Volume 88, Issue 4, Pages 711-721 (October 2015) Mammalian target of rapamycin signaling inhibition ameliorates vascular calcification via Klotho upregulation  Yang Zhao, Ming-Ming Zhao, Yan Cai, Ming-Fei Zheng, Wei-Liang Sun, Song-Yang Zhang, Wei Kong, Jun Gu, Xian Wang, Ming-Jiang Xu  Kidney International  Volume 88, Issue 4, Pages 711-721 (October 2015) DOI: 10.1038/ki.2015.160 Copyright © 2015 International Society of Nephrology Terms and Conditions

Figure 1 Rapamycin inhibits the mammalian target of rapamycin (mTOR) pathway and reduces vascular calcification. (a) Western blot analyses of phospho-p70 S6 kinase (P-S6K) protein levels in rat aorta from the control (Ctrl), chronic renal failure (CRF), and CRF plus oral rapamycin (CRF+Rapa) groups. β-actin was a loading control. (b) Von Kossa staining of rat abdominal aortas of Ctrl, CRF, and CRF+Rapa rats. (c) Calcium content of abdominal aorta was measured and normalized by dried tissue weight. (d) Immunohistochemical staining of α-actin and Cbfα-1 in the abdominal aortas of Ctrl, CRF, and CRF+Rapa rats. (e) Thoracic aorta mRNA was extracted from Ctrl, CRF, and CRF plus rapamycin (CRF+Rapa) groups, and real-time PCR analyzed the mRNA levels of osteogenic/chondrogenic genes core-binding factor α-1 (Cbfα-1), msh homeobox 2 (Msx2), osterix, aggrecan, and Sox9; calcification inhibitors matrix gla protein (Mgp) and osteopontin (Opn); vascular smooth muscle cell (VSMC) lineage markers SM22α and α-actin. Data are relative to β-actin level. (f) Serum pyrophosphate (PPi) levels were measured. n=13–16 rats. *P<0.05, **P<0.01, ***P<0.001 vs. Ctrl; #P<0.05 vs. CRF. (g) Human aortic rings were cultured and treated with inorganic phosphate (Pi; 3.0 mmol/l) and/or rapamycin (rapa, 30 nmol/l) for 7 days. Von Kossa staining was performed. Kidney International 2015 88, 711-721DOI: (10.1038/ki.2015.160) Copyright © 2015 International Society of Nephrology Terms and Conditions

Figure 2 Activation of mammalian target of rapamycin (mTOR) mediates inorganic phosphate (Pi)-induced calcification in bovine aortic smooth muscle cells (BASMCs). (a) Western blotting analyzed endogenous P-mTOR and phospho-p70 S6 kinase (P-S6K) in BASMCs after exposure to Pi (3.0 mmol/l) for the indicated time. β-actin was a loading control. (b) Effects of leucine (Leu, 4 mmol/l) and rapamycin (Rapa, 30 nmol/l) on Pi (24 h)-induced P-S6K level in BASMCs. (c,d) BASMCs were treated with rapamycin (c) or leucine (d) at the indicated concentrations plus Pi for 7 days; then, calcium content was measured. Data were normalized by total protein. n=3, *P<0.05 vs. Ctrl. #P<0.05 vs. Pi. (e) BASMCs were transfected with GFP vector (GFP) or mTOR-expression plasmid (mTOR). mTOR overexpression was validated by increased P-S6K level (upper panel). The cells were then treated with Pi for 7 days, and calcium content was determined. (f) BASMCs were transfected with GFP vector (GFP) or kinase-dead mTOR-expression plasmid (mTOR KD); mTOR signaling inhibition was validated by decreased P-S6K level (upper panel). The cells were then treated with Pi for 7 days, and calcium content was determined; *P<0.05 vs. Ctrl, #P<0.05 vs. GFP. (g) Aortic smooth muscle cells were isolated and cultured from TSC1 flox-flox mice and treated with Ad-GFP or Ad-Cre at the indicated multiplicity of infection for 24 h, and then treated with Pi (3.0 mmol/l) for 10 days. Calcium content was measured. n=3, *P<0.05 vs. Ad-GFP, #P<0.05 vs. Ad-GFP+Pi. Kidney International 2015 88, 711-721DOI: (10.1038/ki.2015.160) Copyright © 2015 International Society of Nephrology Terms and Conditions

Figure 3 Activation of mammalian target of rapamycin (mTOR) mediates inorganic phosphate (Pi)-induced calcification in human aortic smooth muscle cells (HASMCs). (a) Western blotting analyzed P-mTOR and phospho-p70 S6 kinase (P-S6K) levels in HASMCs after exposure to Pi (3 mmol/l) for the indicated time. mTOR and S6K are controls for P-mTOR and P-S6K, respectively. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was a loading control. (b) Effects of leucine (Leu, 4 mmol/l) and rapamycin (Rapa, 32 nmol/l) on Pi (24 h)-induced P-S6K level in HASMCs. (c,d) HASMCs were treated with rapamycin (c) or leucine (d) at the indicated concentrations plus Pi for 7 days; then, calcium content was measured. (e) HASMCs were treated with Pi and/or rapamycin for a duration of 10 days (1–10), or treated with Pi for 3 days, and then treated with rapamycin plus Pi for an additional 7 days (4–10); then, calcium content was measured. Data were normalized by total protein. n=3, *P<0.05 vs. Ctrl, #P<0.05 vs. Pi. Kidney International 2015 88, 711-721DOI: (10.1038/ki.2015.160) Copyright © 2015 International Society of Nephrology Terms and Conditions

Figure 4 The mammalian target of rapamycin (mTOR) signaling regulates vascular smooth muscle cell differentiation. Bovine aortic smooth muscle cells (BASMCs) and human aortic smooth muscle cells (HASMCs) were treated with inorganic phosphate (Pi), leucine (Leu), or rapamycin (Rapa) as indicated for 7 days. Real-time PCR analyzed the mRNA levels of osteogenic genes Msx2 and Cbfα-1 (a,b), and vascular smooth muscle cell (VSMC) lineage markers SM22α and α-actin (c,d). Data are relative to β-actin level. n=4, *P<0.05 vs. Ctrl, #P<0.05 vs. Pi. Kidney International 2015 88, 711-721DOI: (10.1038/ki.2015.160) Copyright © 2015 International Society of Nephrology Terms and Conditions

Figure 5 The role of autophagy in rapamycin-inhibited vascular calcification. (a) Vascular smooth muscle cells (VSMCs) were treated with inorganic phosphate (Pi), rapamycin, or 3-MA as indicated for 12 h; then, LC3I/II protein levels were detected by western blot analyses. (b) VSMCs were treated with Pi, 3-MA, or rapamycin at the indicated concentrations for 7 days; then, calcium content was measured. Data were normalized by total protein. (c) VSMCs were treated with Pi, rapamycin, or 3-MA as indicated for 3 days; total cell lysates were subjected for caspase 3/7 activity assay. n=3, *P<0.05. Kidney International 2015 88, 711-721DOI: (10.1038/ki.2015.160) Copyright © 2015 International Society of Nephrology Terms and Conditions

Figure 6 Mammalian target of rapamycin (mTOR) signaling mediates inorganic phosphate (Pi)-decreased Klotho expression. (a) Western blot analyses showed Klotho level in bovine aortic smooth muscle cells (BASMCs) after exposure to Pi (3.0 mmol/l) for 3 days. (b) Western blot analyses showed the levels of P-mTOR and Klotho in BASMCs after exposure to Pi for 7 days. (c) BASMCs were treated with Pi for 24 h and 72 h, and then Klotho mRNA level was analyzed by real-time PCR (n=3, *P<0.01 vs. Ctrl). (d) BASMCs were overexpressed with GFP or mTOR, and then treated with Pi (3.0 mmol/l) for 24 h. Klotho mRNA was measured by real-time PCR (n=3, *P<0.01 vs. GFP). (e) Mouse TSC1 flox-flox smooth muscle cells were infected with Ad-GFP or Ad-Cre, and then Klotho mRNA level was measured (n=3, *P<0.01 vs. Ad-GFP). (f,g) BASMCs and human aortic smooth muscle cells (HASMCs) were treated with leucine (Leu) or rapamycin (Rapa) at the indicated concentrations for 24 h, and then Klotho mRNA level was measured by real-time PCR (n=3, *P<0.01 vs. "0"). (h,i) BASMCs were treated with Pi and/or Rapa (15 nmol/l) for 7 days. Klotho protein level was analyzed and quantified by western blotting and enzyme-linked immunosorbant assay (ELISA; n=3, *P<0.01 vs. Ctrl. #P<0.01 vs. Pi). (j,k) Proteins of abdominal aorta from Ctrl, CRF, and CRF+Rapa rats were analyzed by western blotting for P-mTOR and Klotho levels; serum Klotho was measured by ELISA (n=6–8 rats, *P<0.01 vs. Ctrl, #P<0.01 vs. CRF). Kidney International 2015 88, 711-721DOI: (10.1038/ki.2015.160) Copyright © 2015 International Society of Nephrology Terms and Conditions

Figure 7 Klotho suppresses vascular calcification in vitro. (a,b) Bovine aortic smooth muscle cells (BASMCs) were transfected with vector or Klotho-expression plasmid for 2 days, and Klotho overexpression was validated by (a) real-time PCR and (b) western blotting. GAPDH was a loading control. (c,d) BASMCs were transfected with vector or Klotho-expression plasmid and treated with inorganic phosphate (Pi) for 7 days, and (c) calcium content and (d) mRNA levels of Cbfα-1, MSX2, α-actin, and SM22α were determined (n=3, *P<0.01 vs. vector or, #P<0.01 vs. vector+Pi). Kidney International 2015 88, 711-721DOI: (10.1038/ki.2015.160) Copyright © 2015 International Society of Nephrology Terms and Conditions

Figure 8 Rapamycin reduces vascular calcification through Klotho upregulation. (a,b) T/G human aortic smooth muscle cells (HASMCs) were transfected with scramble siRNA (C-siR) and Klotho siRNA (Si-Klotho) for 2 days; Klotho mRNA (a) and protein (b) levels were measured (n=3, *P<0.01 vs. C-siR). (c) HASMCs were transfected with C-siR or Si-Klotho, and then treated with inorganic phosphate (Pi) and/or rapamycin (Rapa) at the indicated concentrations for 10 days. Calcium content was measured (n=3, *P<0.01 vs. C-siR, #P<0.01 vs. C-siR+Pi). (d) Aortic rings of wild-type (WT) mice were cultured and treated with Pi and/or rapa (30 nmol/l) for 3 days. Klotho expression was measured by real-time PCR (*P<0.01 vs. Ctrl, #P<0.05). (e) Aortic rings of WT mice were cultured and treated with Pi and/or rapa (30 nmol/l) for 7 days. Calcium content was measured (*P<0.01 vs. Ctrl, #P<0.05 vs. Pi). (f) Klotho level in aortas of Klotho knockout (kl−/−) mice and WT littermates were measured by western blot analyses (*P<0.01 vs. WT). (g) Aortic rings of kl−/− mice (<3 weeks old) were cultured and treated with Pi and/or rapa (30 nmol/l) for 7 days. Calcium content was measured (n=3 mice, *P<0.01 vs. Ctrl. (h) Saline or rapa (1.2 mg/kg·per day) was administered intragastrically to the 3-week-old kl−/− and WT mice for 4 weeks. Aortic calcium content was measured (n=4, *P<0.01 vs. WT). Kidney International 2015 88, 711-721DOI: (10.1038/ki.2015.160) Copyright © 2015 International Society of Nephrology Terms and Conditions