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Nephrol Dial Transplant (2003) 18: III81-III85
© 2003 European Renal Association-European Dialysis and Transplant Association

Original Article

Sevelamer hydrochloride, a calcium-free phosphate binder, inhibits parathyroid cell proliferation in partially nephrectomized rats

Nobuo Nagano1,, Sonoe Miyata1, Sachiko Obana1, Nami Kobayashi1, Megumi Abe1, Naoshi Fukushima2 and Michihito Wada1

1 Pharmaceutical Development Laboratories, Kirin Brewery Co., Ltd, Takasaki and 2 Fuji Gotenba Research Laboratory, Chugai Pharmaceutical Co., Ltd, Gotemba, Japan



   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Background. Secondary hyperparathyroidism characterized by hyperplasia of the parathyroid gland (PTG) is a consequence of chronic renal insufficiency (CRI). Dietary phosphate restriction and sevelamer hydrochloride, a calcium-free phosphate binder, suppress parathyroid hormone (PTH) secretion and PTG hyperplasia in experimental animals with CRI, independently of serum calcium and 1,25(OH)2D3 concentrations. In the present study, the effect of sevelamer on PTG cell proliferation in rats with CRI was investigated.

Methods. Seven weeks after a 5/6 nephrectomy, rats were fed a diet containing 0, 1 or 3% sevelamer for 4 weeks, and sham-operated rats were fed a normal diet. Serum calcium, phosphorus, PTH and 1,25(OH)2D3 concentrations were measured. The number of cells positive for proliferating cell nuclear antigen (PCNA) in the maximal two-dimensional PTG area was counted at the end of study.

Results. Sevelamer inhibited increases in serum phosphorus, calcium–phosphorus product and PTH concentrations without affecting serum calcium or 1,25(OH)2D3. Sevelamer also suppressed the maximal PTG area and PCNA-positive cells. There was also a strong correlation between the maximal PTG area and serum PTH concentration, and between PCNA-positive cells and the maximal PTG area, as well as between serum phosphorus concentration and PCNA-positive cells.

Conclusions. These results indicate that sevelamer treatment reduces serum phosphorus concentration and could inhibit PTG cell proliferation and prevent PTG hyperplasia.

Keywords: parathyroid cell proliferation; parathyroid gland hyperplasia; serum phosphorus; sevelamer hydrochloride



   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Secondary hyperparathyroidism (2HPT) characterized by parathyroid gland (PTG) hyperplasia is a common consequence of chronic renal insufficiency (CRI). It has been reported that lowered blood calcium and 1,25(OH)2D3 concentrations affect the development of 2HPT and PTG cell proliferation [1–3]. Recent studies have shown the direct effect of phosphorus on PTG function and cell proliferation which is independent of calcium and 1,25(OH)2D3 concentrations. A high phosphorus diet increases parathyroid hormone (PTH) mRNA and PTH secretion and causes PTG hyperplasia in both normal rats and those with CRI [4–6]. Conversely, a low phosphorus diet reverses 2HPT in uraemic dogs [7], and inhibits PTH mRNA and PTG hyperplasia in rats with CRI [6,8,9]. In vitro studies have demonstrated that a high phosphate medium stimulates PTH secretion in PTG tissue preparations [9,10].

Sevelamer hydrochloride is a calcium-free phosphate-binding polymer used to treat hyperphosphataemia in patients undergoing haemodialysis [11]. We have demonstrated that sevelamer treatment arrests PTG hyperplasia in rats with CRI induced by Adriamycin [12]. The mechanism for the inhibitory effect of sevelamer on PTG hyperplasia could be inhibition of PTG cell proliferation rather than apoptosis, because apoptosis rarely occurs in experimental animals [1–3,6]. The present study examined whether sevelamer could inhibit PTG cell proliferation in partially nephrectomized rats, which is the commonly used experimental animal model of CRI.



   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Experimental protocol
The procedure was approved by the Experimental Animal Ethical Committee of Kirin Brewery Co., Ltd. Male Sprague–Dawley rats (9 weeks of age, CLEA Japan) were fed a standard powder diet containing 0.85% phosphorus and 1.12% calcium (CE-2, CLEA Japan) throughout the experiment. Two branches of the left renal artery were ligated and, 1 week later, the right kidney was removed (Nx rats). Another group of 24 rats underwent a sham operation (Sham rats). Seven weeks after the 5/6 nephrectomy, a blood sample was collected from the tail artery to measure serum parameters. The Sham and Nx rats were divided into two and four groups, respectively, each containing 12 rats that were matched by body weight and serum parameters. Ether was used to anaesthetize one group of both the Sham and Nx rats, blood was collected by abdominal aortic puncture, and then the thyroid–parathyroid complexes were resected (these were the baseline groups of rats). On the same day (day 0), the other three groups of Nx rats were given free access to a diet containing 0, 1 or 3% sevelamer for 4 weeks. The remaining Sham group was fed the normal diet. Blood samples were collected from the tail artery during the course of the study. At the end of the study (day 28), blood samples were taken from the abdominal aorta, and thyroid–parathyroid complexes were resected.

Serum chemistry
Serum phosphorus and calcium concentrations, and blood urea nitrogen (BUN) were measured by standard calorimetric methods with commercial test kits (Wako Pure Chemical). Serum creatinine, PTH and 1,25(OH)2D3 concentrations were measured using enzyme assay (CRE-EN, Kynos), rat PTH-(1–34) immunoradiometric assay kit (IRMA) (Nichols Institute Diagnostics) and radioreceptor assay kit (RRA) (SRL, Yamasa), respectively.

Maximal PTG area
The maximal two-dimensional PTG area was measured by a previously reported method [12]. The thyroid–parathyroid complexes were fixed with Bouin's fixative, embedded in paraffin and cut into 3 µm serial sections. They were stained with haematoxylin–eosin and examined using a light microscope (Axiophoto, Carl Zeiss) connected to a television monitor (PVM-1454Q, Sony). The outside edge of the PTG was traced and the area inside the circle was calculated by an image analysis system (IBAS, Carl Zeiss). The maximal PTG area was determined from ~10 serial sections per animal, selected to include the largest section.

Immunohistochemistry for proliferating cell nuclear antigen (PCNA)
After sequential dewaxing and rehydration, sections close to the maximal PTG area were incubated with anti-proliferating cell nuclear antigen (PCNA) mouse monoclonal antibody (PC10, Santa Cruz Biotechnology), and anti-mouse IgG biotinylated horse IgG (Vector Laboratories) was used as secondary antibody. Signals were amplified with indirect TSA (NEN Life Science Product) and visualized with a VECTASTAIN Elite ABC Standard Kit (Vector Laboratories) and DAB Substrate Kit (Vector Laboratories). The slides were counterstained with haematoxylin. The number of PCNA-positive cells was counted using the television monitor and expressed as a percentage of the total PTG cell number, having counted >1000 cells per animal.

Drug
Sevelamer hydrochloride [active ingredient of Renagel®, cross-linked poly(allylamine hydrochloride)] is synthesized by The Dow Chemical Company and was supplied via Chugai Pharmaceutical Co., Ltd.

Statistics
All values are expressed as the mean±SEM. The data obtained from Sham and Nx rats on the normal diet were compared using Student's t-test. Multiple comparisons were performed among three Nx groups using parametric Dunnett's test. The correlation analyses were performed using Pearson's correlation test. P<0.05 indicated statistical significance.



   Results
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 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Body weight and food intake
Sevelamer did not affect food intake or body weight. The mean doses of sevelamer administered throughout the study are shown in Table 1Go. One Nx rat on the normal diet, four Nx rats in the 1% sevelamer group and one Nx rat in the 3% sevelamer group died during the study from severe uraemia.


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  		Table 1.  Body weight, food intake volume, sevelamer dose, serum calcium (Ca), serum CaxP, blood urea nitrogen (BUN), serum creatinine and serum 1,25(OH)2D3 in Sham and Nx rats at baseline and effects of dietary treatment with sevelamer on these parameters at the end of the study

 

Serum parameters
Serum phosphorus concentration progressively increased in Nx rats as the study progressed (Figure 1AGo). However, 1% sevelamer suppressed the occurrence of hyperphosphataemia, and 3% sevelamer initially maintained serum phosphorus concentration below the Sham level; however, they reached that concentration towards the end of the experiment. The time course changes in serum calcium–phosphorus product (CaxP) were similar to those of serum phosphorus because sevelamer had no significant effect on serum calcium concentration at any point. Serum calcium and CaxP measured at baseline and at the end of study are shown in Table 1Go. Serum PTH concentrations in Nx rats were already elevated at baseline and then progressively increased in Nx rats (Figure 1BGo). In contrast, 1% sevelamer maintained PTH concentrations at baseline, and 3% sevelamer decreased PTH concentrations below the baseline concentration throughout the study. Serum creatinine and BUN gradually increased in Nx rats as the study progressed and were not significantly affected by 1 or 3% sevelamer (Table 1Go). Serum 1,25(OH)2D3 concentration was significantly reduced in Nx rats at baseline and at the end of study, and was not affected by sevelamer (Table 1Go).



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  		Fig. 1.  Effects of dietary treatment of sevelamer on serum concentrations of phosphorus (A) and PTH (B) in partially nephrectomized (Nx) rats. Values are the mean±SE (n=8–12), and missing SEM bars are hidden within the symbols. Sham rats were fed a normal diet (filled circles) and Nx rats were fed a normal diet (open circles), or a diet containing 1% (open triangle) or 3% sevelamer (open squares) for 4 weeks. #P<0.05, ##P<0.01 vs Sham rats. *P<0.05, **P<0.01, ***P<0.001 vs Nx rats fed a normal diet.

 

Maximal PTG area and PCNA-positive cells
The maximal PTG area in Nx rats was significantly larger at baseline and showed further increases by the end of the study (Figure 2AGo). However, 1% sevelamer tended to suppress this enlargement, and 3% sevelamer significantly suppressed it to the equivalent of the baseline measurement. Histological observations of PCNA-positive cells in the PTG, identified by brown staining in the nuclei, are shown in Figure 3Go. The PCNA-positive cells in Nx rats were ~2-fold larger than those in Sham rats at baseline and showed further increases at the end of the study (Figure 2BGo). We observed that 1% sevelamer maintained PCNA-positive cells at approximately baseline level, and 3% sevelamer markedly suppressed them to lower than baseline. PCNA-positive cells in the 3% sevelamer group were reduced below the Sham level, although the difference was not statistically significant.



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  		Fig. 2.  Maximal PTG area (A) and PCNA-positive cells (B) in Sham and partially nephrectomized (Nx) rats at baseline, and effects of dietary treatment with sevelamer for 4 weeks on maximal PTG area and PCNA-positive cells at the end of the study. Values are the mean±SE (n=8–12). #P<0.05, ##P<0.01, ###P<0.001 vs Sham rats. *P<0.01, ***P<0.001 vs Nx rats fed a normal diet.

 


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  		Fig. 3.  PCNA-positive cells in the PTG of Sham rats (A), and partially nephrectomized (Nx) rats fed a normal diet (B), or a diet containing 1% sevelamer (C) or 3% sevelamer (D). Immunohistochemistry for PCNA combined with counterstaining with haematoxylin.

 

Correlation analysis
There was a strong positive correlation between PCNA-positive cells and maximal PTG area (Figure 4Go). The maximal PTG area correlated positively with serum PTH concentration (r=0.743, P<0.001). Serum phosphorus concentration correlated positively with PCNA-positive cells. In contrast, serum calcium concentration correlated weakly and negatively, and serum 1,25(OH)2D3 concentration did not correlate with PCNA-positive cells.



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  		Fig. 4.  Correlation analysis between PCNA-positive cells and maximal PTG area (A), and serum parameters and PCNA-positive cells (B–D). Sham rats fed a normal diet (filled circles), and partially nephrectomized (Nx) rats fed a normal diet (open circles), or a diet containing 1% (open triangles) or 3% sevelamer (open squares). r, correlation coefficient with corresponding P-value in parentheses. NS, not significant.

 



   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
Recent in vitro and in vivo studies have demonstrated the direct effect of phosphorus on PTG function and cell proliferation, independent of calcium and 1,25(OH)2D3. In the present study, sevelamer inhibited the progressive increase of serum phosphorus and PTH concentrations without affecting serum calcium or 1,25(OH)2D3 concentrations in Nx rats with CRI. In addition, sevelamer suppressed the maximal PTG area and PCNA-positive cells, and serum phosphorus concentration correlated significantly with PCNA-positive cells.

The results of the present study indicate that the reduced serum phosphorus concentration achieved by sevelamer treatment contributed to reduced PTG cell proliferation and prevention of PTG hyperplasia. The present study also showed a strong positive correlation between the maximal PTG area and serum PTH concentration, in agreement with published reports relating to patients [1,2] and rats with CRI [13,14]. The suppression of PTG hyperplasia by sevelamer treatment resulted in an ongoing reduction in concentration of serum PTH.

By switching the diet from high phosphorus to low phosphorus in remnant kidney rats, the PTG remains enlarged and there is no apoptosis in the PTG cells [3]. The present study demonstrated that 3% sevelamer maintained PTG size at baseline measurements, but could not decrease the size of an enlarged PTG. We have demonstrated that PTG enlargement is caused predominantly by PTG cell hyperproliferation in Nx rats [13,14]. The strong positive correlation between PCNA-positive cells and maximal PTG area observed in the present study suggests that sevelamer treatment could inhibit PTG cell proliferation and prevent PTG hyperplasia. In the 3% sevelamer group, the number of PCNA-positive cells was less than the Sham level, but the difference was not statistically significant. However, this result suggests that if serum phosphorus concentration can be maintained below normal, PTG cell proliferation can be limited to less than the normal level regardless of uraemia and deficiency of 1,25(OH)2D3. Further investigation of inhibition of PTG cell proliferation as a result of phosphorus restriction is necessary.

Serum calcium and 1,25(OH)2D3 concentrations are other factors causing 2HPT and PTG hyperplasia [1–3]. Serum calcium negatively correlated, but 1,25(OH)2D3 did not correlate with PCNA-positive cells in the present study. Sevelamer did not affect serum calcium or 1,25(OH)2D3 concentrations. Therefore, it may be concluded that the serum phosphorus concentration [not that of serum Ca or 1,25(OH)2D3] is the main factor that determines the PTG cell proliferation in Nx rats being treated with sevelamer.

Sevelamer did not affect the increase of BUN or serum creatinine. It has been reported that dietary phosphate restriction partly protects renal function in experimental animals with CRI. We have observed that sevelamer treatment begun immediately after the injection of nephrotoxic serum slows deterioration of renal function in Wistar Kyoto rats with CRI [15]. The reason for the lack of a renal protective effect by sevelamer in Nx rats is unclear. A possible explanation is that irreversible changes occurred and became established during the 7-week interval between nephrectomy and the start of sevelamer treatment. In addition, Nx rats are initiated by acute surgical reduction of renal function, whereas antibody-induced CRI rats have a gradually decreasing renal function. The inhibitory effect of sevelamer on PTG cell proliferation observed in the present study was independent of renal function.

In conclusion, the lowered serum phosphorus concentration achieved by sevelamer treatment apparently inhibits PTG cell proliferation, which results in prevention or retardation of PTG hyperplasia. Secondary hyperparathyroidism is initiated at an early stage of CRI, and PTG hyperplasia is accompanied by hypersecretion of PTH. In addition, PTG hyperplasia is rarely reversible and often develops into nodular hyperplasia, which is refractory to vitamin D therapy. The use of sevelamer from the early stages of CRI could prevent PTG hyperplasia and facilitate later management of 2HPT.



   Notes
 
Correspondence and offprint requests to: Nobuo Nagano, PhD, Pharmaceutical Development Laboratories, Kirin Brewery Co., Ltd 3 Miyahara, Takasaki, Gunma 370-1295, Japan. Email: n-nagano{at}kirin.co.jp Back



   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

  1. Parfitt AM. The hyperparathyroidism of chronic renal failure: A disorder of growth. Kidney Int 1997; 52:3–9[Web of Science][Medline]
  2. Fukagawa M. Cell biology of parathyroid hyperplasia in uremia. Am J Med Sci 1999; 317:377–382[CrossRef][Web of Science][Medline]
  3. Slatopolsky E, Dusso A, Brown AJ. The role of phosphorus in the development of secondary hyperparathyroidism and parathyroid cell proliferation in chronic renal failure. Am J Med Sci 1999; 317:370–376[CrossRef][Web of Science][Medline]
  4. Hernández A, Concepción MT, Rodríguez M, Salido E, Torres A. High phosphorus diet increases preproPTH mRNA independent of calcium and calcitriol in normal rats. Kidney Int 1996; 50:1872–1878[Web of Science][Medline]
  5. Wang Q, Paloyan E, Parfitt AM. Phosphate administration increases both size and number of parathyroid cells in adult rats. Calcif Tissue Int 1996; 58:40–44[Web of Science][Medline]
  6. 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. J Clin Invest 1995; 96:1786–1793
  7. Lopez-Hilker S, Dusso AS, Rapp NS, Martin KJ, Slatopolsky E. Phosphorus restriction reverses hyperparathyroidism in uremia independent of changes in calcium and calcitriol. Am J Physiol 1990; 259:F432–F437[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 Dis 1996; 28:596–602[Web of Science][Medline]
  9. Slatopolsky E, Finch J, Denda M et al. Phosphorus restriction prevents parathyroid gland growth: high phosphorus directly stimulates PTH secretion in vitro. J Clin Invest 1996; 97:2534–2540[Web of Science][Medline]
  10. Nielsen PK, Feldt-Rasmussen U, Olgaard K. A direct effect in vitro of phosphate on PTH release from bovine parathyroid tissue slices but not from dispersed parathyroid cells. Nephrol Dial Transplant 1996; 11:1762–1768[Abstract/Free Full Text]
  11. Amin N. The impact of improved phosphorus control: use of sevelamer hydrochloride in patients with chronic renal failure. Nephrol Dial Transplant 2002; 17:340–345[Abstract/Free Full Text]
  12. Nagano N, Miyata S, Obana S et al. Sevelamer hydrochloride (Renagel®), a non-calcaemic phosphate binder, arrests parathyroid gland hyperplasia in rats with progressive chronic renal insufficiency. Nephrol Dial Transplant 2001; 16:1870–1878[Abstract/Free Full Text]
  13. 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 Int 2000; 57:50–58[CrossRef][Web of Science][Medline]
  14. 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 Nephrol 2000; 11:903–911[Abstract/Free Full Text]
  15. Nagano N, Miyata S. Obana et al. Renagel® (PB-94), a non-calcemic phosphate binder prevents the deterioration of renal function in rats with progressive renal insufficiency. J Am Soc Nephrol 2000; 11:565A

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