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© 2004 The American Society for Nutritional Sciences J. Nutr. 134:637-640, March 2004

Nutrient Interactions and Toxicity
Research Communication

Nephrocalcinosis Caused by Dietary Calcium:Phosphorus Imbalance in Female Rats Develops Rapidly and Is Irreversible1,2

Kevin A. Cockell3 and Bartholomeus Belonje

Nutrition Research Division, Food Directorate, Health Products and Food Branch, Health Canada, Ottawa, ON, Canada K1A 0L2

3To whom correspondence should be addressed. E-mail: kevin_cockell{at}hc-sc.gc.ca.


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
Female rats fed the standardized AIN-76A diet develop kidney calcium deposits (nephrocalcinosis, NC). A low dietary Ca:P molar ratio is a primary factor in this disorder. The AIN-93G diet has a lower P content and higher Ca:P molar ratio and lowers the incidence of NC. To examine the early stages of NC induced by dietary Ca:P imbalance and the potential reversibility of this disorder, weanling female Sprague-Dawley rats were fed a modified AIN-93G test diet containing Ca:P at AIN-76A levels (NC-inducing diet) for 0.5–16 wk before necropsy, or were switched to AIN-93G control diet after 0.5–4 wk until necropsy at 16 wk. A dramatic increase in incidence and severity of NC was noted after 2 wk of feeding the test diet. NC was not reversible by switching to the control diet. As little as 0.5 wk of exposure to the test diet followed by 15.5 wk of consuming the control diet resulted in increased incidence and severity of NC compared with 16 wk of consuming the control diet. Short-term (as little as several days) feeding of an NC-inducing diet to female rats can lead to NC even if they are switched to an optimal diet.

KEY WORDS: • nephrocalcinosis • female rats • irreversible • potentiation • early onset

Nephrocalcinosis (NC) in female rats has been observed for many years (1). A number of dietary variables are involved in the development of this disorder (2), and a low molar ratio of dietary Ca:P is an important contributing factor (3,4). The female rat provides a sensitive model for examining the potential adverse effects of Ca and P on the kidney (5).

In the 1970s, the American Institute of Nutrition established a standardized rodent diet (AIN-76A) that would meet the nutritional requirements of rats and provide a consistent control diet for nutritional, toxicological, and regulatory purposes. However, female rats fed this diet commonly developed kidney calcium deposits typical of NC. In the early 1990s, a new standardized rodent diet, AIN-93G, was developed (4). One of the aims of the reformulation was to improve the ratio of Ca:P in the diet, thereby reducing the incidence of NC in female rats. Some evidence of NC in female rats was shown to persist in longer-term studies using the AIN-93G diet formulation (6,7).

NC in female rats can complicate the interpretation of nutritional or toxicological studies involving the kidney (8,9). Thus it is important to understand the etiology of NC, including dietary factors. NC occurred with cereal-based diets as well as semipurified diets, which led the National Toxicology Program to reformulate their standard cereal-based diet to conform to the AIN recommendation regarding the Ca:P molar ratio (10). NC was shown to be irreversible once it was well established, after 8 wk of feeding female rats a diet imbalanced in Ca:P (11). It was suggested that the onset of diet-induced NC must be rapid (12). However, the short-term onset and potential for reversibility of early stages in the progression of NC has not been thoroughly investigated to date.

The present study was conducted using the AIN-93G control diet formulation and a test diet differing only in phosphorus concentration, to mimic the proportions of Ca:P found in the AIN-76A formulation (positive nephrocalcinogenic diet). Early stages (days to weeks) in the development of diet-induced NC were investigated, as was the potential for reversibility from these early stages by switching to the AIN-93G control diet formulation. The results of this study are relevant to short-term feeding of diets with a suboptimal Ca:P ratio, for example, during acclimation to laboratory conditions or for other reasons.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 LITERATURE CITED
 
    Animals and diets. Weanling (21 d old) female Sprague-Dawley rats (Charles River Canada) were randomly assigned to one of two groups fed diets differing in the ratio of Ca to P. The control diet was very similar to the standard AIN-93G formulation, with 0.5% Ca and 0.3% P and a calculated Ca:P molar ratio of 1.3 (4), differing only in the chemical forms of Ca and P added (see below). The test diet formulation was further modified from AIN-93G to contain 0.5% Ca and 0.5% P by weight, to mimic the Ca:P levels and molar ratio (~0.75 by calculation) in the AIN-76A formulation.4 Diets were formulated to be equal in Ca, K, and all other minerals except for P. The P in casein was considered in calculating these formulations.

An initial group of 8 rats was killed at wk 0 for assessment of pretreatment kidney histology and kidney Ca concentration. The remaining rats were fed the control diet or the test diet for 16 wk or switched from the test diet to the control diet after 0.5–4 wk (Table 1). At each time point from 0.5 to 4 wk, two groups of 8 rats fed either the control or test diet were killed and tissues collected as described below; an additional group of 8 rats was then switched from the test diet to the control diet for the balance of the 16-wk feeding experiment. At the end of the 16 wk, all remaining rats were killed and tissues collected. Rats were maintained in accordance with the guidelines of the Canadian Council on Animal Care, in stainless steel wire-bottomed cages in a temperature- and humidity-controlled room with a 12-h light:dark cycle. The experimental protocol was approved by the institutional Animal Care Committee of the Health Products and Food Branch of Health Canada.


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  		TABLE 1 Outline of the experimental design1

 
    Tissue sampling and histopathology. At scheduled sampling times as noted above, rats were killed by exsanguination under isoflurane anesthesia. Kidneys were removed and cut in half (left kidney cut longitudinally, right kidney cut transversely). One half of each kidney was immediately placed in buffered formalin for subsequent processing and paraffin-embedding following routine methods for histopathology (Histology Services Laboratory, Department of Pathology and Laboratory Medicine, University of Ottawa). Adjacent 5-µm sections were cut and stained with hematoxylin and eosin for routine examination or von Kossa’s stain for calcium phosphate salts (13). With the von Kossa staining procedure, precipitates of calcium phosphate salts were apparent as black granules on a pink background of tissue (7). NC was scored histochemically by counting granules on one longitudinal and one transverse kidney section on each von Kossa-stained slide. Because rats of different ages (and consequently different sizes) were used in this study, granule counts were normalized per cm2 of kidney section examined, and a histochemical grading scale was used as follows: 0 = 0 granules/cm2; + = 1–10 granules/cm2; ++ = 11–40 granules/cm2; +++ = 41–80 granules/cm2; ++++ = >80 granules/cm2. The other half of each kidney was frozen at -80°C for subsequent mineral analyses.

    Mineral analyses. Samples of diets and kidneys were dry ashed at 450°C using concentrated nitric acid as an oxidizing agent before analysis for Ca by flame atomic absorption spectrophotometry (Perkin Elmer 5100PC, Perkin-Elmer) (14). P content was assayed colorimetrically (15). Analytical standards were prepared from certified single-element stock solutions (SPEX Chemical). These analytical methods were verified in multilaboratory quality control studies (16) and analysis of NBS Bovine Liver (1577 or 1577a; National Institute of Standards and Technology, Gaithersburg, MD) gave results within 5% of the certified values.

    Statistical analyses. Initial body weights across all groups of rats, final body weights of groups remaining at wk 16, and total feed intake at wk 16 were analyzed by ANOVA. Dietary Ca and P concentrations, and final body weights and total feed intakes of groups killed at each intermediate sampling time (0.5–4 wk) were analyzed by t test. Because the variances of kidney Ca and P concentrations were unequal (significant Levene’s test), assumptions were not satisfied for parametric ANOVA. Standard transformations were unable to overcome these limitations; therefore, the nonparametric Mann-Whitney U test was used to assess the onset of NC (Table 2); Kruskal-Wallis ANOVA was used to assess reversibility (Table 3). The threshold of significance used in these tests was P < 0.05. When the Kruskal-Wallis ANOVA was significant, post-hoc analysis using Dunn’s Multiple Comparison Test was used to determine which means differed, using a threshold of {alpha} = 0.15, which yielded an experiment-wise error rate threshold of significance of 0.0125 (17). Statistical analyses were conducted using Statistica for Windows, version 5.1 (StatSoft). Results are presented as means ± SD, with ranges also specified for kidney Ca concentrations.


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  		TABLE 2 Early onset of nephrocalcinosis caused by dietary calcium:phosphorus imbalance in female rats1

 

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  		TABLE 3 Irreversibility of nephrocalcinosis caused by dietary calcium:phosphorus imbalance in female rats1

 

   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 LITERATURE CITED
 
The Ca and P concentrations of the test diets were (dry weight basis) as follows: control diet, 5.2 ± 0.1 g Ca and 3.7 ± 0.2 g P/kg diet, molar ratio = 1.08; test diet 5.1 ± 0.1 g Ca and 5.5 ± 0.4 g P/kg diet, molar ratio = 0.72. Body weight gains and food consumption rates were not affected by the dietary treatments (results not shown).

Exposure for 2 wk to the NC-inducing test diet was sufficient to induce NC in susceptible rats (Table 2). This was apparent through either histochemical score or kidney Ca concentration. With longer term exposure (4–16 wk in this experiment), a mild degree of NC was apparent even in some control rats. Kidney P concentration was elevated less than Ca, and only after 4–16 wk of exposure to the NC-inducing diet (Table 2).

The nephrocalcinogenic effects of 2 wk of exposure to the test diet were not reversible by 14 wk of subsequent feeding of the control (AIN-93G) diet (Table 3). Some individual rats exposed to the test diet for as little as 0.5–1 wk followed by 15–15.5 wk consuming the control diet had kidney Ca levels in excess of 3 times the normal level (i.e., >25 µmol Ca/g dry weight). There was considerable variation among rats within a treatment group in the extent of kidney Ca accumulation and the severity of NC. For example, in the group fed the test diet for 2 wk, followed by the control diet for 14 wk (Table 3), one rat had normal kidney Ca (7.58 µmol Ca/g dry weight), whereas another had extremely elevated kidney Ca (976 µmol Ca/g dry weight). Kidney P concentration was less responsive than kidney Ca. Kidney P was elevated only in the group fed the NC-inducing diet for 16 wk, whereas individual rat kidney P concentrations in excess of 2 times the normal level occurred only in the treatment group fed the NC-inducing diet for 2 wk followed by the control diet for 14 wk (Table 3). Because of the very high variability of kidney Ca and P concentrations in this group, they did not differ significantly from those in any other group.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
LITERATURE CITED
 
No discernible morbidity was associated with NC in this study, and neither feed intake nor growth was significantly affected. This is in agreement with previous reports (7,12,18) that NC was not associated with growth disruption or clinical morbidity.

NC resulted in the accumulation of calcium phosphate precipitates in the outer stripe of the outer medulla of the kidney; with von Kossa staining, these appeared as dark granules against a pink background. As noted previously (7), there was a strong correlation between our grading scheme of histochemical score and the concentration of calcium in the kidney tissue. This was true even after adjusting our method of histochemical scoring to reflect differences in the area of tissue examined on each slide so as to compare rats of different ages. Spearman’s rank order correlation coefficient in the present study was r = 0.85 (P < 0.00001, n = 119), which is very similar to our observation in the previous study (7). No correlation was found for kidney P concentration vs. histochemical score (P = 0.44, n = 119) in the present study, in contrast to our previous work.

The results of the present study indicate that exposure to a nephrocalcinogenic diet, imbalanced in Ca:P, for as little as 0.5 wk can potentiate the subsequent development of NC in female rats, even if the rats are switched to the AIN-93G control diet. Clear indication of NC induced by a diet imbalanced in Ca:P was evident by 2 wk. This is consistent with the early onset observed for dietary Mg deficiency-induced NC (19), in which kidney concretions and tissue damage were evident by electron microscopy within 1–5 d of initiation of feeding a severely Mg-deficient diet to male rats. Susceptibility to NC induced by Mg deficiency differs from that for Ca:P imbalance, i.e., Ca:P imbalance readily causes NC in female rats, whereas male rats are much less sensitive. NC caused by Mg deficiency occurs in both male and female rats, although it may be more severe in females (2). Despite this potential for differences in etiology of the disorder, the early onset appears to follow similar timelines. To our knowledge, the "potentiation" of NC in the present study in rats fed the test diet for 0.5–1 wk followed by 15–15.5 wk of consuming the control diet is an observation not previously reported.

A mild degree of NC was observed in some female rats fed the control diet for 4–16 wk in this study. A similar observation in our previous work prompted the conclusion that a Ca:P molar ratio of 1.07 (very similar to the 1.08 in the control diet in the present study) might not be quite high enough to prevent NC completely (7). However, NC was observed even in female rats fed for 16 wk the AIN-93G diet with a Ca:P molar ratio of 1.3 or a cereal-based diet with a Ca:P molar ratio of 1.6 (6).

The irreversibility of NC induced by dietary Ca:P imbalance is consistent with literature reports, although no previous studies apparently examined the potential for reversibility from the earliest stages of development as reported here. NC induced by dietary Ca:P imbalance in female Sprague Dawley rats fed for 8 wk was not reversible with up to 25 wk of follow-up feeding a diet with a higher Ca:P molar ratio (11). NC produced in female Sprague-Dawley rats through the use of a diet based on the AIN-76 mineral mix (and thus presumably imbalanced in Ca:P) for 8 wk did not regress with 6 wk of feeding a similar diet supplemented with Mg and F at levels that had previously been shown to prevent the onset of NC (20). NC produced by 29 d of feeding a high phosphorus diet to female Wistar rats was not reversible by switching to a low phosphorus diet for 91 d (18), causing those authors to conclude that "... the composition of the pre-experimental diet is as crucial as that of the experimental diet" in minimizing NC. Others have thus provided evidence that NC, once well established, may not readily be reversed. The results of the present study have extended this conclusion to earlier stages in the development of NC. Our observation of "potentiation" of NC emphasizes this concern, even with regard to short-term feeding of diets imbalanced in Ca:P, for example, during acclimation to laboratory conditions or for other reasons.

Short-term feeding of a diet imbalanced in Ca and P can lead to NC in female rats, even if the rats are subsequently fed a diet that is optimally balanced with regard to these two mineral nutrients. Because rats are a standard laboratory species used in toxicity testing for regulatory purposes (21), NC in rats can have widespread implications for human safety and risk assessment activities. This dictates that appropriate care should be taken in the choice of rodent diets, even during short-term feeding, particularly if conducting experiments with female rats.


   FOOTNOTES
 
1 Portions of this work were presented in poster form at the 45th Annual Meeting of the Canadian Federation of Biological Societies, June 12–15, 2002, Montreal, Canada [Cockell, K.A. & Belonje, B. (2002) Early onset and irreversibility of diet-induced nephrocalcinosis in female rats. Abstract F010]. This is publication no. 588 of the Bureau of Nutritional Sciences, Food Directorate, Health Products and Food Branch, Health Canada. Back

2 Funded by the Food Directorate, Health Canada. Back

4 Both diets contained (g/kg diet): cornstarch (BestFoods) 344; sucrose (Lantic Sugar Limited) 100; vitamin-free casein 200; dextrinized cornstarch 132; fiber (Solka Floc) 50; vitamin mix AIN-93-VX, 10; modified AIN-93-MX mineral mix (without calcium carbonate, potassium phosphate monobasic or potassium citrate monohydrate), 13.2 (Harlan Teklad); macromineral premix (see below) 75; L-cystine 3.0; choline bitartrate 2.5; and soybean oil (containing t-butylhydroquinone at 0.2 g/kg oil to provide 0.014 g/kg diet) 70 (ICN Biomedicals). For the control diet, the macromineral premix supplied (g/kg diet) CaHPO4 9.28; CaCO3 5.66; tripotassium citrate monohydrate 7.93; and cornstarch 52.13, to provide 5g Ca and 3g P/kg diet. For the test diet, the macromineral premix supplied (g/kg diet) CaHPO4 16.97; KH2PO4 1.09; tripotassium citrate monohydrate 7.06; and cornstarch 49.88, to provide 5g Ca and 5g P/kg diet. Macromineral premix ingredients were from Fisher Scientific, VWR Canlab, and BDH. Back

Manuscript received 29 October 2003. Initial review completed 17 November 2003. Revision accepted 9 December 2003.


   LITERATURE CITED
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 

1. Cousins, J. B. & Geary, C.P.M. (1966) A sex-determined renal calcification in rats. Nature (Lond.) 211:980-981.

2. Ritskes-Hoitinga, J. & Beynen, A. C. (1992) Nephrocalcinosis in the rat: a literature review. Prog. Food Nutr. Sci. 16:85-124.[Medline]

3. Shah, B. G. & Belonje, B. (1991) Different calcium levels required to prevent nephrocalcinosis in male and female rats. Nutr. Res. 11:385-390.

4. Reeves, P. G., Nielsen, F. H. & Fahey, G. C., Jr (1993) AIN-93 purified diets for laboratory rodents: final report of the American Institute of Nutrition ad hoc writing committee on the reformulation of the AIN-76A rodent diet. J. Nutr. 123:1939-1951.

5. Burgess, N. A., Reynolds, T. M., Williams, N., Pathy, A. & Smith, S. (1995) Evaluation of four animal models of intrarenal calcium deposition and assessment of the influence of dietary supplementation with essential fatty acids on calcification. Urol. Res. 23:239-242.[Medline]

6. L’Abbé, M. R., Trick, K. D. & Belonje, B. (1997) The AIN-93G diet: nephrocalcinosis, kidney calcium and tissue trace element levels. Fischer, P.W.F. L’Abbé, M. R. Cockell, K. A. Gibson, R. S. eds. Trace Elements in Man and Animals - 9: Proceedings of the Ninth International Symposium on Trace Elements in Man and Animals 1997:401-402 NRC Research Press Ottawa, Canada. .

7. Cockell, K. A., L’Abbé, M. R. & Belonje, B. (2002) The concentration and ratio of dietary calcium and phosphorus influence development of nephrocalcinosis in female rats. J. Nutr. 132:252-256.[Abstract/Free Full Text]

8. 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.

9. Lina, B.A.R., Bos-Kuipers, 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]

10. 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.

11. Peterson, C. A., Baker, D. H. & Erdman, J. W., Jr (1996) Diet-induced nephrocalcinosis in female rats is irreversible and is induced primarily before the completion of adolescence. J. Nutr. 126:259-265.

12. Geary, C. P. & Cousins, J. B. (1969) An oestrogen-linked nephrocalcinosis in female rats. Br. J. Exp. Pathol. 50:507-515.[Medline]

13. Kiernan, J. A. (1990) Methods for inorganic ions. Histological and Histochemical Methods: Theory and Practice 2nd ed. 1990:221-234 Pergamon Press Oxford, UK.

14. 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]

15. 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.

16. 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.

17. Daniel, W. W. (1978) Applied Nonparametric Statistics 1978:202-214 Houghton Mifflin Company Boston, MA.

18. Soeterboek, S.J.A.J., Ritskes-Hoitinga, J., Lemmens, A. G. & Beynen, A. C. (1991) Phosphorus-induced nephrocalcinosis in female rats: a study on regression and clinical abnormalities. Lab. Anim. 25:258-262.[Abstract/Free Full Text]

19. Bunce, G. E., Saacke, R. G. & Mullins, J. (1980) The morphology and pathogenesis of magnesium deficiency-induced nephrocalcinosis. Exp. Mol. Pathol. 33:203-210.[Medline]

20. Shah, B. G., Trick, K. D. & Belonje, B. (1991) Dietary factors in rat nephrocalcinosis. Trace Elem. Med. 8:154-160.

21. Organization for Economic Co-operation and Development (1993) Summary of considerations in the report from the OECD Expert Groups on short term and long term toxicology. OECD Guidelines for the Testing of Chemicals, Volume 2, Section 4: Health Effects 1993:1-12 Organization for Economic Cooperation and Development Paris, France.





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