Effects of simultaneous increases in dietary phosphorus and magnesium concentrations on nephrocalcinosis and kidney function in female rats

ARTICLE

Auteur(s) : Hiroshi Matsuzaki¹, Ritsuko Masuyama², Mariko Uehara², Kahoru Nakamura¹, Kazuharu Suzuki²

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

Introduction

Rats fed a high-phosphorus (P) diet have been reported to exhibit an increased incidence of nephrocalcinosis [1-3], and calcium (Ca) deposition is primarily observed in the corticomedullary junction of the kidney [4]. Intraluminal and intracellular Ca deposits as well as necrotic cells have been reported in the proximal tubules, Henle's loops and collecting ducts of rats fed a high-P diet [5-7], and it is clear that in such rats nephrocalcinosis is developing, resulting in changes in kidney function. Ritskes-Hoitinga et al. [4] reported that urinary albumin excretion, which is positively correlated with kidney Ca concentration, is elevated in rats fed a high-P diet. Furthermore, we found that rats fed a high-P diet display an increase in N-acetyl-β-D-glucosaminidase activity in the urine as well as increased urinary β2-microglobulin excretion [6, 7]. These studies suggest that a high-P diet diminishes kidney function in rats [4, 6, 7]. Dietary magnesium (Mg) concentration also affects nephrocalcinosis and kidney function. A Mg-deficient diet induces nephrocalcinosis, which can be seen on histological examination [8, 9]. Urinary albumin excretion and N-acetyl-β-D-glucosaminidase activity in the urine were increased in rats fed the Mg-deficient diet [10].

The results of a large number of previous studies thus indicate that dietary P and Mg concentrations play an important role in the development of nephrocalcinosis and diminished kidney function in rats [1-10]. Most of these studies have been conducted on the effects of high-P or low-Mg diet administration on nephrocalcinosis and kidney function [1-10]. However, there have been few studies that have investigated either the effects of P and Mg on nephrocalcinosis and kidney function by simultaneously varying P and Mg concentrations, or the interaction between the two minerals [11, 12]. Furthermore, no study has yet assessed the effects of various dietary P:Mg ratios. Therefore, numerous points remain unclear, and clarifying the optimal P and Mg concentrations as well as the optimal P:Mg ratio for the prevention of nephrocalcinosis and diminished kidney function is a very important goal for nutritional research using rats. Accordingly, the present study investigated the effects of simultaneous increases in dietary P and Mg concentrations while maintaining the P:Mg molar ratio on nephrocalcinosis and kidney function.

Materials and methods

Animals and diets

Four-week-old female 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%.

The compositions of the experimental diets are shown in table I.

Experimental diets were based on the AIN-93G diet [13]. P and Mg concentrations in the control diet were 3 g and 0.5 g per kg diet, respectively. P and Mg concentrations in the other two experimental diets were adjusted to 3 times the control concentrations (3-fold diet; 9 g P and 1.5 g Mg per kg diet) and 5 times the control concentrations (5-fold diet; 15 g P and 2.5 g Mg per kg diet) by adding potassium dihydrogenphosphate and magnesium oxide. The three experimental diets all had the same P:Mg molar ratio. All experimental diets were stored at 4 °C until used.

Before the study period began, there was a 7-d acclimatization period during which all rats were given free access to the control diet and demineralized water. After the acclimatization period, rats were divided into three groups of 6 rats, with each group having a similar mean body weight. One of the experimental diets was assigned to each group and rats were given free access to the assigned experimental diet as well as demineralized water throughout the experimental period. At the end of the 21-d experimental period, all rats were killed by exsanguination from the carotid artery. The study protocols were approved by the Animal Use Committee at Tokyo University of Agriculture, and animals were maintained in accordance with the university's guidelines for the care and use of laboratory animals.

Collection of samples

From days 20 to 21 of the experiment, rats were housed individually in stainless-steel metabolic cages, and urine was collected from each rat for 24 h. Blood was collected in tubes at the time of exsanguination, and was centrifuged to obtain the serum. The right kidney was removed and weighed after the renal capsule was discarded. Samples were stored at – 40 °C until needed for analysis.

Chemical analysis

The right 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) [14], while phosphorus was analyzed using the method of Gomori [15]. Creatinine in serum and urine was determined with a Wako Creatinine Test kit (Wako Pure Chemical Industries, Osaka, Japan). Albumin and β₂-microglobulin in urine were respectively determined with Panatest Rat Albumin and Panatest Rat β₂-microglobulin kits (Panapharm Laboratories Co., Kumamoto, Japan). Because a positive correlation has been reported between kidney mineral concentrations and histological severity of nephrocalcinosis [16, 17], kidney mineral concentrations were used as indicators for the onset and severity of nephrocalcinosis in the present study.

Statistical analysis

Data are expressed as mean ± SD. Data were analyzed by one-way ANOVA. Tukey's test was used to evaluate the significance of differences in multiple comparisons among groups, with differences being considered significant at p < 0.05. All statistical analyses were performed using the SPSS package program Ver. 11.0 J.

Results

P and Mg concentrations and P:Mg ratios of experimental diets

P and Mg concentrations as determined by analysis of the experimental diets were as follows: control diet, 3.12 g P and 0.51 g Mg per kg diet; 3-fold diet, 9.25 g P and 1.42 g Mg per kg diet; 5-fold diet, 14.97 g P and 2.37 g Mg per kg diet. The P:Mg molar ratios of the three experimental diets were 4.81, 5.11 and 4.96 for the control, 3-fold and 5-fold diets, respectively.

Body weight, food intake and serum mineral levels

Body weight, food intake and serum mineral levels are shown in table II.

Final body weight was significantly lower in rats fed the 5-fold diet than in rats fed the control or 3-fold diets. Food intake also was significantly decreased in rats fed the 5-fold diet. No significant differences in serum Ca, Mg and P levels were observed among the three groups.

Kidney analysis

Kidney weight and mineral concentrations are shown in table III. Kidney wet and dry weights were significantly higher in rats fed the 5-fold diet than in rats fed the control or 3-fold diets. Kidney Ca, Mg and P concentrations were significantly higher in rats fed the 5-fold diet, however the 3-fold diet had no significant influence on kidney weight or kidney mineral concentration.

Biochemical indicators of kidney function

Table IV. Biochemical indicators of kidney function in rats fed diets differing in phosphorus and magnesium concentrations¹

Discussion

A large number of studies indicate that dietary P and Mg concentrations are important in the etiology of nephrocalcinosis [1-9]. This study investigated the effects of increasing dietary P and Mg concentrations while maintaining the P:Mg molar ratio on nephrocalcinosis.

In the present study, the 3-fold diet had no influence on kidney Ca, Mg or P concentrations when compared to the control diet. The significance of this observation is that although P concentration in the 3-fold diet was 3 times that of the control diet, there was no effect on kidney mineral levels. With regard to the effect of dietary P concentrations on kidney mineral concentration, increased kidney Ca and P concentrations are induced in rats fed the high-P diet [1-7]. In addition, our previous study reported that rats fed a high-P diet containing approximately 9 g P per kg diet increased kidney Ca and P concentrations in female rats [18]. Dietary P in that was similar to 3-fold diet in the present study, but still resulted in increased kidney mineral concentrations. This contrast with the results of the present study may be related to the relative Mg concentrations in the diet. It has also been reported that increasing dietary Mg concentration prevents nephrocalcinosis in rats fed a high-P diet [11, 12] and the Mg concentration in the 3-fold diet used in this study was 3 times that in the control diet. We therefore believe that the contrasts between the present observations and those previously reported [1-7, 18] can be ascribed to the preventive effects of high dietary Mg concentrations.

This study also investigated the effects of a P concentration 5 times higher than in the control diet. The results showed that kidney Ca, Mg and P concentrations were elevated in rats fed the 5-fold diet, and demonstrate that elevating dietary P concentrations can induce nephrocalcinosis. Furthermore, the Mg concentration in the 5-fold diet was 5 times that in the control diet. We suggested above that the lack of change in kidney mineral concentration in rats fed the 3-fold diet was due to increased dietary Mg. This implies that the 5-fold diet will have no effect on kidney mineral concentration, but despite elevated Mg concentrations in the diet, the 5-fold diet resulted in elevated kidney mineral concentrations. These findings suggest that, when dietary concentrations of P and Mg are increased without altering the P:Mg ratio, the preventative effects of increased dietary Mg concentrations on nephrocalcinosis can be expected up to a threshold concentration of dietary P, but not above it.

Previous studies reported that a high-P diet induces diminished kidney function [4, 6, 7]. With regard to the relationship between increasing dietary P and Mg concentrations at a constant P:Mg molar ratio and kidney function, with the 3-fold diet, no effect was detected by the biochemical indicators used in this study. This suggests that a diet with 3 times the P and Mg levels as the control diet has no influence on kidney function or on kidney mineral concentration.

On the other hand, urinary β2-microglobulin excretion was higher in rats fed the 5-fold diet than in those fed the control or 3-fold diets. This result indicates that proximal tubular function was diminished in rats fed the 5-fold diet. It has previously been reported that Ca deposition was evident in the proximal tubules of rats fed a high-P diet [7]. This study showed that the 5-fold diet induces increased kidney Ca, Mg and P concentrations. This suggests that Ca deposition was induced in the proximal tubules of rats fed the 5-fold diet, and as a result, proximal tubular function was diminished. Furthermore, rats fed the 5-fold diet exhibited increased urinary albumin excretion, and this may also be due to changes in proximal tubular function. Albumin resorption occurs primarily in the proximal tubules [19]. A high-P diet diminished proximal tubular function but did not induce injury to the renal glomerular basement membrane [6, 7]. We therefore suggest that increased urinary albumin excretion in rats fed the 5-fold diet is due to obstruction of proximal tubular albumin resorption as a result of diminished proximal tubular function.

Conclusion

This study investigated the effects of simultaneous increases in dietary P and Mg concentrations on nephrocalcinosis and kidney function in female rats. The P:Mg ratio for the three diets used in the present study all had the same P:Mg molar ratio. While the 3-fold diet, which contained 3 times the P and Mg concentrations of the control diet, showed no marked effects on kidney mineral concentrations or kidney function, the 5-fold diet, which contained 5 times the P and Mg concentrations of the control diet, increased kidney mineral concentrations and inhibited kidney function. These findings suggest that absolute concentrations of dietary P and Mg are important for prevention of nephrocalcinosis and diminished kidney function, and may provide valuable information when preparing diets in animal studies using rats.

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