Data showed positive stained renal tubular epithelial 
cells in wild-type mice (WT+Sham), but not in A2AR knockout mice (KO+Sham). Scale bar = 50 mm, 400x. (C) Demonstration of the renal levels of A2AR mRNA in non-UUO mice and at day 3, 7 and 14 post-UUO 22948146 in mice. Data are mean 6 SD. n = 10 per time point. * P,0.05, vs non-UUO WT mice; P,0.05; vs WT day 3; # P,0.05, vs WT day 7, accordingly. doi:10.1371/journal.pone.0060173.gAdenosine A2AR and Renal Interstitial FibrosisMedChemExpress BTZ043 Figure 2. A2AR activity affected UUO-induced deposition of collagen I. (A) Representative immunohistochemistry of renal collagen I (Col I) from the A2AR KO and WT mice, at day 3, 7 and 14 post-UUO or sham surgery (Sham), following treatment of CGS21680 (CGS) or vehicle (Veh). Scale bar = 50 mm, 4006. (B) Demonstration of Col I deposition in the post-UUO WT animals received treatment of vehicle (WT+UUO+Veh) or A2AR agonist CGS21680 (WT+UUO+CGS), and in the A2AR post-UUO KO mice received treatment of vehicle (KO+UUO+Veh), or CGS21680 (KO+UUO+CGS), at day 3, 7 and 14 post-UUO, along with that in sham control animals (WT+Sham and KO+Sham)(n = 10 per group). Data are mean 6 SD. * P,0.05 between two compared groups; NS, no significance. doi:10.1371/journal.pone.0060173.gWestern blotThe Western blot was performed according as previously described [24] with modification. Briefly, 
mouse kidneys were first homogenized in tissue protein extraction reagent (Thermo scientific, cat# MD156494) with a protease inhibitor cocktail (Thermo scientific, cat# ME156994) according to the manufacturer’s instructions. Forty mg of protein ��-Sitosterol ��-D-glucoside site extracts from each sample were loaded on and separated by 10 10457188 SDS-PAGE, then transferred onto nitrocellulose membrane. The blots were probed overnight at 4uC with primary antibodies against E-cadherin (ab76055, 1:1000, Abcam), a-SMA (ab7817, 1:200, Abcam), and b-actin (a2228, 1:2000, Sigma-Aldrich), respectively, followed by the respective horseradish peroxidase-linked secondary antibody (a4416, 1:5000, Sigma-Aldrich). Horseradish peroxidase activity was visualized via an enhanced chemiluminescence kit (20-500120, Biolind, Israel). Images were scanned and processed for densitometric quantification by the Image analysis program (Labworks 4.0, UVP).Results 1. A2AR activation attenuated collagen deposition in matrix accumulationTo evaluate the effect of A2AR on renal fibrosis, we applied the UUO model to mice combined with A2AR agonist CGS21680 and genetic A2AR inactivation (as aforementioned paradigm in Methods). Pathology assessment using H E staining and immunohistostaining of Col I and Col III deposition were evaluated at day 3, 7, and 14 after UUO. Our H E data demonstrated the successfulness of UUO modeling with featured pathological changes, e.g. progressively aggravated tubular dilatation and leukocytes infiltration (Figure 1 A). Our A2AR immunochemistry data demonstrated that positive stained renal tubular epithelial cells were seen in WT mice (WT+Sham), but devoid in KO mice (KO+Sham) (Figure 1B). Furthermore, we used RT-qPCR to detect the temporal changes of A2AR mRNA expression in the progress of UUO-induced RIF. We showed that the mRNA level of A2AR was significantly increased at day 3 through day 14 postUUO, in a time-dependent manner. WT mice in WT+UUO+Veh group displayed an increase of 156 , 529 and 816 at day 3, 7 and 14, respectively, compared to non-UUO mice (F = 541.22, P,0.05, n = 10 per time point, Figure 1C). Conversely, A2AR mRNA level in.Data showed positive stained renal tubular epithelial cells in wild-type mice (WT+Sham), but not in A2AR knockout mice (KO+Sham). Scale bar = 50 mm, 400x. (C) Demonstration of the renal levels of A2AR mRNA in non-UUO mice and at day 3, 7 and 14 post-UUO 22948146 in mice. Data are mean 6 SD. n = 10 per time point. * P,0.05, vs non-UUO WT mice; P,0.05; vs WT day 3; # P,0.05, vs WT day 7, accordingly. doi:10.1371/journal.pone.0060173.gAdenosine A2AR and Renal Interstitial FibrosisFigure 2. A2AR activity affected UUO-induced deposition of collagen I. (A) Representative immunohistochemistry of renal collagen I (Col I) from the A2AR KO and WT mice, at day 3, 7 and 14 post-UUO or sham surgery (Sham), following treatment of CGS21680 (CGS) or vehicle (Veh). Scale bar = 50 mm, 4006. (B) Demonstration of Col I deposition in the post-UUO WT animals received treatment of vehicle (WT+UUO+Veh) or A2AR agonist CGS21680 (WT+UUO+CGS), and in the A2AR post-UUO KO mice received treatment of vehicle (KO+UUO+Veh), or CGS21680 (KO+UUO+CGS), at day 3, 7 and 14 post-UUO, along with that in sham control animals (WT+Sham and KO+Sham)(n = 10 per group). Data are mean 6 SD. * P,0.05 between two compared groups; NS, no significance. doi:10.1371/journal.pone.0060173.gWestern blotThe Western blot was performed according as previously described [24] with modification. Briefly, mouse kidneys were first homogenized in tissue protein extraction reagent (Thermo scientific, cat# MD156494) with a protease inhibitor cocktail (Thermo scientific, cat# ME156994) according to the manufacturer’s instructions. Forty mg of protein extracts from each sample were loaded on and separated by 10 10457188 SDS-PAGE, then transferred onto nitrocellulose membrane. The blots were probed overnight at 4uC with primary antibodies against E-cadherin (ab76055, 1:1000, Abcam), a-SMA (ab7817, 1:200, Abcam), and b-actin (a2228, 1:2000, Sigma-Aldrich), respectively, followed by the respective horseradish peroxidase-linked secondary antibody (a4416, 1:5000, Sigma-Aldrich). Horseradish peroxidase activity was visualized via an enhanced chemiluminescence kit (20-500120, Biolind, Israel). Images were scanned and processed for densitometric quantification by the Image analysis program (Labworks 4.0, UVP).Results 1. A2AR activation attenuated collagen deposition in matrix accumulationTo evaluate the effect of A2AR on renal fibrosis, we applied the UUO model to mice combined with A2AR agonist CGS21680 and genetic A2AR inactivation (as aforementioned paradigm in Methods). Pathology assessment using H E staining and immunohistostaining of Col I and Col III deposition were evaluated at day 3, 7, and 14 after UUO. Our H E data demonstrated the successfulness of UUO modeling with featured pathological changes, e.g. progressively aggravated tubular dilatation and leukocytes infiltration (Figure 1 A). Our A2AR immunochemistry data demonstrated that positive stained renal tubular epithelial cells were seen in WT mice (WT+Sham), but devoid in KO mice (KO+Sham) (Figure 1B). Furthermore, we used RT-qPCR to detect the temporal changes of A2AR mRNA expression in the progress of UUO-induced RIF. We showed that the mRNA level of A2AR was significantly increased at day 3 through day 14 postUUO, in a time-dependent manner. WT mice in WT+UUO+Veh group displayed an increase of 156 , 529 and 816 at day 3, 7 and 14, respectively, compared to non-UUO mice (F = 541.22, P,0.05, n = 10 per time point, Figure 1C). Conversely, A2AR mRNA level in.
