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Volume 69, Issue 10, Pages 1780-1785 (May 2006) Indoxyl sulfate stimulates proliferation of rat vascular smooth muscle cells H. Yamamoto, S. Tsuruoka, T. Ioka, H. Ando, C. Ito, T. Akimoto, A. Fujimura, Y. Asano, E. Kusano Kidney International Volume 69, Issue 10, Pages 1780-1785 (May 2006) DOI: 10.1038/sj.ki.5000340 Copyright © 2006 International Society of Nephrology Terms and Conditions

Figure 1 Effect of IS on the proliferation of rat VSMCs by 4-[3-[4-lodophenyl]-2-4(4-nitrophenyl)-2H-5-tetrazolio-1,3-benzene disulfonate] proliferation assay. Growth-arrested VSMCs were stimulated by IS and PDGF for 24 h. 4-[3-[4-lodophenyl]-2-4(4-nitrophenyl)-2H-5-tetrazolio-1,3-benzene disulfonate] was added for the last 4 h. PDGF (10 ng/ml) was used as a positive control. Absorbance was measured by an enzyme-linked immunosorbent assay reader. Values are mean±s.e.m. (n=8). *P<0.05 compared with the vehicle. Kidney International 2006 69, 1780-1785DOI: (10.1038/sj.ki.5000340) Copyright © 2006 International Society of Nephrology Terms and Conditions

Figure 2 Effect of IS on the proliferation of rat VSMCs assessed by DNA synthesis. (a) Growth-arrested VSMCs were stimulated by IS and PDGF for 24 h. [3H]Thymidine was added for the last 6 h. PDGF (10 ng/ml) was used as a positive control. (b) Effect of IS on the proliferation of rat VSMCs in the presence of 4 g/dl albumin in the medium assessed by DNA synthesis. Values are mean±s.e.m. (n=8). *P<0.05 compared with the vehicle. Kidney International 2006 69, 1780-1785DOI: (10.1038/sj.ki.5000340) Copyright © 2006 International Society of Nephrology Terms and Conditions

Figure 3 Effect of the removal of tryptophan on IS-induced increase of DNA synthesis in rat VSMCs. Growth-arrested VSMCs were incubated for 24 h in DMEM or tryptophan (-) medium with or without IS (250 μM). [3H]Thymidine was added for the last 6 h. Values are mean±s.e.m. (n=8). *P<0.05 compared with vehicle. Kidney International 2006 69, 1780-1785DOI: (10.1038/sj.ki.5000340) Copyright © 2006 International Society of Nephrology Terms and Conditions

Figure 4 Effect of probenecid on IS-induced increase of DNA synthesis in rat VSMC. Growth-arrested VSMCs were pretreated for 1 h with probenecid (0.5 mM) and then stimulated by IS (250 μM) for 24 h. Values are mean±s.e.m. (n=8). *P<0.05. Kidney International 2006 69, 1780-1785DOI: (10.1038/sj.ki.5000340) Copyright © 2006 International Society of Nephrology Terms and Conditions

Figure 5 Immunohistochemistry of rat OAT3 in rat VSMCs. (a) VSMCs were stained with polyclonal antibodies against rat OAT3 (b) There was no staining in the negative control with rabbit immunoglobulin fraction. Bar=200 μm. Kidney International 2006 69, 1780-1785DOI: (10.1038/sj.ki.5000340) Copyright © 2006 International Society of Nephrology Terms and Conditions

Figure 6 Effect of IS on p44/42 MAPK phosphorylation. Growth-arrested VSMCs were stimulated by IS (250 μM) for the indicated times. (a) Representative immunoblots are shown with antibodies that recognize phosphorylated p44/42 MAPK and total p44/42 MAPK. (b) Densitometric analysis of phosphorylated p44/42 MAPK. Values are mean±s.e.m. (n=5). *P<0.05. Kidney International 2006 69, 1780-1785DOI: (10.1038/sj.ki.5000340) Copyright © 2006 International Society of Nephrology Terms and Conditions

Figure 7 Effect of PD98059 on IS-induced phosphorylation of p44/42 MAPK. Growth-arrested VSMCs were pretreated with PD98059 (10 μM) for 1 h and then stimulated by IS (250 μM) for 5 min. (a) Representative immunoblots are shown with antibodies that recognize phosphorylated p44/42 MAPK and total p44/42 MAPK. (b) Densitometric analysis of phosphorylated p44/42 MAPK. Values are mean±s.e.m. (n=5). *P<0.05. Kidney International 2006 69, 1780-1785DOI: (10.1038/sj.ki.5000340) Copyright © 2006 International Society of Nephrology Terms and Conditions

Figure 8 Effect of PD98059 on IS-induced proliferation of rat VSMCs assessed by DNA synthesis. Growth-arrested VSMCs were pretreated for 1 h with PD98059 (10 μM) and then stimulated with IS (250 μM) for 24 h. Values are mean±s.e.m. (n=8). *P<0.05. Kidney International 2006 69, 1780-1785DOI: (10.1038/sj.ki.5000340) Copyright © 2006 International Society of Nephrology Terms and Conditions

Figure 9 Effect of IS on mRNA expressions of PDGF-A and -C chains and PDGF-β receptor. Growth-arrested VSMCs were stimulated with IS (250 μM) for 6 h and then total RNA was isolated. Real-time quantitative PCR was performed as described in Materials and Methods. Values are mean±s.e.m. of two identical samples performed in at least triplicate (n=4 in each). *P<0.05 compared with the vehicle. Kidney International 2006 69, 1780-1785DOI: (10.1038/sj.ki.5000340) Copyright © 2006 International Society of Nephrology Terms and Conditions