NDT Advance Access originally published online on September 16, 2005
Nephrology Dialysis Transplantation 2006 21(3):634-643; doi:10.1093/ndt/gfi137
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© The Author [2005]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org
Original Articles: Experimental Nephrology
Sevelamer hydrochloride reverses parathyroid gland enlargement via regression of cell hypertrophy but not apoptosis in rats with chronic renal insufficiency
1 Pharmaceutical Development Laboratories, Kirin Brewery Co., Ltd, Takasaki, Japan and 2 Medical Affairs Section, Pharmaceutical Division, Kirin Brewery Co., Ltd, Tokyo, Japan
Correspondence and offprint requests to: Nobuo Nagano, PhD, Medical Affairs Section, Pharmaceutical Division Kirin Brewery Co. Ltd., 26-1, Jingumae 6-chome, Shibuya-ku, Tokyo, 150-8011, Japan. Email: n-nagano{at}kirin.co.jp
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Abstract |
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Background. Dietary phosphate restriction suppresses parathyroid hormone (PTH) secretion, synthesis, and parathyroid cell proliferation in experimental animals with chronic renal insufficiency (CRI), independently of serum calcium and 1,25(OH)2D3 levels. This study was conducted to examine whether sevelamer hydrochloride (sevelamer), a metal-free phosphate binder, could regress an advanced parathyroid gland (PTG) hyperplasia and enlargement in rats with CRI.
Methods. Male Sprague–Dawley rats were fed a diet containing adenine for 6 weeks to establish CRI. Normal rats and adenine-treated rats were sacrificed to obtain the PTG (baseline group). The adenine diet was changed to a normal diet or diet containing 1 or 3% sevelamer for another 4 weeks. Time course changes of serum levels of calcium, phosphorus, and PTH were measured. At the end of the study, the PTG was weighed and examined histologically.
Results. Adenine-treated rats developed severe CRI with marked elevation of serum phosphorus and PTH. The PTG weight markedly increased with enlarged cell volume (i.e. cell hypertrophy) at baseline. Sevelamer treatment rapidly lowered serum phosphorus and PTH levels within 6 days, and after 4 weeks, reduced the PTG weight by 38% compared to adenine-treated rats at baseline. The reduction in PTG weight was due to regression of cell hypertrophy, but not to decreased cell number by apoptosis. Decreased expression of calcium receptor in the PTG at baseline was partially recovered by the sevelamer treatment.
Conclusions. The sevelamer treatment can reduce the PTG weight with a reduction in serum PTH levels via regression of cell hypertrophy but not apoptosis in rats with CRI. Reduced PTG function might contribute to the regression of cell hypertrophy.
Keywords: cell hypertrophy; parathyroid gland hyperplasia; parathyroid hormone (PTH); phosphorus; sevelamer hydrochloride
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Introduction |
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Secondary hyperparathyroidism (2HPT), which is characterized by increased cell proliferation (i.e. hyperplasia) of the parathyroid gland (PTG), is a common consequence of chronic renal insufficiency (CRI) [1]. PTG hyperplasia is positively associated with high levels of serum parathyroid hormone (PTH) [2]. In addition, PTG hyperplasia is rarely reversible and often develops into nodular hyperplasia, which is refractory to vitamin D therapy, clinically [3].
It is widely accepted that lowered blood calcium and 1,25(OH)2D3 levels play pivotal roles in the development of 2HPT and PTG cell proliferation [1–3]. However, direct effects of phosphorus on PTG function and cell proliferation have been demonstrated to occur independently of calcium and 1,25(OH)2D3 [4]. A high phosphorus diet increases PTH secretion and PTH mRNA and causes PTG hyperplasia in normal and CRI rats [4,5]. Conversely, low phosphorus diet inhibits PTH secretion, PTH mRNA, and PTG hyperplasia in uraemic dogs and rats [4,5]. In addition, the direct effect of phosphorus has also been confirmed by in vitro studies demonstrating that high phosphate medium stimulates PTH secretion in PTG tissue preparations [4].
Sevelamer hydrochloride (cross-linked poly[allylamine hydrochloride], hereafter referred to as sevelamer) is a metal-free phosphate binder for the treatment of hyperphosphataemia in patients on dialysis [6]. We reported previously that sevelamer treatment prevents the progression of PTG hyperplasia in rats with CRI by inhibiting PTG cell proliferation [7,8]. However, these studies showed that the sevelamer treatment maintained the original size of the PTG from the start of the sevelamer treatment. Thus, it is still unclear whether sevelamer could regress PTG hyperplasia that is already advanced.
Dietary overloaded adenine is converted to 2,8-dihydroxyadenine, a less water-soluble substance, and then impairs renal function due to intratubular and interstitial precipitations such as acicular crystals [9]. A severe CRI with hyperphosphataemia and 2HPT is also observed in adenine-treated rats [10]. In the present study, we examined whether sevelamer treatment could regress PTG hyperplasia and enlargement once established, and if so, we aimed to investigate the possible mechanism by histological analysis. The results indicate that the sevelamer treatment can reduce the PTG weight with a concomitant reduction in serum PTH levels via regression of cell hypertrophy but not through apoptosis.
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Subjects and methods |
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Experimental protocol
The experimental protocol was approved by the Experimental Animal Ethical Committee of Kirin Brewery Co. Ltd., and is shown in Table 1. Male Sprague–Dawley rats, 8 weeks of age, were purchased from Charles River Japan (Japan) and fed a standard powder diet containing 1.02% phosphorus, 1.08% calcium, 25.1% crude protein, and 2.4 IU/g vitamin D3 (CE-2; CLEA Japan, Japan). Rats were kept singly in cages and allowed free access to food and water. After an acclimatization period of 11 days, a blood sample was collected from the tail artery to measure serum phosphorus, calcium, creatinine, blood urea nitrogen (BUN) and PTH levels on experimental day –42. The rats were divided into two matched groups with respect to body weight. One group was fed a normal diet and the other group was fed a powder diet containing 0.75% adenine for 2 weeks (day –42 to –29) and 0.5% adenine for another 4 weeks (day –28 to –1). After the adenine treatment for a total of 6 weeks, normal control rats were divided into two groups and adenine-treated rats were divided into four groups of seven animals each, matched with respect to body weight, BUN, serum creatinine, phosphorus, and PTH levels on day –1. After collecting 24-hour urine samples with metabolic cages, each group of normal control rats (baseline-normal group) and adenine-treated rats (baseline-adenine group) was sacrificed to obtain blood, thyroid-PTG complexes, and kidneys on day –1. The other group of normal control rats was fed a normal diet until the end of the study (normal control group). On day 0, the adenine diet was switched to a normal diet (disease control group) or a diet containing 1 or 3% sevelamer for another 4 weeks (day 0 to 29). Body weight and food intake volume were measured every week and blood samples were collected on days 6, 14, 20, and 29 to measure time course changes in serum parameters. Rats were kept singly in metabolic cages to collect 24-hour urine samples on days 27 and 28. At the end of the study (day 29), blood was collected by abdominal aortic puncture under ether anaesthesia to measure serum 1,25(OH)2D3 levels and then the thyroid-PTG complexes and kidneys were immediately removed by dissection.
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Serum and urinary chemistries
Serum phosphorus, calcium, and BUN levels were measured with commercial kits (Wako Pure Chemical Industries, Japan). Serum and urinary creatinine was measured by enzymatic assay (CRE-EN; Kainos Laboratories, Japan). Serum PTH and 1,25(OH)2D3 were measured with a Rat PTH IRMA kit (Immutopics, CA, USA) and a 1,25(OH)2D RIA kit (TFB; Immunodiagnostic System, UK), respectively. All parameters except PTH and 1,25(OH)2D3 were determined by the standard colorimetric method employing a microplate spectrophotometer (SpectraMax 250; Molecular Devices, CA, USA).
Histological analysis
The thyroid-PTG complexes and kidneys were fixed in 10% Formalin Neutral Buffer Solution (Wako Pure Chemical Industries) at 4°C overnight. The left PTG was carefully dissected out from the complexes and the weight was measured using an electronic ultramicrobalance (Sarutorius Supermicro S4; Sartorius AG, Germany). The right thyroid-PTG complexes and kidneys were embedded in paraffin and cut into 3 µm serial sections. After sequential dewaxing and rehydration, sections were stained with haematoxylin and then examined with a light microscope (Axiophoto; Carl Zeiss, Germany) connected to a television monitor (PVM-1454Q; Sony, Japan). The PTG cell nucleus density in the section of maximal PTG area defined as we previously reported [7] was calculated from the number of haematoxylin-stained nuclei existing within four square areas measuring 200 x 200 µm per animal.
Immunohistochemistry and detection of apoptosis
To analyse the expression of calcium receptor (CaR), sections close to the maximal PTG area were pre-treated with Target Retrieval Solution (DAKO, CA, USA). Anti-CaR mouse monoclonal antibody (ADD; a gift of Dr E. Nemeth at NPS Pharmaceuticals Inc., Toronto, Canada) and anti-mouse IgG biotinylated horse IgG (Vector Laboratories, CA, USA) were used as the primary and secondary antibodies, respectively. Signals were amplified with a TSA Biotin System (PerkinElmer Life Sciences, MA, USA) and visualized with a vectastain abc-ap standard kit (Vector Laboratories) and an alkaline phosphatase substrate kit I (Vector Laboratories). No positive finding was confirmed when normal mouse IgG was used instead of the primary antibody (data not shown).
To identify apoptosis, nuclear DNA fragmentation was detected in situ by the TUNEL method using an ApopTag Plus Peroxidase In Situ Apoptosis Detection Kit (Intergen, NY, USA).
Drugs
Sevelamer hydrochloride (cross-linked poly[allylamine hydrochloride], the active ingredient of Renagel®) was synthesized by The Dow Chemical Company (MI, USA) and supplied via Chugai Pharmaceuticals Co., Ltd (Japan). Adenine was obtained from Sigma Chemical Co. (MO, USA).
Statistical analysis
All values are expressed as mean±SEM. The data between the normal control group and the disease control group and between the baseline-normal group and the baseline-adenine group were compared using the Student's t-test. Multiple comparisons were performed among adenine-treated groups receiving normal, 1% and 3% sevelamer-containing diets using the parametric Dunnett's test. Statistical significance was defined as P<0.05, two-sided.
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Results |
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Body weight and food intake volume
During and after the adenine treatment, body weight gain was retarded in adenine-treated rats compared to the normal control group (Figure 1). Mean food intake volumes, measured weekly during the sevelamer treatment for 4 weeks are shown in Table 2. The 1% and 3% sevelamer treatments did not significantly affect the body weight and food intake volume.
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BUN and serum creatinine levels
BUN and serum creatinine levels progressively increased during the adenine treatment for 6 weeks (day –42 to –1) (Figures 2A and 2B). After the end of adenine treatment, BUN and serum creatinine levels remained at high levels. The 1% and 3% sevelamer treatments did not affect BUN or serum creatinine levels during the study.
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Serum phosphorus and calcium levels
Serum phosphorus levels gradually increased during the adenine treatment for 6 weeks (day –42 to –1) (Figure 3A). A marked hyperphosphataemia developed at the baseline (day 0) and then serum phosphorus levels were maintained high levels until the end of the study in the disease control group. Six days after switching from the adenine diet to sevelamer treatment (day 6), serum phosphorus levels lowered in a dose-dependent manner. The 1% sevelamer treatment continued to reduce serum phosphorus levels to around the normal control levels, while 3% sevelamer kept serum phosphorus levels below the normal control levels until the end of the study.
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In the latter part of the study, a significant hypocalcaemia was observed in the disease control group (Figure 3B). The initiation of 1% and 3% sevelamer treatments significantly increased serum calcium levels within 14 days and 3% sevelamer kept the levels high throughout the study.
Serum PTH levels
Serum PTH levels dramatically and steeply increased towards the end of study (Figure 4). After the start of the 1% sevelamer treatment, the elevation rate of serum PTH levels was partly attenuated. In contrast, the 3% sevelamer treatment rapidly decreased serum PTH levels to almost the normal control levels 6 days after the treatments started (day 6), and then maintained normal levels until the end of the study.
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Serum 1,25(OH)2D3 levels and creatinine clearance (CCr)
Serum 1,25(OH)2D3 levels and CCr were markedly lowered in the baseline-adenine group compared to those in the baseline-normal group on day –1 (Table 2). The 1% and 3% sevelamer treatments did not significantly affect these parameters by the end of the study.
PTG weight
The PTG weight already showed a 5.3-fold increase in the baseline-adenine group compared to the baseline-normal group (Figure 5A). At the end of the study, a further 6.8-fold increase was observed in the disease control group compared to the normal control group. The 1% sevelamer treatment for 4 weeks maintained the baseline PTG weight, while the 3% sevelamer treatment decreased the PTG weight by 38% compared to the baseline-adenine group. The difference between disease control group and 3% sevelamer group was statistically significant.
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Histological examinations
The kidneys in the baseline-normal and the normal control groups were histologically normal (data not shown). In the kidney of the baseline-adenine group, acicular crystals supposed to be 2,8-dihydroxyadenine were numerous in the dilated tubules with deciduation of the brush border membrane and interstitium at the cortex and medulla (Figure 6). Interstitial fibrosis, cast formation, and infiltration of mononuclear cells were also observed while glomeruli were almost intact. At the end of the study, similar lesions were observed in the disease control group (data not shown). The sevelamer treatments had no influence on the tubular and interstitial lesions or on the precipitation of acicular crystal.
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The PTG cell nucleus density was significantly lowered in the baseline-adenine group compared to the baseline-normal group (Figures 5B and 7), indicating enlarged cell volume (i.e., cell hypertrophy). The 1% sevelamer treatment had no effect on the PTG cell nucleus density, but 3% sevelamer significantly increased this parameter above the normal control levels, indicating a decreased mean cell volume (i.e. regression of cell hypertrophy).
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The expression of CaR protein in the normal control group was extensively and abundantly observed in the PTG and in parafollicular C cells in the thyroid (Figure 7). The section close to the maximal PTG area was markedly enlarged and the CaR expression lowered in the baseline-adenine group compared to the baseline-normal group. Treatment with 1% sevelamer did not affect CaR expression, but 3% sevelamer tended to recover the CaR expression in the PTG at the end of the study.
We detected no apoptosis in the PTG cells of any normal control, adenine-treated, or sevelamer-treated rats while apoptosis was clearly observed in the mammary gland of female rats after weaning, which was used as a positive control specimen stained simultaneously (data not shown).
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Discussion |
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Dietary adenine-treated rats were used as a CRI animal model in this study because this model shows stable and long-lasting severe uraemia with hyperphosphataemia and 2HPT even after the discontinuation of adenine feeding [10]. The tubulo-interstitial lesions and precipitation of acicular crystals at baseline were unchanged 4 weeks after the discontinuation of adenine treatment, supporting persistent high levels of BUN and serum creatinine levels and reduced CCr. Sevelamer treatment did not affect renal function in adenine-treated rats. It is known that dietary phosphate restriction prevents deterioration of renal function in experimental animal models with CRI [11]. In fact, we have observed that sevelamer treatment begun immediately after the injection of nephrotoxic serum, slows deterioration of renal function in Wistar Kyoto rats with progressive CRI by maintaining kidney calcium content at a low level [12]. The reason for the lack of a renal protective effect by sevelamer in this study might be attributed to the irreversible extensive changes already established during the adenine treatment 6 weeks. In addition, it is also likely that the tubulo-interstitial lesions caused by adenine might block the renal protective effect of sevelamer, since nephrocalcinosis primarily occurs at these sites. Nevertheless, it is clear that the lowering effect of sevelamer on serum phosphorus and PTH levels and PTG weight observed in this study is independent of renal function.
In adenine-treated rats, the PTG hyperplasia, confirmed by marked increased weight, was accompanied by each cell hypertrophy, which was indicated by both the histological observation and decreased cell nucleus density. Our previous studies using stereologic techniques have demonstrated that PTG cell hypertrophy also contributes to the PTG enlargement, although its role is not predominant compared to that of increased cell number (i.e. hyperplasia), adding about 20–40% to the total increase in PTG volume in partially nephrectomized rats [13,14]. It has been reported that phosphate load increases PTG cell size and proliferation, consistent with PTH hyper-secretion, but the changes are completely reversible when high phosphorus diet is removed in normal rats [15,16]. Although the mechanism of PTG cell hypertrophy is not fully understood, electron microscopic observations demonstrate that the PTG cells in an active state of PTH hyper-secretion show the enlargement and increased number of intracellular organelles, such as the Golgi apparatus, rough endoplasmic reticulum, tortuous plasma membrane, mitochondrion, and secretory granules, indicating enhanced capacity for PTH synthesis, packaging, storage, transport, and membrane synthesis [17,18]. In this study, the sevelamer treatment markedly lowered serum phosphorus levels and regressed the PTG cell hypertrophy accompanied by a reduction in serum PTH levels. In addition, we have shown that the sevelamer treatment can decrease the number of proliferating cell nuclear antigen-positive cells below the normal control levels in the PTG of partially nephrectomized rats [8]. Therefore, phosphorus appears to regulate the PTG cell size by controlling PTG function such as PTH secretion, synthesis, and cell proliferation. Further investigation using electron microcopy is clearly needed.
Of interest, the 3% sevelamer treatment for 4 weeks resulted in the reduction of PTG weight by 38% compared to the baseline-adenine group. Although it is controversial, apoptosis rarely occurs in PTG cells of experimental animals [2–4,18] and we observed no apoptosis in the previous [13] and present studies. In addition, a similar result has been reported that the PTG hyperplasia (increased cell number) is rarely reversible, 6 weeks after withdrawal of high phosphorus diet [16]. Therefore, we concluded that the sevelamer treatment decreased the PTG weight without affecting the cell number. In other words, sevelamer could not regress the PTG hyperplasia as long as the hyperplasia is defined as an increased cell number. Combined with the decreased PTG weight and increased cell nucleus density, the reduced PTG weight during 4 weeks of sevelamer treatment is due to regression of cell hypertrophy. However, it should be noted that sevelamer treatment can decrease PCNA-positive cells [8]. Therefore, the sevelamer treatment appears to prevent the PTG hyperplasia from progression to a more severe form.
The sevelamer treatment also increased serum calcium levels. The elevation of serum calcium induced by the sevelamer would partly result from the marked reduction in serum phosphorus levels (i.e. ion-counter action). In addition, intestinal phosphate binding by sevelamer might account for an increase in free calcium ion, which in turn would result in increased intestinal calcium absorption since we have observed that the sevelamer treatment decreases faecal calcium excretion associated with increased calcium absorption in a balance study with normal rats [19]. This elevated serum calcium induced by sevelamer might partly contribute to regression of PTG cell hypertrophy, because we have demonstrated that the stimulation of cell surface CaR by calcimimetics can reduce PTG cell hypertrophy to normal cell size in partially nephrectomized rats [13,14]. In addition, there was a trend towards up-regulation of CaR expression by the sevelamer treatment in this study, although the quantification of CaR expression by a Western blot analysis is necessary for further confirmation. A similar result was reported that the decreased PTG CaR expression in partially nephrectomized rats fed a high phosphorus diet can be normalized by reducing the dietary phosphorus content [20]. Therefore, it is likely that increased serum calcium levels and CaR expression would synergistically act and result in regression of cell hypertrophy.
Another possibility is that 1,25(OH)2D3 might contribute to the regression of PTG cell size, because 1,25(OH)2D3 is also known to regulate PTG function and cell proliferation [1,5,18]. However, marked depletion of serum 1,25(OH)2D3 levels was observed in this study and the sevelamer treatment had no influence on this depletion. Therefore, the contribution of 1,25(OH)2D3 to regression of PTG cell hypertrophy is excluded, at least in the present study.
In conclusion, the sevelamer treatment, despite being initiated after the establishment of PTG hyperplasia, has the potential to inhibit PTG hyper-function including PTH secretion, cell proliferation, and cell hypertrophy. If serum phosphorus levels are maintained below normal levels, the PTG function could be normalized despite having PTG hyperplasia.
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Acknowledgments |
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We are grateful to Dr Edward F. Nemeth (NPS Pharmaceuticals, Inc., Toronto, Canada) for generously providing the monoclonal antibody against CaR.
Conflict of interest statement. All authors are employees of Kirin Brewery Co., Ltd. Kirin Brewery Co., Ltd manufactures and sells sevelamer hydrochloride with Chugai Pharmaceuticals, Inc. in Asia.
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Accepted in revised form: 12. 8.05
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