Volume 90, Issue 3, Pages 598-609 (September 2016) Hypoxia-inducible factor-1 plays a role in phosphate-induced vascular smooth muscle cell calcification  Sophie Mokas, Richard Larivière, Laurent Lamalice, Stéphane Gobeil, David N. Cornfield, Mohsen Agharazii, Darren E. Richard  Kidney International  Volume 90, Issue 3, Pages 598-609 (September 2016) DOI: 10.1016/j.kint.2016.05.020 Copyright © 2016 International Society of Nephrology Terms and Conditions

Figure 1 Hypoxia-inducible factor-1 (HIF-1) activation in calcifying aortas from a chronic kidney disease (CKD) rat model. Aortas from nephrectomized rats receiving a normal diet (CKD) or undergoing mineral imbalance (CKD+MI) were surgically isolated. (a) Representative images of immunohistochemical analysis performed using a HIF-1α antibody. Arrowheads indicate HIF-1α positive nuclear staining. Bar = 100 μm. (b) Quantification of HIF-1α positive nuclei from immunohistochemical analyses (arrowheads in a). (c) RNA was extracted and real-time quantitative reverse transcriptase–polymerase chain reaction was performed to determine mRNA expression levels for smooth muscle actin alpha-2 (ACTA2), vascular endothelial growth factor A (VEGFA), glucose transporter type 1 (GLUT-1), and HPRT1 (reference gene). Results are an average ± SEM. CKD, n = 6; CKD+MI, n = 6. ∗P < 0.05; ∗∗P <0.01 as compared to CKD animals. Kidney International 2016 90, 598-609DOI: (10.1016/j.kint.2016.05.020) Copyright © 2016 International Society of Nephrology Terms and Conditions

Figure 2 Elevated inorganic phosphate (HiPO4) induces vascular smooth muscle cells mineralization. Vascular smooth muscle cells were maintained in either control or HiPO4 (2.5 mM inorganic phosphate) conditions for 2 to 10 days. (a) Ca2+ content was assessed by atomic absorption spectrometry. Results are presented as fold changes of the calcium content (μg/mg protein) between treatment conditions and the corresponding control. †P < 0.05, and ††P < 0.01 as compared to HiPO4-treated cells under 20.9% O2. (b) Representative images of von Kossa staining after 6 days of treatment with control (left panels) and HiPO4 (right panels) conditions. Ca2+-containing deposits (in black) where assessed using light microscopy at ×20 and ×60 magnification. Bars = 100 μm (upper panels) and 10 μm (lower panels). Kidney International 2016 90, 598-609DOI: (10.1016/j.kint.2016.05.020) Copyright © 2016 International Society of Nephrology Terms and Conditions

Figure 3 Hypoxia strongly enhances elevated inorganic phosphate (HiPO4)-induced vascular smooth muscle cell mineralization. Vascular smooth muscle cells were maintained in control or HiPO4 conditions under either normal oxygen (20.9% O2) or hypoxic conditions (1% O2) for 6 days. (a) Ca2+ content was assessed by atomic absorption spectrometry. Results are presented as fold changes of the calcium content (μg/mg protein) between treatment conditions and the corresponding control. ∗∗P < 0.01 as compared to control cells under 20.9% O2. †P < 0.05 as compared to HiPO4-treated cells under 20.9% O2. (b) Representative images of von Kossa staining after 6 days of HiPO4 treatment under (left) 20.9% O2 or (right) 1% O2. Ca2+-containing deposits (in black) were assessed using light microscopy at ×10 magnification. Bar = 10 mm. (c) RNA was extracted and real-time quantitative reverse transcriptase–polymerase chain reaction was performed to determine mRNA expression levels for runt-related transcription factor 2 (RUNX2), bone morphogenetic protein-2 (BMP-2), and 18S (reference gene). ∗∗∗P < 0.001 as compared to control cells under 20.9% O2. ††P < 0.01, †††P < 0.001 as compared to HiPO4-treated cells under 20.9% O2. (d) Representative Western blot performed using nuclear protein extracts and RUNX2 and Histone H3 antibodies. Kidney International 2016 90, 598-609DOI: (10.1016/j.kint.2016.05.020) Copyright © 2016 International Society of Nephrology Terms and Conditions

Figure 4 Hypoxia-inducible factor prolyl hydroxylases inhibition increases elevated inorganic phosphate (HiPO4)-induced vascular smooth muscle cell mineralization. Vascular smooth muscle cells were maintained in control or HiPO4 conditions under either 20.9% O2 or 1% O2, or they were treated with 50 μM deferoxamine (DFO) or 50 μM FG-4592 for 6 days. (a) Ca2+ content was assessed by atomic absorption spectrometry. Results are presented as fold changes of the calcium content (μg/mg protein) between treatment conditions and the corresponding control. ∗P < 0.05, ∗∗P < 0.01 as compared to control cells under 20.9% O2. †P < 0.05 as compared to HiPO4-treated cells under 20.9% O2. (b) RNA was extracted and real-time quantitative reverse transcriptase–polymerase chain reaction was performed to determine mRNA expression levels for runt-related transcription factor 2 (RUNX2), bone morphogenetic protein-2 (BMP-2), and 18S (reference gene). ∗P < 0.05, ∗∗P < 0.01 as compared to control cells under 20.9% O2. †P < 0.05, ††P < 0.01 as compared to HiPO4-treated cells under 20.9% O2. (c) Representative images of von Kossa staining after 6 days of HiPO4 treatment under 20.9% O2 (left panels) or HiPO4 (right panels). Ca2+-containing deposits (in black) where assessed using light microscopy at ×20 magnification. Bar = 200 μm. DMSO, dimethylsulfoxide. Kidney International 2016 90, 598-609DOI: (10.1016/j.kint.2016.05.020) Copyright © 2016 International Society of Nephrology Terms and Conditions

Figure 5 Hypoxia-inducible factor-1 (HIF-1) deletion decreases elevated inorganic phosphate (HiPO4)-induced vascular smooth muscle cell mineralization. Wild-type (WT) and SM22Cre/HIF-1αfl/fl vascular smooth muscle cells were maintained in control or HiPO4 conditions under either 20.9% O2 or 1% O2 for 6 days. (a) Results are presented as fold changes of the calcium content (μg/mg protein) between treatment conditions and the corresponding control. ∗∗∗P < 0.001 as compared to control cells under 20.9% O2. †P < 0.05 as compared to HiPO4-treated cells under 20.9% O2. (b) Representative images of von Kossa staining after 6 days of HiPO4 treatment under (left) 20.9% O2 or (right) 1% O2. Ca2+-containing deposits (in black) were assessed using light microscopy at ×10 magnification. Bar = 1 mm. (c) Representative Western blot using total cell extracts with runt-related transcription factor 2 (RUNX2) and β-actin antibodies. Kidney International 2016 90, 598-609DOI: (10.1016/j.kint.2016.05.020) Copyright © 2016 International Society of Nephrology Terms and Conditions

Figure 6 Elevated inorganic phosphate (HiPO4) increases hypoxia-inducible factor-1 (HIF-1) activity in vascular smooth muscle cells (VSMCs). VSMCs were maintained under (a) control conditions or treated with HiPO4 for time periods indicated or (b) control conditions or treated with HiPO4 under either 20.9% O2 or 1% O2 for 4 hours. Representative Western blots using total protein extracts with HIF-1α and α-tubulin antibodies. Western blots in (b) were quantitated with the Odyssey Infrared Imaging system. ∗P < 0.05 and ∗∗P < 0.01, as compared to control cells under 20.9% O2. (c) VSMC were transfected with 2 μg pGL3 (R2.2)3HRE-tk reporter plasmid and 50 ng Renilla reniformis luciferase expression vector. VSMC were maintained under control conditions or treated with HiPO4 under either 20.9% O2 or 1% O2 for 6 hours. Results are presented as fold changes of luciferase activity over R reniformis luciferase activity and are an average ± SEM of 4 independent experiments performed in triplicate. ∗P <0.05; ∗∗P < 0.01, and ∗∗∗P < 0.001 as compared to control cells under 20.9% O2. (d) VSMC were maintained under control conditions or treated with HiPO4 for 6 hours under either 20.9% O2 or 1% O2. RNA was extracted and real-time quantitative reverse transcriptase–polymerase chain reaction was performed to determine the mRNA expression levels of vascular endothelial growth factor A (VEGFA), glucose transporter type 1 (GLUT-1), and 18S (reference gene). Results are an average ± SEM of 5 independent experiments. ∗P < 0.05; ∗∗P < 0.01, and ∗∗∗P < 0.001 as compared to control cells under 20.9% O2. ††P < 0.01, †††P < 0.001 as compared to HiPO4-treated cells under 20.9% O2. Kidney International 2016 90, 598-609DOI: (10.1016/j.kint.2016.05.020) Copyright © 2016 International Society of Nephrology Terms and Conditions

Figure 7 Elevated inorganic phosphate (HiPO4) increases hypoxia-inducible factor-1 (HIF-1) stability. (a) Vascular smooth muscle cells (VSMCs) were transfected with 2 μg CMV-LUC-HIF-1α-ODDD and 50 ng Renilla reniformis luciferase expression vector. VSMC were maintained under 20.9% O2 or treated with either HiPO4 or 200 μM CoCl2 for 4 hours. (Upper) Results are presented as fold changes of luciferase activity over R reniformis luciferase activity and are an average ± SEM of 4 independent experiments performed in triplicate. ∗P < 0.05 and ∗∗P < 0.01 as compared to control cells under 20.9% O2. (Lower) Representative Western blots using total protein extracts with HIF-1α and α-tubulin antibodies. (b) VSMC were maintained under 20.9% O2 or treated with either HiPO4 or 200 μM CoCl2 for 4 hours. Cytoplasmic extracts were incubated with glutathione S-transferase (GST)-HIF-1α followed by an incubation with in vitro translated von Hippel-Lindau tumor suppressor gene (VHL). (Lower) Western blots were performed using hemagglutinin (VHL) and GST antibodies. (Upper) Western blots were quantitated with the Odyssey Infrared Imaging system. Results are an average ± SEM of 5 independent experiments. ∗∗P < 0.01 and ∗∗∗P < 0.001 as compared to control cells under 20.9% O2. VSMC were treated with (c) 1 μM stigmatellin (STIG) and 0.5 μM SkQ1 and (d) 5 μM ascorbate (ASC) and maintained under 20.9% O2 or treated with HiPO4 for 4 hours. Western blots using total protein extracts with HIF-1α and α-tubulin antibodies. CMV, cytomegalovirus; LUC, luciferase; ODDD, oxygen-dependent degradation domain. Kidney International 2016 90, 598-609DOI: (10.1016/j.kint.2016.05.020) Copyright © 2016 International Society of Nephrology Terms and Conditions

Figure S1 Reduced vascular gene expression in calcifying aortas from a chronic kidney disease (CKD) rat model. Aortas from nephrectomized rats receiving a normal diet (CKD) or undergoing mineral imbalance (CKD+MI) were isolated and RNA was extracted. Real-time quantitative reverse transcriptase–polymerase chain reaction was performed to determine mRNA expression levels for CNN1, SM22, MYH11, and HPRT (reference gene). Results are an average ± SEM. CKD, n = 6; CKD+MI, n = 6. ∗∗P < 0.01; ∗∗∗P < 0.001 as compared to CKD animals. Kidney International 2016 90, 598-609DOI: (10.1016/j.kint.2016.05.020) Copyright © 2016 International Society of Nephrology Terms and Conditions

Figure S2 Hypoxia-inducible factor-1α (HIF-1α) levels and enhanced elevated inorganic phosphate (HiPO4)-induced osteocalcin (OC) mRNA expression in vascular smooth muscle cells. (A) Vascular smooth muscle cells were maintained under either 20.9% O2 or decreasing levels of O2 for 4 hours. Representative Western blot performed using total protein extracts with anti-HIF-1α and anti-α-tubulin antibodies. (B) Vascular smooth muscle cells were maintained under control conditions or treated with HiPO4 for 6 hours under either 20.9% O2 or 1% O2. RNA was extracted and real-time quantitative reverse transcriptase–polymerase chain reaction was performed to determine mRNA expression levels for OC and 18S (reference gene). ∗∗P < 0.01 as compared to control cells under 20.9% O2. †††P < 0.001 as compared to HiPO4-treated cells under 20.9% O2. Kidney International 2016 90, 598-609DOI: (10.1016/j.kint.2016.05.020) Copyright © 2016 International Society of Nephrology Terms and Conditions

Figure S3 Deferoxamine (DFO) and FG-4595 induced hypoxia-inducible factor-1 (HIF-1) and calcification in vascular smooth muscle cells (VSMCs). (A) VSMCs were maintained under either 20.9% O2, 1% O2, or treated with 50 μM DFO for 6 days. Representative Western blot performed using total protein extracts with anti-HIF-1α and anti-α-tubulin antibodies. (B) VSMCs were maintained under 20.9% O2 or treated with indicated concentrations of FG-4592 for 6 days. Representative Western blot performed using total protein extracts with anti-HIF-1α and anti-α-tubulin antibodies. (C) Quantification, using Metamorph, of von Kossa staining in Figure 3. VSMCs were maintained at 20.9% O2 under control or elevated inorganic phosphate (HiPO4) conditions and treated with or without 50 μM FG-4592 for 6 days. ∗∗P < 0.01 as compared to control cells under 20.9% O2. ††P < 0.01 as compared to HiPO4-treated cells under 20.9% O2. Kidney International 2016 90, 598-609DOI: (10.1016/j.kint.2016.05.020) Copyright © 2016 International Society of Nephrology Terms and Conditions

Figure S4 Hypoxia-inducible factor-1 (HIF-1) deletion blocked HIF-1 activity in vascular smooth muscle cells. Vascular smooth muscle cells from wild-type mice (WT) and mice containing a vascular smooth muscle cell–specific HIF-1 inactivating mutation (SM22Cre/HIF-1αfl/fl) were maintained under either 20.9% O2 (-) or hypoxic conditions (1% O2) or treated with 200 μM CoCl2, an HIF-1 inducer, for 4 hours. (A) Representative Western blots performed using total protein extracts with anti-HIF-1α and anti-α-tubulin antibodies. (B) Total RNA was extracted and real-time quantitative reverse transcriptase–polymerase chain reaction was performed to determine the mRNA expression levels of vascular endothelial growth factor A (VEGFA) and 18S (reference gene). Kidney International 2016 90, 598-609DOI: (10.1016/j.kint.2016.05.020) Copyright © 2016 International Society of Nephrology Terms and Conditions

Figure S5 Hypoxia-inducible factor-1 (HIF-1) depletion decreased elevated inorganic phosphate (HiPO4)-induced vascular smooth muscle cell mineralization. Vascular smooth muscle cells were infected with 2 different short hairpin RNA (shRNA) against short hairpin HIF-1α (shHIF1A) or a control shRNA (shCtrl). Vascular smooth muscle cells were maintained in control or HiPO4 conditions under either 20.9% O2 or 1% O2 for 6 days. (A) Results are presented as fold changes of the calcium content (μg/mg protein) between treatment conditions and the corresponding control. ∗∗∗P < 0.001 as compared to control cells under 20.9% O2. †P < 0.05 as compared to HiPO4-treated cells under 20.9% O2. (B) Representative Western blot performed using total protein extracts with anti-HIF-1α and anti-α-tubulin antibodies. Kidney International 2016 90, 598-609DOI: (10.1016/j.kint.2016.05.020) Copyright © 2016 International Society of Nephrology Terms and Conditions

Figure S6 Elevated inorganic phosphate (HiPO4)-induced hypoxia-inducible factor-1 (HIF-1) activity and intracellular ascorbate measurements. (A) Vascular smooth muscle cells were transfected with a small, interfering RNA (siRNA) against small, interfering HIF-1α (siHIF1) or a control siRNA (siCtrl). Vascular smooth muscle cells were maintained under control conditions or treated with HiPO4 for 6 hours under either 20.9% O2 or 1% O2. (Upper) Total RNA was extracted and real-time quantitative reverse transcriptase–polymerase chain reaction analysis was performed to determine the mRNA expression levels of vascular endothelial growth factor A (VEGFA), glucose transporter type 1 (GLUT-1), and 18S (reference gene). (Lower) Representative Western blots performed using total protein extracts with anti-HIF-1α and anti-α-tubulin antibodies. (B) Vascular smooth muscle cells were pretreated with 250 μM ascorbate prior to being maintained under control conditions or treated with HiPO4 for 6 hours under 20.9% O2 in the presence or absence of 0.5 μM SkQ1. Cellular ascorbate levels were determined using a commercial assay. Kidney International 2016 90, 598-609DOI: (10.1016/j.kint.2016.05.020) Copyright © 2016 International Society of Nephrology Terms and Conditions