Endocrinol Metab.  2017 Mar;32(1):115-123. 10.3803/EnM.2017.32.1.115.

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

• Bae KH
• Seo JB
• Jung YA
• Seo HY
• Kang SH
• Jeon HJ
• Lee JM
• Lee S
• Kim JG
• Lee IK
• Jung GS
• Park KG

• KMID: 2413295
• DOI: http://doi.org/10.3803/EnM.2017.32.1.115

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.

Keyword

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

MeSH Terms

Actins
Animals
Atrophy
Blotting, Western
Collagen Type I
Diabetic Nephropathies
Fibroblasts
Fibrosis*
In Vitro Techniques
Mesangial Cells
Mice*
Peroxisomes*
Phosphorylation
Plasminogen Activator Inhibitor 1
Polymerase Chain Reaction
Rats
Renal Insufficiency, Chronic
Reverse Transcription
Transforming Growth Factor beta
Transforming Growth Factors
Up-Regulation
Ureter*
Ureteral Obstruction*
Actins
Collagen Type I
Plasminogen Activator Inhibitor 1
Transforming Growth Factor beta
Transforming Growth Factors

Figure

• 

  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.

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