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Onset of nephrocalcinosis depends on dietary phosphorus concentration in male rats fed a magnesium-deficient diet

Magnesium Research. Volume 19, Numéro 4, 255-60, December 2006, Original article

DOI : 10.1684/mrh.2006.0069

Summary

Auteur(s) : H Matsuzaki, S Katsumata, M Uehara, M Miwa, K Suzuki , Department of Nutrition, Junior College of Tokyo University of Agriculture, 1-1-1 Sakuragaoka, Setagaya-ku, Tokyo 156-8502, Japan, Department of Nutritional Science, Faculty of Applied Bioscience, Tokyo University of Agriculture, 1-1-1 Sakuragaoka, Setagaya-ku, Tokyo 156-8502.

ARTICLE

Auteur(s) : H Matsuzaki¹, S Katsumata², M Uehara², M Miwa¹, K Suzuki²

¹Department of Nutrition, Junior College of Tokyo University of Agriculture, 1-1-1 Sakuragaoka, Setagaya-ku, Tokyo 156-8502, Japan
²Department of Nutritional Science, Faculty of Applied Bioscience, Tokyo University of Agriculture, 1-1-1 Sakuragaoka, Setagaya-ku, Tokyo 156-8502

Nephrocalcinosis induced by a magnesium (Mg)-deficient diet was first reported by Cramer [1]. It is known that one of the typical effects of a Mg-deficient diet is induction of nephrocalcinosis. While our previous study [2] observed that, although kidney Ca and P concentrations were elevated in female rats, the Mg-deficient diet had no effects on kidney Ca and P concentration in male rats. An Mg-deficient diet is generally known to increase kidney Ca and P concentrations and to induce nephrocalcinosis in male rats [3-5], so it was very noteworthy that our previous study [2] showed that, despite the administration of a Mg-deficient diet, kidney Ca and P concentrations were not increased in male rats. We speculated that this result was due to the experimental diet used in our previous study which was based on the AIN-93G formula. The AIN-93G diet was developed as a standard diet for rodents by the American Institute of Nutrition, and one of the major differences between the AIN-93G diet and the conventional standard diet (AIN-76) for rodents is that the P concentration in AIN-93G diet is low in order to prevent the onset of nephrocalcinosis [6]. In other words, increases in kidney Ca and P concentrations in male rats fed on Mg-deficient diet were not seen in our previous study [2] because the suitable P concentration in the diet based on the AIN-93G formula prevented the onset of nephrocalcinosis [6]. Subsequently, we hypothesized that even with the AIN-93G diet, an increase in dietary P concentration may induce nephrocalcinosis in male rats fed on Mg-deficient diet.In the present study, the experimental diets with two different Mg concentrations and three different P concentrations were used in order to confirm our hypothesis that the Mg-deficient diet based on AIN-93G formula would not induce and that the Mg-deficient diet based on AIN-93G formula with increased dietary P concentrations would induce nephrocalcinosis in male rats. As mentioned above, the kidney Ca and P concentration was not increased in male rats fed on the Mg-deficient diet [2]. Therefore, the present study used only male rats as experimental animals, and focused on the effects of increased dietary P concentration on onset of nephrocalcinosis in male rats fed on the Mg-deficient diet.

Materials and methods

Animals and diets

Three-week-old male Wistar rats (Clea Japan, Tokyo, Japan) were housed in individual stainless-steel wire-mesh cages. During the experiment, cages were located in a room with controlled lighting under a 12-h light:dark cycle (light, 0800-2000 h), a temperature of 22±1°C and relative humidity of 60-65%. Experimental diets were based on AIN-93G diets (table 1( Table 1 )) [6]. Six experimental diets contained the two different Mg concentrations [0.5 g per kg diet (normal-Mg) or Mg-free (Mg-deficient)] and three different P concentrations [3 (3-P), 5 (5-P) or 7 (7-P) g per kg diet]. After experimental diets were made, a sample for the measurement of Ca, Mg and P concentrations was collected from eight points of each experimental diet. The Ca, Mg and P concentrations of the experimental diets are shown in table 2( Table 2 ). Before the study period began, there was a 7-days acclimatization period during which all rats were given free access to the normal-Mg diet containing 3-P and deionized water. After the acclimatization period, rats were randomly divided into six groups and fed one of six experimental diets differing in Mg and P concentrations for 28 days. Rats fed on the other experimental diets were fed the mean weight of food consumed by the rats fed on the Mg-deficient diet containing 7-P on the previous day. The rats were given free access to deionized water. At the end of the experimental period, all rats were sacrificed, and both kidneys were collected for analysis. The right kidney was used for a histological examination, and the left for a mineral analysis. Animals were treated in accordance with the guidelines of the National Research Council for the Care and Use of Laboratory Animals (1985).
Table 1 Composition of the experimental diets.

	Normal-Mg diet			Mg-deficient diet
	3-P	5-P	7-P	3-P	5-P	7-P
Ingredients	g/kg diet
Corn starch	528.657	519.870	511.083	529.486	520.699	511.912
Casein	200.0	200.0	200.0	200.0	200.0	200.0
Sucrose	100.0	100.0	100.0	100.0	100.0	100.0
Soybean oil	70.0	70.0	70.0	70.0	70.0	70.0
Cellulose powder	50.0	50.0	50.0	50.0	50.0	50.0
Mineral mix^a	35.0	35.0	35.0	35.0	35.0	35.0
Vitamin mix^b	10.0	10.0	10.0	10.0	10.0	10.0
L-cystine	3.0	3.0	3.0	3.0	3.0	3.0
Choline bitartrate	2.5	2.5	2.5	2.5	2.5	2.5
Tert-butylhydroquinone	0.014	0.014	0.014	0.014	0.014	0.014
MgO	0.829	0.829	0.829	–	–	–
KH₂PO₄	–	8.787	17.574	–	8.787	17.574

^aThe mineral mix is a modification of the AIN-93G mineral mix without magnesium oxide.

^bAIN-93 vitamin mix.

Table 2 Mineral concentrations of the experimental diets.

	Normal-Mg diet			Mg-deficient diet
3-P	5-P	7-P	3-P	5-P	7-P
Ca (g/100 g dry weight)	0.510 ± 0.002	0.505 ± 0.003	0.506 ± 0.004	0.513 ± 0.002	0.520 ± 0.003	0.511 ± 0.003
Mg (g/100 g dry weight)	0.053 ± 0.001	0.053 ± 0.000	0.054 ± 0.001	0.004 ± 0.000	0.004 ± 0.000	0.004 ± 0.000
P (g/100 g dry weight)	0.316 ± 0.002	0.518 ± 0.002	0.715 ± 0.004	0.315 ± 0.002	0.515 ± 0.003	0.709 ± 0.003

Mineral analysis

The left kidney was weighed after the renal capsule was discarded. The left kidney was dried overnight at 100°C, and the dry weight was measured. Samples of the experimental diets and kidney were ashed at 550°C for 48 h in a muffle furnace, and minerals were extracted in 1 mol/L of HCl for analysis. Ca and Mg were determined by atomic absorption spectrometry (Hitachi A-2000) according to the method of Gimblet et al. [7]. P was analyzed using the method of Gomori [8].

Histological examination

Immediately after the right kidney was removed, and cut in half, half of the kidney was fixed in 10% formalin for subsequent processing and paraffin embedding following routine methods for histopathology. Approximately 3 μm sections were cut and stained with von Kossa’s. The degree of nephrocalcinosis was graded on a scale from - (not detected) to +++ (severe).

Statistical analysis

Results are expressed as means ± SE. Data were analyzed by two-way ANOVA. Two-way ANOVA was used to determine the effect of dietary Mg concentration and effect of dietary P concentration. Fisher’s PLSD was used to determine the significant differences of multiple comparisons among groups. Differences were considered significant at p < 0.05.

Results

Body weight

Final body weight was significantly decreased in rats fed on the Mg-deficient diet compared with the rats fed on the normal-Mg diet, irrespective of the dietary P concentration (table 3( Table 3 )).
Table 3 Body weight in rats fed on six experimental diets.

	Normal-Mg diet			Mg-deficient diet			Two-way ANOVA (P-values)¹
	3-P	5-P	7-P	3-P	5-P	7-P	Mg	p	Mg×P
Body weight
Initial (g)	79.4 ± 2.4	78.8 ± 2.2	78.5 ± 1.8	79.4 ± 1.6	78.0 ± 1.7	79.1 ± 1.7	NS	NS	NS
Final (g)	210.4 ± 1.3^a	213.8 ± 4.0^a	199.7 ± 1.8^b	164.2 ± 3.4^c	158.7 ± 2.5^c	167.0 ± 4.8^c	< 0.0001	NS	< 0.01

Serum Mg level

Serum Mg level was significantly decreased in rats fed on the Mg-deficient diet compared with the rats fed on the normal-Mg diet (table 4( Table 4 )). In rats fed on the Mg-deficient diet, serum Mg level was significantly decreased in rats fed on the diet containing 7-P compared with the rats fed on the diet containing 3-P and 5-P.
Table 4 Serum Mg level in rats fed on six experimental diets.

	Normal-Mg diet			Mg-deficient diet			Two-way ANOVA (P-values)¹
3-P	5-P	7-P	3-P	5-P	7-P	Mg	p	Mg × P
Mg (mg/dL)	2.31 ± 0.06^a	2.20 ± 0.04^a	2.21 ± 0.04^a	0.71 ± 0.05^b	0.63 ± 0.02^b	0.46 ± 0.01^c	< 0.0001	< 0.01	NS

Kidney mineral concentration and degree of nephrocalcinosis

In rats fed on the diet containing 5-P and 7-P, kidney Ca and P concentrations were significantly increased in rats fed on the Mg-deficient diet compared with rats fed on the normal-Mg diet (table 5( Table 5 )). Kidney Ca and P concentrations in the Mg-deficient groups were significantly increased with increased dietary P concentration, while no significant difference in kidney Ca and P concentrations were observed between the normal-Mg diet containing 3-P and the Mg-deficient diet containing 3-P groups. Dietary P concentration also had no significant influence on kidney Ca and P concentrations of the normal-Mg groups. In the Mg-deficient group, nephrocalcinosis appeared with increased dietary P concentration. Namely, nephrocalcinosis was observed in rats fed on the Mg-deficient diet containing 5-P and 7-P. The degree of nephrocalcinosis was severe in rats fed on the Mg-deficient diet containing 7-P compared with rats fed on the Mg-deficient diet containing 5-P. On the other hand, there was no nephrocalcinosis in the normal-Mg groups, irrespective of the dietary P concentration. It is especially worthy of notice that despite the Mg-deficient diet feeding, nephrocalcinosis was not observed in rats fed on the Mg-deficient diet containing 3-P.
Table 5 Kidney mineral concentration and degree of nephrocalcinosis in rats fed on six experimental diets.

	Normal-Mg diet			Mg-deficient diet			Two-way ANOVA (P-values)¹
3-P	5-P	7-P	3-P	5-P	7-P	Mg	p	Mg × P
Ca (mmol/100 g dry weight)	1.21 ± 0.02^a	1.23 ± 0.02^a	1.30 ± 0.04^a	1.26 ± 0.06^a	6.01 ± 0.78^b	20.06 ± 3.04^c	< 0.0001	< 0.0001	< 0.0001
P (mmol/100 g dry weight)	42.1 ± 0.1^a	43.0 ± 0.2^a	42.9 ± 0.2^a	42.8 ± 0.3^a	46.1 ± 0.6^b	51.7 ± 2.4^c	< 0.0001	< 0.001	< 0.001
Degree of nephrocalcinosis²	–	–	–	–	+	++ to +++

Discussion

Our previous study [2] observed that despite the administration of a Mg-deficient diet, the kidney Ca and P concentrations were not increased in male rats. We speculated that this result was due to the P concentration in the experimental diet based on the AIN-93G formula. Therefore, the present study was focused on the effects of increased dietary P concentration on onset of nephrocalcinosis in male rats fed on a Mg-deficient diet, and was conducted to determine whether a Mg-deficient diet based on the AIN-93G diet with increased dietary P concentrations would induce nephrocalcinosis in male rats.

The previous studies [3-5] indicated that a Mg-deficient diet induces nephrocalcinosis in male rats. However, the present study found that the Mg-deficient diet containing 3-P had no significant influence on kidney Ca and P concentrations, and by using histological examination, nephrocalcinosis was not observed in rats fed on the Mg-deficient diet containing 3-P. This result was in agreement with our previous study by using the AIN-93G diet [2]. However, this result seems to be contradictory to the results in previous studies using the AIN-76 diet [3-5] on nephrocalcinosis in male Mg-deficient rats. This contradiction may be explained by the difference of P concentration in the experimental diet, since it is well known that dietary P concentration is an important factor in the etiology of nephrocalcinosis [9-12]. The experimental diet used in the present study was based on the AIN-93G formula, while the experimental diets used in previous studies [3-5] were based on the AIN-76 formula. Thereby, P concentrations in the experimental diet used in the present study and previous studies [3-5] were different as follows: dietary P concentration is lower in the AIN-93G diet (3 g per kg diet) than in the AIN-76 diet (5 g per kg diet) in order to prevent the onset of nephrocalcinosis [6]. In fact, the values of P concentration analyzed in the experimental diet used in previous studies [3-5] is approximately 5 g per kg diet, and that in present study is approximately 3 g per kg diet. Consequently, we suggest that the difference between the results in the present study and previous studies was due to the P concentration in the experimental diet used. A Mg-deficient diet containing 5-P and 7-P induced an increase in kidney Ca and P concentrations and nephrocalcinosis. Results in the present study confirmed our hypothesis that the Mg-deficient diet based on the AIN-93G formula did not induce the onset of nephrocalcinosis, however the Mg-deficient diet based on AIN-93G formula with increased dietary P concentrations induced the onset of this disorder in male rats. In other words, no nephrocalcinosis in male rats fed the Mg-deficient diet based on the AIN-93G formula was due to the P concentration in the experimental diet based on the AIN-93G diet. Furthermore, the degree of nephrocalcinosis was severe in rats fed on the Mg-deficient diet containing 7-P compared with rats fed on the Mg-deficient diet containing 5-P. From the results in the present study, we suggest that the development of nephrocalcinosis depends on dietary P concentration in male rats fed on a Mg-deficient diet.

The previous studies [9-12] reported that the development of nephrocalcinosis was induced in rats fed on high P diet. However, the present study observed that increased dietary P concentration in the normal-Mg groups had no influence on kidney mineral concentration and the onset of nephrocalcinosis. In the previous study [13], the effect of high-P diet on kidney mineral concentration was examined in the male rats fed on a diet containing 3 (control), 6, 9, 12 and 15 g P per kg diet, consequently although kidney Ca and P concentrations were increased in male rats fed on the diet containing 12 and 15 g P, male rats fed on the diet containing 6 and 9 g P did not induce increase in kidney Ca and P concentrations. Therefore, we suggest that the nephrocalcinosis in male rats is not induced at less than 9 g P per kg diet when dietary P concentration increased with normal-Mg concentration. The present study showed that in the Mg-deficient groups, increased dietary P concentration induced increases in kidney Ca and P concentrations and onset of nephrocalcinosis. Thus, a difference in the effect of increased dietary P concentration between normal-Mg groups and Mg-deficient groups was exhibited. The difference of effect of dietary P concentration in the present study may be related to Mg deficiency. Serum Mg level was decreased in Mg-deficient groups, indicating that the Mg-deficient diet induces Mg deficiency. From the results of the present study, we suggest that the effect of increased dietary P concentration appears strongly with concomitant Mg deficiency. Subsequently, nephrocalcinosis was observed in rats fed on the Mg-deficient diet containing 5-P and 7-P. Furthermore, the serum Mg level of Mg-deficient groups was decreased by a Mg-deficient diet containing 7-P. This result indicates that the increased dietary P concentration deteriorates the degree of Mg deficiency in rats fed on the Mg-deficient diet. Consequently, the degree of nephrocalcinosis was severe in rats fed on the Mg-deficient diet containing 7-P compared with rats fed on the Mg-deficient diet containing 5-P.

Although urinary mineral excretion was not measured in the present study, changes in urinary mineral excretion may affect the onset of nephrocalcinosis. A Mg-deficient diet [14, 15] and increasing dietary P concentration [10, 16, 17] resulted in increased urinary P excretion. These results indicate that a Mg-deficient diet and high dietary P concentration induce an increase in P concentration in the lumen of tubule and in the tubular cells. High P concentration in the lumen of tubule and in the tubular cells enhances the risk for nephrocalcinosis [18]. Furthermore, urinary Mg excretion was decreased in rats fed on the Mg-deficient diet [4, 15] and with increasing dietary P concentration [10, 16, 17]. A low urinary Mg excretion may stimulate the development of nephrocalcinosis. Rayssiguier and Larvor [19] also reported that modification of the urinary Ca/P ratio could prevent kidney calcification.

The detail of the effects of dietary P and dietary Mg on the onset of nephrocalcinosis remains unclear. Further studies are needed to elucidate the interaction between dietary P and dietary Mg on the onset of nephrocalcinosis. However, the present study indicates that P concentration in the AIN-93G diet is the optimum condition for prevention of nephrocalcinosis in male rats. Therefore, it may provide valuable information when preparing diets in animal studies using rats.

Conclusion

In the present study, the experimental diets with two different Mg concentrations and three different P concentrations were used in order to confirm our hypothesis that the Mg-deficient diet based on the AIN-93G formula would not induce, and that the Mg-deficient diet based on AIN-93G formula with increased dietary P concentrations would induce nephrocalcinosis in male rats. Nephrocalcinosis was not observed in male rats fed on the Mg-deficient diet containing 3-P. Whereas nephrocalcinosis was appeared in male rats fed on the Mg-deficient diet containing 5-P and 7-P. These results confirmed our hypothesis. Furthermore, we suggest that the onset of nephrocalcinosis could depend on the dietary P concentration in male rats fed on Mg-deficient diet.

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