Uremia-related vascular calcification: More than apatite deposition
2006; Elsevier BV; Volume: 71; Issue: 4 Linguagem: Inglês
10.1038/sj.ki.5002028
ISSN1523-1755
AutoresSteven Verberckmoes, Veerle P. Persy, Geert J. Behets, Ellen Neven, Ann-Sophie Hufkens, Hong Zebger‐Gong, Dominik N. Müller, Dieter Haffner, Uwe Querfeld, Sylvain Bohic, Marc E. De Broe, Patrick C. D’Haese,
Tópico(s)Magnesium in Health and Disease
ResumoIn the present study, we characterized and compared the mineral phase deposited in the aortic wall of two different frequently used chronic renal failure rat models of vascular calcification. Vascular calcification was induced in rats by either a 4-week adenine treatment followed by a 10-week high-phosphate diet or 5/6 nephrectomy followed by 6 weeks of 0.25 μg/kg/day calcitriol treatment and a high-phosphate diet. Multi-element mapping for calcium and phosphate together with mineral identification was performed on several regions of aortic sections by means of synchrotron X-ray-μ-fluorescence and diffraction. Bulk calcium and magnesium content of the aorta was assessed using flame atomic absorption spectrometry. Based on the diffraction data the Von Kossa-positive precipitate in the aortic regions (N=38) could be classified into three groups: (1) amorphous precipitate (absence of any diffraction peak pattern, N=12); (2) apatite (N=16); (3) a combination of apatite and magnesium-containing whitlockite (N=10). The occurrence of these precipitates differed significantly between the two models. Furthermore, the combination of apatite and whitlockite was exclusively found in the calcitriol-treated animals.These data indicate that in adenine/phosphate-induced uremia-related vascular calcification, apatite is the main component of the mineral phase. The presence of magnesium-containing whitlockite found in addition to apatite in the vitamin D-treated rats, has to be seen in view of the well-known vitamin D-stimulated gastrointestinal absorption of magnesium. In the present study, we characterized and compared the mineral phase deposited in the aortic wall of two different frequently used chronic renal failure rat models of vascular calcification. Vascular calcification was induced in rats by either a 4-week adenine treatment followed by a 10-week high-phosphate diet or 5/6 nephrectomy followed by 6 weeks of 0.25 μg/kg/day calcitriol treatment and a high-phosphate diet. Multi-element mapping for calcium and phosphate together with mineral identification was performed on several regions of aortic sections by means of synchrotron X-ray-μ-fluorescence and diffraction. Bulk calcium and magnesium content of the aorta was assessed using flame atomic absorption spectrometry. Based on the diffraction data the Von Kossa-positive precipitate in the aortic regions (N=38) could be classified into three groups: (1) amorphous precipitate (absence of any diffraction peak pattern, N=12); (2) apatite (N=16); (3) a combination of apatite and magnesium-containing whitlockite (N=10). The occurrence of these precipitates differed significantly between the two models. Furthermore, the combination of apatite and whitlockite was exclusively found in the calcitriol-treated animals. These data indicate that in adenine/phosphate-induced uremia-related vascular calcification, apatite is the main component of the mineral phase. The presence of magnesium-containing whitlockite found in addition to apatite in the vitamin D-treated rats, has to be seen in view of the well-known vitamin D-stimulated gastrointestinal absorption of magnesium. Cardiovascular events are responsible for almost 50% of the mortality in hemodialysis patients. In patients with chronic kidney disease, vascular disease is often accompanied by arterial calcifications.1.Foley R.N. Parfrey P.S. Sarnak M.J. Epidemiology of cardiovascular disease in chronic renal disease.J Am Soc Nephrol. 1998; 9: S16-S23Crossref PubMed Scopus (30) Google Scholar Several investigators have shown that coronary artery calcifications are already present in young dialysis patients2.Oh J. Wunsch R. Turzer M. et al.Advanced coronary and carotid arteriopathy in young adults with childhood-onset chronic renal failure.Circulation. 2002; 106: 100-105Crossref PubMed Scopus (611) Google Scholar,3.Eifinger F. Wahn F. Querfeld U. et al.Coronary artery calcifications in children and young adults treated with renal replacement therapy.Nephrol Dial Transplant. 2000; 15: 1892-1894Crossref PubMed Scopus (50) Google Scholar tend to progress rapidly4.Goodman W.G. Goldin J. Kuizon B.D. et al.Coronary-artery calcification in young adults with end-stage renal disease who are undergoing dialysis.N Engl J Med. 2000; 342: 1478-1483Crossref PubMed Scopus (2331) Google Scholar and are associated with decreased survival related to the vascular site containing calcifications.5.London G.M. Pannier B. Guerin A.P. et al.Alterations of left ventricular hypertrophy in and survival of patients receiving hemodialysis: follow-up of an interventional study.J Am Soc Nephrol. 2001; 12: 2759-2767PubMed Google Scholar,6.Blacher J. Guerin A.P. Pannier B. et al.Arterial calcifications, arterial stiffness, and cardiovascular risk in end-stage renal disease.Hypertension. 2001; 38: 938-942Crossref PubMed Scopus (1180) Google Scholar Elevated serum phosphate levels, hyperparathyroidism, and an increased Ca × P product are identified as independent risk factors for coronary artery calcifications.2.Oh J. Wunsch R. Turzer M. et al.Advanced coronary and carotid arteriopathy in young adults with childhood-onset chronic renal failure.Circulation. 2002; 106: 100-105Crossref PubMed Scopus (611) Google Scholar,4.Goodman W.G. Goldin J. Kuizon B.D. et al.Coronary-artery calcification in young adults with end-stage renal disease who are undergoing dialysis.N Engl J Med. 2000; 342: 1478-1483Crossref PubMed Scopus (2331) Google Scholar Treatment of secondary hyperparathyroidism with calcitriol is currently recommended in adult and pediatric patients with chronic kidney disease.7.Eknoyan G. Levin A. Levin N.W. K/DOQI clinical practice guidelines for bone metabolism and disease in chronic kidney disease – foreword.Am J Kidney Dis. 2003; 42: S7-S201Abstract Full Text PDF Google Scholar,8.Klaus G. Watson A. Edefonti A. et al.Prevention and treatment of renal osteodystrophy in children on chronic renal failure: European guidelines.Pediatr Nephrol. 2006; 21: 151-159Crossref PubMed Scopus (139) Google Scholar However, the suppressive effect of calcitriol on parathyroid hormone release goes along with an increased calcium/phosphorus gastrointestinal absorption, favoring ectopic calcification. Vascular calcification is a tightly regulated process driven by specific cellular events9.Reynolds J.L. Joannides A.J. Skepper J.N. et al.Human vascular smooth muscle cells undergo vesicle-mediated calcification in response to changes in extracellular calcium and phosphate concentrations: a potential mechanism for accelerated vascular calcification in ESRD.J Am Soc Nephrol. 2004; 15: 2857-2867Crossref PubMed Scopus (689) Google Scholar,10.Chen N.X. O'Neill K.D. Duan D. et al.Phosphorus and uremic serum up-regulate osteopontin expression in vascular smooth muscle cells.Kidney Int. 2002; 62: 1724-1731Abstract Full Text Full Text PDF PubMed Scopus (286) Google Scholar and particular components present in the serum.11.Ketteler M. Gross M.L. Ritz E. Calcification and cardiovascular problems in renal failure.Kidney Int. 2005; 67: S120-S127Abstract Full Text Full Text PDF Google Scholar Transdifferentiation of vascular smooth muscle cells towards cells with an osteogenic phenotype, expressing the major bone-specific proteins, is a key event in the process of medial calcification.12.Yang H. Curinga G. Giachelli C.M. Elevated extracellular calcium levels induce smooth muscle cell matrix mineralization in vitro.Kidney Int. 2004; 66: 2293-2299Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar Based on these observations it is generally assumed that the mineral deposited in the vascular wall has the physicochemical properties of hydroxyapatite, the mineral compound of bone. However, owing to the limited availability of appropriate techniques data on the identity and ultrastructural composition of the mineral phase deposited during vascular calcification are scarce.13.Lomashvili K.A. Cobbs S. Hennigar R.A. et al.Phosphate-induced vascular calcification: role of pyrophosphate and osteopontin.J Am Soc Nephrol. 2004; 15: 1392-1401Crossref PubMed Scopus (239) Google Scholar,14.LeGeros R.Z. Formation and transformation of calcium phosphates: relevance to vascular calcification.Z Kardiol. 2001; 90: 116-124PubMed Google Scholar By the application of synchrotron radiation based X-ray μ-fluorescence in combination with μ-diffraction analysis, we studied the ultrastructural spatial composition of the mineral deposited in the vessel wall of calcitriol- and non-calcitriol-treated models of uremia-induced vascular calcification. Both rat models, that is, the combination of (i) adenine treatment with a high-phosphate diet and (ii) 5/6 nephrectomy, a high-phosphate diet, and oral dosing of calcitriol resulted in mineral deposition in the vascular wall, as could be visualized by Von Kossa staining (data not shown).15.Haffner D. Hocher B. Muller D. et al.Systemic cardiovascular disease in uremic rats induced by 1,25(OH)2D3.J Hypertens. 2005; 23: 1067-1075Crossref PubMed Scopus (94) Google Scholar,16.Persy V.P. Postnov A. Neven E. et al.Detection of vascular calcifications by high resolution X-ray microtomography in living rats with chronic renal failure.Arterioscler Thromb Vasc Biol. 2006; 26: 2110-2116Crossref PubMed Scopus (27) Google Scholar X-ray-μ-fluorescence analysis was performed on aorta samples obtained from rats of both models with a positive Von Kossa staining. In all investigated microscopic aortic regions of interest (N=38) a positive X-ray fluorescence signal for phosphorus and calcium was observed (Figure 1b and c). By recording the X-ray diffraction patterns of several calcium- and phosphorus-positive spots in the investigated aortic regions three different types of mineral were identified (Table 1): (i) amorphous calcium phosphate precipitate in the absence of any diffraction peak pattern (N=12), or (ii) poorly crystalline apatite (microcrystalline or non-stoechiometric apatite) (N=16), or (iii) a combination of apatite and whitlockite (N=10) (Figure 1d). The occurrence of the various types significantly (P<0.05) differed between the two models and the combination of apatite and whitlockite mineral was exclusively found in vitamin D-treated animals (Table 1).Table 1Distribution of the different mineral phases in the investigated aortic regionsAmorphous precipitateApatiteApatite/whitlockiteAdenine model170Calcitriol-treated remnant kidney model11910χ2 analysis revealed significant proportional differences (P=0.012) in the occurrence of the mineral phases between the two rat models. Open table in a new tab χ2 analysis revealed significant proportional differences (P=0.012) in the occurrence of the mineral phases between the two rat models. The spatial heterogeneity of the apatite and apatite/whitlockite mineral deposition in the aorta was further investigated by performing 10 μm step size line scans through the vascular wall. At each point, the calcium fluorescence (Figure 2, upper panels) and diffraction pattern (Figure 2, lower panels) were recorded simultaneously. In all samples positive for poorly crystalline apatite or a combination of apatite and whitlockite, amorphous precipitate was found at the edges of the calcified region (upper panels of Figure 2a and b, shaded zone) as indicated by a positive calcium signal in the absence of a diffraction pattern (Figure 1d). Moreover, in samples where the mineral was identified as a mixture of whitlockite and apatite, the fluorescence/diffraction line scan analysis showed areas positive (Figure 2b, left diffraction pattern) and negative (Figure 2b, right diffraction pattern) for the presence of whitlockite indicating a heterogenic distribution of this mineral phase in the vessel wall of calcitriol-treated animals. As the formation of whitlockite has been associated with the presence of magnesium,17.Hamad M. Heughebaert J. The growth of whitlockite.J Crystal Growth. 1986; 79: 192-197Crossref Scopus (33) Google Scholar,18.Lagier R. Baud C.A. Magnesium whitlockite, a calcium phosphatecrystal of special interest in pathology.Pathol Res Pract. 2003; 199: 329-335Crossref PubMed Scopus (107) Google Scholar serum and mineralized aortas of both models were analyzed for their calcium and magnesium content. No difference in serum magnesium concentration was found (0.037±0.019 vs 0.032±0.019 μg/ml magnesium in adenine treated vs calcitriol treated 5/6 nephrectomized rats, respectively). In contrast, the calcified areas of the aorta obtained from subtotally nephrectomized animals loaded with calcitriol showed a significantly (P<0.001) higher magnesium/calcium ratio compared to rats with adenine-induced renal failure receiving a high-phosphate diet (median: 0.63, range: 0.12–1.28 (mean±s.d.: 0.48±0.23) vs median: 0.22, range: 0.09–0.69 (mean±s.d.: 0.32±0.18); Figure 3). Recent in vitro studies have shown that vascular calcification is a tightly regulated process in which the vascular smooth muscle cell undergoes a trans-differentiation towards an osteogenic phenotype.12.Yang H. Curinga G. Giachelli C.M. Elevated extracellular calcium levels induce smooth muscle cell matrix mineralization in vitro.Kidney Int. 2004; 66: 2293-2299Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar In vitro data further suggest that the mineral compound present in the vascular wall is microcrystalline apatite,9.Reynolds J.L. Joannides A.J. Skepper J.N. et al.Human vascular smooth muscle cells undergo vesicle-mediated calcification in response to changes in extracellular calcium and phosphate concentrations: a potential mechanism for accelerated vascular calcification in ESRD.J Am Soc Nephrol. 2004; 15: 2857-2867Crossref PubMed Scopus (689) Google Scholar, 13.Lomashvili K.A. Cobbs S. Hennigar R.A. et al.Phosphate-induced vascular calcification: role of pyrophosphate and osteopontin.J Am Soc Nephrol. 2004; 15: 1392-1401Crossref PubMed Scopus (239) Google Scholar, 19.Moe S.M. Duan D. Doehle B.P. et al.Uremia induces the osteoblast differentiation factor Cbfa1 in human blood vessels.Kidney Int. 2003; 63: 1003-1011Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar as it naturally occurs in bone. Indeed, the presence of hydroxyapatite in the aorta of uremic rats was suggested as early as 1979 by the application of X-ray fluorescence micro analysis.20.Ejerblad S. Ericsson J.L. Ultrastructure of the aorta in experimental uraemia.Acta Chir Scand. 1979; 145: 331-343PubMed Google Scholar This technique, however, only provides information concerning the elemental composition, and does not allow the identification of the mineral phase. In an autopsy study of patients with end-stage renal disease, hydroxyapatite and calcium phosphate were found in calcified atherosclerotic plaques by the application of X-ray diffraction.21.Schwarz U. Buzello M. Ritz E. et al.Morphology of coronary atherosclerotic lesions in patients with end-stage renal failure.Nephrol Dial Transplant. 2000; 15: 218-223Crossref PubMed Scopus (509) Google Scholar Although these observations20.Ejerblad S. Ericsson J.L. Ultrastructure of the aorta in experimental uraemia.Acta Chir Scand. 1979; 145: 331-343PubMed Google Scholar,22.Massy Z.A. Ivanovski O. Nguyen-Khoa T. et al.Uremia accelerates both atherosclerosis and arterial calcification in apolipoprotein E knockout mice.J Am Soc Nephrol. 2005; 16: 109-116Crossref PubMed Scopus (163) Google Scholar have provided valuable information on the nature of severe atherosclerotic lesions, much less is known of the physicochemical properties of mineral deposits in the vascular media in uremia. Such information might further contribute to the unraveling of the mechanisms leading to the formation/possible reversal of vascular calcification in renal disease. In the present study, two frequently used models of uremia-related vascular calcification were compared at the level of deposited mineral. Interestingly, between the investigated models a significantly difference in distribution of mineral phases (both amorphous calcium phosphate and whitlockite) was found. Calcium phosphate precipitating from a supersaturated solution in vitro is initially an amorphous compound which is converted into the thermodynamically more stable microcrystalline apatite form.17.Hamad M. Heughebaert J. The growth of whitlockite.J Crystal Growth. 1986; 79: 192-197Crossref Scopus (33) Google Scholar This process occurs also in biological mineralization such as in bone formation.23.Rey C. Hina A. Tofighi A. et al.Maturation of poorly crystalline apatites: chemical and structural aspects in vivo and in vitro.Cells Mater. 1995; 5: 345-356Google Scholar,24.Rey C. Shimizu M. Collins B. et al.Resolution-enhanced Fourier transform infrared spectroscopy study of the environment of phosphate ion in the early deposits of a solid phase of calcium phosphate in bone and enamel and their evolution with age: 2. Investigations in the nu3PO4 domain.Calcif Tissue Int. 1991; 49: 383-388Crossref PubMed Scopus (253) Google Scholar Hence, in the frame of the current study it is not surprising that both amorphous calcium phosphate and poorly crystalline apatite were present in the calcified aorta of uremic rats exposed to either calcitriol and/or a high-phosphate diet. The application of synchrotron X-ray-μ-diffraction with an X-ray probe size of 2 × 10 μm enabled us to study the spatial heterogeneity of the mineral phase in the vascular media at the ultrastructural micrometer-scale level. This evaluation showed the presence of amorphous calcium phosphate at the edges of the mineral phases. This observation fits well with previous findings in that the amorphous calcium phosphate precipitate is an early deposit which matures over time under the formation of calcium apatite.17.Hamad M. Heughebaert J. The growth of whitlockite.J Crystal Growth. 1986; 79: 192-197Crossref Scopus (33) Google Scholar Moreover, as amorphous calcium phosphate is a labile precipitate its presence suggests that the mineral deposition in vascular calcification may be reversible at these sites. Surprisingly, in 1/3 of the aortic regions of animals exposed to calcitriol, the mineral phase consisted not only of apatite but contained also whitlockite, which is a calcium-magnesium orthophosphate, that is, (Ca,Mg)3(PO4)2.18.Lagier R. Baud C.A. Magnesium whitlockite, a calcium phosphatecrystal of special interest in pathology.Pathol Res Pract. 2003; 199: 329-335Crossref PubMed Scopus (107) Google Scholar,17.Hamad M. Heughebaert J. The growth of whitlockite.J Crystal Growth. 1986; 79: 192-197Crossref Scopus (33) Google Scholar Already at low concentrations, magnesium ions have a marked effect on nucleation and growth of calcium phosphates. These ions delay the conversion of amorphous calcium precipitates to the more stable apatite phase and promote the formation of whitlockite.17.Hamad M. Heughebaert J. The growth of whitlockite.J Crystal Growth. 1986; 79: 192-197Crossref Scopus (33) Google Scholar, 18.Lagier R. Baud C.A. Magnesium whitlockite, a calcium phosphatecrystal of special interest in pathology.Pathol Res Pract. 2003; 199: 329-335Crossref PubMed Scopus (107) Google Scholar, 25.Ennever J. Vogel J.J. Magnesium inhibition of apatite nucleation by proteolipid.J Dent Res. 1981; 60: 838-841Crossref PubMed Scopus (18) Google Scholar As (i) X-ray diffraction only offers information on mineral lattice characteristics, but not of the mineral composition, and (ii) the applied X-ray-μ-fluorescence technique was not suitable to detect chemical elements with low atomic number, such as magnesium, flame atomic absorption spectroscopy was used to determine the bulk magnesium content in calcified aortas of both models of vascular calcification. The presence of a significantly higher magnesium/calcium ratio in the aortas of calcitriol-treated animals vs the adenine-treated rats strongly supports the presence of whitlockite. These new findings are in line with previous observations showing that calcitriol supplementation not only stimulates calcium and phosphorus uptake but also favors the gastrointestinal absorption of magnesium.26.Hardwick L.L. Jones M.R. Brautbar N. et al.Magnesium absorption: mechanisms and the influence of vitamin D, calcium and phosphate.J Nutr. 1991; 121: 13-23PubMed Google Scholar,27.Schweigel M. Martens H. Magnesium transport in the gastrointestinal tract.Front Biosci. 2000; 5: D666-D677Crossref PubMed Google Scholar As under normal physiological conditions the kidney plays a central role in the metabolism of magnesium it is not surprising that magnesium accumulation occurs in the presence of renal failure.28.Lindeman R.D. Chronic renal failure and magnesium metabolism.Magnesium. 1986; 5: 293-300PubMed Google Scholar The observed difference in mineral composition between the two investigated models of uremia-related vascular calcification cannot be ascribed to the methodology applied inducing chronic renal failure as the degree of renal impairment and other biochemical parameters (Ca × P product and parathyroid hormone) were comparable. The only significant difference between both models was the duration of uremia, with a three weeks longer period in renal insufficiency in the adenine-treated rats. This has the consequence that the deposited precipitate in the adenine-treated rats had a longer maturation time. This, together with the possible effects of magnesium delaying the formation of stable apatite in the calcitriol-treated animals can explain the difference in distribution of the amorphous phase between both models. Recently, a link between the status of bone metabolism and vascular calcification has been made.29.Moe S.M. Vascular calcification and renal osteodystrophy relationship in chronic kidney disease.Eur J Clin Invest. 2006; 36: 51-62Crossref PubMed Scopus (106) Google Scholar In this context it has to be noted that it has been reported that adenine treatment not only induces chronic renal failure but also affects steroidogenesis and reduces bone metabolism resulting in a reduced bone mineral density.30.Ogirima T. Tano K. Kanehara M. et al.Sex difference of adenine effects in rats: renal function, bone mineral density and sex steroidogenesis.Endocr J. 2006; 53: 407-413Crossref PubMed Scopus (11) Google Scholar Although the male gender and a reduced bone mineral density have been considered as risk factors for the development of vascular calcification, such an effect can be excluded as histomorphometric analysis of bone samples of the adenine-treated animals did not show a reduced bone area or signs of a reduced bone mineral density. Instead a histological picture of secondary hyperparathyroidism was found (Persy et al. SA-PoO950, ASN, Renal Week 2004). Because of the high-phosphate diet in combination with the severe renal failure, the vitamin D-treated animals also showed the histological features of secondary hyperparathyroidism. The physiopathological role and clinical importance of whitlockite is much less studied compared to apatite and its relevance may have been largely underestimated as the classical Von Kossa staining for calcified tissues does not allow to differentiate between whitlockite and apatite. Techniques such as X-ray or electron diffraction and infrared spectrometry that allow discrimination between mineral phases are not routinely applied on biological/pathological samples. To the best of our knowledge only one publication is available in literature that mentions the presence of whitlockite in the vascular media of autopsy samples of subjects of the general population.31.Reid J.D. Andersen M.E. Medial calcification (whitlockite) in the aorta.Atherosclerosis. 1993; 101: 213-224Abstract Full Text PDF PubMed Scopus (55) Google Scholar In contrast with our findings, however, in the latter paper no microanalysis was performed, nor was the heterogeneity of the mineral phase investigated. Moreover, the occurrence of whitlockite in arterial calcifications in uremic patients and its association with calcitriol treatment has not been described previously. Our results indicate that the process of uremia-related vascular calcification is not solely owing to the deposition of calcium-apatite and that a role for magnesium in the pathogenesis of uremia-related calcification should be considered – especially during calcitriol treatment. This study was performed as an extension of two previous studies on either the effect of adenine treatment and a high-phosphate diet16.Persy V.P. Postnov A. Neven E. et al.Detection of vascular calcifications by high resolution X-ray microtomography in living rats with chronic renal failure.Arterioscler Thromb Vasc Biol. 2006; 26: 2110-2116Crossref PubMed Scopus (27) Google Scholar or a 5/6 nephrectomy and calcitriol-treatment15.Haffner D. Hocher B. Muller D. et al.Systemic cardiovascular disease in uremic rats induced by 1,25(OH)2D3.J Hypertens. 2005; 23: 1067-1075Crossref PubMed Scopus (94) Google Scholar on the induction of vascular calcification. Medical vascular calcifications were induced in two models of chronic renal failure. Chronic renal failure was induced by feeding male Wistar rats a diet containing 0.75%. adenine and 0.92% phosphorus for 4 weeks, which has been shown to induce a stable, moderate to severe renal function impairment.32.Okada H. Kaneko Y. Yawata T. et al.Reversibility of adenine-induced renal failure in rats.Clin Exp Immunol. 2005; 3: 82-88Google Scholar Following adenine treatment the animals (N=13) were fed a diet containing 1.06% calcium and 1.03% phosphorus during 6 weeks until killing.16.Persy V.P. Postnov A. Neven E. et al.Detection of vascular calcifications by high resolution X-ray microtomography in living rats with chronic renal failure.Arterioscler Thromb Vasc Biol. 2006; 26: 2110-2116Crossref PubMed Scopus (27) Google Scholar Male Sprague–Dawley rats underwent a 5/6 nephrectomy to induce chronic renal failure. Two days after surgery (day 0), the animals received 0.25 μg/kg. of 1,25(OH)2D3 (Rocaltrol™, Roche, Mannheim, Germany) postoperatively daily for 6 weeks. Animals (N=12) received a standard commercial diet containing 1.2% phosphorus, 0.9% calcium (Altromin™, Altromin Co., Lage, Germany).15.Haffner D. Hocher B. Muller D. et al.Systemic cardiovascular disease in uremic rats induced by 1,25(OH)2D3.J Hypertens. 2005; 23: 1067-1075Crossref PubMed Scopus (94) Google Scholar Through a combined midline laparotomy and thoracotomy the vascular tree was exposed and the large vessels were removed 'en bloc'. The aorta was divided in different pieces; one piece was used for general histology after paraffin embedding, another was embedded with methyl methacrylate for mineral identification, whereas a third part was used for bulk analysis. Methyl methacrylate embedding of the aorta samples was performed using a standard procedure as reported elsewhere.33.Behets G.J. Dams G. Damment S. et al.An assessment of the effects of lanthanum on bone in a chronic renal failure (CRF) rat model.J Am Soc Nephrol. 2001; 12: 740AGoogle Scholar A 10-μm thick methyl methacrylate-embedded aorta section was used for evaluation of the mineral deposition in the vessel wall by Von Kossa staining and counterstaining with hematoxylin and eosin. Another 10 μm section (adjacent to the Von Kossa-stained section) was dried unstained between two glass cover slides at 60°C, and used for synchrotron X-ray-μ-fluorescence and X-ray-μ-diffraction analysis. Unfixed tissue samples were weighed with a precision balance and subsequently digested in 65% nitric acid at 65°C overnight. Serum and tissue calcium and magnesium content was measured by flame atomic absorption spectrometry (Model 3110, Perkin-Elmer, Norwalk, CT, USA) after appropriate further dilution of the samples in 0.1% lanthanum nitrate to avoid chemical interferences. Unstained 10-μm thick methyl methacrylate-embedded aorta sections adjacent to those stained positive for Von Kossa were investigated by X-ray-μ-analysis. Several regions of interest were defined on each sample which were investigated by synchrotron X-ray-μ-fluorescence and X-ray-μ-diffraction on beam line ID18F of the European Synchrotron Radiation Facility (Grenoble, France)34.Vekemans B. Vincze L. Somogyi A. et al.Quantitative X-ray fluorescence analysis at the ESRF ID18F microprobe.Nuclear Instruments & Methods in Physics Research Section B-Beam Interactions with Materials and Atoms. 2003; 199: 369-401Crossref Scopus (19) Google Scholar applying a same beamline set-up as published before.35.Verberckmoes S.C. Behets G.J. Oste L. et al.Effects of strontium on the physicochemical characteristics of hydroxyapatite.Calcif Tissue Int. 2004; 75: 405-415Crossref PubMed Scopus (77) Google Scholar The optimum step-size for either raster or line scans is determined by the size of the focused beam (2 × 10 μm) and was set to 10 μm in both directions (horizontally and vertically). Calcium and phosphorus mapping were obtained by integrating the fluorescence intensities defined by the appropriate energy windows for the respective element (calcium: 3.47–4.24 keV and phosphorus: 1.81–2.13 keV) during a recording time of 1 s. Integration of the obtained circular X-ray-μ-diffraction patterns to d-spacing format (d=interplanar or interatomic distance) was carried out using the X-ray diffraction software package FIT2D developed by Hammersley et al.36.Hammersley A.P. Svensson S.O. Hanfland M. et al.Two-dimensional detector software: from real detector to idealised image or two-theta scan.High Press Res. 1996; 14: 235-248Crossref Google Scholar Comparison with the reference diffraction spectra for synthetic hydroxyapatite (Ca10(PO4)6(OH)2: PDF #86-0740) and whitlockite ((Ca,Mg)3(PO4)2: PDF #70-2064) from the powder diffraction database PCPDFWIN version 2.1 (International Centre of Diffraction Data, USA) was performed after background correction of the integrated X-ray diffraction spectra. Results of the bulk analysis of the magnesium/calcium ratio in the rat aorta were analyzed for significant differences using the non-parametric Mann–Whitney test. Comparison of the prevalence of the various mineral phases in the two models was carried out by χ2 analysis. P-values <0.05 (two tailed) were considered as significant. We acknowledge the European Synchrotron Radiation Facility for provision of synchrotron radiation facilities and for assistance in using beamline ID18F.
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