How dietary phosphate, renal failure and calcitriol administration affect the serum calcium–phosphate relationship in the rat

Aquiles Jara1,, Cecilia Chacón1 and Arnold J. Felsenfeld2

1 Department of Nephrology, Pontificia Universidad Católica de Chile, Santiago, Chile and 2 Department of Medicine, West Los Angeles VA Medical Center and UCLA, Los Angeles, California, USA



   Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Background. The effect of hyperphosphataemia on serum calcium regulation in renal failure has not been well studied in a setting in which hypercalcaemia is not parathyroid hormone (PTH) mediated. In azotemic rats with a normal serum calcium concentration, an increased dietary phosphate burden affects serum calcium regulation because of its effects on skeletal resistance to PTH, calcitriol production, and possibly intestinal calcium absorption. Our goal was to determine how hyperphosphataemia affected the development of hypercalcaemia during calcitriol-induced hypercalcaemia and PTH suppression in azotemic rats with established hyperparathyroidism.

Methods. Rats underwent a two-stage 5/6 nephrectomy or corresponding sham operations. After surgery, rats were given a high phosphate diet (P 1.2%) for 4 weeks to exacerbate hyperparathyroidism and were then changed to a normal diet (P 0.6%) for 2 weeks to normalize serum calcium values in the azotemic rats. At week 7, rats were divided into five groups and sacrificed after receiving three intraperitoneal doses of calcitriol (CTR, 500 pmol/100 g) or vehicle at 24 h intervals. The five groups and dietary phosphate content were: group 1, normal renal function (NRF)+0.6% P+vehicle; group 2, NRF+0.6% P+CTR; group 3, renal failure (RF)+0.6% P+vehicle; group 4, RF+1.2% P+CTR; and group 5, RF+0.6% P+CTR. Both the 0.6% and 1.2% phosphate diets contained 0.6% calcium.

Results. Serum creatinine values were increased (P<0.05) in 5/6 nephrectomized rats (groups 3, 4 and 5), as were serum calcium values (P<0.05) in CTR-treated rats (groups 2, 4 and 5) and serum phosphate values (P<0.05) in CTR-treated azotemic rats (groups 4 and 5). Serum PTH values were suppressed (P<0.05) in CTR-treated hypercalcemic rats (groups 2, 4 and 5) and increased (P<0.05) in azotemic rats not given CTR (group 3). In the azotemic groups (groups 3, 4 and 5), an inverse correlation was present between serum calcium and phosphate in each group, despite a wide variation in serum calcium values. The slope of the inverse relationship between serum calcium and phosphate was steeper in CTR-treated azotemic rats on a 1.2% phosphate (group 4) diet than on a 0.6% phosphate (group 5) diet (P=0.02). Thus, for a similar increase in the serum phosphate concentration, serum calcium values decreased more in group 4 than in group 5. The independent effect of dietary phosphate on serum calcium values was also confirmed by analysis of covariance. Finally, the serum calcium concentration was shown to be greater for any given serum phosphate value in CTR-treated rats than in those not on CTR.

Conclusions. In azotemic rats with calcitriol-induced hypercalcaemia, the magnitude of hypercalcaemia is affected by: (i) the serum phosphate concentration; and (ii) differences in dietary phosphate content. Calcitriol administration also acts to shift upwards the relationship between serum calcium and phosphate so that a higher serum calcium concentration can be maintained for any given serum phosphate value.

Keywords: calcitriol; calcium; hyperparathyroidism; parathyroid hormone; phosphate; renal failure



   Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In parathyroid hormone (PTH)-induced increases in the serum calcium concentration, the presence of a reciprocal relationship between serum calcium and phosphate has often been noted. This interesting observation was probably first recognized by Fuller Albright [1] who noted that: (i) during studies of PTH infusion in hypoparathyroid humans, a decrease in serum phosphate values preceded any increase in the serum calcium concentration; and (ii) during PTH-induced hypercalcaemia, the serum calcium concentration stopped increasing when the serum phosphate concentration failed to decrease further. In a recent study in parathyroidectomized rats in which different doses of PTH were infused, the presence of a reciprocal relationship between serum calcium and phosphate was also shown to be present [2]. Thus, these and other studies [14] suggest that the pivotal role of PTH in lowering serum phosphate concentration is important in enhancing the calcemic action of PTH.

In the 1960s, phosphate administration was used to treat hypercalcaemia and lower the serum calcium concentration, even in situations in which hypercalcaemia was not PTH-mediated or likely to be mediated by PTH-related peptide (PTHrP) [5]. Moreover, in hypercalcaemia due to vitamin D intoxication or hyperthyroidism, serum phosphate values are often in the high normal range or even elevated [6,7] and thus conceivably could be acting to reduce further increases in the serum calcium concentration. In such causes of hypercalcaemia, PTH suppression induced by hypercalcaemia reduces urinary phosphate excretion and contributes to increases in the serum phosphate concentration. Whether the reduction in serum calcium concentration during phosphate administration results from precipitation of calcium–phosphate complexes or because of increased skeletal resistance to PTH has not been resolved [24,8,9]. Moreover, in hypercalcaemia not mediated by PTH or PTHrP, whether hyperphosphataemia affects the resulting serum calcium value in a predictable manner has not been well studied.

In the present study, hypercalcaemia was induced in normal and azotemic rats with high doses of calcitriol. In the two groups of azotemic rats given calcitriol, the dietary phosphate load was different. Our goal was to determine in azotemic rats with established hyperparathyroidism how hyperphosphataemia affected the development of hypercalcaemia during calcitriol administration when PTH, due to its suppression, was not contributing to the regulation of serum calcium.



   Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The study was performed in male Sprague-Dawley rats, which weighed 140–160 g at the start of the study. All rats underwent two sham operations or a two-stage 5/6 nephrectomy in which two of the three main renal arteries in the hilum of the left kidney were ligated; 1 week later, a right nephrectomy was performed. During surgery, rats were anaesthetized with administered intraperitoneally with ketamine 7.5 mg/100 g (Ketaset; Fort Dodge Laboratories, Fort Dodge, IA, USA) and xylazine 0.5 mg/100 g (AnaSed; Lloyd Laboratories, Shenandoah, IA, USA). Rats were housed in individual cages, given 15 g of food daily and allowed free access to water. Rats ingesting <12 g of food daily were removed from the study.

To exacerbate the magnitude of secondary hyperparathyroidism in sham-operated and 5/6 nephrectomized rats, all rats were placed for the first 4 weeks of the study on a high phosphate diet (P 1.2%, Ca 0.6%), which contained 100 IU of vitamin D per 100 g of diet (ICN, Cleveland, OH). After 4 weeks on the high phosphate diet, all rats were changed to a normal diet (P 0.6%, Ca 0.6%) for a further 2 weeks to normalize serum calcium values in the azotemic rats. At the start of week 7, rats were divided into five groups, given their study diet for 1 day, and then received either three i.p. doses of calcitriol (500 pmol/100 g of body weight per dose) (Abbott, Chicago, IL) or its vehicle at 72, 48 and 24 h before sacrifice. A high dose of calcitriol (CTR) was used to induce hypercalcaemia because the study was designed to determine whether CTR induced apoptosis of parathyroid cells [10]. The five study groups and the phosphate content of the diets given during the 96 h of week 7 before sacrifice were: (i) group 1 (NRF+0.6% P, n=12), sham-operated rats with normal renal function (NRF)—received vehicle; (ii) group 2 (NRF+0.6% P+CTR, n=13), sham-operated rats with normal renal function—received i.p. CTR; (iii) group 3 (RF+0.6% P, n=12), rats with renal failure—received vehicle; (iv) group 4 (RF+1.2% P+CTR, n=13), rats with renal failure—received i.p. CTR; and (v) group 5 (RF+0.6% P+CTR, n=12), rats with renal failure—received i.p. CTR. Both the 0.6% and 1.2% phosphate diets contained 0.6% calcium. Twenty-four hours after the last dose of CTR or vehicle, rats were anaesthetized and then sacrificed by exsanguination after puncture of the abdominal aorta.

Serum calcium was measured with an autoanalyser, serum phosphate with a specific kit (Sigma, St Louis, MO, USA), serum creatinine with a creatinine analyser (Beckman, Fullerton, CA, USA) and intact PTH with an immunoradiometric assay for the rat (Nichols, San Clemente, CA, USA). Due to technical problems, PTH values could not be measured in one rat each in groups 2 and 5.

Statistics
One-way analysis of variance (ANOVA) was used to compare three or more groups. Post-hoc analysis was performed using the Fisher LSD. For correlation between two variables, Pearson's correlation was used. The comparison of the slopes of two or more correlations was performed with the software program GraphPad Prism version 3.0. Because the data for the slopes were not normally distributed, a logarithmic transformation of the data was performed. To determine whether dietary phosphate affected the relationship between serum calcium and serum phosphate independently, analysis of covariance (ANCOVA) was used. A P value <0.05 was considered significant. Results are shown as the mean±standard error (SE).



   Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In Figure 1Go, the biochemical data of the five groups of rats are shown. Serum creatinine was greater in the three groups subjected to 5/6 nephrectomy (groups 3, 4 and 5). As compared with group 3, however, serum creatinine was greater in groups 4 and 5, in which hypercalcaemia and hyperphosphataemia were present. Serum calcium was greater in the three groups that received CTR (groups 2, 4 and 5). Among these groups, serum calcium was less in the group on the high phosphate diet (group 4). Serum phosphate was greater in groups 4 and 5, but was higher in group 4 (high phosphate diet) than group 5. Parathyroid hormone was greatest in the azotemic group (group 3) that did not receive CTR, and was also greater in the non-azotemic group which did not receive CTR (group 1) compared with the three hypercalcemic groups given CTR. The weight of the azotemic rats in group 4 (274±4 g), which had received both CTR and a high phosphate diet, was less (P<0.05) than the other four groups: 302±8 g (group 1), 301±4 g (group 2), 301±8 g (group 3) and 289±6 g (group 5).



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Fig. 1.  Shown are the serum creatinine, calcium, phosphate and PTH values for each of the five groups. NRF=normal renal function; CTR=calcitriol; RF=renal failure; and P=dietary phosphate. The ANOVA P values among the five groups for serum creatinine, calcium, phosphate and PTH are <0.001. Differences between individual groups were determined by post-hoc testing. a, the value is not different from other groups marked with a; b, the value is not different from other groups marked with b and the P value is <0.05 vs a; c, the value is not different from other groups marked with c and the P value is <0.05 vs a and b.

 
The relationships between serum calcium, phosphate and PTH for the five groups are shown in the three graphs of Figure 2Go. In Figure 2BGo, a sharp, inverse relationship was present between serum calcium and PTH with hypercalcaemia, resulting in suppression of PTH secretion. Because of the wide variation in biochemical values among the five groups, it was difficult to show relationships common to all groups. In the three groups with CTR-induced hypercalcaemia, an inverse correlation was present between serum calcium and phosphate (r=-0.76, P<0.001).



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Fig. 2.  Shown for the five separate groups are the individual data points for the comparisons between (A) serum calcium and phosphate, (B) serum PTH and calcium, and (C) serum PTH and phosphate.

 
In each of the azotemic groups (groups 3, 4 and 5), the inverse correlation between serum calcium and phosphate was significant or closely approached significance (Figure 3Go). Differences were also present in the slopes of the inverse correlations, which in group 4 was steeper than in group 5 (P=0.02) and approached significance vs group 3 (P=0.08). Thus, as serum phosphate values increased, the serum calcium concentration decreased progressively in all groups, but for a similar increase in serum phosphate values, the decrease in serum calcium values was greater in group 4 than in group 5.



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Fig. 3.  Shown for the three azotemic groups (groups 3, 4 and 5) are the individual data points for the comparisons between serum calcium and phosphate. The regression lines are drawn for the individual correlations in the separate groups (group 3, solid line; group 4, dotted line; group 5, dashed line). Serum calcium vs phosphate correlations for individual groups are: RF, y=11.6-0.125x (r=-0.57, P=0.05); RF+CTR+1.2% P, y=17.6-0.322x (r=-0.79, P=0.001); and RF+CTR+0.6% P, y=16.8-0.147x (r=-0.54, P=0.07). CTR=calcitriol; RF=renal failure; P=dietary phosphate.

 
To determine better whether dietary phosphate directly affected serum calcium regulation independent of the serum phosphate concentration, ANCOVA was performed in: (i) all five groups; (ii) the three azotemic groups; (iii) the three CTR-treated groups; and (iv) the two azotemic groups treated with CTR. As shown in Table 1AGo, ANCOVA showed not only that CTR treatment had an independent effect on serum calcium, as would be expected, but also that dietary phosphate separate from the serum phosphate concentration had an independent effect on the serum calcium concentration. A similar independent effect of dietary phosphate was observed when only azotemic groups (groups 3, 4 and 5) were evaluated (Table 1BGo). When only CTR-treated groups (groups 2, 3 and 5) were evaluated by ANCOVA (Table 2AGo), dietary phosphate again was shown to have an independent effect on the serum calcium concentration. In this analysis, PTH was not included as a covariate because as the magnitude of hypercalcaemia increased in CTR-treated rats, PTH values progressively decreased. Thus, in this setting, PTH cannot be considered to be a regulator of the serum calcium concentration. Finally, the effect of dietary phosphate was evaluated in the two azotemic groups treated with CTR and given different phosphate diets (groups 4 and 5). As shown in Table 2BGo, dietary phosphate was again shown to have an independent effect on the serum calcium concentration.


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Table 1.  Separate effect of calcitriol administration and of dietary phosphate on serum calcium values as determined by ANCOVA

 

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Table 2.  Effect of differences in dietary phosphate on serum calcium values as determined by ANCOVA

 
In Figure 4Go, the relationship between the serum calcium–phosphate product and the serum phosphate concentration is shown. For any given serum phosphate value, the calcium–phosphate product was greater in the two CTR-treated groups on a 0.6% phosphate diet (groups 2 and 5) than in the two groups on a 0.6% phosphate diet that were not given CTR (groups 1 and 3). One group in each subset had renal failure (groups 3 and 5). Figure 4Go also indirectly shows that for any given serum phosphate value, the serum calcium concentration was greater in the CTR-treated groups. For example, at a serum phosphate value of 8 mg/dl, the serum calcium concentration would be 15.75 mg/dl in CTR-treated rats (calcium–phosphate product of 126 divided by the serum phosphate value of 8 mg/dl) in contrast to a value of 10.4 mg/dl in non-CTR treated rats.



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Fig. 4.  The correlation between the serum calcium–phosphate product and serum phosphate is shown in the two calcitriol-treated groups on a 0.6% phosphate diet (groups 2 and 5) and in the two groups not given calcitriol (groups 1 and 3). At any given serum phosphate value, the serum calcium concentration is greater by >=5 mg/dl in the calcitriol-treated groups (serum calcium–phosphate product divided by the serum phosphate concentration). The correlation in the two calcitriol-treated groups is y=12.8x+23.1 (r=0.98, P<0.001). In the two groups not given calcitriol, the correlation is y=10.1x+3.49 (r=0.98, P<0.001). CTR=calcitriol.

 



   Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In hypercalcaemia due to CTR administration, we were able to show in azotemic rats that: (i) the magnitude of hypercalcaemia was affected by hyperphosphataemia; (ii) the magnitude of hypercalcaemia for a similar elevation in serum phosphate was independently affected by dietary phosphate; and (iii) CTR administration increased the serum calcium concentration for any given serum phosphate value. Thus, similar to PTH-mediated hypercalcaemia, a reciprocal relationship between serum calcium and phosphate was present in CTR-mediated hypercalcaemia, but dietary phosphate also affected this latter relationship.

Since all rats received the same diets for the first 6 weeks of the study, the biochemical values at the onset of week 7 would be expected to be similar in the two non-azotemic groups (groups 1 and 2) and in the three azotemic groups (groups 3, 4 and 5). Thus, differences in serum calcium, phosphate, PTH and creatinine values between groups 1 and 2 and among groups 3, 4 and 5 must have developed during the 72–96 h before sacrifice. Moreover, the biochemical values at sacrifice in group 1 (non-azotemic) and group 3 (azotemic) should reflect the biochemical values present in non-azotemic (group 2) and azotemic (groups 4 and 5) groups before CTR was given or dietary phosphate was increased (group 4).

In the two azotemic groups with hypercalcaemia, an inverse correlation was present between serum calcium and phosphate. In group 4, the slope was steeper than in group 5. Thus, for similar increases in serum phosphate values, the serum calcium concentration decreased more in group 4 than in group 5. The difference in slopes between groups 4 and 5 appeared to result from the enhanced dietary influx of phosphate in group 4, and such a conclusion was supported by the ANCOVA analysis performed in Table 2Go. A greater gut transfer of phosphate in group 4 could have increased the amount of phosphate presented for bone buffering and thus could have acted to retard CTR-induced bone release of calcium [3,4]. Another possibility is that the higher dietary phosphate content reduced calcium absorption. There are conflicting data on this subject [11,12], but if present, such an effect may be small and could be due to an associated decrease in serum CTR levels [12]. Another consideration is whether the reduced slope in group 4 could have resulted from extravascular precipitation of calcium–phosphate complexes.

The use of the serum calcium–phosphate product and serum phosphate relationship (Figure 4Go) improved on the correlation between serum calcium and phosphate because the serum phosphate value influenced the calcium–phosphate product more than the serum calcium concentration. This is so because the increase in serum phosphate was much greater than the corresponding decrease in serum calcium. Between the serum phosphate extremes of 6 and 19 mg/dl present in the two CTR-treated groups, the serum calcium concentration decreased from 16.67 to 14 mg/dl. Thus, serum calcium decreased 0.205 mg/dl for every 1 mg/dl increase in serum phosphate. The approach using the serum calcium–phosphate product eliminates much of the variability that was present for the correlation between the serum calcium and phosphate concentrations, and thus allows for a better comparison between CTR-treated and non-CTR-treated groups on a 0.6% phosphate diet. At the same serum phosphate value, the serum calcium–phosphate product was greater in the CTR-treated groups because the serum calcium value was greater. Thus, at any serum phosphate concentration between the 6 and 10 mg/dl values seen in the non-CTR treated rats, the serum calcium concentration was greater by >5 mg/dl (serum calcium=serum calcium-phosphate product divided by serum phosphate).

Important questions are: (i) the extent to which hypercalcaemia in the CTR-treated rats was due to enhanced intestinal calcium absorption or efflux from bone [13,14], and (ii) whether the lower serum calcium values in azotemic, CTR-treated rats on a high phosphate diet (group 4) resulted from reduced intestinal calcium absorption, decreased calcium efflux from bone, or extravascular precipitation of calcium and phosphate. For the most part, these questions cannot be answered by the design of our study. But with respect to whether calcium–phosphate precipitation could be the cause of the lower serum calcium concentration in group 4 as compared with group 5, an interesting observation is that for a serum phosphate value of 19 mg/dl in group 5, the serum calcium was still ~14 mg/dl (product 266). Conversely, for the same serum phosphate concentration, the serum calcium value in group 4 was only ~11 mg/dl (product 209). Such a finding would seem to suggest that precipitation of calcium and phosphate might not be the primary cause of the lower serum calcium concentration in group 4.

In our study, the magnitude of hypercalcaemia in CTR-treated, azotemic rats was less for the same serum phosphate concentration in the rats on a high phosphate diet. Why, in the normal state, serum calcium values are similar between species and between the young and adults of any species while serum phosphate values vary considerably between such species as the rat and human and are also higher in the young than in the adult remain unanswered and important questions [1517]. For example, in the adult human, a serum phosphate value of 8 mg/dl is expected to perturb serum calcium regulation. But the same serum phosphate value in the rat or human neonates [18] does not seem to affect serum calcium regulation. Whether, in such settings, inhibitors of soft tissue calcium deposition such as matrix-GLA protein in arteries and cartilage of the growth plate protect against calcium–phosphate deposition [19,20] in these locations, and whether the resulting high calcium–phosphate provides a gradient for deposition in the growth plate of rapidly growing bones and in teeth are important considerations.

In conclusion, we showed during calcitriol-induced hypercalcaemia in azotemic rats that the magnitude of hypercalcaemia was affected by both the serum phosphate concentration and differences in dietary phosphate. Calcitriol administration also acted to shift upwards the relationship between serum calcium and phosphate so that a higher serum calcium concentration could be maintained for any given serum phosphate value.



   Acknowledgments
 
The authors would like to thank Drs Armando Torres and Alejandro Jimenez for their help with the comparisons of the slopes and the analysis of covariance, and Drs Mariano Rodriguez and Barton Levine for helpful suggestions during the writing of the manuscript. This work was supported by a grant from Fondo Nacional de Ciencias y Tecnología de Chile (FONDECYT No. 1960785).



   Notes
 
Correspondence and offprint requests to: Aquiles Jara, Department of Nephrology, Hospital Clinico, Pontificia Universidad de Católica de Chile, Marcoleta 345, Santiago, Chile. Email: ajara{at}med.puc.cl Back



   References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received for publication: 27. 3.01
Accepted in revised form: 18.12.01





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