Volume 85, Issue 5, Pages 1103-1111 (May 2014) Fibroblast growth factor 23 accelerates phosphate-induced vascular calcification in the absence of Klotho deficiency  Rika Jimbo, Fumiko Kawakami-Mori, Shengyu Mu, Daigoro Hirohama, Bohumil Majtan, Yuichiro Shimizu, Yutaka Yatomi, Seiji Fukumoto, Toshiro Fujita, Tatsuo Shimosawa  Kidney International  Volume 85, Issue 5, Pages 1103-1111 (May 2014) DOI: 10.1038/ki.2013.332 Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 1 Expression profiling of fibroblast growth factor (FGF) receptors and Klotho in rat vascular smooth muscle cells and the aorta. (a) Results of reverse transcription–PCR (RT–PCR) analysis show mRNA expression of FGFR1, FGFR2, FGFR3, and Klotho in rat aortas and vascular smooth muscle cells (VSMCs). Purification of VSMCs was confirmed by the presence of transgelin and myocardin and the absence of endothelial marker vascular endothelial (VE)-cadherin. (b) FGFR1 and Klotho protein expression in the rat aorta was confirmed by western blotting. (c) FGFR1 and Klotho proteins were detected in the medial layer of rat aorta sections by immunohistochemistry. Aorta sections incubated without primary antibodies were used as the negative control (original magnification × 400). Bar=100μm. Kidney International 2014 85, 1103-1111DOI: (10.1038/ki.2013.332) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 2 Fibroblast growth factor 23 (FGF23) increased phosphate-induced vascular calcification in the rat model of chronic kidney disease (CKD). (a) Rat aortic rings from uremic rats were cultured for 6 days in control medium or high-phosphate (HP) medium (4mmol/l) in the absence or presence of recombinant FGF23 (10ng/ml). Representative microphotographs of aortic sections after immunostaining with Klotho antibody and staining with Alizarin Red or von Kossa. Calcium deposition is indicated by black arrows. Bars=50μm. (b) Quantification of calcium deposition. Results are expressed as mean±s.e.m. *P<0.05 versus control, **P<0.05 versus HP medium without FGF23. Kidney International 2014 85, 1103-1111DOI: (10.1038/ki.2013.332) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 3 Extracellular signal–related kinase 1/2 (ERK1/2) was phosphorylated by fibroblast growth factor 23 (FGF23) in a dose-dependent manner in Klotho-overexpressing vascular smooth muscle cells (VSMCs). (a) Overexpression of Klotho in VSMCs. VSMCs were transfected with a vector expressing membrane Klotho or an empty vector (negative control). After 48h, cell lysates were collected and analyzed by western blotting with anti-Klotho antibodies. (b, c) ERK1/2 was phosphorylated by FGF23 in Klotho-overexpressing VSMCs. VSMCs overexpressing Klotho were serum-starved for 24h and treated with (b) recombinant FGF23 (0–10ng/ml) for 30min or (c) recombinant FGF23 (5ng/ml) for the indicated time. Cell lysates were analyzed by western blotting with antibodies against phosphorylated ERK (p-ERK) and total ERK (t-ERK). Results are expressed as mean±s.e.m. *P<0.05 versus control. Kidney International 2014 85, 1103-1111DOI: (10.1038/ki.2013.332) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 4 Fibroblast growth factor 23 (FGF23) increased phosphate-induced calcium deposition. (a) Klotho-overexpressing vascular smooth muscle cells were cultured for 6 days in control medium (CON) or high-phosphate (HP) medium (5mmol/l) in the absence or presence of recombinant FGF23 (2–10ng/ml). Representative microphotographs of Alizarin Red S staining of control cells, cells incubated in HP medium, and cells incubated in HP medium with FGF23 (2–10ng/ml). (b) Quantification of calcium deposition. FGF23 dose dependently increased phosphate-induced calcium deposition. Results are expressed as mean±s.e.m. *P<0.05 versus control, **P<0.05 versus HP medium without FGF23. Kidney International 2014 85, 1103-1111DOI: (10.1038/ki.2013.332) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 5 Effect of fibroblast growth factor 23 (FGF23) on the expression of calcification regulatory factors. Real-time reverse transcription–PCR (RT–PCR) analysis of osteoblastic markers (a) MSX2 and (b) OSX. Klotho-overexpressing vascular smooth muscle cells (VSMCs) were incubated in control medium or high-phosphate (HP) medium for 48h in the absence or presence of FGF23 (5ng/ml). Results are expressed as mean±s.e.m. (c) Synergic effects of FGF23 and phosphate on extracellular signal–related kinase (ERK) phosphorylation. Klotho-overexpressing VSMCs were cultured in control medium or HP medium in the absence or presence of FGF23 (5ng/ml) for 24h. *P<0.05 versus control, **P<0.05 versus HP medium without FGF23. p-ERK, phosphorylated ERK; t-ERK, total ERK. Kidney International 2014 85, 1103-1111DOI: (10.1038/ki.2013.332) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 6 Effect of MEK inhibition on osteoblastic differentiation. (a, b) Klotho-overexpressing VSMCs were cultured in control medium, HP medium alone, or HP medium with FGF23 (5ng/ml) for 48h with the MEK inhibitor U0126 (5μmol/l) or inactive compound U0124 (5μmol/l). Results are expressed as mean±s.e.m. *P<0.05. U0126 treatment also inhibited FGF23-induced Msx2 and Osx expression. (c) Proposed mechanisms underlying FGF23 acceleration of phosphate-induced vascular calcification. ERK, extracellular signal–regulated kinase; FGF23, fibroblast growth factor 23; FGFR1, fibroblast growth factor receptor 1; HP, high-phosphate; MEK, mitogen-activated protein kinase kinase; Na, sodium; P, phosphate; PIT-1, sodium-dependent phosphate transporter 1; VSMC, vascular smooth muscle cell. Kidney International 2014 85, 1103-1111DOI: (10.1038/ki.2013.332) Copyright © 2014 International Society of Nephrology Terms and Conditions