Breast arterial calcification in chronic kidney disease: absence of smooth muscle apoptosis and osteogenic transdifferentiation  W. Charles O'Neill, Amy L. Adams  Kidney International  Volume 85, Issue 3, Pages 668-676 (March 2014) DOI: 10.1038/ki.2013.351 Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 1 Patterns of calcification in breast arteries shown by von Kossa staining. (a) Diffuse punctate staining in the medial layer (patient 17). L indicates lumen. (b) Linear calcification of the internal elastic lamina (IEL) in patient 1. There is no intimal thickening. (c) Both diffuse medial and linear IEL calcification in the same artery (patient 12). Intimal thickening is present. (d) Severe confluent calcification (patient 1). (e) Two adjacent arteries with and without calcification (patient 8). (f) Calcification of small arteries and arterioles (patient 8). Bar=100μm in all images. Kidney International 2014 85, 668-676DOI: (10.1038/ki.2013.351) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 2 Histology of smooth muscle cells in calcified arteries. (a) Hematoxylin and eosin staining of a heavily calcified artery from patient 1 showing normal-appearing smooth muscle cells. (b) Staining for SM22α in the same artery. There was fracturing of the artery during sectioning. (c) Ductal epithelial cells do not stain for SM22α, whereas underlying smooth muscle is positive. (d) Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining of a large, heavily calcified artery from patient 18 demonstrating no apoptosis adjacent to the calcifications (C). There is some staining of cells in the adventitia (arrows) and some artifactual staining of the calcifications. Most of the calcification was lost during sectioning. L indicates the lumen. (e) The von Kossa staining pattern corresponding to the region shown in d. (f) Positive TUNEL staining of neointimal cells. Bars=100μm. Kidney International 2014 85, 668-676DOI: (10.1038/ki.2013.351) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 3 Immunohistochemical analysis of matched sections of small breast arteries from two patients. (a) There is calcification within the media and of the internal elastic lamina (von Kossa staining). (b) All cells within the media stained for SM22α. There was no staining for osteocalcin (c) or Runx2 (d). Kidney International 2014 85, 668-676DOI: (10.1038/ki.2013.351) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 4 Immunohistochemical analysis of matched sections of a large breast artery from patient 12. Left hand column: low magnification; bars=100μm. Right hand column: higher magnification of region indicated by box. (a) The von Kossa staining. (b) SM22α. (c) Osteocalcin. (d) Runx2. Bars=100μm. There is no staining for osteocalcin or Runx2. Kidney International 2014 85, 668-676DOI: (10.1038/ki.2013.351) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 5 Staining for osteogenic/chondrogenic markers. (a) Staining for osteocalcin in patient 17 showing a granular pattern within the media and in the internal elastic lamina. (b) Osteocalcin staining of heavy calcifications in patient 18. The vessel wall has fractured during sectioning. (c) The von Kossa staining of artery from patient 13. (d) Osterix immunostaining of the same artery. (e) The von Kossa staining of artery from patient 12. (f) SOX-9 immunostaining of the same artery. Bars=100μm. Kidney International 2014 85, 668-676DOI: (10.1038/ki.2013.351) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 6 Positive controls for immunohistochemical analysis. (a) Sample of bone showing cellular staining of the osteocytes for osteocalcin. (b) Runx2 staining of placenta. (c) Osterix immunostaining of neonatal mouse spine. (d) SOX-9 immunostaining of neonatal mouse spine. Bars=100μm. Kidney International 2014 85, 668-676DOI: (10.1038/ki.2013.351) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 7 Osteogenic markers in calcified arteries from lower extremities. Arrows indicate the internal elastic lamina with the intima (I) to the right, the media (M) to the left, and the adventitia (A) to the far left. (a) Decalcified section of a tibial artery showing staining for osteocalcin of the medial calcifications and some smooth muscle cells within the calcifications. (b) Section of a tibial artery showing no staining for osteocalcin. The artery fractured at the calcifications during sectioning, with loss of the intima. (c) Decalcified section of a tibial artery showing no staining for Runx2. The intima was lost due to a fracture at the internal elastic lamina during sectioning. (d) Decalcified section of a femoral artery from a patient with chronic kidney disease showing no staining for osterix. Bars=100μm. Kidney International 2014 85, 668-676DOI: (10.1038/ki.2013.351) Copyright © 2014 International Society of Nephrology Terms and Conditions