Lobeglitazone, a Novel Peroxisome Proliferator-Activated Receptor γ Agonist, Attenuates Renal Fibrosis Caused by Unilateral Ureteral Obstruction in Mice

Abstract

Background: Renal tubulointerstitial fibrosis is a common feature of the final stage of nearly all cause types of chronic kidney disease. Although classic peroxisome proliferator-activated receptor γ (PPARγ) agonists have a protective effect on diabetic nephropathy, much less is known about their direct effects in renal fibrosis. This study aimed to investigate possible beneficial effects of lobeglitazone, a novel PPARγ agonist, on renal fibrosis in mice.

Methods: We examined the effects of lobeglitazone on renal tubulointerstitial fibrosis in unilateral ureteral obstruction (UUO) induced renal fibrosis mice. We further defined the role of lobeglitazone on transforming growth factor (TGF)-signaling pathways in renal tubulointerstitial fibrosis through in vivo and in vitro study.

Results: Through hematoxylin/eosin and sirius red staining, we observed that lobeglitazone effectively attenuates UUO-induced renal atrophy and fibrosis. Immunohistochemical analysis in conjunction with quantitative reverse transcription polymerase chain reaction and Western blot analysis revealed that lobeglitazone treatment inhibited UUO-induced upregulation of renal Smad-3 phosphorylation, α-smooth muscle actin, plasminogen activator inhibitor 1, and type 1 collagen. In vitro experiments with rat mesangial cells and NRK-49F renal fibroblast cells suggested that the effects of lobeglitazone on UUO-induced renal fibrosis are mediated by inhibition of the TGF-β/Smad signaling pathway.

Conclusion: The present study demonstrates that lobeglitazone has a protective effect on UUO-induced renal fibrosis, suggesting that its clinical applications could extend to the treatment of non-diabetic origin renal disease.

Keywords: Lobeglitazone; Renal tubulointerstitial fibrosis; Transforming growth factor beta; Unilateral ureteral obstruction.

Fig. 1. Effects 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. 2. Effects 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. 3. Effects 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. 4. Effects 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.