Fig 1.
PR-619 improves renal histopathological changes in mice with UUO.
Mice were treated daily with 100 μg PR-619 in 10 μL DMSO or an equal volume of vehicle by intraperitoneal injection. Paraffin-embedded sections of UUO kidneys were stained with HE and MT. (A) Representative images of HE (upper panel) and MT (lower panel) staining of kidney sections from sham-operated mice and mice with UUO treated with or without PR-619. (B) Quantification of the interstitial cell density (left panel) and interstitial fibrosis (right panel). HE staining was used to assess the interstitial cell count. MT staining was used to detect connective tissue as indicated by the blue staining. Values are expressed as the mean ± SD. Statistical analysis was performed using ANOVA followed by Tukey’s post hoc test. *P < 0.05, n = 5 mice per group. DMSO, dimethyl sulfoxide; UUO, unilateral ureteral obstruction; HE, hematoxylin-eosin; MT, Masson’s trichrome; SD, standard deviation; ANOVA, analysis of variance.
Fig 2.
PR-619 ameliorates expression of mesenchymal markers in the kidneys of mice with UUO.
Kidney samples were collected from mice with UUO after vehicle or PR-619 treatment and examined for expression of α-SMA and FSP-1 as mesenchymal markers. (A) α-SMA mRNA levels were determined by qRT-PCR in mice with UUO with or without PR-619 administration. GAPDH was used as an internal control. (B) Typical western blot analysis demonstrating the level of α-SMA protein expression. The graph shows the expression level quantified by densitometry and normalized to GAPDH. (C) Representative images showing immunostaining for α-SMA and FSP-1. Quantification is shown in the right panel. Values are expressed as the mean ± SD. Statistical analysis was performed using ANOVA followed by Tukey’s post hoc test. *P < 0.05, n = 5 mice per group. UUO, unilateral ureteral obstruction; α-SMA, α-smooth muscle actin; FSP-1, fibroblast-specific protein-1; qRT-PCR, quantitative real-time RT-PCR; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; SD, standard deviation; ANOVA, analysis of variance.
Fig 3.
PR-619 suppresses UUO-induced ECM deposition.
The same samples in Fig 2 were used to examine collagen 1, collagen 3, and fibronectin as ECM proteins. (A) Collagen 1, collagen 3, and fibronectin mRNA levels were determined by qRT-PCR in mice with UUO with or without PR-619 administration. GAPDH was used as an internal control. (B) Typical western blot analysis demonstrating the level of fibronectin protein expression. The graph shows the expression level quantified by densitometry and normalized to GAPDH. (C) Representative images showing immunostaining for collagen 1, collagen 3, and fibronectin. Quantification is shown in the right panel. Values are expressed as the mean ± SD. Statistical analysis was performed using ANOVA followed by Tukey’s post hoc test. *P < 0.05, n = 5 mice per group. ECM, extracellular matrix; qRT-PCR, quantitative real-time RT-PCR; UUO, unilateral ureteral obstruction; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; SD, standard deviation; ANOVA, analysis of variance.
Fig 4.
PR-619 reduces UUO-induced MMP expression, apoptosis, and inflammation.
The same samples in Figs 2 and 3 were used to investigate UUO-induced tissue damage including expression of MMPs, TUNEL-positive apoptotic cell death, CD68-positive macrophage infiltration, and TGF-β1 mRNA levels. (A) MMP2 and MMP9 mRNA levels were determined by qRT-PCR in mice with UUO with or without PR-619 administration. GAPDH was used as an internal control (upper panel). Typical western blot analysis demonstrating the levels of MMP2 and MMP9 protein expression. The graphs show the expression level quantified by densitometry and normalized to GAPDH (lower panel). (B) Representative photomicrograph of TUNEL staining in kidney sections. Quantification of TUNEL-positive cells is shown in the right panel. (C) Representative images showing CD68 immunostaining. Quantification is shown in the right panel. (D) TGF-β1 mRNA levels were determined by qRT-PCR. GAPDH was used as an internal control. Values are expressed as the mean ± SD. Statistical analysis was performed using ANOVA followed by Tukey’s post hoc test. *P < 0.05, n = 5 mice per group. UUO, unilateral ureteral obstruction; MMP, matrix metalloproteinase; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; TGF-β1, transforming growth factor-β1; qRT-PCR, quantitative real-time RT-PCR; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; SD, standard deviation; ANOVA, analysis of variance.
Fig 5.
PR-619 decreases Smad4 expression in UUO mice.
The same protein lysates in Figs 2–4 were used to examine expression of TGF-β1-Smad signaling molecules. (A) Typical western blot analysis demonstrating the total levels of Smad2, Smad3, Smad4, TGF-βRI, and TGF-βRII protein expression in mice with UUO with or without PR-619 administration. (B) Quantification of the expression of these proteins on a relative scale. The band intensities were normalized to GAPDH. Values are expressed as the mean ± SD. Statistical analysis was performed using ANOVA followed by Tukey’s post hoc test. *P < 0.05, n = 5 mice per group. TGF-β1, transforming growth factor-β1; TGF-βRI, Type I TGF-β receptor; TGF-βRII, Type II TGF-β receptor; UUO, unilateral ureteral obstruction; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; SD, standard deviation; ANOVA, analysis of variance.
Fig 6.
PR-619 attenuates TGF-β1-induced myofibroblastic changes and reduces Smad4 expression in NRK-49F cells.
NRK-49F renal interstitial fibroblasts were incubated with PR-619 (3 μmol/L) for 60 min and then stimulated with TGF-β1 (10 ng/mL) for 24 h. Cell lysates were subjected to western blot analysis using antibodies against α-SMA or Smad4. Typical western blot analysis demonstrating the expression levels of (A) α-SMA and (B) Smad4. Graphs show the expression levels quantified by densitometry and normalized to β-actin. Values are expressed as the mean ± SD. Statistical analysis was performed using ANOVA followed by Tukey’s post hoc test. *P < 0.05, n = 5 samples per group. NRK-49F, normal rat kidney-49F; TGF-β1, transforming growth factor-β1; α-SMA, α-smooth muscle actin; SD, standard deviation; ANOVA; analysis of variance.