1. Volume 83, Issue 6, Pages 1042-1051 (June 2013)
Phosphate-induced autophagy counteracts vascular calcification by reducing matrix vesicle release 
Xiao-Yan Dai, Ming-Ming Zhao, Yan Cai, Qing-Cong Guan, Ying Zhao, Youfei Guan, Wei Kong, Wei-Guo Zhu, Ming-Jiang Xu, Xian Wang 
Kidney International 
Volume 83, Issue 6, Pages (June 2013)
DOI: /ki
Copyright © 2013 International Society of Nephrology Terms and Conditions

2. Figure 1
Phosphate (Pi) induces autophagy in vascular smooth muscle cells (VSMCs). (a) Western blot analysis of light-chain 3-II (LC3II) levels in bovine aortic smooth muscle cells (BASMCs) treated with Pi at the indicated concentrations for 12h and (b) in BASMCs pretreated with chloroquine (Chq, 25μmol/l) for 30min, then with Pi for 12h. (c) Confocal microscopy of green fluorescent protein (GFP) in BASMCs transfected with GFP–LC3 plasmids, cultured for 36h, and then treated with Pi for 12h. Autophagosomes are indicated by fluorescence puncta. (d) Percentage of BAMSCs with GFP–LC3 puncta after Pi treatment for the indicated durations. *P<0.05 versus corresponding control. (e) Electron microscopy of autophagic structures (arrows) in cells exposed to Pi for 12h. n=3–4, one representative image is shown. (f) Confocal microscopy of GFP–LC3 puncta in human aortic VSMCs (HA-VSMCs) transfected with GFP–LC3 plasmids and treated with Pi (3mmol/l) for 12h. (g) Immunofluorescence analysis of LC3 puncta in renal artery wall from human organ donor and recipient with chronic kidney disease during renal transplantation. CKD, chronic kidney disease.
Kidney International  , DOI: ( /ki )
Copyright © 2013 International Society of Nephrology Terms and Conditions

3. Figure 2
Reactive oxygen species (ROS) mediate phosphate (Pi)-induced autophagy in vascular smooth muscle cells (VSMCs). (a) Confocal microscopy of fluorescence with control and Pi (3mmol/l) induction in bovine aortic smooth muscle cells (BASMCs) incubated with MitoTracker Red CM-H2XRos probe for 30min to indicate mitochondrial ROS level. (b) Western blot analysis of light-chain 3 (LC3)I/II level in BASMCs pretreated with MnTMPyP for 30min, or (c) infected with adenovirus-expressing green fluorescent protein (GFP) or superoxide dismutase 2 (SOD2) for 24h, and then treated with Pi for 12h. (d) Immunofluorescence and percentage of cells with GFP–LC3 puncta in BASMCs treated with Pi (3mmol/l) and/or MnTMPyP (25μmol/l) for 12h. (e) Western blot analysis of LC3 protein level in abdominal aortas of rats with chronic renal failure (CRF) with control, vehicle (CRF), and CRF plus MnTMPyP (CRF+MnTMPyP) treatment and (f) immunofluorescence of LC3 puncta and MitoTracker Red CM-H2XRos probe (200nmol/l) for mitochondrial ROS in rat abdominal aorta walls. n=3–5, *P<0.05.
Kidney International  , DOI: ( /ki )
Copyright © 2013 International Society of Nephrology Terms and Conditions

4. Figure 3
Type III sodium-dependent phosphate cotransporter Pit1 mediates phosphate (Pi)-induced autophagy in vascular smooth muscle cells (VSMCs). (a) Reverse transcriptase PCR analysis of mRNA levels of Pit1 and Pit2 in human VSMCs (HA-VSMCs) transfected with scramble or Pit1 siRNA for 48h. (b) Western blot analysis of Pit1/2 protein levels in HA-VSMCs transfected with scramble or Pit1 siRNA for 48h. (c) Light chain 3 (LC3)I/II protein levels in HA-VSMCs transfected with scramble or Pit1 siRNA for 48 h, then treated with Pi (3mM) for 12h. (d) Calcium content assay of HA-VSMCs transfected with scramble or Pit1 siRNA for 48 h, then treated with Pi (3mM) for 10 days. n=3, *P<0.05.
Kidney International  , DOI: ( /ki )
Copyright © 2013 International Society of Nephrology Terms and Conditions

5. Figure 4
Phosphate (Pi)-induced autophagy is abolished by autophagy inhibition. (a) Western blot analysis of light-chain 3 (LC3)I/II protein levels in bovine aortic smooth muscle cells (BASMCs) pretreated with autophagy inhibitor 3-methyladenine (3-MA 5mmol/l) for 30min, and then with Pi (3mmol/l) for 12h. (b) Percentage of cells with green fluorescent protein (GFP)–LC3 puncta in BASMCs treated with Pi (3mmol/l) or 3-MA+Pi for 12h. (c) Western blot analysis of protein level of autophagy protein 5 (Atg5) in rat vascular smooth muscle cells (VSMCs) transfected with scramble or Atg5 small interfering RNA (siRNA) for 48h. (d) Percentage of GFP–LC3 puncta in rat VSMCs transfected with scramble or Atg5 siRNA for 48h, and then with Pi for 12h. n=3, *P<0.05.
Kidney International  , DOI: ( /ki )
Copyright © 2013 International Society of Nephrology Terms and Conditions

6. Figure 5
Blockage of autophagy exacerbates phosphate (Pi)-induced calcification. (a–d) Cells were treated with Pi (3mmol/l) or Pi+3-methyladenine (3-MA) (5mmol/l) for the indicated days, and then alizarin red S staining (a) and calcium content assay were performed (b–d). (e) Calcium content assay of rat vascular smooth muscle cells (VSMCs) and (f) A7r5 cells transfected with scramble or autophagy protein 5 (Atg5) small interfering RNA (siRNA) for 48h, and then treated with Pi for 10 days; (g) rat aortic ring explants treated with Pi (3mmol/l) and/or 3-MA (5mmol/l) for 7 days. n=3–5, *P<0.05. BASMCs, bovine aortic smooth muscle cells; HA-VSMCs, human aortic VSMCs.
Kidney International  , DOI: ( /ki )
Copyright © 2013 International Society of Nephrology Terms and Conditions

7. Figure 6
Autophagy inducer valproic acid (VPA) reduces calcification. (a) Calcium content assay of rat aortic ring explants and (b) bovine aortic smooth muscle cells (BASMCs) treated with phosphate (Pi) (3mmol/l) and/or VPA (1mmol/l) for 7 days. n=3, *P<0.05.
Kidney International  , DOI: ( /ki )
Copyright © 2013 International Society of Nephrology Terms and Conditions

8. Figure 7
The pro-calcification effect of autophagy inhibition does not involve reactive oxygen species (ROS)-mediated vascular smooth muscle cell (VSMC) apoptosis. (a) Cleaved caspase-3 and (b) caspase-3/7 activity detected in rat VSMCs transfected with scramble or autophagy protein 5 (Atg5) small interfering RNA (siRNA) for 48h, and then treated with MnTMPyP and/or phosphate (Pi) as indicated for 10 days. (c) Quantification of terminal transferase dUTP nick-end labeling (TUNEL) staining of apoptotic cells in bovine aortic smooth muscle cells (BASMCs) treated with Pi and/or 3-methyladenine (3-MA) and MnTMPyP as indicated for 7 days. (d) Caspase-3/7 activity and (e) calcium content assay in BASMCs treated with 3-MA, MnTMPyP, Z-VAD-FMK, and/or Pi as indicated for 7 days. n=3, *P<0.05 versus corresponding group 1 or as indicated; #P<0.05 versus corresponding group 2; !P<0.05 versus corresponding group as indicated.
Kidney International  , DOI: ( /ki )
Copyright © 2013 International Society of Nephrology Terms and Conditions

9. Figure 8
Blocking autophagy promotes matrix vesicle (MV) release. (a–c) Bovine aortic smooth muscle cells (BASMCs) were treated with phosphate (Pi) (3mmol/l) and/or 3-methyladenine (3-MA) (5mmol/l) for 3 days, MVs were isolated by centrifugation, and then (a) MV release was assessed by total protein content. (b) Western blot analysis of protein level of annexin II (MV marker) in MVs. (c) Alkaline phosphatase (ALP) activity shown by ELF 97 phosphatase substrate in cultured BASMCs. (d) Assay of ALP activity in MVs. n=4, *P<0.05.
Kidney International  , DOI: ( /ki )
Copyright © 2013 International Society of Nephrology Terms and Conditions