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Nutrition Research Division, Food Directorate, Health Products and Food Branch, Health Canada, 2203C Banting Research Centre, Ottawa, ON, Canada K1A 0L2
2To whom correspondence should be addressed. E-mail: kevin_cockell{at}hc-sc.gc.ca.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • dietary calcium • dietary phosphorus • nephrocalcinosis • female rats
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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) and includes a number of dietary factors in its etiology. A low molar ratio of dietary Ca:P is an important contributing factor (2
,3
). In the 1970s, the American Institute of Nutrition established a standardized semipurified rodent diet (AIN-76A) that would meet the nutritional requirements of rodents and provide a consistent control diet for nutritional, toxicological and regulatory purposes (4
). However, female rats fed this diet commonly developed NC. In the American Institute of Nutrition’s revised standard diet (AIN-93G), the chemical forms of Ca and P in the mineral premix were changed and the P content was decreased, thereby increasing the Ca:P molar ratio (3
). These changes led to a significantly reduced incidence of NC in female rats compared with the AIN-76A formulation (3
,5
). Male rats generally do not show the same degree of susceptibility to diet-induced NC (1
,3
), although there are studies documenting NC in male rats fed Mg-deficient (6
) or high P diets (7
).
The occurrence of NC can complicate the interpretation of nutritional or toxicological studies involving the kidney. Thus it is of interest to fully understand the etiology of this condition, including dietary factors. Although the AIN-93G diet provides a standardized diet for rodent studies, its use is not yet universal. Changes in diet formulations involving, for example, protein sources other than casein may also involve changes in Ca and P levels. The female Sprague-Dawley rat provides a sensitive experimental model for investigation of intrarenal calcification or NC (8
). The present study was conducted to determine the effect of increasing dietary concentrations of Ca and P, while maintaining the protective Ca:P ratio found in the AIN-93G diet, on the incidence of NC in female and male rats.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Weanling (21-d-old) female and male Sprague-Dawley rats (Charles River Canada, St. Constant, Canada) were randomly assigned to one of five groups (8 rats per sex per group) and fed modified AIN-93G diets differing in Ca and P contents (Table 1
). The negative control diet (AIN93) contained Ca and P at the AIN-93G diet amounts (5 g Ca + 3 g P per kg diet, Ca:P molar ratio 1.3:1) (3
). Three diets (1.5x, 2.5x and 4.0x) contained the same proportions of Ca:P, at concentrations of 1.5, 2.5 and 4.0 times those found in the negative control diet. A fifth diet (AIN76) with 5 g Ca + 5 g P/kg diet, was included as a positive control containing Ca and P at the concentrations of the AIN-76A formulation, which has been shown to cause NC in female rats. All five diets included 10 g celite/kg to provide sufficient acid-insoluble ash (9
) for measurement in digestibility assays (to be reported elsewhere). Preliminary assays showed this level of dietary celite to be well tolerated (data not shown). A sixth group of 8 male and 8 female rats was killed at wk 0 for assessment of pretreatment kidney histology and kidney Ca concentration.
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Tissue sampling and histopathology.
Rats were killed by exsanguination under isoflurane anesthesia. Kidneys were dissected out and cut in half (left kidney cut longitudinally, right kidney cut transversely). One half of each was preserved in formalin for subsequent processing and paraffin embedding following routine methods for histopathology. Adjacent 5-µm sections were cut and stained with hematoxylin and eosin (H&E) or by the von Kossa technique (10
). Black granular precipitates on von Kossa–stained sections were manually counted and a histochemical score was assigned according to the number of granules found in one transverse plus one longitudinal kidney section per rat. A score of 0 was given for no granules found in the sections, + for 1–25 granules, ++ for 26–75 granules, +++ for 76–150 granules and ++++ for >150 granules in the sections. The other half of each kidney was frozen at -80°C for subsequent mineral analyses.
Mineral analyses.
Samples of tissues and diets were dry ashed at 450°C using concentrated nitric acid as an oxidizing agent before analysis for Ca by flame atomic absorption spectrometry (Perkin-Elmer 5100PC, Perkin-Elmer, Norwalk, CT) (11
) and P by a colorimetric method (12
). Analytical standards were prepared from certified single-element stock solutions (SPEX Chemical, Metuchen, NJ). These analytical methods have been checked in multilaboratory quality control studies (13
) and analysis of NBS Bovine Liver (1577 or 1577a, National Institute of Standards and Technology, Gaithersburg, MD) gave results within 5% of certified values.
Statistical analyses.
Growth results were analyzed by ANOVA followed by Scheffé’s test for difference of means (comparing Ca + P concentration effects, diets AIN93, 1.5x, 2.5x and 4.0x) or t test for independent samples (comparing Ca:P ratio effects, AIN93 vs. AIN76 diets), using P < 0.05 as the threshold of significance. Kidney Ca results were analyzed by nonparametric methods (Kruskal-Wallis ANOVA followed by Dunn’s multiple comparison or Mann-Whitney U test) due to profound heterogeneity of variances, which could not be overcome by standard transformation procedures. For Kruskal-Wallis ANOVA and Mann-Whitney U test, P < 0.05 was used as the threshold of significance. For Dunn’s multiple comparison, which was performed only where Kruskal-Wallis ANOVA achieved significance, a threshold of 
= 0.15 was used, yielding an experiment-wise error rate threshold for significance of 0.0125 (14
). The association between kidney Ca or P concentration and histological score was investigated using the Spearman Rank Order correlation. Statistical analyses were conducted using Statistica for Windows, version 5.1 (StatSoft, Tulsa, OK). Results are presented as mean ± SD, with ranges also specified for kidney Ca concentrations.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Kidney Ca concentration in the group of weanling rats killed at the outset of the study was 7.53 ± 0.40 µmol/g dry kidney (range 6.90–8.18) for female rats and 7.05 ± 0.30 µmol/g dry kidney (range 6.65–7.50) for male rats. All weanling rats killed at the outset of the study had histochemical scores of zero, indicating no evidence of black precipitates on von Kossa–stained kidney sections.
Male rats showed almost no histochemical evidence of NC with any of the diets and had less kidney Ca accumulation than female rats (Table 2
). In female rats, increased kidney Ca concentration and incidence of histochemically determined NC was found after 16 wk of feeding increasing amounts of dietary Ca and P at the optimal molar ratio (comparing diets AIN93 through 4.0x, Table 2
), although more extensive Ca accumulation and more severe NC resulted from imbalance in dietary Ca and P intakes (comparing diet AIN93 and AIN76;Table 2
and Fig. 1
).
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. For categorization of kidney calcium score in our laboratory, the "normal" level for rats is 7.50 ± 0.63 µmol Ca/g dry kidney (mean ± SD) based on historical data from several studies. This value is similar to the analyzed value for female rats killed at the outset of the present study. With increasing dietary Ca and P concentrations, a higher proportion of female rats had "elevated" kidney calcium score (>2 SD above "normal") and 1 of 8 female rats fed diet 4.0x had a kidney calcium score indicative of NC, >10 times the normal level (i.e., Ca concentration >75 µmol/g dry kidney). Kidney P concentration was significantly increased only in female rats fed diet AIN76 in comparison to AIN93 (445 ± 80 vs. 351 ± 11 µmol P/g dry kidney, respectively).
The location of mineral precipitation in kidney sections, shown by von Kossa staining, was similar with elevated concentrations of Ca and P at the optimal ratio (e.g., diet 4.0x, Fig. 1
B) to that seen with Ca:P imbalance (diet AIN76, Fig. 1
C) although the degree of precipitation was generally more severe in the latter group. Most of the female rats fed diet AIN93 had no histochemical evidence of mineral precipitation after 16 wk of feeding (Fig. 1
A). There was considerable variation among rats within a treatment group in the extent of kidney Ca accumulation and the severity of NC (Table 2)
. Even with diet 2.5x, there were some female rats with kidney Ca concentrations in the normal range and no histochemical evidence of NC. With diet 4.0x, all female rats showed elevated kidney Ca, although the range of responses was still very large. Higher incidence and severity of NC was seen with the suboptimal dietary Ca:P molar ratio (diet AIN76) than when these elements were present at 4 times the normal concentrations but "optimally" balanced (diet 4.0x) (Fig. 1
and Table 2
).
There was a significant positive association between kidney histochemical score and kidney Ca concentration in female rats (Fig. 2
) which yielded a Spearman Rank Order correlation coefficient r = 0.87 (P < 0.00001, n = 40). A similar positive association between kidney histochemical score and kidney P concentration in female rats (data not shown) yielded a Spearman Rank Order correlation coefficient r = 0.65 (P < 0.00001, n = 40).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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) examined the effect of increasing dietary Ca and P concentrations, comparing 0.25% Ca + 0.20% P or 0.25% Ca + 0.4% P to diets containing twice the content of each (0.5% Ca + 0.4% P or 0.5% Ca + 0.8% P, respectively). In both cases, the diets with the higher Ca + P content yielded more severe NC. Low incidence and severity of NC was found using a diet containing 0.75% Ca + 0.4% P (15
), which is similar in Ca and P composition to our diet 1.5x.
It has been suggested that the Ca:P molar ratio must be >1 to prevent kidney calcification in female rats (16
). Although this was achieved in our AIN93 through 4.0x diets, the higher concentrations of Ca and P intake at the recommended ratio still produced NC. Studies described by Reeves et al. (16
) indicated that a Ca:P molar ratio of 0.97 (5 g Ca + 4 g P/kg diet) did not completely eliminate kidney calcification in female rats, although normal kidney Ca concentrations were found in female rats fed a purified diet with a Ca:P molar ratio of 1.3 for 16 wk. The present finding of slightly elevated kidney Ca in one of eight female rats fed diet AIN93 suggests that a Ca:P molar ratio of 1.07 does not completely prevent Ca accumulation in kidneys. Earlier work in our laboratory indicated that even with a diet formulated according to AIN-93G specifications, with an analyzed Ca:P molar ratio of 1.31, some female rats had elevated kidney Ca after 16 wk of feeding, and one of eight female rats had a kidney Ca concentration consistent with NC (5
). It is important to note that kidney Ca concentration in female rats in the present study varied widely within a treatment group, as did the severity of histochemically assessed NC. Similar results have been reported by others (2
,5
,15
).
The typical histological appearance of diet-induced NC shows calcification primarily in the corticomedullary junction region (1
) and this was the pattern seen in the present study. The von Kossa histochemical procedure detects principally phosphate and carbonate ions, which are the counterions with which Ca is commonly associated in normal and pathologically calcified tissues, rather than being specific for Ca (10
). Others have demonstrated that the kidney precipitates found in rats fed diets varying in Ca and/or P content, such as ours, are typically composed of calcium and phosphate (17
,18
). The significant association between histochemical score and kidney Ca concentration using the Spearman rank correlation indicates the robustness of this histological grading scheme. A similar correlation was shown previously in rats fed diets varying in Ca and P content (15
,19
,20
) and in human renal biopsy specimens (21
). A similar association between histochemical score and kidney P concentration has also been reported (15
). Together, these associations support the identification of the kidney concretions found in female rats in the present study as calcium phosphate.
NC in female rats can complicate the interpretation of toxicity studies (22
–24
). This problem has been noted in cereal-based diets as well as semipurified diets, and has led the National Toxicology Program to reformulate their standard cereal-based diet to conform to the AIN recommendation regarding Ca:P molar ratio (25
). Because rats are one of the standard laboratory animal species used in toxicity testing for regulatory purposes (26
), NC in rats can have widespread implications for human safety and risk assessment activities. Researchers should be cognizant of Ca and particularly P concentrations and adjust mineral mixes accordingly when substituting different protein sources in modified AIN-93G diets.
Although the reformulation leading to the AIN-93G diet was largely successful in reducing the incidence and severity of nephrocalcinosis in female rats, it must be remembered that the absolute concentrations of dietary Ca and P, and not just their ratio, influence the development of nephrocalcinosis in female rats.
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FOOTNOTES |
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3 Abbreviations used: AIN93, modified AIN-93G diet containing Ca and P at the concentrations specified in the AIN-93G formulation; 1.5x, 2.5x and 4.0x, diets containing Ca and P at 1.5, 2.5 and 4.0 times, respectively, the concentrations in the AIN-93G diet; AIN76, modified AIN-93G diet containing Ca and P at the concentrations specified in the AIN-76A formulation; H&E, hematoxylin and eosin stain; NC nephrocalcinosis.
Manuscript received 6 September 2001. Initial review completed 28 September 2001. Revision accepted 27 November 2001.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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1. Cousins, J. B. & Geary, C.P.M. (1966) A sex-determined renal calcification in rats. Nature (Lond.) 211:980-981.[Medline]
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11. Cockell, K. A., Fischer, P.W.F. & Belonje, B. (1999) Elemental composition of anatomically distinct regions of rat liver. Biol. Trace Elem. Res. 70:251-263.[Medline]
12. Murphy, J. & Riley, J. P. (1962) A modified single solution method for the determination of phosphate in natural waters. Anal. Chim. Acta 27:31-36.
13. Health Protection Branch Laboratories (1985) Sample preparation by dry ashing for the determination of various elements by flame atomic absorption spectroscopy. Laboratory Procedure LPFC-137 1985 Bureau of Nutritional Sciences, Health and Welfare Canada Ottawa, Canada .
14. Daniel, W.W. (1978) Applied Nonparametric Statistics 1978:202-214 Houghton Mifflin Company Boston, MA. .
15. Hoek, A. C., Lemmens, A. G., Mullink, J.W.M.A. & Beynen, A. C. (1988) Influence of dietary calcium:phosphorus ratio on mineral excretion and nephrocalcinosis in female rats. J. Nutr. 118:1210-1216.
16. Reeves, P. G., Rossow, K. L. & Lindauf, J. (1993) Development and testing of the AIN-93 purified diets for rodents: results on growth, kidney calcification and bone mineralization in rats and mice. J. Nutr. 123:1923-1931.
17. Woodard, J. C. (1971) A morphologic and biochemical study of nutritional nephrocalcinosis in female rats fed semi-purified diets. Am. J. Pathol. 65:253-268.[Medline]
18. Stonard, M. D., Samuels, D. M. & Lock, E. A. (1984) The pathogenesis and effect on renal function of nephrocalcinosis induced by different diets in female rats. Food Chem. Toxicol. 22:139-146.[Medline]
19. Mars, Y.W.H.M., Lemmens, A. G. & Beynen, A. C. (1980) Dietary phosphorus and nephrocalcinosis in female rats. Nutr. Rep. Int. 38:249-258.
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Marsman, G., Pastoor, F.J.H., Mathot, J.N.J.J., Theuns, H. M. & Beynen, A. C. (1991) Vitamin D, within its range of fluctuation in commercial rat diets, does not influence nephrocalcinogenesis in female rats. Lab. Anim. 25:330-336.
21. Giminez, L. F., Solez, K. & Walker, W. G. (1987) Relation between renal calcium content and renal impairment in 246 human renal biopsies. Kidney Int 31:93-99.[Medline]
22. Forbes, R. M. (1971) Attempts to alter kidney calcification in the magnesium-deficient rat. J. Nutr. 101:35-44.
23. Meyer, O., Blom, L. & Olsen, P. (1978) Influence of diet and strain of rat on kidney damage observed in toxicity studies. Arch. Toxicol. Suppl. 1:355-358.
24. Lina, B.A.R., Bos-Kuijpers, M.H.M., Til, H. P. & Bär, A. (1996) Chronic toxicity and carcinogenicity study of erythritol in rats. Regul. Toxicol. Pharmacol. 24:S264-S279.[Medline]
25. Rao, G. N. (1997) New nonpurified diet (NTP-2000) for rodents in the National Toxicology Program’s toxicology and carcinogenesis studies. J. Nutr. 127:842S-846S.
26. OECD (1993) Summary of considerations in the report from the OECD Expert Groups on short term and long term toxicology. Section 4 Health Effects OECD Guidelines for the Testing of Chemicals 2:1-12 Organization for Economic Co-operation and Development Paris, France. .
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