Volume 89, Issue 3, Pages 601-611 (March 2016) Vitamin K antagonism aggravates chronic kidney disease–induced neointimal hyperplasia and calcification in arterialized veins: role of vitamin K treatment?  Emma Zaragatski, Jochen Grommes, Leon J. Schurgers, Stephan Langer, Lieven Kennes, Miriam Tamm, Thomas A. Koeppel, Jennifer Kranz, Tina Hackhofer, Karen Arakelyan, Michael J. Jacobs, Maria Kokozidou  Kidney International  Volume 89, Issue 3, Pages 601-611 (March 2016) DOI: 10.1038/ki.2015.298 Copyright © 2015 International Society of Nephrology Terms and Conditions

Figure 1 Vitamin K2 supplements change intimal and medial proportion and ratio in AVF vein (a) and vena femoralis (b) of CKD animals, whereas they do not affect the proportions of healthy animals. Upper panels show intima and media percentage contributing to the vessel wall radius in AVF or vena femoralis. Intima and media percentage and ratio was pooled for sacrifice age and vein levels. Quantification was performed as illustrated in the histologic figures shown later. CKD increased intima percentage of the arterialized veins (c) without affecting media percentage in non-operated, femoral veins of rats. Vitamin K2 did not affect the intima and/or media of the arterialized (d) and non-operated femoral veins of the rats. Both vitamin K2 treatment alterations (e, f) reduced intima percentage of CKD rats without affecting media percentage in non-operated, femoral veins of the animals. In addition, CKD diet caused significant intima/media ratio increase while vitamin K2 diminished intima/media ratio in healthy, and CKD AVF veins. The columns represent mean values ± SD. *Significant differences (P < 0.01); **significant differences (P < 0.001). Bars = 100 μm. A, artery; AVF, arteriovenous fistula; CKD, chronic kidney disease; V, vein. Kidney International 2016 89, 601-611DOI: (10.1038/ki.2015.298) Copyright © 2015 International Society of Nephrology Terms and Conditions

Figure 2 Diet supplements and time change total AVF vein calcification in animals (a, average of levels L1-L6). Detection of calcified venous tissue was performed via qualitative von Kossa histomorphometry as shown in representative AVF vein sections (b, c). Calcification increases with time after surgery in every group; CKD treatment developed extensive calcification in the AVF vein (b). Both vitamin K2 treatment alterations reduced calcification in CKD animals after 42 and 63 days while vitamin K2 treatment of healthy animals reduced the calcification even more significantly compared with the nontreated animals (c). *Significant differences (P < 0.0001). Bars = 100 μm. A, artery; AVF, arteriovenous fistula; CKD, chronic kidney disease; V, vein. Kidney International 2016 89, 601-611DOI: (10.1038/ki.2015.298) Copyright © 2015 International Society of Nephrology Terms and Conditions

Figure 3 Diet supplements change intimal and medial proportion and ratio in AVF vein (a) and vena femoralis (b) of animals. Upper panels show intima and media percentage contributing to the vessel wall radius in AVF or vena femoralis. Intima and media percentage and ratio were pooled for sacrifice age and vein levels. Quantification was performed as illustrated in the histologic figures below. Diet alterations caused significant intimal increase and medial lowering. Vitamin K2 diminished intima in healthy, VKA-treated AVF veins. All treatment alterations except CKD reduced intima percentage without affecting media percentage in non-operated, femoral veins of rats. In addition, all diet alterations caused significant intima/media ratio increase while vitamin K2 diminished intima/media ratio in healthy, VKA-treated AVF veins. In human samples, the percent intimal area and the intima/media ratio were more than twofold higher in arterialized veins as compared with normal veins, whereas the percent medial area was approximately 50% lower in arterialized veins. No significant differences in intima/media ratio were observed between the 2 AVF vein groups (group 1 and 2) and the 2 native vein groups (group 3 and 4), respectively (c). Arrow points at exemplary rupture of elastica interna. The columns represent mean values ± SD. Alternatively, AVF intima/media ratio and human intima/media ratio figure list a standard error of 5%. n ≥ 10 for each animal and n ≥ 4 for each human bar. *Significant differences (P < 0.01); **significant differences (P < 0.001). Bars = 100 μm. AVF, arteriovenous fistula; CKD, chronic kidney disease; NIH, neointimal hyperplasia; VKA, vitamin K antagonist. Kidney International 2016 89, 601-611DOI: (10.1038/ki.2015.298) Copyright © 2015 International Society of Nephrology Terms and Conditions

Figure 4 Diet supplements change apoptosis ratio in AVF vein of animals (a, average of all sacrifice time points). When measured between L4 and L5, TUNEL (apoptosis)/PCNA (proliferation) ratio in intima and media of AVF vein decreased in almost all diet alterations, which implies less apoptosis or more proliferation. Precisely, a VKA diet elevated proliferation in animals with normal renal function while apoptosis rarely changed. In addition, CKD led to dramatically enhanced apoptosis ratio in native venae femorales, affected by proliferation decline in this group. Representative histological sections revealed apoptosis and proliferation around heavily calcified venous tissue but not at the site of beginning calcification (b). Von Kossa staining gave evidence of calcium phosphate; TUNEL- or PCNA-positive cells are indicated by arrows. No apoptosis was found in and around early calcification formation as indicated by the circles. Furthermore, arterialization changed apoptotic ratio in intima (but not significantly in media) of human samples (c). Likewise, CKD or calcification did not elevate apoptotic ratio in intima or media. n ≥ 10 for each animal and n ≥ 4 for each human bar. *Significant differences (P < 0.01); **significant differences (P < 0.001). Bars = 100 μm. AVF, arteriovenous fistula; CKD, chronic kidney disease; NIH, neointimal hyperplasia; PCNA, proliferating cell nuclear antigen; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; VKA, vitamin K antagonist. Kidney International 2016 89, 601-611DOI: (10.1038/ki.2015.298) Copyright © 2015 International Society of Nephrology Terms and Conditions

Figure 5 Diet supplements and time change total AVF vein calcification in animals (a, average of levels L1-L6). Detection of calcified venous tissue was performed via qualitative vK histomorphometry as shown in representative AVF vein sections (b). Calcification increases with time after surgery in every group; all groups with VKA or CKD treatment developed extensive calcification in the AVF vein. Simultaneous CKD+VKA therapy reduced calcification compared with CKD or VKA groups after 84 days. K2 supplementation reduced calcification in healthy and CKD animals treated with VKA after 42 and 84 days. By contrast, calcification was only present in group 1 of human vein tissue (c), as shown in representative human vein histology after vK staining. n ≥ 6 for each animal group and sacrifice time point. The columns represent mean values ± SD. *Significant differences (P < 0.0001) compared with the chow diet. Bars = 100 μm or 1 μm. AVF, arteriovenous fistula; CKD, chronic kidney disease; NIH, neointimal hyperplasia; vK, von Kossa; VKA, vitamin K antagonist. Kidney International 2016 89, 601-611DOI: (10.1038/ki.2015.298) Copyright © 2015 International Society of Nephrology Terms and Conditions

Figure 6 Diet supplements change MGP carboxylation in AVF veins and venae femorales in animals (a, average of all sacrifice time points). When measured between L4 and L5, cMGP/ucMGP ratio in intima and media of AVF vein decreased with CKD indicating local vitamin K deficiency in the vessel wall. K2 supplementation increased MGP carboxylation in AVF veins and native venae femorales of healthy and CKD animals treated with VKA. Furthermore, representative histological and immunohistochemical sections of an AVF vein with calcification and NIH pointed at MGP accumulation around calcified tissue (b). A, B, C, and D show fistula vein belonging to the VKA group and E, F, G, and H to the VKA+K2 group and sacrifice day 84. Both cMGP and ucMGP still express in calcified areas (A, B, E, F) while there is increased ucMGP positivity in the surrounding tissue of the VKA group (D) in comparison with the VKA+K2 group (H) and, respectively, reduced cMGP in the VKA group (C) in comparison with the VKA+K2 group where the cMGP expression improves dramatically in the surrounding tissue (E, G). cMGP and ucMGP were detected in intima and media. However, arterialization, calcification, and CKD did not change cMGP/ucMGP carboxylation ratio in human veins (c). MGP carboxylation increased between AVF veins with NIH and calcification and AVF veins with NIH but without calcification remained not significant. n ≥ 10 for each animal and n ≥ 4 for each human bar. *Significant differences (P < 0.01); **significant differences (P < 0.001). Bars = 100 μm. AVF, arteriovenous fistula; CKD, chronic kidney disease; cMGP, carboxylated matrix Gla protein; MGP, matrix Gla protein; NIH, neointimal hyperplasia; ucMGP, uncarboxylated matrix Gla protein; VKA, vitamin K antagonist. Kidney International 2016 89, 601-611DOI: (10.1038/ki.2015.298) Copyright © 2015 International Society of Nephrology Terms and Conditions

Figure S1 Diagram of an inta-operative image of an arteriovenous fistula (AVF) between the femoral artery (A) and femoral vein (V). Kidney International 2016 89, 601-611DOI: (10.1038/ki.2015.298) Copyright © 2015 International Society of Nephrology Terms and Conditions

Figure S2 Animal study group design of the pilot group (a) and the VKA treated study group. Kidney International 2016 89, 601-611DOI: (10.1038/ki.2015.298) Copyright © 2015 International Society of Nephrology Terms and Conditions

Figure S3 Serial section of the fistula vein of a CKD rat. Kidney International 2016 89, 601-611DOI: (10.1038/ki.2015.298) Copyright © 2015 International Society of Nephrology Terms and Conditions

Figure S4 Percentages of cMGP and ucMGP positive cells within the different groups. Kidney International 2016 89, 601-611DOI: (10.1038/ki.2015.298) Copyright © 2015 International Society of Nephrology Terms and Conditions

Figure S5 Group treatment in relation to the body weight variation and creatinine and phosphate serum levels in the course of experimental time points. Kidney International 2016 89, 601-611DOI: (10.1038/ki.2015.298) Copyright © 2015 International Society of Nephrology Terms and Conditions

Figure S6 In group four to six of the VKA treated study group blood was taken from five animals once a week between surgery and sacrifice. Kidney International 2016 89, 601-611DOI: (10.1038/ki.2015.298) Copyright © 2015 International Society of Nephrology Terms and Conditions

Figure S7 Human study group design (a) and human study medical demographic data (b). Kidney International 2016 89, 601-611DOI: (10.1038/ki.2015.298) Copyright © 2015 International Society of Nephrology Terms and Conditions

Figure S8 Example of serial section staining of a fistula vein and a femoral non-operated vein stained with EvG, SMA, actin and desmin. Kidney International 2016 89, 601-611DOI: (10.1038/ki.2015.298) Copyright © 2015 International Society of Nephrology Terms and Conditions