Volume 75, Issue 12, Pages 1297-1307 (June 2009) Phosphate feeding induces arterial medial calcification in uremic mice: role of serum phosphorus, fibroblast growth factor-23, and osteopontin  Mohga M. El-Abbadi, Ashwini S. Pai, Elizabeth M. Leaf, Hsueh-Ying Yang, Bryan A. Bartley, Krystle K. Quan, Carly M. Ingalls, Hung Wei Liao, Cecilia M. Giachelli  Kidney International  Volume 75, Issue 12, Pages 1297-1307 (June 2009) DOI: 10.1038/ki.2009.83 Copyright © 2009 International Society of Nephrology Terms and Conditions

Figure 1 Aortic calcium content in severely uremic mice and sham controls under normal and high-phosphate feeding conditions. Dotted lines indicate mean values for each group. (P<0.05, analysis of variance); Sham/NP, n=5; Sham/HP, n=6; SU/NP, n=4; SU/HP, n=7. HP, high phosphate; NP, normal phosphate; SU, severe uremia. Kidney International 2009 75, 1297-1307DOI: (10.1038/ki.2009.83) Copyright © 2009 International Society of Nephrology Terms and Conditions

Figure 2 Histological analyses of AMC in uremic mouse arteries. (a) Alizarin red staining of vascular tree whole mount. B-M, Representative micrographs of alizarin red–stained paraffin sections of arteries in the different study groups; (b) abdominal aorta; (c) carotid; (d) iliac; and (e), mesenteric artery in uremic, high phosphate–fed mice (scale bar=100 μm for (b–d), and 50 μm for (e)). Extensive mineralization exclusively in the tunica media was observed in the SU/HP group (f, aortic arch and g, aortic arch; scale bar=30 μm, aortic arch). Early mineralization foci were often observed in the elastic lamella (h, aortic arch) and in the extracellular matrix between lamellae (i, aortic arch) in the MU/HP group (scale bar=30 μm). No arterial calcification was observed in the aorta of mice from the Sham/NP group (j), Sham/HP group (k), SU/NP group (l), or MU/NP group (m) (scale bar=25 μm). L=lumen. HP, high phosphate; Mu, moderate uremia; NP, normal phosphate; SU, severe uremia. Kidney International 2009 75, 1297-1307DOI: (10.1038/ki.2009.83) Copyright © 2009 International Society of Nephrology Terms and Conditions

Figure 3 Serum phosphorus, calcium, and calcium × phosphorus in severely uremic mice. Serum phosphorus (a), calcium (b), and calcium × phosphorus (c) linear regression between serum phosphorus and aortic calcium (d) in severely uremic mice and sham controls under normal and high phosphate-feeding conditions. Data represent the mean±s.e.m. Statistically significant differences (P<0.05) were observed between groups with same alphabetical notations; Sham/NP, n=5; Sham/HP, n=6; SU/NP, n=6; SU/HP, n=13. Kidney International 2009 75, 1297-1307DOI: (10.1038/ki.2009.83) Copyright © 2009 International Society of Nephrology Terms and Conditions

Figure 4 Aortic calcium content in moderately uremic mice and sham controls under normal and high phosphate–feeding conditions. Dotted lines indicate mean values for each group. (P<0.05, analysis of variance); Sham/NP, n=7; Sham/HP, n=7; MU/NP, n=10; MU/HP, n=10. Kidney International 2009 75, 1297-1307DOI: (10.1038/ki.2009.83) Copyright © 2009 International Society of Nephrology Terms and Conditions

Figure 5 Serum phosphorus, calcium, and calcium X phosphorus in moderately uremic mice. Serum phosphorus (a), calcium (b), and calcium × phosphorus (c) linear regression between serum phosphorus and aortic calcium (d) in moderately uremic mice and sham controls under normal and high phosphate–feeding conditions. Data represent the mean±s.e.m. Statistically significant differences (P<0.05) were observed between groups with same alphabetical notations; Sham/NP, n=7; Sham/HP, n=7; SU/NP, n=10; SU/HP, n=10. Kidney International 2009 75, 1297-1307DOI: (10.1038/ki.2009.83) Copyright © 2009 International Society of Nephrology Terms and Conditions

Figure 6 Serum FGF-23 and osteopontin levels in moderately uremic mice. Serum FGF-23 (a); correlation between serum FGF-23 and aortic calcium (b); serum osteopontin (OPN) (c); correlation between serum OPN and aortic calcium (d); correlation between serum OPN and FGF-23 (e) levels in moderately uremic mice and sham controls under normal and high phosphate-feeding conditions. Data represent the mean±s.e.m. Statistically significant differences (P<0.05) were observed between groups with same alphabetical notations; Sham/NP, n=7; Sham/HP, n=7; SU/NP, n=6; SU/HP, n=6. Kidney International 2009 75, 1297-1307DOI: (10.1038/ki.2009.83) Copyright © 2009 International Society of Nephrology Terms and Conditions

Figure 7 Histochemical staining of mildly calcified and non-calcified arteries. (a–d) are adjacent sections capturing an early mineralization focus in the aorta of a uremic, high phosphate–fed mouse. (a) alizarin red–positive foci of elastin calcification (arrowhead); (b) osteopontin staining (brown) was detected both in SMC surrounding the calcification focus (methyl green counterstained nuclei, arrows) as well as along the calcified elastic lamina (arrowhead); (c) SM22α staining (brown) was absent or greatly reduced in the SMC (methyl green counterstained nuclei) near the calcified focus. (d) Hemotoxylin and eosin (H&E)–stained adjacent section indicates intact SMC in the calcified areas e–h are adjacent aortic sections of an U/NP mouse showing (e), the absence of alizarin red–positive staining; (f) lack of osteopontin staining (brown); (g) abundant SM22α staining (brown) in the medial SMC; (h), normal cellular and matrix morphology by H&E staining; (i) and (j), Runx2/cbfa1-positive nuclear staining (brown) in the aorta of an U/HP mouse (i) or Sham/NP mouse (j). Scale bar 30 μm. Kidney International 2009 75, 1297-1307DOI: (10.1038/ki.2009.83) Copyright © 2009 International Society of Nephrology Terms and Conditions

Figure 8 Immunochemical staining of a severely calcified artery showing evidence of SMC loss. (a) H&E staining (scale bar=200 μm), boxed area is shown at higher magnification in (b–d); (b) Alizarin red staining (scale bar=30 μm); (c) H&E staining (scale bar=30 μm); (d) SM22α immunostaining (brown) with methyl green nuclear counterstaining (scale bar=30 μm). Note the absence of both SM22α staining as well as cell nuclei in the calcified area (dashed rectangle). Kidney International 2009 75, 1297-1307DOI: (10.1038/ki.2009.83) Copyright © 2009 International Society of Nephrology Terms and Conditions