Effect of aluminium load on parathyroid hormone synthesis

Carmen Díaz-Corte1, Jose Luis Fernández-Martín1, Susana Barreto1, Carlos Gómez1, Teresa Fernández-Coto2, Socorro Braga2 and Jorge B. Cannata,2

1 Bone and Mineral Research Unit, Instituto Reina Sofía de Investigación and 2 Biochemistry Unit, Hospital Central de Asturias, Oviedo, Spain



   Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Background. Aluminium overload leads to parathyroid hormone (PTH) suppression. However, it is unclear whether a decrease in synthesis or release of the hormone is mainly involved. The aim of this study was to assess the effect of an acute administration of aluminium on PTH synthesis and release in rats with chronic renal failure and secondary hyperparathyroidism.

Methods. The study was performed using 100 adult male Wistar rats (body weight 443±54 g). 7/8 nephrectomy was performed and the rats were maintained on a high dietary phosphorous intake. Five weeks after surgery, the rats were randomly divided into two groups, one loaded with aluminium (AlCl3) and the other given placebo. Aluminium or placebo were administered i.p. for two consecutive days. The placebo group received saline at the same pH as the aluminium solution. After 2 weeks, serum calcium, phosphorous, creatinine, PTH, and aluminium were measured. The parathyroid glands were removed and PTH messenger RNA (mRNA) was measured by northern blot. Intact PTH was measured by IRMA (Rat PTH®, Nichols Institute).

Results. No differences in serum PTH levels were found between the two groups after 5 weeks of renal failure. At the end of the study the rats given aluminium had higher aluminium levels than the placebo group and lower PTH levels. No significant differences were found for calcium, phosphorous, renal function, or body weight. PTH mRNA expression was lower in the aluminium group than in the placebo group.

Conclusion. The administration of aluminium in rats with chronic renal failure resulted in reductions in serum PTH and PTH mRNA. Thus far, previous studies had demonstrated that aluminium suppressed PTH release. The present findings suggest that PTH synthesis is also reduced.

Keywords: aluminium; chronic renal failure; hyper-parathyroidism; mRNA of pre-proPTH; PTH; rats



   Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
A number of clinical and experimental studies have demonstrated that aluminium overload reduces circulating parathyroid hormone (PTH) levels [17]. Aluminium suppresses PTH either indirectly by increasing serum calcium levels, or directly influencing parathyroid synthesis, degradation, or release. The bulk of the evidence suggests that the direct effect is the most frequent and relevant mechanism of inhibition. However, it is unclear whether aluminium inhibits the synthesis or release of PTH. Studies carried out before molecular biology techniques were available suggested that aluminium interferes with PTH release and degradation. In contrast, preliminary results from our laboratory indicated that aluminium-overloaded rats had a reduction in pre-proPTH messenger RNA (mRNA) [8]. The present study was designed to determine whether aluminium induces hypoparathyroidism by suppressing PTH at the message level.



   Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The study was performed using 100, 2-month-old male Wistar rats, weighing 443±54 g. The rats were housed in wire cages (four rats per cage). They received a standard diet (Panlab A-04®: calcium 0.6%, phosphorous 0.6%), and drinking water ad libitum. The study was initiated after 1 week of acclimatization. Chronic renal failure was induced surgically (7/8 nephrectomy, week 0) using the technique modified by Ormrod and Miller [9], which creates moderate to severe chronic renal failure with secondary hyperparathyroidism. The rats received phosphorus supplementation in the drinking water during the first 4 weeks of the study (4.5 mg/ml from week -1 to week 0, and 1.5 mg/ml from week 0 to week 3). At 5 weeks after nephrectomy, urine was collected for the measurement creatinine. Blood was obtained from the jugular vein for measurement of serum creatinine, calcium, phosphorous, parathyroid hormone and aluminium [3]. Creatinine clearances were used to divide animals into groups with similar renal function. The rats assigned to the aluminium groups received AlCl3 (8.94 mg AlCl3x6H2O= 1 mg Al) i.p. during two consecutive days. The placebo groups received the same volume of saline adjusted to the pH of AlCl3 (pH=2.3). After 2 weeks, urine was collected and blood was obtained by cardiac puncture to obtain repeated urinary and serum measurements. The animals were then sacrificed and the parathyroid glands were removed and stored at -70°C until processing to measurement of either aluminium or mRNA pre-proPTH concentrations. The study plan is detailed in Figure 1Go.



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Fig. 1. Scheme of the study. W, week; Nx, nephrectomy; Sx, sacrifice.

 
Serum calcium, phosphorous, and creatinine were measured with a multichannel autoanalyser (Hitachi 717®, Boehringer Mannheim, Germany), and PTH was measured using IRMA Rat PTH®, Nichols Institute. Aluminium was measured in blood and parathyroid glands using atomic absorption spectrometry using previously described methodology [10,11]. The pre-proPTH mRNA was measured using Northern blot analysis [12], and the radioactive signal was analysed with an Instant Imager®, Packard.

Statistical analysis was performed using SPSS 8.0. Comparisons between groups were performed using non-parametric tests (Mann-Whitney and Wilcoxon tests) and Student's t-tests. A P value lower than 0.05 was considered statistically significant.

Study chronology and experimental groups
The study was divided in two phases. In phase I (54 rats), 3 groups of rats were formed: group 1 received 20 mg/kg aluminium, group 2 received 10 mg/kg aluminium and group 3 received saline (placebo). At the end of the experiment the parathyroid glands were removed and pooled to measure aluminium concentration in parathyroid tissue. Parathyroid glands from three rats were needed to obtain enough tissue to measure the aluminium concentration in the glands.

In phase II (46 rats) two groups of rats were formed using information gained from phase I: group 1 received 32 mg/kg aluminium and group 2 received saline (placebo). At the end of the experiment, the parathyroid glands were removed to measure pre-proPTH mRNA.



   Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Thirty-six rats completed phase I: 11 from group 1 (20 mg/kg of Al), 13 from group 2 (10 mg/kg of Al) and 12 from group 3 (placebo). Before forming these groups (week 5), there were no significant differences in weight, mean intake of drinking water, phosphorous and food intake, creatinine clearance, and serum biochemical parameters (PTH, calcium, phosphorous, and aluminium). At the end of the experiment (week 7), there were no significant differences in any of the parameters except in serum aluminium and PTH. Serum aluminium levels were 4.9±2.0, 184±83 and 406±86 g/l in groups 3, 2, and 1, respectively (P<0.001), and serum PTH fell from 24.5±10.4 to 16.3±10.4 pg/ml (33%) in group 1 (P=0.007).

The serum aluminium levels were correlated with the percentage fall in PTH (r=0.47, P<0.05). The aluminium content in the parathyroid glands displayed a remarkably strong correlation with serum aluminium levels (r=0.96) (Figure 2Go).



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Fig. 2. Correlation between serum aluminium levels and aluminium content in parathyroid glands. Each value along the x axis depicts the aluminium content of a pool of parathyroid glands from three rats; each value along the y axis depicts the mean±SD of serum aluminium from the three rats.

 
In phase II, and because of the results observed in phase I, we formed two groups and used a higher dose of aluminium. Thirty rats completed the study: 14 from group 1 (aluminium group) and 16 from group 2 (placebo group). Before dividing rats into these groups (week 5), there were no differences in the parameters studied (detailed in methodology of phase I), except for weight (group 1 weighted 440±44 and group 2 weighted 471±54 g, P=0.036) (Table 1Go). At the end of the experiment (week 7), we observed differences in serum aluminium and PTH levels (Table 2Go).


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Table 1. Body weight and biochemical parameters (mean±SD) in the two groups from phase II before aluminium or placebo administration. Values with statistical differences are in bold (Mann–Whitney test) (CCr, creatinine clearance)

 

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Table 2. Biochemical parameters (mean±SD) in the two groups from phase II (placebo and aluminium) at the end of the study. Values with statistical differences are shown in bold (Mann–Whitney test)

 
The placebo group showed a decrease in serum calcium from 11.49±0.38 to 11.2±0.25 (P=0.16), without a change in PTH levels (Figure 3Go). They also gained weight from 440±44 to 453±44 g (P<0.001). The aluminium group had significantly higher serum aluminium levels, no changes in serum calcium, and a significant fall in serum PTH, decreasing from 24.5±18.3 to 12.2±7.1 pg/ml (48%) (P=0.002) (Figure 3Go). This group also showed a decrease in weight, from 471±54 to 452±62 g (P=0.003).



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Fig. 3. Evolution of serum PTH levels in the two groups before and after administration of aluminium or placebo *P<0.05, 5 vs 7 weeks (Wilcoxon test).

 
In the aluminium loaded rats, the reduction in the mRNA of pre-proPTH was inversely proportional to serum aluminium. A more marked reduction in mRNA expression was obtained in the rats with the highest serum aluminium level (P=0.033, Student's t-test) (Figure 4Go).



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Fig. 4. Auto-radiography of prepro PTH mRNA and 28S rRNA. Colour bars show the relation between the two signals: preproPTH RNA/28sRNA (P=0.033). The numbers below depict the aluminium levels of the rats of each lane (two rats in each lane).

 



   Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Since the early 1980s, several in vitro [7] and in vivo [1,13] studies have confirmed the inhibitory effect of aluminium on parathyroid function [14,15]. Thus far, all the gathered evidence came from studies using indirect methods to evaluate the effect of aluminium on parathyroid function. The possibility of measuring PTH mRNA provides the opportunity of using a more direct approach to this matter.

As suggested in our preliminary study [8], our study confirmed that aluminium reduces the synthesis of PTH. The acute aluminium load reduced pre-proPTH of mRNA. Serum PTH levels were also reduced, demonstrating that the low PTH levels observed after in aluminium overload are at least partly because of a reduction in the production of PTH.

In our study, the inhibitory effect of aluminium on the parathyroid gland was observed even in the presence of mild-moderate hyperparathyroidism (PTH values 1.5–2 times higher than normal values). The degree of PTH suppression was proportional to the degree of aluminium overload (assessed by the serum aluminium levels), and was also correlated with the concentration of aluminium found in the parathyroid glands (Figure 2Go). The highest PTH suppression was observed in the group of rats with the highest serum aluminium levels, and the rats with the lowest serum aluminium levels showed the least degree of PTH suppression (Figure 4Go).

The observed reduction in the pre-proPTH mRNA gene expression (up to 34%) could have resulted from a decrease in the transcription induced by the aluminium load; however, the present study could not rule out post-transcriptional effects of aluminium. Further studies are needed to clarify this aspect.

In summary, high levels of aluminium in serum and parathyroid tissue caused a decrease in PTH production, but at the same time they might also have reduced PTH release, as has been suggested by others [47]. The observed reductions in pre-proPTH mRNA and in serum PTH levels have demonstrated for the first time that the production of PTH is reduced in the presence of aluminium overload. Despite the fact that rats did not have severe secondary hyperparathyroidism, the magnitude of PTH inhibition observed suggests that hypoparathyroidism in presence of aluminium overload occurs, at least in part, because of reduced PTH synthesis.



   Acknowledgments
 
This study has been partly supported by Fundación Renal Iñigo Alvarez de Toledo.



   Notes
 
Correspondence and offprint requests to: Dr J. B. Cannata Andía, Bone and Mineral Research Unit, Hospital Central de Asturias, C/ Julián Clavería s/n. E-33006, Oviedo, Spain. Back



   References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received for publication: 28.12.99
Revision received 10.11.00.