Animals and diets
Renal insufficiency was induced in female Sprague-Dawley rats by 5/6 nephrectomy. This procedure involves ligation of most of the branches of the left renal artery followed by right nephrectomy. In protocol 1, rats made uremic were placed immediately on a high phosphate diet (HP; 0.9% P, 0.6% Ca) that promotes secondary hyperparathyroidism, and then studied at 1, 2, 4 and 7 days. In protocol 2, rats made uremic were placed for 1 week on a low phosphate diet (LP; 0.2% P, 0.5% Ca) that inhibits development of secondary hyperparathyroidism, and then switched to a HP diet (1.2% P, 0.5% Ca) for 0, 1, 2, 3, 4, 7 and 14 days. Control (intact) rats in each experiment were fed a normal chow diet.
At each time point, the animals were exsanguinated by aortic puncture and the blood analyzed for clinical determinations. The PTGs were weighed and fixed in 10% formalin for subsequent immunostaining. Diets were obtained from Dyets, Inc (Bethlehem, PA, USA). The Animal Studies Committee of Washington University approved all animal protocols.
Clinical determinations
Plasma ionized calcium was measured using a specified electrode (Model ICA-1; Radiometer, Copenhagen, Denmark). Serum creatinine and phosphorus were determined by an autoanalyzer (COBAS MIRA Plus, Branchburg, NJ, USA). Serum PTH was measured by an immunoradiometric assay specific for intact rat PTH (Immutopics, San Clemente, CA, USA). Parathyroid glands were weighed on a microbalance (CAHN C-31; CAHN Instruments, Inc., Cerritos, CA, USA) and results reported as a ratio of 
g gland weight per gram body weight.
Immunohistochemistry
Calcium-sensing receptor protein in the rat parathyroid glands was measured by immunostaining and quantified by computer-assisted image analysis as previously described7. Briefly, immunostaining was performed on formalin-fixed, paraffin-embedded parathyroid glands using our primary antibody and a commercial staining kit (Histostain-Plus, rabbit; Zymed Laboratories, Inc., South San Francisco, CA, USA). An IgG fraction of a polyclonal antibody developed in rabbit against a 23-amino acid peptide ("ADD" peptide) contained in the extracellular domain of the CaR that is conserved in human, bovine, and rat (Research Genetics, Huntsville, AL, USA) was used as the primary antibody. Non-specific staining (negative control) was obtained by substituting rabbit pre-immune IgG for the primary antibody. The tissue was deparaffinized, rehydrated, and endogenous peroxidase quenched using 0.6% hydrogen peroxide in methanol. Tissue was blocked with 10% pre-immune goat serum and incubated with an IgG fraction of the primary antibody (6.4 g/mL) or pre-immune IgG (6.4 g/mL) for one hour at room temperature. Biotinylated secondary antibody was applied, followed by a streptavidin-horseradish peroxidase conjugate. The immune complexes were visualized with AEC substrate-chromagen.
Immunostaining of the CaR protein was quantified using a Nikon Diaphot-TMD microscope coupled to a camera and an image analysis system. Images of stained tissue sections (
100 magnification) were acquired using a DAGE-330 color camera and captured with a Pentium P-166 IBM-compatible computer equipped with a Coreco frame-grabber. The digitized images were converted to gray scale and analyzed using Image-Pro Plus software (Media Cybernetics, Silver Spring, MD, USA). All components were obtained from Boyce Scientific, Inc. (St. Louis, MO, USA). The intensity of staining of the CaR was then quantified using the optical density function of the software. The average optical density per section of tissue was calculated by dividing the sum integrated optical density by the sum area. Background staining (the average optical density of the negative control tissue) was subtracted from this value. Approximately 6 to 10 sections of tissue from each parathyroid gland were immunostained. On each day of immunostaining, tissue sections from 3 to 5 animals per experimental group were analyzed together. The optical densities of the staining intensities from each of the animals in the experimental groups were calculated as a percentage of optical densities obtained from control animals for that day of staining. The percentages obtained for any one animal on different days of staining were then averaged, resulting in a single value per animal. The coefficients of variation for the groups in which CaR immunostaining was measured averaged 22.8 
2.7% (SEM); therefore, a 20% change (lower limit) in CaR could be detected statistically with confidence using this method. The Student unpaired t test was used for statistical evaluation.
To examine parathyroid cell proliferation, sections of PTGs were immunostained for proliferative cell nuclear antigen (PCNA) and/or Ki-67, two proteins that are found in the nuclei of dividing cells. While PCNA has been widely used for many years as an indicator of cell proliferation, Ki-67 is becoming more accepted as a superior marker of proliferation. Not only is Ki-67 tightly association with the cell cycle, it is not found in non-cycling cells or in cells that are undergoing DNA repair, and its expression decreases rapidly after mitosis. Briefly, after deparaffinization and rehydration, PCNA and Ki-67 antigens were unmasked by microwaving the slides in 10 mmol/L citric acid, pH 6.0 for 14 minutes at high power (1100 watts) using a Sharp Carousel microwave. After cooling at room temperature for 10 minutes, the slides were processed using anti-PCNA primary antibody (PC10; Zymed Laboratories) and mouse Ki-67 primary antibody (MIB-5; Beckman Coulter, Inc., Fullerton, CA, USA) with a commercial staining kit (Histostain-Plus mouse kit; Zymed Laboratories) per the manufacturer's instructions. One to two slides, each consisting of one to two sections of rat parathyroid tissue, were assayed per nuclear antigen per animal. To insure objectivity, counting of positive nuclei was performed in a "blind" fashion, that is, the label on each slide was covered with opaque tape, leaving the observer unaware of which experimental group was being counted. For Ki-67, all clearly stained nuclei were designated as positive. For PCNA, heavily stained nuclei were designated as positive as determined by the observer. Quantification of parathyroid cell proliferation was obtained by counting the total number of Ki-67- or PCNA-positive nuclei in the entire section of tissue. This value was then divided by the area of the entire section of tissue (as obtained from a digitized computer image using the Image-Pro Plus software described above), giving a value of the number of positive nuclei per unit area (1000 pixels). Quantitating in this manner, in which the total number of nuclei stained for PCNA/Ki-67 in the entire section of tissue is corrected by the area of the entire section of tissue, provides a direct evaluation of overall cell proliferation in that tissue section, not just in randomly selected areas. On each day of staining, the number of PCNA- or Ki-67-positive cells per unit area for each animal in each experimental group was reported as the percent of control values obtained on that day. (As an example, one rat on the high phosphate diet for 2 days had 85 positive Ki-67 nuclei in the entire cross-section of tissue; the area of that cross-section of tissue was 22,791 pixels. Therefore, it had a labeling index of 3.73 nuclei per unit area of 1000 pixels. Compare this to a normal rat that had 14 positive Ki-67 nuclei per 15,837 pixels, which resulted in a labeling index of 0.884 nuclei per unit area of 1000 pixels. The labeling index for each experiment rat was compared to the labeling index of the control rats and reported as % control). The percentages obtained for any one animal on different days of staining were then averaged, resulting in a single value per animal. The Student unpaired t test was used for statistical evaluation.
RESULTS
Protocol 1
Rats made uremic were placed immediately on a HP diet for 1, 2, 4 and 7 days; intact control rats were fed a normal chow diet. Serum chemistries, PTH and PTG weights are shown in Table 1. Of particular interest were fluctuations of the parameters measured (except PTG weight) at 1 day post-nephrectomy. Most likely these could be attributed to the stress of surgery, as both ionized calcium (ICa) and P levels returned to normal levels by day 2, and the increases in PTH and creatinine levels were not as extreme at day 2. Nephrectomized rats at each time point exhibited uremia as indicated by significant increases in serum creatinine levels compared with controls. Serum phosphorus levels were elevated at 1 day post-nephrectomy compared with controls, returned to normal levels at 2 days of uremia and then significantly increased at 4 and 7 days of uremia. Ionized calcium levels in uremic rats, despite an initial decrease at 1 day, were not different from controls at 2, 4 and 7 days of uremia. PTH levels in nephrectomized rats were elevated at day 1 and remained higher than controls for the duration of the time course. Parathyroid gland weights at 1 and 2 days of uremia were not different from controls, but were significantly higher at 4 and at 7 days of uremia.
Cell proliferation (PCNA) and CaR content were examined by immunohistochemical staining of sections of formalin-fixed, paraffin-embedded parathyroid glands. Representative images of the time courses showing the increase in cell proliferation in relation to the fall in CaR expression are shown in Figure 1. PCNA-positive cells exhibited punctate staining of the nuclei, which increased early in the time course. CaR expression in the controls and at days 1 and 2 post-nephrectomy was homogeneous, but was decreased and more heterogeneous at 4 and 7 days of uremia and a HP diet.
Figure 1.
Representative images of the changes in proliferative cell nuclear antigen (PCNA) and calcium-sensing receptor (CaR) expression in uremic rats maintained on a high phosphate diet that promotes parathyroid hyperplasia. PCNA and CaR content were examined by immunohistochemical staining of sections of formalin-fixed, paraffin-embedded parathyroid glands (magnification, PCNA 400, CaR 100).
Quantification of parathyroid cell proliferation was obtained by counting the total number of PCNA-positive nuclei in the entire section of tissue; this value was divided by the area of that tissue as obtained from a digitized computer image using the Image-Pro Plus software. The number of PCNA-positive cells per unit area was then reported as the percent of control values. Quantification of the CaR immunostaining was performed by computer assisted image analysis and reported as percent of control values. As shown in Figure 2, an increase in cell proliferation was observed early in uremia: PCNA immunostaining at 2 days of uremia was 2.4-fold higher than controls and remained elevated throughout the time course. CaR content at 1 and 2 days of uremia was not different from controls. However, by 4 days of uremia, the CaR content had significantly dropped to 63 22% of controls, and then to 61 19% at 7 days of uremia. These data indicate that parathyroid cell proliferation precedes the down-regulation of the CaR in uremic rats.
Figure 2.
Time course of changes in PCNA (
) and CaR (
) expression for protocol 1. Rats were made uremic and placed immediately on a high phosphate (HP) diet that promotes parathyroid hyperplasia. Intact control rats (day 0) were fed a regular chow diet. CaR protein content was assessed by immunohistochemical staining and quantified by computer-assisted analysis and reported as optical density, % of control values. Cell proliferation was determined as the number of PCNA-positive cells per unit area (1000 pixels), and reported as % of control values. Data are expressed as mean SE, P 
0.01.
Protocol 2
In the initial experiment, rats were nephrectomized and placed immediately on a high phosphate diet. While this initiated a rapid proliferative response and down-regulation of the CaR in the parathyroid gland, it rendered the individual effects of uremia and phosphate loading on both events indistinguishable. In addition, perturbations of serum chemistries were evident at 1 day post-surgery. Therefore, a second experiment was performed to allow for recovery from the surgery before initiating the time course to examine the changes in CaR expression and parathyroid proliferation in uremic rats in response to the HP diet. In protocol 2, rats were nephrectomized and placed on a low phosphate diet (LP; 0.2% P, 0.5% Ca), which prevents both down-regulation of the CaR and parathyroid hyperplasia in uremic rats7. After 1 week on the LP diet, the rats were switched to the HP diet, and changes in CaR expression and parathyroid proliferation examined as in the previous protocol. Serum chemistries, PTH and PTG weights are shown in Table 2. Nephrectomized rats maintained on the LP diet and at each time point after being switched to the HP diet exhibited uremia as indicated by significant elevations in serum creatinine levels compared with controls. PTH levels in nephrectomized rats kept on the LP diet for 1 week were not significantly different than controls. However, PTH levels increased significantly in nephrectomized rats by 1 day of being switched to the HP diet and remained elevated throughout the time course. Parathyroid gland weights of nephrectomized rats were consistently higher by 3 days on the HP diet compared to controls.
Quantitation of the changes in cell proliferation (PCNA immunostaining) in relation to changes of the CaR expression (CaR immunostaining) from protocol 2 is shown in Figure 3. Again, an increase in parathyroid cell proliferation was observed rapidly after uremic rats were switched to the HP diet: PCNA immunostaining at 2 days on the HP diet was 2.4-fold higher than controls, and 3.7-fold higher at 3 days. This increase in parathyroid cell proliferation at 2 days was verified by immunostaining for Ki-67, another nuclear antigen associated with cell proliferation. Ki-67 immunostaining was within normal levels at 1 day on the HP diet, but increased significantly at 2 days (3.3-fold higher than controls, P < 0.0001) and remained significantly elevated at 3 days on the HP diet (2.6-fold higher, P < 0.001). Parathyroid gland CaR content of uremic rats on the LP diet (day 0) was not different from controls; CaR content of uremic rats switched to the HP diet remained within control levels through 7 days, but was down-regulated by 14 days. Therefore, in protocol 2, we again found that cell proliferation in uremic rats on a HP diet, as assessed by both PCNA and Ki-67, increased very early in uremia and preceded down-regulation of the CaR.
Figure 3.
Time course of changes in PCNA () and CaR () expression for protocol 2. Uremic rats were maintained on a low phosphate (LP) diet for one week before being switched to a high phosphate (HP) diet that promotes parathyroid hyperplasia. Intact control rats were maintained on a regular chow diet. CaR protein content was assessed by immunohistochemical staining, quantified by computer-assisted analysis, and reported as optical density, % of control values. Cell proliferation was determined as the number of PCNA-positive cells per unit area (1000 pixels), and reported as % of control values. Data are expressed as mean SE, P 0.01.
DISCUSSION
It is generally accepted that decreases in circulating calcium and 1,25 dihydroxyvitamin D3, and retention of phosphate are the main systemic factors responsible for the parathyroid hyperplasia associated with uremia. The cellular and molecular mechanisms by which these factors regulate parathyroid cell proliferation, however, remain unclear. The relationship between the calcium-sensing receptor (CaR) and parathyroid cell proliferation has been investigated. Parathyroid hyperplasia is found in neonatal severe hyperparathyroidism, a disease attributed to loss-of-function mutations in the CaR11, and in the CaR-nullizygous mouse12, suggesting that total loss of the CaR leads to parathyroid cell proliferation. Several groups have noted that the CaR expression is decreased in parathyroid gland adenomas, in hyperplastic parathyroid glands of patients with chronic renal disease1,2, and in hyperplastic parathyroid glands of uremic rats7. Down-regulation of the CaR is associated with high proliferative activity of parathyroid glands in patients with secondary hyperparathyroidism and in uremic rats6,7. In addition, NPS-R568, an allosteric enhancer of the calcium-sensing receptor suppressed parathyroid cell proliferation in rats with renal insufficiency13,14.
Although loss-of-function mutations in the CaR have not been detected in hyperplastic glands from uremic patients, is has been suggested that a reduction in CaR expression may be an initiating event in the hyperplastic response. To investigate the nature of the temporal relationship between these two events, the current study compared the time courses for the onset of parathyroid gland proliferation and down-regulation of the CaR in the uremic rat model. In our initial protocol, rats were nephrectomized and immediately placed on a high phosphate diet. An increase in parathyroid cell proliferation was detected very early at 2 days post-nephrectomy. CaR expression was not changed at 2 days, but was decreased by 4 days post-nephrectomy. This window of time between which changes in cell proliferation and CaR expression were detected was relatively small and could have merely reflected the differences in the precision of the two measurements (that is, it is possible that it was easier to detect the initial increase in the cell proliferation than it was to detect the initial quantitative loss of CaR). However, in a separate experiment we were able to expand the time between these two events. In Protocol 2, rats were allowed to recover from nephrectomy while being maintained on a low phosphate diet that prevents parathyroid hyperplasia and the fall in CaR expression. This low phosphate diet also minimizes the fall in serum calcium and the rise in serum phosphorus observed at 1 day post-surgery. (These fluctuations, not seen in patients with slowly progressing renal disease, could have an impact on both parathyroid cell hyperplasia and CaR down-regulation). After 1 week on the low phosphate diet, the rats were switched to a high phosphate diet. Within 2 days of the switch, an increase in parathyroid cell proliferation was detected. CaR content, however, did not decrease until after day 7. As in the initial experiment, therefore, parathyroid cell proliferation preceded down-regulation of the CaR in rats responding to the combination of renal insufficiency and dietary phosphate loading.
We and other have previously shown that down-regulation of the CaR is associated with areas of parathyroid cell proliferation6,7. However, as illustrated in Figure 1, one may note that the number of cells traversing the cell cycle (PCNA-positive immunostaining) in response to the high phosphate diet appears to be minor compared to the more diffuse decrease in CaR. We have found that when uremic rats are fed a high phosphate diet, the parathyroid CaR becomes down-regulated and only returns to normal levels when the high phosphate stimulus is withdrawn (unpublished data). In the current study, uremic rats were fed a continuous diet of high phosphate. Under these circumstances, the number of cells proliferating at any one time is going to appear less than the accumulating areas of decreased CaR. Also, it is possible that cell proliferation/dedifferentiation triggers a cascade of events that leads (directly or indirectly) to down-regulation of the CaR in surrounding cells. As discussed below, autocrine/growth factors such as transforming growth factor-
(TGF-), acidic-fibroblast growth factor (acidic-FGF), and endothelin-1 (ET-1) have been found in hyperplastic parathyroid tissue. As yet, their functions on parathyroid proliferation and/or the CaR are not fully defined. If a potentiation of CaR down-regulation does occur in surrounding cells, it would appear that fewer cells were dividing in relation to a widening area of decreased CaR.
Our data indicate that parathyroid proliferation precedes down-regulation of the CaR in uremic rats fed a high phosphate diet. This is supported by a new report by Imanishi et al15, who generated transgenic mice in which cyclin D1 is specifically overexpressed in the parathyroid gland. Since cyclin D1 is integral in driving cells from the G1 to S phase of the cell cycle, these mice developed parathyroid hyperplasia. They found that the enlarged parathyroid glands contained decreased levels of CaR. Their study establishes a definite connection between parathyroid proliferation and down-regulation of the CaR, and supports our finding that proliferation precedes down-regulation of the CaR.
The question persists as to what factors at the cellular and molecular levels are involved in enhancing parathyroid tissue growth. The stimulatory effect of phosphorus on parathyroid hyperplasia has been well established16,17,18,19, and there is growing evidence for a direct action of phosphate on parathyroid tissue in vitro17,20, possibly involving a phospholipase A2-arachidonic acid signaling pathway21. We have previously shown that high dietary phosphate stimulates both parathyroid cell proliferation and a fall in CaR7. However, whether phosphate has a direct effect on the down-regulation of the CaR is unknown. Gogusev et al have shown that de novo expression of TGF- and strong expression of its receptor (epidermal growth factor receptor) are found in hyperplastic parathyroid glands of patients with secondary hyperparathyroidism22. This is supported by a recent study by Dusso et al, who found an increase in TGF- expression in hyperplastic parathyroid glands from uremic rats maintained on a high phosphate diet23. Other factors have been proposed as mediators of parathyroid cell growth. Endothelin-1 appears to act as an autocrine inducer of parathyroid cell proliferation24, and a negative correlation between expression of parathyroid hormone–related peptide (PTHrP) and parathyroid cell proliferation in patients with secondary hyperparathyroidism has recently been reported25. Also, in a parathyroid clonal cell model, an acidic fibroblastic growth factor autocrine system has been proposed as a mediator of calcium-regulated parathyroid cell growth26, and an increased expression of the immediate early gene c-myc has been suggested to be involved in parathyroid hyperplasia in uremic rats27.
In this study, we find that parathyroid cell proliferation precedes down-regulation of the CaR in uremic rats. The cellular and molecular events responsible for the initiation of parathyroid hyperplasia associated with secondary hyperparathyroidism and the factors responsible for the subsequent down-regulation of the CaR are obviously complex and require further investigation.
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Acknowledgments
The research was supported by NIH grant no. DK-53774. The Editors are grateful to Dr. Drueke, who served as Guest Editor during the review of this article.
