Volume 65, Issue 3, Pages 961-971 (March 2004) PPARγ ligands attenuate mesangial contractile dysfunction in high glucose  Maki Ueta, Masanori Wakisaka, Tetsuro Ago, Takanari Kitazono, Udai Nakamura, Mototaka Yoshinari, Masanori Iwase, Mitsuo Iida  Kidney International  Volume 65, Issue 3, Pages 961-971 (March 2004) DOI: 10.1111/j.1523-1755.2004.00474.x Copyright © 2004 International Society of Nephrology Terms and Conditions

Figure 1 Effects of TNF-α (left) and TPA (right) on peroxisome proliferator–activated receptor γ (PPARγ)1 protein of rat mesangial cells in 5mmol/L glucose. The cells were treated with TNF-α for 1day and with TPA for 2days, respectively. Relative density of the PPARγ1 protein compared to that in 5mmol/L glucose was determined as described in Methods. Upper panel: Western blots of PPARγ1 protein. Lower panel: Relative density of PPARγ1 protein. TNF-α, tumor necrosis factor-α; TPA, 12-O-tetradecarbonyl 13-acetate; Pio, pioglitazone. Data are mean ± SEM. N = 4 in each condition. *P < 0.01 vs. control. Kidney International 2004 65, 961-971DOI: (10.1111/j.1523-1755.2004.00474.x) Copyright © 2004 International Society of Nephrology Terms and Conditions

Figure 2 Serial changes in peroxisome proliferator–activated receptor γ (PPARγ)1 protein of rat mesangial cells during incubation in 20mmol/L glucose for 5days. Upper panel: Western blots of PPARγ1 protein. Lower panel: Relative density of PPARγ1 protein. Data are mean ± SEM. N = 16 in each condition. *P < 0.01 vs. 5mmol/L glucose. Jurcut cell: used as an internal standard for the PPARγ1 protein. Kidney International 2004 65, 961-971DOI: (10.1111/j.1523-1755.2004.00474.x) Copyright © 2004 International Society of Nephrology Terms and Conditions

Figure 3 Effects of peroxisome proliferator–activated receptor γ(PPARγ) ligand, antiinflammatory agents, and PD98059 on PPARγ1 protein of rat mesangial cells after 5days incubation in 20mmol/L glucose. Pio, 10-6 mol/L pioglitazone, 15dPGJ2, 10-5 mol/L 15 deoxy-12, 14 delta-prostaglandin J2, indo, 2 × 10-5 mol/L indomethacin, Asp, 10-4 mol/L acetyl salicylic acid, PD98059, 4 × 10-6 mol/L mitogen-activated kinase kinase inhibitor. Upper panel: Western blots of PPARγ1 protein. Lower panel: Relative density of PPARγ1 protein. Data are mean ± SEM. N = 8 of each condition *P < 0.01 vs. 5mmol/L glucose. #P < 0.01 vs. 20mmol/L glucose. Kidney International 2004 65, 961-971DOI: (10.1111/j.1523-1755.2004.00474.x) Copyright © 2004 International Society of Nephrology Terms and Conditions

Figure 4 Effect of PD98058 or pioglitazone on the reduced peroxisome proliferator–activated receptor γ (PPARγ)1 mRNA under 20mmol/L glucose, TNF-α, and TPA condition. The cells were treated with 20mmol/L glucose for 5days, TNF-α for 1day, and TPA for 2days in the presence and absence of PD98058 or pioglitazone, respectively. Upper panel: PCR-products for PPARγ1 (418bp) and GAPDH (307bp) in a 2% agarose gel. N, 5mmol/L glucose, H, 20mmol/L glucose. Lower panel: Relative PPARγ1/GAPDH ratio compared to that in 5mmol/L glucose. N = 6 of each condition. Data are mean ± SEM. *P < 0.01 vs. 5mmol/L glucose. #P < 0.01 vs. 20mmol/L glucose. Kidney International 2004 65, 961-971DOI: (10.1111/j.1523-1755.2004.00474.x) Copyright © 2004 International Society of Nephrology Terms and Conditions

Figure 5 Effects of peroxisome proliferator–activated receptor γ (PPARγ) ligand, 10-6 mol/L pioglitazone (Pio), 2 × 10-5 mol/L indomethacin (Indo), 10-4 mol/L acetyl salicylic acid (Asp) on α-smooth muscle actin of rat mesangial cells after 5days incubation in 20mmol/L glucose. Upper panel: Western blots of PPARγ1 protein. Lower panel: Relative density of PPARγ1 protein. N = 4 of each condition. Data are mean ± SEM. *P < 0.01 vs. 5mmol/L glucose. #P < 0.01 vs. 20mmol/L glucose. Kidney International 2004 65, 961-971DOI: (10.1111/j.1523-1755.2004.00474.x) Copyright © 2004 International Society of Nephrology Terms and Conditions

Figure 6 Representative morphologic changes of cultured rat mesangial cells under various conditions after exposure to angiotensin II. Normal glucose condition before (A) and after (B), high glucose condition before (C) and after (D), high glucose with 10-7mol/L pioglitazone before (E) and after (F), and high glucose with 10-6 mol/L pioglitazone before (G) and after (H) exposure to 10-7 mol/L angiotensin II. Kidney International 2004 65, 961-971DOI: (10.1111/j.1523-1755.2004.00474.x) Copyright © 2004 International Society of Nephrology Terms and Conditions

Figure 7 Changes in surface area of rat mesangial cells in the presence of different concentrations of angiotensin II (A) and effect of high glucose on 10-7 mol/L angiotensin II-induced contraction of cells during incubation in 20mmol/L glucose for 5days (B). Data are mean ± SEM of 20 to 30 cells from 3 separate experiments. ‡P < 0.01 vs. 5mmol/L glucose (no agonist) *P < 0.01 vs. 5mmol/L glucose + angiotensin II. #P < 0.01 vs. 20mmol/L glucose + angiotensin II. Kidney International 2004 65, 961-971DOI: (10.1111/j.1523-1755.2004.00474.x) Copyright © 2004 International Society of Nephrology Terms and Conditions

Figure 8 Effect of pioglitazone on angiotensin II-induced contraction (A) and cell viability (B) of cells cultured in either 5mmol/L or 20mmol/L glucose for 5days. The changes in cell surface area in response to 10-7 mol/L angiotensin II were analyzed. Data are mean ± SEM of 20 to 30 cells from 3 separate experiments. *P < 0.01 vs. 5mmol/L glucose + angiotensin II. #P < 0.01 vs. 20mmol/L glucose + angiotensin II. (B) The cell viability was analyzed using MTT assay as described in Methods.N = 6 of each condition. Data are mean ± SEM *P < 0.01 vs. 5mmol/L glucose. #P < 0.01 vs. 20mmol/L glucose. Kidney International 2004 65, 961-971DOI: (10.1111/j.1523-1755.2004.00474.x) Copyright © 2004 International Society of Nephrology Terms and Conditions

Figure 9 Effects of 10-5 mol/L 15 deoxy-12, 14 delta-prostaglandin J2 (15dPDJ2), 2 × 10-5 mol/L indomethacin (indo), 10-4 mol/L acetyl salicylic acid (Asp), and 4 × 10-6 mol/L PD98059 on angiotensin II-induced contraction and change in surface area of rat mesangial cells after 5days incubation in 20mmol/L glucose. Data are mean ± SEM of 20 to 30 cells from 3 separate experiments. *P < 0.01 vs. 5mmol/L glucose + angiotensin II. #P < 0.01 vs. 20mmol/L glucose + angiotensin II after 5days incubation. Kidney International 2004 65, 961-971DOI: (10.1111/j.1523-1755.2004.00474.x) Copyright © 2004 International Society of Nephrology Terms and Conditions

Figure 10 Effects of antisense DNA on peroxisome proliferator–activated receptor γ(PPARγ)1 protein, α-smooth muscle actin, and contractile response to angiotensin II of rat mesangial cells in 5mmol/L glucose. (A) The cells were treated with antisense and sense DNA for 6hours, and then incubated in 5mmol/L glucose for 2days. Upper panel: Western blots of PPARγ1 protein. Lower panel: Relative density of PPARγ1 protein. Data are mean ± SEM. N = 4 in each condition. *P < 0.05 vs. control. **P < 0.01 vs. control. (B) Upper panel: Western blots of α-smooth muscle actin. Lower panel: Relative density of α-smooth muscle actin. Data are mean ± SEM. N = 4 in each condition. *P < 0.05 vs. 0.5 μmol/L and 1.0 μmol/L sense DNA. (C) Data are mean ± SEM of 40 to 50 cells. *P < 0.001 before vs. after angiotensin II stimulation in each condition. #P < 0.05 vs. 5mmol/L glucose alone. ##P < 0.01 vs. 5mmol/L glucose alone. †P < 0.01 vs. 0.5 μmol/L and 1.0 μmol/L sense DNA. Kidney International 2004 65, 961-971DOI: (10.1111/j.1523-1755.2004.00474.x) Copyright © 2004 International Society of Nephrology Terms and Conditions