Calcium and vitamin D have a synergistic role in a rat model of kidney stone disease

Vitamin D supplementation in humans should be accompanied by calcium administration to avoid bone demineralization through vitamin D receptor signaling. Here we analyzed whether long-term exposure of rats to vitamin D supplementation, with or without a calcium-rich diet, would promote kidney stone formation. Four groups of rats received vitamin D alone (100,000 UI/kg/3 weeks), a calcium-enriched diet alone, both vitamin D supplementation and calcium-enriched diet, or a standard diet (controls) for 6 months. Serum and urine parameters and crystalluria were monitored. Kidney stones were assessed by 3-dimensional micro-computed tomography, infrared spectroscopy, von Kossa/Yasue staining, and field emission scanning electron microscopy. Although serum calcium levels were similar in the 4 groups, rats receiving vitamin D had a progressive increase in urinary calcium excretion over time, especially those receiving both calcium and vitamin D. However, oral calcium supplementation alone did not increase urinary calcium excretion. At 6 months, rats exposed to both calcium and vitamin D, but not rats exposed to calcium or vitamin D alone, developed significant apatite kidney calcifications (mean volume, 0.121 mm3). Thus, coadministration of vitamin D and increased calcium intake had a synergistic role in tubular calcifications or kidney stone formation in this rat model. Hence, one should be cautious about the cumulative risk of kidney stone formation in humans when exposed to both vitamin D supplementation and high calcium intake. Vitamin D supplementation in humans should be accompanied by calcium administration to avoid bone demineralization through vitamin D receptor signaling. Here we analyzed whether long-term exposure of rats to vitamin D supplementation, with or without a calcium-rich diet, would promote kidney stone formation. Four groups of rats received vitamin D alone (100,000 UI/kg/3 weeks), a calcium-enriched diet alone, both vitamin D supplementation and calcium-enriched diet, or a standard diet (controls) for 6 months. Serum and urine parameters and crystalluria were monitored. Kidney stones were assessed by 3-dimensional micro-computed tomography, infrared spectroscopy, von Kossa/Yasue staining, and field emission scanning electron microscopy. Although serum calcium levels were similar in the 4 groups, rats receiving vitamin D had a progressive increase in urinary calcium excretion over time, especially those receiving both calcium and vitamin D. However, oral calcium supplementation alone did not increase urinary calcium excretion. At 6 months, rats exposed to both calcium and vitamin D, but not rats exposed to calcium or vitamin D alone, developed significant apatite kidney calcifications (mean volume, 0.121 mm3). Thus, coadministration of vitamin D and increased calcium intake had a synergistic role in tubular calcifications or kidney stone formation in this rat model. Hence, one should be cautious about the cumulative risk of kidney stone formation in humans when exposed to both vitamin D supplementation and high calcium intake. Vitamin D severe deficiency during infancy leads to rickets and may promote secondary hyperparathyroidism and accelerated bone mass loss in adults.1Reid I.R. What diseases are causally linked to vitamin D deficiency?.Arch Dis Child. 2016; 101: 185-189Crossref PubMed Scopus (28) Google Scholar Serum 25-hydroxyvitamin D (25[OH]D) depends on nutritional intakes and absorption of vitamin D2 and vitamin D3 (including supplementation) and solar exposure.2Mazahery H. von Hurst P.R. Factors affecting 25-hydroxyvitamin D concentration in response to vitamin D supplementation.Nutrients. 2015; 7: 5111-5142Crossref PubMed Scopus (133) Google Scholar The synthesis of 1,25-dihydroxyvitamin D (1,25[OH]2D) or calcitriol by the kidney increases calcium absorption and bone remodeling.3Carmeliet G. Dermauw V. Bouillon R. Vitamin D signaling in calcium and bone homeostasis: a delicate balance.Best Pract Res Clin Endocrinol Metab. 2015; 29: 621-631Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar Actually, 1,25(OH)2D binds its receptor (VDR) in enterocytes, increasing the calcium digestive absorption via the transcellular pathway. It has long been described that calcium-dependent kidney stone formers have high 1,25(OH)2D serum levels and/or increased sensitivity to 1,25(OH)2D, possibly due to VDR overexpression or activation.4Kaplan R.A. Haussler M.R. Deftos L.J. et al.The role of 1 alpha, 25-dihydroxyvitamin D in the mediation of intestinal hyperabsorption of calcium in primary hyperparathyroidism and absorptive hypercalciuria.J Clin Invest. 1977; 59: 756-760Crossref PubMed Scopus (216) Google Scholar, 5Broadus A.E. Insogna K.L. Lang R. et al.Evidence for disordered control of 1,25-dihydroxyvitamin D production in absorptive hypercalciuria.N Engl J Med. 1984; 311: 73-80Crossref PubMed Scopus (154) Google Scholar, 6Li X.Q. Tembe V. Horwitz G.M. et al.Increased intestinal vitamin D receptor in genetic hypercalciuric rats. A cause of intestinal calcium hyperabsorption.J Clin Invest. 1993; 91: 661-667Crossref PubMed Scopus (150) Google Scholar Bushinsky et al. have extensively studied the main murine model of kidney stone formation, the genetic hypercalciuric stone–forming (GHS) rat, and evidenced an increased expression of VDR in several tissues including gut and bone.6Li X.Q. Tembe V. Horwitz G.M. et al.Increased intestinal vitamin D receptor in genetic hypercalciuric rats. A cause of intestinal calcium hyperabsorption.J Clin Invest. 1993; 91: 661-667Crossref PubMed Scopus (150) Google Scholar, 7Frick K.K. Asplin J.R. Favus M.J. et al.Increased biological response to 1,25(OH)(2)D(3) in genetic hypercalciuric stone-forming rats.Am J Physiol Renal Physiol. 2013; 304: F718-F726Crossref PubMed Scopus (23) Google Scholar Vitamin D supplementation, with or without increased calcium intake, has been proposed for a long time to prevent bone mass loss, especially in postmenopausal women.8Zerwekh J.E. Sakhaee K. Glass K. Pak C.Y. Long term 25-hydroxyvitamin D3 therapy in postmenopausal osteoporosis: demonstration of responsive and nonresponsive subgroups.J Clin Endocrinol Metab. 1983; 56: 410-413Crossref PubMed Scopus (36) Google Scholar The main goal of vitamin D supplementation is to increase calcium digestive absorption. Because the net bone calcium flux stands near the equilibrium in adults, increased calcium digestive absorption should necessarily result in increased calcium urinary excretion and therefore increase the risk of stone formation. Nevertheless, the role of vitamin D supplementation in kidney stone formation in humans remains controversial. Actually, epidemiologic studies did not demonstrate a link between 25(OH)D serum levels and kidney stone formation or urinary calcium excretion.9Eisner B.H. Thavaseelan S. Sheth S. Haleblian G. Pareek G. Relationship between serum vitamin D and 24-hour urine calcium in patients with nephrolithiasis.Urology. 2012; 80: 1007-1010Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 10Tang J. McFann K.K. Chonchol M.B. Association between serum 25-hydroxyvitamin D and nephrolithiasis: the National Health and Nutrition Examination Survey III, 1988-94.Nephrol Dial Transplant. 2012; 27: 4385-4389Crossref PubMed Scopus (32) Google Scholar, 11Nguyen S. Baggerly L. French C. et al.25-Hydroxyvitamin D in the range of 20 to 100 ng/mL and incidence of kidney stones.Am J Public Health. 2014; 104: 1783-1787Crossref PubMed Scopus (25) Google Scholar One study found no difference in 25(OH)D serum levels between stone formers and control individuals but identified higher 25(OH)D serum levels in hypercalciuric stone formers than in normocalciuric stone formers.12Netelenbos J.C. Jongen M.J. van der Vijgh W.J. et al.Vitamin D status in urinary calcium stone formation.Arch Intern Med. 1985; 145: 681-684Crossref PubMed Scopus (44) Google Scholar Another study identified a correlation between 25(OH)D serum levels and urinary calcium excretion in kidney stone formers.13Berlin T. Björkhem I. Collste L. et al.Relation between hypercalciuria and vitamin D3-status in patients with urolithiasis.Scand J Urol Nephrol. 1982; 16: 269-273Crossref PubMed Scopus (25) Google Scholar To date, only 1 interventional study has been dedicated to the role of vitamin D supplementation in urinary calcium excretion.14Leaf D.E. Korets R. Taylor E.N. et al.Effect of vitamin D repletion on urinary calcium excretion among kidney stone formers.Clin J Am Soc Nephrol. 2012; 7: 829-834Crossref PubMed Scopus (62) Google Scholar Twenty-nine stone formers received 50,000 IU of ergocalciferol weekly for 2 months. Despite an increase in 25(OH)D serum levels, no evidence of a statistical increase in urinary calcium excretion was observed, although some individuals had an increase in urinary calcium excretion (attributed at least in part to increased protein and salt intake). The body calcium balance was not analyzed in this study. In contrast, the Women's Health Initiative study is a large double-blind, randomized study the results of which raise concerns. During an average of 7 years, 36,282 postmenopausal women received 1 g calcium and 400 IU vitamin D daily or a placebo.15Jackson R.D. LaCroix A.Z. Gass M. et al.Women's Health Initiative Investigators. Calcium plus vitamin D supplementation and the risk of fractures.N Engl J Med. 2006; 354: 669-683Crossref PubMed Scopus (1502) Google Scholar Although no significant reduction in bone fractures was observed, an increased kidney stone risk was described in the intervention group, suggesting a cumulative role of calcium and vitamin D supplementation. We hypothesized that the combination of high calcium intakes and vitamin D supplementation would increase the risk of kidney stone formation and/or kidney tissue calcifications in a murine model. We describe here the evolution of urine composition and the appearance of kidney stones in rats receiving calcium, vitamin D, or both for 6 months, defining thereby a model of kidney stone disease. The 4 groups of rats (controls, calcium, vitamin D, and calcium plus vitamin D) had similar baseline serum levels of calcium, magnesium, phosphate, creatinine, and urea (Table 1). Six months later, no difference was observed between the 4 groups with the exception of a small but significant increase in serum creatinine levels in the vitamin D group but not in the calcium plus vitamin D group (Table 1). Vitamin D serum levels were significantly higher in the 2 groups of rats receiving vitamin D compared with controls (Table 1). Rat growth and weight were similar in all groups (not shown).Table 1Biological parameters, serumControlsCalciumVitamin DCalcium + vitamin DBaseline Calcium, mmol/l2.51 ± 0.022.53 ± 0.032.56 ± 0.032.45 ± 0.03 Magnesium, mmol/l0.87 ± 0.020.92 ± 0.030.83 ± 0.020.87 ± 0.03 Phosphate, mmol/l2.32 ± 0.162.64 ± 0.102.05 ± 0.182.6 ± 0.10 Creatinine, μmol/l22.5 ± 0.919.7 ± 1.024.3 ± 1.419.4 ± 0.9 Urea, mmol/l4.97 ± 0.195.53 ± 0.514.55 ± 0.234.8 ± 0.27At 6 months Calcium, mmol/l2.57 ± 0.042.59 ± 0.052.67 ± 0.052.64 ± 0.03 Magnesium, mmol/l0.90 ± 0.040.94 ± 0.041.03 ± 0.090.85 ± 0.03 Phosphate, mmol/l1.92 ± 0.111.93 ± 0.092.05 ± 0.091.94 ± 0.07 Creatinine, μmol/l27.9 ± 1.326.3 ± 1.034.2 ± 2.2aP < 0.05 versus control and calcium groups at 6 months.28.0 ± 2.1 Urea, mmol/l4.95 ± 0.296.1 ± 0.555.07 ± 0.215.72 ± 0.34 Vitamin D, ng/ml55.2 ± 17.949.9 ± 1.7126.2 ± 9.8aP < 0.05 versus control and calcium groups at 6 months.110.8 ± 21.7aP < 0.05 versus control and calcium groups at 6 months.a P < 0.05 versus control and calcium groups at 6 months. Open table in a new tab Crystalluria revealed the frequent presence of calcium oxalate and calcium phosphate crystals in the fresh urine of rats receiving vitamin D and vitamin D plus calcium (Figure 1a–c ). Overall, crystals were more frequently present in the urine of rats receiving calcium plus vitamin D than in rats receiving vitamin D only. During the first weeks, crystals were mainly calcium oxalate dihydrate (Figure 1b), whereas calcium phosphate crystals predominated after 2 months (Figure 1c). Urinary pH remained alkaline, ∼7, in all groups during the whole protocol (not shown). Calcium urine levels increased gradually during 6 months in rats receiving vitamin D and vitamin D plus calcium. After 8 weeks, the urinary calcium excretion was higher in the vitamin D plus calcium group than in the control group. Urinary calcium excretion increased significantly in the vitamin D group after week 14 (Figure 2a). In addition, urinary calcium levels were higher in the vitamin D plus calcium group than in rats receiving only vitamin D, especially at 14 weeks (P < 0.05). We observed lower oxalate levels in the 2 groups of rats receiving high amounts of calcium (Figure 2b) (P < 0.05). Urinary phosphate and magnesium levels were similar in the 4 groups (Figure 2c and d). X-ray microtomography analyses were performed to assess kidney papillary calcifications and stones (Figure 3a and b ). Rats exposed to vitamin D and calcium had many stones compared with the other groups. In addition, intratubular calcifications were observed. The global volume of the calcifications (including tubular calcifications and stones) was quantified after 3-dimensional kidney reconstruction (Figure 3c–f). Rats exposed to calcium or vitamin D had a mean volume of calcifications of 0.010 mm3 and 0.017 mm3, respectively (P = not significant vs. controls: 0.003 mm3), whereas rats exposed to both vitamin D and calcium had a mean volume of 0.121 mm3 (P < 0.05 vs. controls) (Figure 3g). Interestingly, most of kidney calcifications were stones and to a lesser extent intratubular calcifications (Figure 3g and h). von Kossa and Yasue staining revealed the presence of calcifications in some kidney tubules and mainly stones in the urinary space (Figure 4a–e ). Comparison with X-ray microtomography reconstructions demonstrated that most of kidney stones were removed during the preparation of kidney sections. Nevertheless, some stone fragments were still present in the urinary space, stranded in close contact with the papilla. The addition of acetic acid during Yasue staining procedure removed tubular calcifications and stones, suggesting that they were made of calcium phosphate rather than calcium oxalate (Figure 4b and c). In some cases, adherent stones surrounded by urothelial cells or fibrous caps were observed (Figure 4c). Interestingly, we observed only very sparse interstitial microcalcifications at the tip of renal papillae in some rats receiving both calcium and vitamin D but no structure mimicking Randall plaque, as observed in humans, and no nephrocalcinosis (Figure 4d and e). Field emission–scanning electron microscopy confirmed the topography and the crystalline nature of the von Kossa–positive deposits, especially in the urinary space (Figure 4f). To further characterize the crystalline phases, we performed μ-Fourier transform infrared spectroscopy with an imaging system. The analysis of the absorption spectrum and its second derivative revealed some features specific for the presence of different absorption bands of apatite [Ca5(PO4)3(OH)], especially the ν3 P-O stretching vibration mode measured at 1035 to 1045 cm–1 (Figure 4g and h). Of note, carbonate ions were detected together with apatite by their ν3 C-O stretching vibration mode at ∼1420 cm–1 and the ν2 C-O bending mode at 875 cm–1. The presence of amorphous calcium phosphate, revealed by the partial disappearance of the shoulder of the ν3 P-O absorption band of apatite, was observed in most of stones and intratubular calcifications (Figure 4g and h). In contrast, the presence of calcium oxalate was not evident. Of note, the composition of intratubular calcifications and kidney stones was similar (carbonated apatite and amorphous calcium phosphate). Long-term administration of calcium and vitamin D in rats progressively increases urinary calcium concentration and the onset of urolithiasis. Calcium or vitamin D supplementation alone is not sufficient to promote significant kidney stone growth. These results raise concerns about the combined prescription of calcium and vitamin D supplementation, a frequent setting in postmenopausal women. For a long time, vitamin D has been prescribed to prevent rickets and bone mass loss.16DeLuca H.F. The vitamin D story: a collaborative effort of basic science and clinical medicine.FASEB J. 1988; 2: 224-236Crossref PubMed Scopus (484) Google Scholar More recently, a craze for vitamin D has been generated by the observation of an association between low serum concentrations of 25(OH)D and a variety of nonskeletal diseases, including cancer, cognitive decline, hypertension, mood disorders, multiple sclerosis, cardiovascular diseases, or metabolic syndrome.17Autier P. Boniol M. Pizot C. et al.Vitamin D status and ill health: a systematic review.Lancet Diabetes Endocrinol. 2014; 2: 76-89Abstract Full Text Full Text PDF PubMed Scopus (814) Google Scholar For these reasons, it has been recommended that 25(OH)D serum levels should stand be >30 ng/ml, a level that is not achieved in the absence of supplementation by a large part of the population.18Holick M.F. Binkley N.C. Bischoff-Ferrari H.A. et al.Evaluation, treatment, and prevention of vitamin D deficiency: an Endocrine Society Clinical Practice Guideline.J Clin Endocrinol Metab. 2011; 96: 1911-1930Crossref PubMed Scopus (6848) Google Scholar Nevertheless, systematic review of the intervention studies involving vitamin D supplementation failed to demonstrate any effect of vitamin D supplementation on disease occurrence.17Autier P. Boniol M. Pizot C. et al.Vitamin D status and ill health: a systematic review.Lancet Diabetes Endocrinol. 2014; 2: 76-89Abstract Full Text Full Text PDF PubMed Scopus (814) Google Scholar The discrepancy between observational and intervention studies suggests that a low 25(OH)D serum level is a marker but not a cause of disease. More surprising, recent studies failed to provide evidence of a role of vitamin D supplementation in the reduction of bone mass loss.19Reid I.R. Bolland M.J. Grey A. Effects of vitamin D supplements on bone mineral density: a systematic review and meta-analysis.Lancet. 2014; 383: 146-155Abstract Full Text Full Text PDF PubMed Scopus (408) Google Scholar On the one hand, calcitriol and VDR stimulation may protect against bone mass loss by enhancing calcium digestive absorption. On the other hand, it has been shown that VDR activation may result in increased bone resorption.20Lieben L. Masuyama R. Torrekens S. et al.Normocalcemia is maintained in mice under conditions of calcium malabsorption by vitamin D-induced inhibition of bone mineralization.J Clin Invest. 2012; 122: 1803-1815Crossref PubMed Scopus (257) Google Scholar, 21Ng A.H. Frick K.K. Krieger N.S. et al.1,25(OH)₂D₃ induces a mineralization defect and loss of bone mineral density in genetic hypercalciuric stone-forming rats.Calcif Tissue Int. 2014; 94: 531-543Crossref PubMed Scopus (13) Google Scholar As a consequence, vitamin D administration is accompanied by increased calcium intake to avoid bone demineralization. Calcium prescription is recommended in postmenopausal women to prevent bone mass loss.22North American Menopause SocietyThe role of calcium in peri- and postmenopausal women: 2006 position statement of the North American Menopause Society.Menopause. 2006; 13: 862-877Crossref PubMed Scopus (95) Google Scholar It has been shown that calcium intake within normal ranges, unlike salt and proteins, does not promote hypercalciuria.23Borghi L. Schianchi T. Meschi T. et al.Comparison of two diets for the prevention of recurrent stones in idiopathic hypercalciuria.N Engl J Med. 2002; 346: 77-84Crossref PubMed Scopus (733) Google Scholar, 24Taylor E.N. Curhan G.C. Demographic, dietary, and urinary factors and 24-h urinary calcium excretion.Clin J Am Soc Nephrol. 2009; 4: 1980-1987Crossref PubMed Scopus (59) Google Scholar Higher dietary calcium has even been associated with a lower risk of kidney stones.25Taylor E.N. Curhan G.C. Dietary calcium from dairy and nondairy sources, and risk of symptomatic kidney stones.J Urol. 2013; 190: 1255-1259Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar This might be explained by the intestinal formation of calcium-oxalate complexes reducing oxalate absorption and thereby calcium oxalate urinary supersaturation. We actually observed in our murine model a decrease in oxalate urinary excretion in groups receiving high amounts of calcium. Therefore, normal calcium intake is highly recommended in kidney stone formers because this population is particularly at risk of bone demineralization.26Sakhaee K. Maalouf N.M. Kumar R. et al.Nephrolithiasis-associated bone disease: pathogenesis and treatment options.Kidney Int. 2011; 79: 393-403Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 27Letavernier E. Traxer O. Daudon M. et al.Determinants of osteopenia in male renal-stone-disease patients with idiopathic hypercalciuria.Clin J Am Soc Nephrol. 2011; 6: 1149-1154Crossref PubMed Scopus (39) Google Scholar According to Thacher et al.,28Thacher T.D. Fischer P.R. Pettifor J.M. et al.A comparison of calcium, vitamin D, or both for nutritional rickets in Nigerian children.N Engl J Med. 1999; 341: 563-568Crossref PubMed Scopus (247) Google Scholar normal calcium intake seems to be more important than vitamin D supplementation in Nigerian children to prevent nutritional rickets. Although dietary calcium (at least in normal ranges) is not a risk factor for kidney stone formation, the results of the Women's Health Initiative study in a large population raise questions about the potentially harmful role of the combination of calcium and vitamin D. The calcium plus vitamin D murine model highlights the synergistic role of this association. Interestingly, this model shares similarities with the main murine model of kidney stone disease, the GHS rat. Actually, in GHS rats, stones develop in the urinary space, whereas most murine models developed in mice generate intratubular calcifications and crystalline nephropathies rather than stones mimicking human urolithiasis. Bushinsky et al. have shown that stone formation in GHS rats results from increased intestinal calcium absorption and bone resorption, attributed to VDR and the response to calcitriol.29Bushinsky D.A. Frick K.K. Nehrke K. Genetic hypercalciuric stone-forming rats.Curr Opin Nephrol Hypertens. 2006; 15: 403-418Crossref PubMed Scopus (45) Google Scholar Dietary calcium has a major influence on kidney stone formation in GHS rats.30Bushinsky D.A. Favus M.J. Mechanism of hypercalciuria in genetic hypercalciuric rats. Inherited defect in intestinal calcium transport.J Clin Invest. 1988; 82: 1585-1591Crossref PubMed Scopus (91) Google Scholar GHS rats also form calcium phosphate stones with standard diet.31Bushinsky D.A. Asplin J.R. Grynpas M.D. et al.Calcium oxalate stone formation in genetic hypercalciuric stone-forming rats.Kidney Int. 2002; 61: 975-987Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar The model described here might also be useful to analyze the impact of vitamin D with or without a calcium enriched–diet on calcium intestinal net flux and on bone turnover in future studies. The impact of lower doses of vitamin D in the GHS rat model also deserves further study. Of note, stones were mainly made of calcium phosphate (carbonated apatite and amorphous calcium phosphate) and contained no detectable amount of calcium oxalate. This might be explained in a part by the high urinary pH of rodents (almost always >7), promoting calcium phosphate supersaturation. On the other hand, oxalate excretion was reduced after 14 weeks in rats receiving calcium or both calcium and vitamin D. Interestingly, the prevalence of calcium phosphate stones has been reported to have increased in past 2 decades, and one may hypothesize that the combined administration of calcium and vitamin D in the population could promote calcium phosphate stone prevalence.32Parks J.H. Worcester E.M. Coe F.L. et al.Clinical implications of abundant calcium phosphate in routinely analyzed kidney stones.Kidney Int. 2004; 66: 777-785Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar Interestingly, despite hypercalciuria and calcium phosphate supersaturation, we observed kidney stones and tubular calcifications but no nephrocalcinosis and only very sparse calcium phosphate deposits at the tip of the papilla in some rats exposed to calcium plus vitamin D, but no development of Randall plaque as observed in humans (Figure 4a and b). Most stones present in the urinary space have been removed by histologic procedures but some stones, sometimes covered by urothelial cells and/or fibrous caps, have been observed. It seems likely that crystal clearance previously described in tubular cells may also be performed by urothelial cells.33Vervaet B.A. Verhulst A. Dauwe S.E. et al.An active renal crystal clearance mechanism in rat and man.Kidney Int. 2009; 75: 41-51Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar These calcifications are stones and not plaques. They predominate in the urinary space on the side of the papilla, not at the tip of the papilla, and are massive and homogeneous calcifications, not diffuse interstitial calcium phosphate deposits. One may hypothesize that rodents are protected against kidney interstitial calcifications or that 6-month exposure to hypercalciuria is too brief to induce plaques. Our study has limitations. First, as most murine models of urolithiasis, kidney stones are made of calcium phosphate, not calcium oxalate as frequently observed in humans. Second, we used very high doses of vitamin D to induce kidney stone formation: 100,000 IU/kg i.m. every 3 weeks. These doses are higher than doses used in humans but were necessary to increase urinary calcium excretion in rats. As a matter of comparison, children affected by rickets (weight, 12.3 ± 3.4 kg) are treated by injections of 600,000 IU vitamin D i.m.28Thacher T.D. Fischer P.R. Pettifor J.M. et al.A comparison of calcium, vitamin D, or both for nutritional rickets in Nigerian children.N Engl J Med. 1999; 341: 563-568Crossref PubMed Scopus (247) Google Scholar Interestingly, rats received supraphysiologic doses of vitamin D, and serum vitamin D levels were very high in the 2 groups receiving vitamin D, but the association of these high doses of vitamin D and calcium intake allowed urinary calcium levels similar to those observed in humans affected by kidney stones to be obtained and did not induce hypercalcemia, ruling out the hypothesis of an intoxication to vitamin D. It seems likely that rodents, at least Sprague-Dawley rats, are less sensitive to vitamin D than humans. Finally, we used male rats only to avoid the influence of estrogens in calcium intestinal absorption, and it would be of interest to determine in further studies whether the combination of estrogens and vitamin D influences calcium absorption and thereby urinary calcium levels in this model. In conclusion, the calcium plus vitamin D rat model highlights the synergistic role of calcium intake and vitamin D supplementation in kidney stone formation. Beyond the description of a murine model of calcium phosphate kidney stones, these results raise questions about the widespread prescription of vitamin D supplementation in the general population, especially in addition to calcium supplementation, in the absence of a proven benefit. Sprague-Dawley male rats, 38 weeks old, were purchased from Harlan Laboratories (Gannat, France) and housed for 6 months. All efforts were made to reduce animal suffering. They were housed in similar conditions (2 rats per cage) with a 12-hour dark/light cycle and fed standard rat chow ad libitum. All rats received standard chow containing 600 IU vitamin D3/kg and 0.73% calcium. Twelve rats (control group) had a free access to water containing 80 mg/l calcium. One rat in the control group died of unknown causes at 6 months. Six rats (vitamin D group) received vitamin D (ergocalciferol 100,000 IU/kg, Sterogyl 15H; DB Pharma, La Varenne Saint-Hilaire, France) every 3 weeks during 6 months by i.m. injection and had a free access to water containing 80 mg/l calcium. Six rats (calcium group) had a free access to water containing 2 g/l calcium (calcium gluconate). Six rats (calcium plus vitamin D group) received a vitamin D injection and had a free access to water containing 2 g/l calcium. Calcium and vitamin D doses were chosen to induce hypercalciuria. All animal procedures in the laboratory were performed in accordance with the European Union Guidelines for the Care and Use of Laboratory Animals and with local Institutional Animal Care and Use Committee (Comité d'Éthique en Experimentation Charles Darwin C2EA-05) guidelines. A specific authorization was obtained from health ministry and local ethics committee (number 04557.02). Urine was collected before the first administration of vitamin D and every 3 weeks from week 5 to week 23 before each vitamin D injection. Urine was collected during 24 hours in metabolic cages with free access to water (calcium enriched or not according to the group). Blood (1 ml) was collected before the first injection of vitamin D and at the time that the animals were killed, 10 days after the last injection of vitamin D. The following parameters were measured in urine: diuresis volume, calcium, magnesium, phosphate, creatinine, urea, and oxalate. The blood samples were analyzed for total calcium, phosphate, magnesium, creatinine, urea, and vitamin D (at 6 months). Serum and urinary creatinine and urea levels were analyzed by enzymatic methods and by the Jaffe method on a Konelab 20 analyzer from Thermo Fisher Scientific (Villebon-sur-Yvette, France). Calcium and magnesium serum and urinary levels were measured using the Perkin Elmer 3300 atomic absorption spectrometer (Perkin Elmer, Villebon-sur-Yvette, France). Vitamin D serum levels were measured by the IDS-iSYS 25-hydroxy vitamin D immunoassay. Oxalate urinary levels were measured by ionic chromatography (Dionex ICS-3000, Dionex Corporation, Sunnyvale, CA). Fresh urine was collected after spontaneous voiding to perform crystalluria testing every 3 weeks from week 5 to week 20, before vitamin D injection. The number and type of crystals were analyzed by trained technicians.34Daudon M. Frochot V. Crystalluria.Clin Chem Lab Med. 2015; 53: s1479-s1487PubMed Google Scholar Left kidneys were fixed in formaldehyde, embedded in paraffin, and subjected to X-ray CT imaging at the AST-RX platform of the Museum National d'Histoire Naturelle (Paris, France), using a GE Sensing and Inspection Technologies Phoenix|x-ray v|tome|x L240-180 CT scanner (GE Sensing, Labège, France). We used the nanofocus XR source to obtain a 10-μm resolution scale. Data were reconstructed using datos|x reconstruction software (Phoenix|X-ray, release 2.0) and then exported into a 16-bit Tag Image File Format stack of virtual slices. We used Mimics Innovation suite 16.0 (Materialise, Leuven, Belgium) for the analysis, quantification of calcification volume (stones and tubular calcifications), and 3-dimensional modeling of stones, tubular calcifications, and kidney vessels. The term "stone" refers to concretions from the papilla in the urinary space. The tubular calcification refers to concretions present within the papilla in tubular structures. Randall plaque refers to interstitial calcium phosphate deposits appearing around the loops of Henle or vasa recta in kidney interstitial tissue. Kidney tissues were fixed in acetic formol alcohol and formalin and embedded in paraffin. Tissue sections (4 μm) were performed and stained by the von Kossa and Yasue procedure to reveal calcifications. Tissue sections (4 μm) were studied with a Zeiss SUPRA55-VP Field Emission scanning Electron Microscope (Zeiss France, Marly-le-Roi, France). Measurements were performed at low voltage (1.4 KeV) and without the usual deposits of carbon at the surface of the sample. Microcalcification phases were characterized using μ-Fourier transform infrared spectrometry. Tissue sections (4 μm) were deposited on low-emission microscope slides (MirrIR, Kevley Technologies, Chesterland, OH; Tienta Sciences, Indianapolis, IN). Fourier transform infrared hyperspectral images were recorded with a Spectrum spotlight 400 FT-IR imaging system (Perkin Elmer), with a spatial resolution of 6.25 μm and a spectral resolution of 8 cm–1. The spectra were recorded in the 4000 to 700 cm–1 mid-infrared range. Each spectral image, covering a substantial part of the tissue, consisted of ∼30,000 spectra. Data are expressed as percentage or mean ± SEM. Mann-Whitney U and Fisher exact tests were used to compare the different groups using SAS and Statview software SAS Institute, Cary, NC. The level of significance was set at <0.05. All the authors declared no competing interests. This work was supported by the Agence Nationale de la Recherche (ANR-13-JSV1-0010-01, ANR-12-BS08-0022), the Société de Néphrologie (Genzyme grant), the Académie Nationale de Médecine (Nestlé-Waters award), Convergence-UPMC CVG1205, and CORDDIM-2013-COD130042.