Osteopontin protects against high phosphate-induced nephrocalcinosis and vascular calcification  Neil J. Paloian, Elizabeth M. Leaf, Cecilia M. Giachelli  Kidney International  Volume 89, Issue 5, Pages 1027-1036 (May 2016) DOI: 10.1016/j.kint.2015.12.046 Copyright © 2016 International Society of Nephrology Terms and Conditions

Figure 1 Loss of normal renal structure in the KO+HP group. Hematoxylin and eosin (H&E) staining of the (a) wild-type (WT) + normal phosphate diet (NP), (b) osteopontin knockout (KO) + NP, and (c) WT + high phosphate diet (HP) kidneys are normal. H&E staining of the kidneys of the (d) KO+HP group demonstrate a marked cellular infiltrate (arrows) surrounding both dilated tubular structures and mineral deposits (arrowhead). Periodic acid–Schiff (PAS) staining of the (e) WT+NP and (f) KO+NP kidneys is normal. (g) Occasional tubular mineral deposits are seen on PAS staining of WT+HP kidneys (arrowhead) with otherwise normal morphology. (h) PAS staining of KO+HP kidneys demonstrates cystic glomeruli (arrows) with marked dilatation of Bowman’s capsule. Dilated tubules are also seen. CD45 immunofluorescence of the (i) WT+NP and (j) KO+NP indicates the presence of occasional inflammatory cells. The WT+HP group shows mild subcapsular and interstitial inflammation. (k) In the KO+HP kidney (l) a significant inflammatory infiltrate is present near cystic, dilated glomeruli and calcification sites (orange in figure). Original magnification ×20. Bar = 75 μm. Kidney International 2016 89, 1027-1036DOI: (10.1016/j.kint.2015.12.046) Copyright © 2016 International Society of Nephrology Terms and Conditions

Figure 2 OPN KO mice on HP diet develop abundant renal calcium phosphate deposition. (i) Von Kossa (VK) staining confirms presence of calcium phosphate deposition throughout the kidney of osteopontin (OPN) (b) knockout (KO) + high phosphate diet (HP) mice. Occasional renal mineral deposition is seen in the (a) wild-type (WT) + HP mice. Original magnification ×10. Bar = 150 μm. (ii) VK scoring for mineral granules demonstrates a marked increase in renal calcium deposits in the KO+HP group when compared with the WT+HP group. 0 = no granules found in the section; 1 = 1 to 25 granules; 2 = 26 to 75 granules; 3 = 76 to 150 granules; 4 = >150 granules. Kidney International 2016 89, 1027-1036DOI: (10.1016/j.kint.2015.12.046) Copyright © 2016 International Society of Nephrology Terms and Conditions

Figure 3 OPN immunohistochemistry confirms depletion of OPN protein in KO kidney. Kidney sections of (a) wild-type (WT) + normal phosphate diet (NP) and (c) WT + high phosphate diet (HP) demonstrate abundant renal tubular osteopontin (OPN) expression. OPN expression is absent in the (b) knockout (KO) + NP and (d) KO + high phosphate diet (HP) kidneys. Original magnification ×40. Bar = 30 μm. G, glomerulus; T, tubule. Kidney International 2016 89, 1027-1036DOI: (10.1016/j.kint.2015.12.046) Copyright © 2016 International Society of Nephrology Terms and Conditions

Figure 4 KO+HP mice develop vascular calcification. (i) Aortic arch plus lower abdominal aorta biochemical calcium content expressed as micrograms of calcium normalized to milligrams dry weight (mean ±SEM). *P < 0.05 compared with the wild-type (WT) + normal phosphate diet (NP), knockout (KO) + NP, and WT + high phosphate diet (HP). Calcification was not different among the WT+NP, KO+NP, and WT+HP groups. (ii) Alizarin red S stains of the abdominal aorta show (b) marked calcification in the KO+HP group and (a) absence of mineral in the WT+HP group. Original magnification ×4 (cropped). Bar = 350 μm. (c) Magnification of the mineral (dashed box in b) in the KO+HP aorta shows that the mineral is located entirely in the medial layer of the vessel. Original magnification ×40. Bar = 30 μm. L, lumen. Kidney International 2016 89, 1027-1036DOI: (10.1016/j.kint.2015.12.046) Copyright © 2016 International Society of Nephrology Terms and Conditions

Figure 5 Loss of SM22α expression in the KO+HP aorta confirms phenotypic transformation of VSMCs. SM22α expression is normal throughout the length of the abdominal aorta in the (a) wild-type (WT) + normal phosphate diet (NP), (b) knockout (KO) + NP, and (c) WT + high phosphate diet (HP) groups. (d) SM22α expression is absent in areas of the aorta (arrows) in the KO+HP mice, which is consistent with phenotypic transformation of the vascular smooth muscle cells (VSMCs) seen in vascular calcification. Original magnification ×40. Bar = 30 μm. L, lumen. Kidney International 2016 89, 1027-1036DOI: (10.1016/j.kint.2015.12.046) Copyright © 2016 International Society of Nephrology Terms and Conditions

Figure 6 OPN expression is absent in the calcified vasculature of the KO+HP group. (a) Osteopontin (OPN) immunohistochemistry of the abdominal aorta of the wild-type (WT) + high phosphate diet (HP) mice demonstrates no OPN expression in the vessel as expected in the absence of calcification. (b) Despite the presence of a mineral (arrow), the knockout (KO) + HP aorta is devoid of OPN. (c) An adjacent section of the KO+HP aorta is stained with alizarin red S to highlight the mineral. Original magnification ×40. Bar = 30 μm. L, lumen. Kidney International 2016 89, 1027-1036DOI: (10.1016/j.kint.2015.12.046) Copyright © 2016 International Society of Nephrology Terms and Conditions

Figure 7 FGF-23 expression is increased in OPN+HP femurs. (i) Fibroblast growth factor-23 (FGF-23) expression is elevated in femurs of wild-type (WT) + high phosphate diet (HP), but expression is highest in knockout (KO) + HP. (ii) Hematoxylin and eosin (H&E) staining of WT+NP and KO+NP demonstrates normal architecture (a,b). Widening of the diaphysis and increased cortical porosity (c,d, arrows) is seen in both HP-fed mice. (d) Large protrusions of trabeculae (arrowheads) are noted exclusively in the KO+HP group. Femoral FGF-23 staining in the WT+NP (e) and KO+NP (f) are unremarkable. There is increased FGF-23 detected in the WT+HP femur (g) but markedly more noted in the KO+HP femur (h). There is more new bone forming in the KO+HP group, and therefore more osteocytes expressing FGF-23 per bone area as seen in the immunofluorescence. H&E: original magnification ×4. Bar = 350 μM. FGF-23 immunofluorescence: original magnification ×20. Bar = 75 μM. NP, normal phosphate diet; OPN, osteopontin. Kidney International 2016 89, 1027-1036DOI: (10.1016/j.kint.2015.12.046) Copyright © 2016 International Society of Nephrology Terms and Conditions