Effect of manipulating serum phosphorus with phosphate binder on circulating PTH and FGF23 in renal failure rats

Effect of manipulating serum phosphorus with phosphate binder on circulating PTH and FGF23 in renal failure rats

original article http://www.kidney-international.org & 2006 International Society of Nephrology see commentary on page 425 Effect of manipulating s...

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original article

http://www.kidney-international.org & 2006 International Society of Nephrology

see commentary on page 425

Effect of manipulating serum phosphorus with phosphate binder on circulating PTH and FGF23 in renal failure rats N Nagano1, S Miyata1, M Abe1, N Kobayashi1, S Wakita1, T Yamashita1 and M Wada1 1

Pharmaceutical Development Laboratories, Pharmaceutical Research Laboratories and Medical Affairs Section, Pharmaceutical Division, Kirin Brewery Company Limited, Takasaki and Tokyo, Japan

Phosphorus directly controls parathyroid hormone (PTH) synthesis and secretion. Serum levels of the novel phosphate-regulating hormone, fibroblast growth factor 23 (FGF23), are positively correlated with hyperphosphatemia in patients with chronic renal insufficiency (CRI). We proposed that changes in serum PTH and FGF23 levels might be associated with changes in serum phosphorus levels caused by the phosphate binder sevelamer hydrochloride (sevelamer, i.e. crosslinked poly[allylamine hydrochloride]). Rats were fed a diet containing adenine for 4 weeks to establish CRI. Animals were then offered either a normal diet or a diet containing 1 or 3% sevelamer for 8 weeks continuously, or intermittently with sevelamer diet or a normal diet offered for alternating 2-week periods. Changes in the serum levels of phosphorus, calcium, PTH, FGF23, and 1a,25-dihydroxyvitamin D3 (1,25(OH)2D3) were monitored over time. Adenine-treated rats developed severe CRI, with markedly elevated serum levels of phosphorus, PTH and FGF23, and reduced levels of serum 1,25(OH)2D3. Continuous treatment with sevelamer suppressed these increases throughout the study period. Serum phosphorus, PTH, and FGF23 levels decreased rapidly when sevelamer treatments commenced and recovered rapidly once they were discontinued. However, the changes in serum FGF23 levels began after the onset of changes in serum phosphorus and PTH levels. In conclusion, circulating PTH, and FGF23 levels can be promptly manipulated through the control of serum phosphorus levels. Moreover, phosphate-binder treatment can effectively inhibit the elevation of serum FGF23 levels, as well as PTH levels, under conditions of CRI. Kidney International (2006) 69, 531–537. doi:10.1038/sj.ki.5000020; published online 4 January 2006 KEYWORDS: 1,25(OH)2D3; calcium; FGF23; parathyroid hormone; phosphorus; sevelamer hydrochloride

Correspondence: N Nagano. Current address: Medical Affairs Section, Pharmaceutical Division, Kirin Brewery Company Limited, 26-1, Jingumae 6-chome, Shibuya-ku, Tokyo 150-8011, Japan. E-mail: [email protected] Received 27 March 2005; revised 28 June 2005; accepted 11 August 2005; published online 4 January 2006 Kidney International (2006) 69, 531–537

Hyperphosphatemia is a major complication in hemodialysis patients and plays a key role in the pathogenesis of secondary hyperparathyroidism (2HPT).1–3 It is now widely accepted that phosphate directly stimulates parathyroid hormone (PTH) secretion and synthesis, as well as parathyroid cell proliferation, independently of calcium and 1a,25-dihydroxyvitamin D3 (1,25(OH)2D3). Conversely, restricting phosphate by means of a low phosphorus diet or phosphate-binder treatment can reverse hyperparathyroidism. Fibroblast growth factor 23 (FGF23) was recently identified as a causative factor in phosphate-wasting disorders, such as tumor-induced osteomalacia, autosomal dominant hypophosphatemic rickets and X-linked hypophosphatemic rickets.4 Marked elevations of serum FGF23 levels have been positively correlated with serum levels of phosphorus, calcium and PTH in patients with end-stage renal disease.5–8 Interestingly, total parathyroidectomy (PTx) reduced serum phosphorus levels and simultaneously decreased serum FGF23 levels in patients on dialysis.8 In healthy subjects, serum FGF23 levels decreased on a low phosphorus diet and increased with oral phosphorus load,9 although an alternative study reported that FGF23 levels were not affected by oral phosphorus deprivation or loading.5 In an animal study, a high phosphorus diet increased serum FGF23 levels in 5/6 nephrectomized rats.10 On the other hand, the administration of recombinant FGF23 slightly decreased serum PTH levels in normal mice and reduced serum phosphorus and 1,25(OH)2D3 levels in rats that underwent PTx.11 Conversely, the administration of 1,25(OH)2D3 increased serum FGF23 levels in mice11 and thyroparathyroidectomized rats without correlation with serum phosphorus levels.10 Thus, it remains unclear whether phosphorus, calcium, PTH, 1,25(OH)2D3 or an alternative factor is directly responsible for the marked elevation of circulating FGF23 levels under chronic renal insufficiency (CRI) conditions. Sevelamer hydrochloride (sevelamer, crosslinked poly[allylamine hydrochloride]) is a metal-free phosphate-binding polymer that is marketed for the treatment of hyperphosphatemia in patients on dialysis. Previously, we demonstrated beneficial protective effects of sevelamer on parathyroid cell 531

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Body weight gain was reduced in the adenine-treated rats compared to that in the normal control group (Figure 1a). The sevelamer treatments did not significantly affect body weight during the study, although a slight increase was observed in the 3% sevelamer continuous-treatment group. The mean food intake volumes in g/day were as follows: 25.470.47 for the normal control group; 12.170.63 for the disease control group; 13.370.46 for the 1% sevelamer continuous-treatment group; 15.270.61 for the 3% sevelamer continuous-treatment group; 12.670.50 for the 1% sevelamer intermittent-treatment group; and 14.370.69 for the 3% sevelamer intermittent-treatment group. Blood urea nitrogen and serum creatinine levels

The blood urea nitrogen (BUN) and serum creatinine levels increased rapidly during the 4 weeks of adenine treatment (days 28 to 1; Figure 1b and c). After the end of the adenine treatment period, the BUN, and serum creatinine levels remained high, although they began to decrease gradually towards the end of the study. The sevelamer treatments had no significant effect on the BUN and serum creatinine levels during the study, regardless of whether it was given continuously or intermittently. Serum phosphorus levels

In the disease control group, the serum phosphorus levels increased rapidly during the adenine treatment (days 28 to 1), reached a maximum at the end of this period and showed a slight decrease towards the end of the study (Figure 2). At 1 day after switching from the adenine diet to the 1 or 3% sevelamer diets (day 1), the serum phosphorus levels decreased rapidly to around the normal control levels. In the 3% sevelamer groups, further decreases in serum phosphorus levels were observed both 3 and 7 days after the treatment commenced (days 3 and 7), while in the 1% sevelamer groups, normal levels were maintained. Serum phosphorus levels in the 1% sevelamer continuous-treatment group remained similar to those of the normal control group, while in the 3% sevelamer continuous group, the levels were 532

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proliferation,12 parathyroid hyperplasia,13 renal functional deterioration,14 and high-turnover bone lesions15 in rats with progressive CRI, in addition to its lowering effects on serum phosphorus and PTH levels and on urinary phosphorus excretion in normal rats16 and rats with CRI.12–15 Excess dietary adenine is converted to 2,8-dihydroxyadenine, which is significantly less soluble in water, and impairs renal function by forming intratubular and interstitial precipitates of acicular crystals.17,18 Severe CRI, accompanied by hyperphosphatemia and 2HPT, is seen in adenine-treated rats.15 In the present study, we examined how serum PTH and FGF23 levels were affected by manipulating serum phosphorus levels through the intermittent administration of sevelamer in adenine-treated rats.

N Nagano et al.: Control of PTH and FGF23 by phosphorus

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Figure 1 | Effects of sevelamer on (a) body weight, (b) blood urea nitrogen (BUN), and (c) serum creatinine levels. Normal control, open circle; disease control, filled circle; 1% sevelamer continuous treatment, open triangle; 3% sevelamer continuous treatment, open square; 1% sevelamer intermittent treatment, filled triangle; and 3% sevelamer intermittent treatment, filled square. Statistically significant differences (Po0.001) between the normal and disease control groups were observed in body weight from days 21 to 56 and in both the BUN and the serum creatinine levels from days 15 to 56 (not indicated in the figures). The sevelamer treatments had no statistically significant effects.

below those in the normal controls until the end of the study (Figure 2a). In the 3% sevelamer intermittent-treatment group, the serum phosphorus levels rapidly returned to normal control levels on day 15 (i.e., 1 day after the normal diet started), increased above the disease control levels on day 17 (i.e., 3 days after the normal diet started) and then gradually decreased (Figure 2b). The second cycle of treatment with sevelamer (days 28–42) suppressed serum Kidney International (2006) 69, 531–537

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N Nagano et al.: Control of PTH and FGF23 by phosphorus

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Figure 2 | Effects of (a) continuous treatment and (b) intermittent treatment with sevelamer on serum phosphorus levels. Normal control, open circle; disease control, open triangle; 1% sevelamer treatment, filled triangle; and 3% sevelamer treatment, filled square. The time schedule for the treatment with sevelamer is indicated along the bottom of each graph. The groups and experimental days on which statistically significant differences were observed are indicated in the table below. #Po0.05, ##Po0.01, ###Po0.001 vs the normal control group. *Po0.05, **Po0.01, ***Po0.001 vs the disease control group. Exp. Day -29 Disease control # 1% continuous 3% continuous 1% Intermittent 3% Intermittent

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phosphorus levels, whereas discontinuation of the treatment (day 42) caused them to begin to rise again. Serum calcium levels

Slight hypercalcemia followed by slight hypocalcemia was observed in the disease control group compared to the normal control group (Figure 3). Serum calcium levels rapidly increased within 3 days of the start of the 1 and 3% sevelamer treatments, and the continuous-treatment groups maintained relatively high levels throughout the study (Figure 3a). Three days after switching from the sevelamer diet to the normal diet, marked reductions in serum calcium levels were observed in the intermittent-treatment groups (days 17 and 45) (Figure 3b). Kidney International (2006) 69, 531–537

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Figure 3 | Effects of (a) continuous treatment and (b) intermittent treatment with sevelamer on serum calcium levels. Normal control, open circle; disease control, open triangle; 1% sevelamer treatment, filled triangle; and 3% sevelamer treatment, filled square. The time schedule for the treatment with sevelamer is indicated along the bottom of each graph. The groups and experimental days on which statistically significant differences were observed are indicated in the table below. #Po0.05, ###Po0.001 vs the normal control group. *Po0.05, **Po0.01, ***Po0.001 vs the disease control group. Exp. Day Disease control 1% continuous 3% continuous 1% Intermittent 3% Intermittent

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Serum PTH levels

In the disease control group, serum PTH levels rose progressively and dramatically throughout the study (Figure 4). By contrast, once the 1% sevelamer treatments had commenced, the serum PTH levels began to decrease gradually, while the 3% sevelamer treatments caused the serum PTH levels to decrease rapidly to around the normal control levels just 1 day after they commenced (day 1), and caused further decreases below the normal control levels until day 13. The 1 and 3% sevelamer continuous treatments continued to reduce serum PTH levels dose-dependently until the end of the study (Figure 4a). In particular, the 3% sevelamer continuous treatment kept the serum PTH levels below the normal control levels during the early part of the 533

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N Nagano et al.: Control of PTH and FGF23 by phosphorus

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Figure 4 | Effects of (a) continuous treatment and (b) intermittent treatment with sevelamer on serum parathyroid hormone (PTH) levels. Normal control, open circle; disease control, open triangle; 1% sevelamer treatment, filled triangle; and 3% sevelamer treatment, filled square. The time schedule for the treatment with sevelamer is indicated along the bottom of each graph. The groups and experimental days on which statistically significant differences were observed are indicated in the table below. ##Po0.01, ###Po0.001 vs the normal control group. **Po0.01, ***Po0.001 vs the disease control group. Exp. Day Disease control 1% continuous 3% continuous 1% Intermittent 3% Intermittent

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study. At 1 day after switching from the sevelamer diet to the normal diet, the serum PTH levels rapidly and markedly increased, especially in the 3% sevelamer intermittenttreatment group (day 15) (Figure 4b). The second intermittent treatments of sevelamer (days 28–42) decreased the serum PTH levels, and discontinuation of these treatments (day 42) caused the serum PTH levels to begin to rise again; the magnitude of the changes was greater in the 3% sevelamer intermittent-treatment group than in the 1% group. Serum FGF23 levels

In the disease control group, the serum FGF23 levels progressively and dramatically increased, reaching a maximum on day 15 and then decreasing gradually (Figure 5). 534

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Figure 5 | Effects of (a) continuous treatment and (b) intermittent treatment with sevelamer on serum fibroblast growth factor 23 (FGF23) levels. Normal control, open circle; disease control, open triangle; 1% sevelamer treatment, filled triangle; and 3% sevelamer treatment, filled square. The time schedule for the treatment with sevelamer is indicated along the bottom of each graph. The groups and experimental days on which statistically significant differences were observed are indicated in the table below. ##Po0.01, ### Po0.001 vs the normal control group. **Po0.01, ***Po0.001 vs the disease control group. Exp.Day Disease control 1%c ontinuous 3% continuous 1% Intermittent 3% Intermittent

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Even the high dose (3%) sevelamer treatments did not affect serum FGF23 levels 1 day after they were initiated (day 1), and the levels decreased gradually between days 3 and 13. In the 1 and 3% sevelamer continuous-treatment groups, serum FGF23 levels continued to decrease dose-dependently until the end of the study (Figure 5a). In the high dose (3%) sevelamer intermittent-treatment group, there was no effect on the serum FGF23 levels 1 day after switching from the sevelamer diet to the normal diet on day 15, but it began to rise from day 17 onwards (Figure 5b). The second treatments with sevelamer (days 28–42) decreased the serum FGF23 levels and discontinuation of treatments (day 42) caused the levels to begin to rise again; the magnitude of the changes was greater in the 3% sevelamer intermittent-treatment group than in the 1% group. Kidney International (2006) 69, 531–537

original article

N Nagano et al.: Control of PTH and FGF23 by phosphorus

Serum 1,25(OH)2D3 (pg/ml)

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Figure 6 | Effects of sevelamer on serum 1,25(OH)2D3 levels. Normal control, open circle; disease control, filled circle; 1% sevelamer continuous treatment, open triangle; 3% sevelamer continuous treatment, open square; 1% sevelamer intermittent treatment, filled triangle; and 3% sevelamer intermittent treatment, filled square. The time schedule for the treatment with sevelamer is indicated along the top of the graph. The statistical significant differences were observed between the normal and disease control groups on days –15 (Po0.001), 1 (Po0.001), 13 (Po0.001), 27 (Po0.01) and 56 (Po0.05). The sevelamer treatments had no statistically significant effects.

Serum 1,25(OH)2D3 levels

During the adenine treatment (days 28 to 1), the serum 1,25(OH)2D3 levels decreased markedly, reaching values close to zero, and then increased slightly as the study progressed (Figure 6). The sevelamer treatments had no significant effect on serum 1,25(OH)2D3 levels during the study. DISCUSSION

Rats fed with adenine were chosen as an animal model for CRI in this study because it is simple to produce and shows relatively stable, long-lasting, and severe uremia, with hyperphosphatemia and 2HPT, even after the discontinuation of adenine feeding.15 Intermittent sevelamer treatments were used to manipulate the circulating phosphorus levels dynamically, and the concomitant changes in serum PTH and FGF23 levels were monitored. Prior to this study, a lag period between the start of sevelamer treatment and the onset of the reduction of serum phosphorus levels was predicted due to the mobilization of phosphate from the bones and intracellular pools. However, serum phosphorus levels were found to decrease 1 day after the start of the sevelamer treatments. Conversely, a rise in the serum phosphorus levels was observed 1 day after the sevelamer treatments were discontinued and they returned to their original levels within 3 days. At 3 days after the discontinuation of the high dose (3%) sevelamer treatment, a rebound phenomenon (i.e., marked hyperphosphatemia) was observed, particularly on day 17. This suggested that an intestinal phosphate absorption system, probably comprised of Na/Pi cotransporter type IIb (Na/Pi IIb), might have been upregulated during the phosphorus restriction caused by Kidney International (2006) 69, 531–537

2-week sevelamer treatment. It is of some interest that phosphate depletion might still result in the upregulation of Na/Pi IIb even under conditions of very severe CRI. The sevelamer treatments increased the serum calcium levels dose-dependently. The increased serum calcium levels are partly dependent on the reduction of serum phosphorus levels induced by sevelamer treatment (i.e., a counter-ion effect). In addition to this mechanism, we reported previously that sevelamer treatment decreased fecal calcium excretion and increased intestinal calcium absorption in a balance study with normal rats.19 Thus, it is also likely that intestinal phosphate binding by sevelamer might increase the concentration of free calcium ions, resulting in increased intestinal calcium absorption. Furthermore, the marked reduction in serum PTH levels by sevelamer might decrease the bone capacity to buffer an intestinal extra calcium load and facilitate the elevation of serum calcium levels. Interestingly, serum PTH levels continued to rise progressively during the study in adenine-treated rats receiving the normal diet (disease control group), despite stable or only minor decreases in levels of serum creatinine, phosphorus and calcium levels and the minor increase in 1,25(OH)2D3 levels. This indicated a progressive development of parathyroid hyperfunction, independent of renal function, calcium, phosphorus and 1,25(OH)2D3 levels, suggesting that longlasting uremic conditions per se can promote 2HPT. The elevated serum PTH levels rapidly decreased when the sevelamer treatments commenced, and returned to their original levels soon after discontinuation of the treatments. Along with the rapid recovery of serum phosphorus levels after the discontinuation of the sevelamer treatments, these findings support that clinical treatment with a phosphate binder should not be discontinued abruptly. The time course of the changes in serum PTH levels was similar to that for serum phosphorus levels. An in vitro study demonstrated previously that elevated phosphate concentrations in the culture media increased PTH secretion from the rat parathyroid gland within 3 h.20 Another in vivo study using normal dogs showed that continuous intravenous infusion of phosphate increased serum PTH levels within 90 min under the serum calcium clamp conditions.21 Although we were unable to examine the hourly changes in serum parameters, the onset of the changes in serum PTH levels seemed likely to follow those of the serum phosphorus levels. These observations also support a direct and acute effect of phosphate on PTH secretion in vivo, although part of the serum PTH changes could be explained by the serum calcium changes induced by the sevelamer treatments. A marked elevation in serum FGF23 levels was observed in the adenine-treated rats, which was consistent with previous reports of patients and animals with CRI.5–8,10 FGF23 mRNA is present in many tissues, including the thymus, brain, bone, thyroid/parathyroid gland and heart.22–24 According to a recent study,25 abundant FGF23 expression in the bone seems to be conclusive, but expressions in some other organs are still controversial. In addition, it remains unclear as to which 535

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N Nagano et al.: Control of PTH and FGF23 by phosphorus

of these organs makes the largest contribution to the significantly elevated circulating FGF23 levels in CRI. Furthermore, the presence of FGF23 in urine has suggested that increased FGF23 levels in CRI are partly due to a decreased clearance of FGF23 by the kidney.5 However, the present result that the intermittent sevelamer treatment drastically changed serum FGF23 levels without affecting renal function clearly indicates that declining renal clearance of FGF23 is not solely responsible for its upregulation. This result may also suggest a hypothesis that the FGF23 response is physiologically relevant rather than a nonimportant consequence of CRI. In addition to the renal tubular lesions in adenine-treated rats, it is considered that marked elevation of serum FGF23 levels partly contributes to the depletion of serum 1,25(OH)2D3 levels. At present, with the exception of the potent downregulation of renal 25-hydroxyvitamin D3-1 alpha-hydroxylase and Na/Pi IIa in physiological conditions, the functional significance of the elevated serum FGF23 levels under CRI conditions remains unclear. However, the present study is the first to show that phosphate-binder treatment can effectively inhibit the elevation of serum FGF23 levels, as well as PTH levels, under CRI conditions. Similar to the influences on serum PTH levels, initiating and discontinuing the sevelamer treatments decreased and increased the serum FGF23 levels. However, the onset of the changes in serum FGF23 levels showed a lag period (at least 1–3 days), and followed the changes in serum phosphorus and PTH levels; serum FGF23 levels were not altered 1 day after the sevelamer treatments began or ended. Similar observations were reported in healthy human subjects, whose serum FGF23 levels decreased on a low phosphorus diet and then increased with an increased oral intake; it took 5–7 days to detect any significant changes in these individuals.9 In addition, a high phosphorus diet was shown to enhance, and a low phosphorus diet to inhibit, the elevation of serum FGF23 levels in 5/6 nephrectomized rats, although this result was obtained after 4 weeks of the dietary treatment.10 Taken together, these findings suggest that serum FGF23 is predominantly regulated by phosphorus, although it is unclear why a lag period occurs. The regulatory mechanisms for FGF23 production and/or secretion should be clarified. It is possible that serum PTH levels might determine circulating FGF23 levels, because the PTx can reduce FGF23 levels in patients on dialysis.8 However, despite complete

surgical ablation of the parathyroid glands, the magnitude of the changes in serum FGF23 levels was relatively small and the serum phosphorus levels simultaneously decreased in this report. Further studies using PTx rats should be conducted in order to exclude the PTH-dependent regulation of FGF23. The administration of FGF23 lowers serum 1,25(OH)2D3 levels, and 1,25(OH)2D3 reciprocally increases FGF23 levels in mice11 and rats in the absence of the parathyroid gland,10 suggesting that 1,25(OH)2D3, as well as phosphate, regulates circulating FGF23 levels. However, the sevelamer treatment decreased serum FGF23 levels without influencing serum 1,25(OH)2D3 levels, indicating that the changes in serum FGF23 levels observed in this study were independent of 1,25(OH)2D3. Another possibility remained that serum calcium might have been involved in the regulation and determination of circulating FGF23 levels in this study. Thus, further research will be necessary to determine which factor is directly responsible for the significantly elevated circulating FGF23 levels under CRI conditions. However, the present study suggests that serum phosphorus levels predominantly determine the circulating FGF23 levels. CONCLUSION

Elevated circulating PTH and FGF23 levels were lowered rapidly by the initiation of phosphate-binder treatment, even under severe 2HPT conditions. However, after discontinuation of the phosphate-binder treatment, the PTH and FGF23 levels recovered promptly. This is the first report showing that phosphate-binder treatment can effectively inhibit the elevation of serum FGF23 levels, as well as PTH levels, under CRI conditions. MATERIALS AND METHODS Experimental protocol The experimental protocol shown in Table 1 was approved by the Experimental Animal Ethical Committee of Kirin Brewery Co. Ltd. Male Sprague–Dawley rats (8 weeks old) were purchased from Charles River Japan (Tokyo, Japan) and fed a standard powder diet containing 1.09% phosphorus, 1.17% calcium, 24.9% crude protein and 2.4 IU/g vitamin D3 (CE-2, CLEA Japan, Tokyo, Japan). The rats were kept singly in cages and allowed free access to food and water. After an acclimatization period of 10 days, a blood sample was collected from the tail artery of each animal to measure serum levels of phosphorus, calcium, creatinine, BUN, PTH, FGF23, and 1,25(OH)2D3 on experimental day 29. The rats were divided into

Table 1 | Experimental protocol Normal control Disease control Continuous treatment Continuous treatment Intermittent treatment Intermittent treatment

Normal diet 0.75% adenine 0.5% adenine 0.5% adenine 0.75% adenine 0.75% adenine 0.5% adenine 0.5% adenine 0.75% adenine 0.5% adenine 0.75% adenine 2 weeks

Blood sampling day Experimental day

536

−29 −28

2 weeks −15 −14

1% sevelamer 3% sevelamer 2 weeks −1 1 3 0

7

Normal diet Normal diet 1% sevelamer 3% sevelamer 1% sevelamer Normal diet Normal diet 3% sevelamer 2 weeks 13 15 17 14

2 weeks 21

27 28

31

Normal diet Normal diet 2 weeks

35

42 45 49 42

56 56

Kidney International (2006) 69, 531–537

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N Nagano et al.: Control of PTH and FGF23 by phosphorus

two groups, which were matched with respect to body weight on day 28. The normal control group of nine animals continued to be fed a normal diet throughout the study. The other group was fed a normal powder diet containing 0.75% adenine for 2 weeks. Then, between days 14 and 1, the dietary adenine content was reduced to 0.5% in this group to avoid death due to severe uremia. After 4 weeks, the adenine-treated rats were divided into five groups, each containing nine animals, which were matched with respect to body weight, BUN, serum creatinine, phosphorus, PTH, and FGF23 levels on day 1. On day 0, the disease control group was switched to the normal diet, the continuous-treatment groups to diet containing 1 or 3% sevelamer for a further 8 weeks and the intermittent-treatment groups to a diet containing 1 or 3% sevelamer for 2-week periods, alternating with normal diet, over the 8-week period (Table 1). Body weight and food intake volume were measured weekly, and blood samples were collected on days 1, 3, 7, 13, 15, 17, 21, 27, 31, 35, 42, 45, 49, and 56 in order to measure changes in the serum parameters over time. All of the animals were killed by abdominal aortic puncture under ether anesthesia on day 56. Serum chemistry Serum phosphorus, calcium and BUN levels were measured using commercial kits (Wako Pure Chemical Industries, Osaka, Japan). Serum creatinine was measured using an enzymatic assay (CRE-EN; Kainos Laboratories, Tokyo, Japan). Serum PTH and 1,25(OH)2D3 were measured using a rat PTH IRMA kit (Immutopics, CA, USA) and a 1,25(OH)2D RIA kit (TFB; Immunodiagnostic System Ltd, UK), respectively. Serum FGF23 levels were determined using an FGF23 ELISA Kit (Kainos Laboratories), which we established originally for the detection of biologically active intact human FGF23, using two different monoclonal antibodies specific for the N- and C-terminals of the molecule.26 This enzyme-linked immunosorbent assay also detects rodent FGF23.26 All of the serum parameters, except PTH, and 1,25(OH)2D3, were determined by a standard calorimetric method using a microplate spectrophotometer (SpectraMax 250, Molecular Devices, CA, USA).

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Drugs Sevelamer hydrochloride (which is the active ingredient of Renagels) was synthesized by Dow Chemical Company (Midland, MI, USA) and supplied via Chugai Pharmaceuticals Co. Ltd (Tokyo, Japan). Adenine was obtained from Sigma Chemical Co. (St Louis, MO, USA). Statistical analysis All values were expressed as the mean7s.e.m. The data obtained from the normal control group and the disease control group were compared using the Student’s t-test. Multiple comparisons were performed among the five adenine-treated groups using the parametric Dunnett’s test. Statistical significance was defined as Po0.05, two-sided.

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REFERENCES 1. 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. 2. Rodriguez M. Direct effect of phosphate on parathyroid function. Nephrol Dial Transplant 1999; 14(Suppl 1): 70–72.

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