ABSTRACT The effects of dietarycalcium (Ca) concen tration and calciumrphosphorus (Ca:P) ratio on mineral balance and nephrocalcinosis were studied in ...
Influence of Dietary Calcium:Phosphorus Ratio on Mineral Excretion and Nephrocalcinosis in Female Rats A. C. HOEK,*t
A. G. LEMMENS, * J. W. M. A. AÃŽÃŽ/LL//YK* AHD A. C.
*Department of Laboratory Animal Science, State University, 3508 TD Utrecht and ^Department of Human Nutrition, Agricultural University, 6703 BC Wageningen, The Netherlands
ABSTRACT The effects of dietary calcium (Ca) concen tration and calciumrphosphorus (Ca:P) ratio on mineral balance and nephrocalcinosis were studied in female rats. In the first experiment there were two dietary Ca concen trations (0.25 and 0.50%, wfwt) at two different Ca:P ratios (0.6 and 1.3). In the second experiment the diets were formulated to contain 0.40% P and either 0.13,0.25, 0.50 or 0.75% Ca. The diets contained 0.03% magnesium (Mg). The fecal outputs of Ca, P and Mg were lower (P< 0.01) after feeding low Ca diets than after feeding high Ca diets. Urinary excretion of P decreased with in creasing dietary Ca and increased with increasing P intake. In rats fed the 0.25% Ca diets whole-body retentions of Ca and P were lower than in the rats fed 0.50% Ca. Both increases in dietary Ca from 0.13 to 0.50% and P from 0.20 to 0.40% elevated Ca and P content of kidneys as well as the degree of nephrocalcinosis. However, after feed ing the highest Ca concentration (0.75%) nephrocalcinosis was essentially absent while kidney concentrations of Ca and P were relativelylow. When compared with 0.50% Ca in the diet, 0.75% Ca increased group mean whole-body retention of Ca but lowered that of P. In individualrats the degree of nephrocalcinosis and the concentrations of min erals in kidney were positively correlated. J. Nutr. 118: 1210-1216, 1988.
MATERIALS AND METHODS
Animals and diets. Female, specified-pathogen-free Riv-TOX rats (National Institute for Environmental Health and Hygiene, Bilthoven, The Netherlands), aged about 3 wk, were used throughout the study. On arrival they were individually housed in Makrolon-3 cages (UNO BV, Zevenaar, The Netherlands) with a layer of sawdust as bedding. For 3 d they were fed ad libitum a commercial pelleted nonpurified diet (RMH-B, Hope Farms, Woerden, The Netherlands). Subsequently, all rats received a semipurified diet with calculated con centrations of 0.50% (wt/wt) Ca and 0.40% P and a Ca:P ratio of 1.25. The composition of this pre-experimental diet is given in Table 1. The diet was formu lated according to the nutrient requirements of rats (9), but the calculated Mg concentration of the pre-experimental as well as the experimental diets was decreased from 0.04 to 0.02% to enhance the susceptibility to nephrocalcinosis (3-6). The pre-experimental period lasted 10 d. Two experiments with exactly the same design were performed. Between the experiments there was an in terval of about 5 wk. Separate batches of diet were made for each experiment. On d 0 of the experimental periods of both experiments the rats were divided into four groups of six rats each, so that group mean body weights were similar. Each group was randomly assigned to one of four purified diets: One group was fed the pre-ex perimental diet and three groups were fed experimental
INDEXING KEY WORDS: •rat
•calcium
•excretion
•phosphorus
•magnesium
•nephrocalcinosis
Nephrocalcinosis in the rat can be demonstrated histologically and refers to the deposition of stainable cal cium (Ca) salts in the kidney, usually in the corticomedullary region. The development of this condition has a genetic basis (1), and female rats are more sus ceptible than males (2). The composition of the diet also is an important factor. Diets with a low concen tration of magnesium (Mg) (3-6) or low cal cium: phosphorus (Ca:P) ratios (7, 8) have been shown to induce nephrocalcinosis. Nephrocalcinosis, as demonstrated histologically, 0022-3166/88 $3.00 ©1988 American Institute of Nutrition.
'To whom correspondence
should be addressed.
Received 9 December 1987. Accepted 9 June 1988.
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should be associated with increased concentrations of Ca and/or P in the kidney and increased retention of these minerals. However, we are not aware of any stud ies showing statistical relationships between these pa rameters. This prompted us to investigate the effects of the dietary Ca:P ratio and Ca concentration on the incidence of nephrocalcinosis, Ca and P concentrations in kidney and urinary and fecal excretion of these min erals.
MINERAL EXCRETION AND NEPHROCALCINOSIS
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TABLE 1 Composition of the diets1
1/2:20.50% 2:0.75% 2:0.13% 1:0.25% 1/2:0.25% 1:0.50% Ca0.40% Ca0.40% Ca0.75% Ca0.40% Ca0.20% Ca0.40% PCa:P PCa:P PCa:P PCa:P PCa:P PCa:P 1.25g/JOOg70.331.241.510.6826.241000.52/0.470.03/0.030.47/0.441.1/1.1Experiment = 1.8869.711.861.510.6826. = 1.2571.610.620.501.0326.241000.270.030.241.1Experiment = 0.6370.950.621.510.6826.241000.27/0.250.03/0.030.43/0.390.6/0.6Experiment = 0.6369.001.243.52—26.241000.520.030.840.6Experiment = 0.3371.320.251.510.6826.241000.110.030.400 = ParameterIngredientGlucoseCaCO3NaH2PO4-H2ONa2CO3Constant
componentsTotalChemical analysis-'CalciumMagnesiumPhosphorusCa:PExperiment
diets. The composition of the diets is given in Table 1. In experiment 1 both the concentrations of Ca (0.25 and 0.50%) and P (0.20, 0.40 and 0.75%) in the diet were varied. Experiment 2 involved changes in dietary Ca (0.13, 0.25, 0.50 and 0.75%) at a fixed concentration of P (0.40%). As indicated in Table 1, the pre-experimental diet and the experimental diet with 0.25% Ca and 0.40% P were used in both experiments. The purified diets were stored at 4°Cuntil feeding. The diets were in meal form. The animals had free access to food and demineralized water. Feed con sumption and body weights were recorded. During the experiment (d 0-28) the rats were housed individually in metabolic cages (Tecniplast Gazzada, Buguggiate, Italy). The cages were placed in a room with controlled lighting (light 12 h/d), temperature (20-24°C) and relative humidity (60% ). Analysis of diets, urine, feces and kidneys. From d 13 to 15 and 26 to 28 urine and feces of each rat were collected quantitatively. The tubes for collecting feces or urine had been cleaned extensively with 0. l N HC1. Urine was acidified to pH 1 with 6 N HCl, and centrifuged for 10 min. Feces were freeze-dried, ashed at 500°C for 18 h and dissolved in 6 N HCl. Ca and Mg in urine and feces were analyzed in the presence of 1% lan thanum chloride by atomic absorption spectroscopy with the use of a Varian AA-475 (Varian Techtron, Springvale, Australia). Total P was analyzed by the method of Taussky and Shorr (10). For complete recovery of P in ashed feces, analysis was performed at least 1 wk
after dissolving the material in HCl. Food samples were analyzed for Ca, P and Mg as described for feces. On d 28 the animals were killed by exposure to car bon dioxide. Kidneys were collected, and capsules were removed. The right kidney of each rat was minced and homogenized in 2 vol of demineralized water. After overnight drying at 106°C,the samples were ashed, dis solved and analyzed for Ca, Mg and P as described for feces. Histological methods. The left kidneys were fixed in Bouin's fluid (11) after being sectioned longitudinally. Subsequently, they were processed by conventional histological methods and embedded in paraffin wax (11). Sections of 5 (¿mthickness were stained with the method of Von Kossa as described by Mallory (12). The severity of nephrocalcinosis was graded on a scale from 0 (ab sence of calcium deposits) to 3 (severe calcinosis). To aid in scoring, four reference slides were used. Four sections randomly selected from the medial to the lat eral aspects of each kidney were scored in random order and blind by two workers. The score of each rat was the average score of the four sections and of the two assessors. Statistics. Because the severity scores for nephro calcinosis were not distributed normally, the parame ter-free Kruskal-Wallis rank statistics test was used throughout to compare group means. In the first ex periment both dietary Ca and P were variables. There fore analysis of variance was performed to disclose in teractions between Ca level and Ca:P ratio. In the second
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'Calculated concentrations of calcium (Ca) and phosphorus (P). 2Pre-experimental diet. JThe constant components consisted of (g/100 g diet|: casein, 15.1; corn oil, 2.5; coconut fat, 2.5; cellulose, 3.0; magnesium carbonate, 0.07; potassium chloride, 0.1; potassium bicarbonate, 0.77; mineral premix, 1.0; vitamin premix, 1.2. The mineral premix consisted of the following (mg): sodium citrate (Na3C6H5O7-2H2O), 153.5; FeSO4-7H2O, 90; MnO2, 14; KA1 (SO4),-12H2O, 20; ZnSO4-H2O, 12.5; KBr, 2; NiSO,-6H,O, 0.85; NaF, 0.85; CuSO4-5H2O, 10; CoSO4-7H2O, 0.5; Na2Mo4-2H2O, 0.5; KI, 0.5; As,0„ 0.02; Na2B4O7-10H2O, 0.5; Na2SeO,-5H,O, 0.015; cornmeal, 694.265. The vitamin premix consisted of the following (mg): thiamin, 6; riboflavin, 2.25; niacinamide, 15.2; DL-calcium panthothetate, 5.6; pyridoxine, 2.25; vitamin B-12, 0.0015; choline chloride, 200; inositol, 100; folie acid, 0.85; biotin, 0.1; p-aminobenzoic acid, 50; menadione, 0.4; DL-alpha tocopheryl acetate, 5; retinyl acetate and retinyl palmitate, 3 (1500 iu); cholecalciferol, 3 (300 iu); vitamin C, 40; cornmeal, 766.3485.
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experiment the concentration of dietary Ca was the only variable, and Scheffétests and Kruskal-Wallis tests were used to compare group means. The Spearman rankorder correlation coefficient was computed between mineral concentration in kidney and degree of nephrocalcinosis. Statistical analyses were calculated from a statistical package prepared by Nie et al. (13).
RESULTS
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Experiment 1. Final body weights of the rats fed the 0.25% Ca diets were higher than those of rats fed the 0.50% Ca diets (Table 2). Body weight of the group fed the 0.50% Ca/0.80% P diet was lower than that of the group fed the 0.25% Ca/0.20% P diet. There were no significant effects due to differences in the Ca:P ratio. The fecal outputs of Ca, P and Mg were significantly lower in rats fed the 0.25% Ca diets than in those fed the 0.50% Ca diets. An increase in the concentration of dietary Ca caused an increase in the excretion of Ca in feces expressed as a percentage of intake. During the first collection period the concentration of P in the diet did not affect the excretion of Ca and Mg in feces. However, in the second collection period dietary P tended to influence fecal excretion of P, although the stimu latory effect of dietary Ca was greater. Urinary excretion of Ca was similar for the four diet groups. Dietary P concentration largely determined the output of P in urine. An increase in Ca content of the diets containing 0.40% P resulted in a significant de crease of urinary P. In the rats fed the 0.50% Ca diets the excretion of Mg in urine tended to be lower than that in their counterparts fed the 0.25% Ca diets. Ca and P retentions were significantly lower in rats fed the 0.25% Ca diets than in those fed the 0.50% Ca diets. An increase in Ca content of the diets resulted in a significant decrease of Mg retention (Table 2). Kidney dry weight was increased in rats fed the diet containing 0.50% Ca and 0.80% P when compared with the other groups (Table 2). All kidneys in this group were swollen and had a fatty appearance. The kidneys of one rat in the 0.25% Ca/0.40% P group and of one rat in the 0.50% Ca/0.40% P group looked flattened and weak, which was probably due to hydronephrosis. There was considerable variation in Ca and P contents of the kidneys within groups. The average contents of Ca were increased by intakes of Ca and/or P. A similar trend was observed for the kidney concentration of P. Mg concentrations in kidney were not affected by di etary treatments. The severity of nephrocalcinosis increased with an increase in dietary Ca and/or P. In rats fed the 0.25% Ca/0.20% P diet, nephrocalcinosis was essentially ab sent. In severe nephrocalcinosis (score > 2) two co herent Von Kossa-positive zones were present. One band was in the cortex, and the other in the corticomedullary layer. Occasionally, there was Von Kossa-positive ma
terial in the medulla. The deposits were found intracellularly as well as in the basement membranes and interstitium. There was no difference in distribution of the deposits longitudinally across the kidney. Spearman rank-order correlation coefficients were calculated between the score of nephrocalcinosis and the concentrations of minerals in the kidneys of indi vidual rats (n = 24). The correlation coefficients were 0.92 (P < 0.01), 0.90 (P < 0.01) and 0.44 (P < 0.05) for Ca, P and Mg, respectively. Experiment 2. Table 3 summarizes the results of the second experiment. Body weight gain and feed intake did not differ between the four diet groups. In rats fed the diet containing 0.75% Ca, urinary ex cretion of Ca was increased compared with the 0.13 and 0.25% Ca groups. Within the range of 0.13 to 0.50% of dietary Ca there was no clear effect on Ca excretion in urine. The concentration of dietary Ca was inversely related to the urinary output of P. A similar relationship was observed between dietary Ca and urinary output of Mg. An increase in Ca intake was associated with in creases in the fecal excretion of Ca, P and Mg. The efficiency of absorption of these minerals decreased with higher Ca concentrations in the diet. An increase in dietary Ca concentration resulted in an increase in average Ca retentions. Within the range of 0.13 to 0.50% of Ca in the diet, the mean amounts of P retention were positively associated with Ca in take. The 0.75% Ca group showed a decrease in P re tention compared with the 0.50% Ca group. In the sec ond collection period but not in the first period there was a tendency toward lower Mg retentions with in creasing dietary Ca concentrations. Average kidney dry weight was lower in the rats fed the diet containing 0.75% Ca, when compared with the groups fed less Ca (Table 3). However, this difference was not significant at the P < 0.01 level. Similarly, the rats fed the 0.75% Ca diet displayed lower concentra tions of Ca in their kidneys. They also had lower kidney concentrations of P and Mg than the groups fed 0.25 or 0.50% Ca diets. Within the range of 0.13 to 0.50% of Ca in the diet, the concentrations in the kidney of Ca, P and Mg were positively associated with Ca intake. Nephrocalcinosis was detected in all dietary groups (Table 3). The incidence and mean score were lowest in rats fed the 0.75% Ca diet. In rats with severe neph rocalcinosis, two continuous bands were seen with densely packed crystals. The outer band (in the cortex) was not as heavily stained as the inner zone in the corticomedullary junction. Correlation coefficients for the relationship between histological and chemical analysis of the kidneys were 0.69 (P
