Fig. 1Effects of lobeglitazone on unilateral ureteral obstruction (UUO)-induced renopathological changes. (A) Representative images of hematoxylin and eosin (H&E) and sirius red staining of kidney tissue sections from control (CON) mice and UUO mice with or without lobeglitazone (Lobe; 1 mg/kg) treatment. The number of atrophic tubules was determined by measuring abnormal and dilated tubular basement membranes in five random fields of H&E stained sections under high power magnification (×200). Areas of positive staining with sirius red were quantitated by computer-based morphometric analysis. All morphometric data were normalized against the corresponding values in CON animals. Data in all bar graphs are expressed as fold increase relative to the CON (n=6 in each group). (B) Representative images of immunohistochemical staining forp-Smad3, α-smooth muscle actin (α-SMA), plasminogen activator inhibitor 1 (PAI-1), and type I collagen in kidney tissue sections from CON mice or UUO mice with or without lobeglitazone (1 mg/kg). Areas of positive staining with p-Smad3, α-SMA, PAI-1, and type 1 collagen antibodies were quantitated by computer-based morphometric analysis. All data were expressed as the mean±SEM of five random fields from each kidney section (n=6 in each group). aP<0.05; bP<0.01; cP<0.001 vs. CON; and dP<0.05; eP<0.01; fP<0.001 vs. UUO.
Fig. 2Effects of lobeglitazone on profibrotic gene expression in kidneys of unilateral ureteral obstruction (UUO) mice. (A) Representative Western blot analysis of p-Smad3, t-Smad3, α-smooth muscle actin (α-SMA), plasminogen activator inhibitor 1 (PAI-1), and type 1 collagen protein expression in UUO kidneys with or without lobeglitazone (Lobe; 1 mg/kg; n=6 in each group). Data are expressed as the mean±SEM of three independent experiments. (B) Representative real-time reverse transcription polymerase chain reaction analysis of α-SMA, PAI-1, and type 1 collagen mRNA expression in UUO kidneys with or without Lobe (1 mg/kg; n=6 in each group). Data in bar graphs are mean±SEM. aP<0.05; bP<0.01; cP<0.001 vs. control (CON); and dP<0.05; eP<0.01; fP<0.001 vs. UUO.
Fig. 3Effects of lobeglitazone (Lobe) on transforming growth factor β (TGF-β)-induced profibrotic gene expression in cultured kidney cell lines. Representative Western blot analysis (A) of p-Smad3, t-Smad3, α-smooth muscle actin (α-SMA), plasminogen activator inhibitor 1 (PAI-1), and type 1 collagen levels and representative real-time reverse transcription polymerase chain reaction (RT-PCR) analysis (B) of α-SMA, PAI-1, and type 1 collagen expression in TGF-β-stimulated NRK-49F cells. Representative Western blot analysis (C) of p-Smad3, t-Smad3, α-SMA, PAI-1, and type 1 collagen expression and representative real-time RT-PCR analysis (D) of α-SMA, PAI-1, and type 1 collagen expression in TGF-β-stimulated rat mesangial cells. Cells were co-incubated with TGF-β (5 ng/mL) and Lobe (10 µM) after 24 hours serum starvation. Data are the mean±SEM of three independent measurements (three separate experiments). aP<0.05; bP<0.01; cP<0.001 vs. control (CON); and dP<0.05; eP<0.01; fP<0.001 vs. TGF-β alone.
Fig. 4Effects of lobeglitazone (Lobe) on the transforming growth factor β (TGF-β)/Smad3 signaling pathway in kidney cell lines. (A) Effects of Lobe on plasminogen activator inhibitor 1 (PAI-1) promoter activity in NRK-49F cells. Cells were treated with TGF-β (5 ng/mL) with or without Lobe co-treatment (10 µM) for 24 hours (left panel). (B) Effects of Lobe on PAI-1 promoter activity in rat mesangial cells. Cells were treated with TGF-β (5 ng/mL) with or without Lobe co-treatment (10 µM) for 24 hours (left panel). Cells were co-transfected with the PAI-1 promoter and expression vectors for Smad3/4 (pRK5) and ALK5 (pcDNA) with or without Lobe treatment (10 µM) for 24 hours (right panel). Data are the mean±SEM of three independent measurements. aP<0.01; bP<0.001 vs. control; and cP<0.001 vs. TGF-β alone or vs. Smad3/4 and ALK5.