Fig 1.
Effects of Ang-(1–7) on fibrosis and apoptosis in obstructed rat kidneys.
Male Sprague-Dawley rats were subcutaneously implanted with osmotic minipumps to deliver Ang-(1–7) (24 μg/[kg·h]) or vehicle (sterile saline) 3 days before the unilateral ureteral obstruction (UUO) operation. The UUO lasted for 7 days. (a) α-smooth muscle actin (α-SMA) protein expression was assessed in UUO kidneys and unaffected controls. (b) Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assays in kidneys from UUO rats following treatment with Ang-(1–7). (c) Prosurvival and proapoptotic protein expression was assessed in kidneys from UUO rats following treatment with Ang-(1–7). (d) Representative immunohistochemical staining of α-SMA, Bax, and Bcl-2 expression in UUO rats. Each column represents the mean ± SEM. Original magnification, 200×. *P < 0.05, compared with controls. †P < 0.05, compared with UUO rats.
Fig 2.
Effects of Ang-(1–7) on TGF-β1/Smad signaling in obstructed rat kidneys.
(a) Tumor growth factor-β1 (TGF-β1) and Smad signaling was examined by western blot in UUO rat kidneys following treatment with Ang-(1–7). Each column represents the mean ± SEM. *P < 0.05, compared with controls. †P < 0.05, compared with UUO rats. (b) Representative immunohistochemical staining of TGF-β1 expression in UUO rats. Original magnification, 200×.
Fig 3.
Effects of Ang-(1–7) on the ACE/Ang II/AT1R signaling axis in obstructed rat kidneys.
(a) Angiotensin-converting enzyme (ACE), angiotensin II (Ang II), and angiotensin type 1 receptor (AT1R) protein expression was examined in UUO rat kidneys. Each column represents the mean ± SEM. *P < 0.05, compared with controls. †P < 0.05, compared with UUO rats. (b) Representative immunohistochemical staining of AT1R expression in UUO rats. Original magnification, 200×.
Fig 4.
Effects of A779 on angiotensin receptors in Ang II stimulated rat tubular epithelial cells.
(a) NRK-52E cells were exposed to Ang II (1 μM, 16 h) with or without 1 h pretreatment with Ang-(1–7) (1 μM) and Mas receptor antagonist A779 (1 μM). Each column represents mean ± SEM. *P < 0.05. **P < 0.01. Data are representative of at least three independent experiments. (b) AT1R expression (red) was examined in NRK-52E cells after treatment with Ang II, Ang-(1–7), and A779; nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI) for immunofluorescence study. Original magnification, 400×. Scale bar = 50 μm. MasR, Mas receptor.
Fig 5.
Effects of A779 on TGF-β1/Smad signaling and ROS generation in Ang II-stimulated rat tubular epithelial cells.
NRK-52E cells were exposed to Ang II (1 μM, 16 h) with or without 1 h pretreatment with Ang-(1–7) (1 μM) and Mas receptor antagonist A779 (1 μM). (a) TGF-β1/Smad expression was analyzed. (b) The generation of reactive oxygen species (ROS) was detected using the ROS-sensitive fluorescent dye, DCF-DA. Each column represents the mean ± SEM. *P < 0.05. **P < 0.01. Data are representative of at least three independent experiments.
Fig 6.
Effects of A779 on fibrosis and apoptosis in Ang II-stimulated rat tubular epithelial cells.
NRK-52E cells were exposed to Ang II (1 μM, 16 h) with or without 1 h pretreatment with Ang-(1–7) (1 μM) and Mas receptor antagonist A779 (1 μM). (a) α-smooth muscle actin (α-SMA), connective tissue growth factor (CTGF), and fibronectin, as well as (b) the Bax and Bcl-2 protein expressions were then analyzed. (c) Downregulation of Mas receptor protein in NRK-52E cells at 48 h after Mas receptor siRNA transfection. NRK-52E cells were transfected with Mas receptor siRNA and then exposed to Ang II (1 μM, 16 h) with or without 1 h pretreatment with Ang-(1–7) (1 μM). Western blot analyses represented the (d) α-SMA, CTGF and fibronectin, as well as (e) the Bax and Bcl-2 protein expression. Each column represents the mean ± SEM. *P < 0.05. **P < 0.01. Data are representative of at least three independent experiments.
Fig 7.
Effects of A779 on cell cycle arrest in Ang II-stimulated rat tubular epithelial cells.
(a) Ang II (1 μM, 16 h) with or without 1 h pretreatment with Ang-(1–7) (1 μM) and Mas receptor antagonist A779 (1 μM). Each column represents the mean ± SEM. *P < 0.05. Data are representative of at least three independent experiments. (b) Phosphorylated histone H3 (Ser 10) expression (red) was examined in NRK-52E cells after treatment with Ang II, Ang-(1–7), and A779; nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI) for immunofluorescence study. Original magnification, 400×. Scale bar = 20 μm.
Fig 8.
Effects of exogenous Ang-(1–7) on ACE2 and TACE expression in Ang II-stimulated rat tubular epithelial cells.
NRK-52E cells were exposed to Ang II (1 μM, 16 h) with or without 1 h pretreatment with Ang-(1–7) (1 μM) and Mas receptor antagonist A779 (1 μM). Each column represents the mean ± SEM. *P < 0.05. **P < 0.01. Data are representative of at least three independent experiments.