Wild-type mice underwent UUO and were treated with Ang-(1–7) or saline (systemic infusion by osmotic minipumps). (A) A representative western blot showing renal Bax and Bcl-xL expression (upper panel) and the calculation of the Bax/Bcl-xl ratio (lower panel) after 5 days of UUO. Open and black bars represent data of saline- or Ang-(1–7)-infused mice, respectively, expressed as mean±SEM of 6 animals per group. Figures B and C show representative kidney sections (B: cortex with glomerulus; C: medulla) with van Gieson staining and anti-F4/80 immunostaining, respectively (positive immunoreactivity reveals brown signal [arrowheads]). Magnification: 400×. (D) Infiltrating leukocytes were counted based on their morphology and location within renal cortex, medulla, and pelvis in mice infused with saline or Ang-(1–7). (E) Renal NF-κB activation was measured in wild-type mice infused with saline or Ang-(1–7) and in Mas-deficient mice (Mas−/−) and their respective wild-type controls (Mas+/+) at 2 and 5 days of UUO. The figure shows data of renal NF-κB activity expressed as n-fold increase vs. contralateral kidney as mean±SEM of 6 animals per group analyzed in duplicate. (F) Shows a representative EMSA experiment. Competition assay with a 100-fold excess of unlabeled NF-κB shows the specificity of the binding (marked by arrows). The position of free oligonucleotides is indicated. (G) Shows data of gene expression of proinflammatory factors (MCP-1 and IL-6) obtained by real-time PCR experiments and expressed as n-fold increase vs. contralateral kidney as mean±SEM of 8–10 animals per group; C: contralateral, O: obstructed kidneys. * P<0.05 vs. contralateral kidney of the same genotype; # P<0.05 vs. Mas+/+ obstructed kidney; ¥ P<0.05 vs. saline-infused obstructed kidneys.

Mas-deficient mice (Mas−/−) and mice deficient in all three Ang II receptors (AT1A, AT1B and AT2 = TKO) and their wild-type controls (Mas+/+ and TWT) were infused with Ang-(1–7) or saline for 5 days. Part of the Ang-(1–7)-infused mice were co-treated with the specific Ang-(1–7)-antagonist A779. A shows representative kidney slices stained by anti F4/80 antibody of each group and B summarizes the computer analysis of the monocytes/macrophages score. Results are expressed as F4/80 positive cells/mm² as mean±SEM of 6–10 animals per group. (C) NF-κB data of densitometric analysis of EMSA experiments expressed as mean±SEM of 6–10 animals per group analyzed in duplicate were performed in all 4 investigated genotypes under the three different treatment regimes. *P<0.05 vs. saline-infused of each genotype, # P<0.05 vs Ang-(1–7)-infused Mas+/+. Ŧ P<0.05 vs. Ang-(1–7)-infused TWT. ψ P<0.05 vs. Ang-(1–7)-infused TKO.

UUO was set in Mas-deficient (Mas−/−) mice and their wild-type controls (Mas+/+), and animals were studied after 2, 5 and 7 days; Masson staining (A) and Azan blue (B) after 2 days and Masson staining after 7 days (C), respectively; magnification: 100×, 200× and 100×, respectively. (D) Shows the quantification of glomeruli with pathomorphological lesions such as collapse, matrix deposition, microthrombi and cellular infiltrates, in UUO kidneys of Mas-deficient mice and their wild-type controls. (E) Demonstrates lower concentrations of blood urea nitrogen in Mas-deficient mice 2 days after UUO. (F) Shows evaluation of apoptosis-related proteins at 2 days of UUO. Data of renal Bax/Bcl-xl ratio expressed as n-fold of increase vs. contralateral kidneys of each genotype, expressed as mean±SEM of 6–8 animals per group. Open and black bars represent Mas+/+ or Mas−/−, respectively; C: contralateral, O: obstructed kidneys; * P<0.05 vs. contralateral kidney of its own genotype; # P<0.05 vs. Mas+/+ obstructed kidney. (G) Shows the computer analysis of monocytes/macrophages scoring 2, 5 and 7 days of UUO. The presence of inflammatory cell infiltration was determined by immunohistochemistry with anti-F4/80 antibody (specific for monocytes/macrophages; brown staining); magnification: 200×. There were no differences between the number of monocytes/macrophages in contralateral kidneys at 2, 5 and 7 days of UUO (data not shown). Results are expressed as F4/80 positive cells/mm² as mean±SEM of 6–10 animals per group. Open bars show data of Mas +/+ and black bars of Mas−/− kidneys, * P<0.05 vs. contralateral kidney of the same genotype; # P<0.05 vs. Mas+/+ obstructed kidneys; § P<0.05 vs. Mas+/+ mice. (H) Illustrates inflammatory cell infiltration in obstructed kidneys (cortex) of Mas+/+ and Mas−/− mice (representative kidneys of 6–10 studied in each genotype, each time point).

I/R was performed in wild-type mice infused with saline (I/R Saline) or Ang-(1–7) (I/R Ang-(1–7)) and in mice deficient for Mas (I/R Mas−/−) and their wild-type controls (I/R Mas+/+). Animals were studied 3 days after I/R. Figures A and B show Ladewig and PAS staining respectively of group-representing renal sections. Magnification: 400×. (C) Infiltrating leukocytes were counted based on their morphology and location within renal cortex, medulla, and pelvis in mice infused with saline or Ang-(1–7) and in wild-type (Mas+/+) and Mas-deficient mice (Mas−/−). Renal NF-κB activity after I/R was measured by EMSA (D) and calculated (E) as renal NF-κB activity expressed as n-fold increase vs. wild-type controls and shown as mean±SEM of 6–10 animals per group analyzed in duplicate. ¥ P<0.05 vs. saline-infused mice; # P<0.05 vs. Mas+/+ mice. The right diagram (F) shows mRNA levels of MCP-1 and IL-6 expressed as n-fold increase vs. wild-type controls and expressed as mean±SEM of 6–8 animals per group analyzed in duplicate. ¥ P<0.05 vs. saline-infused mice; # P<0.05 vs. Mas+/+ mice.

(A) Growth arrested cells were treated with 10⁻⁷ mol/L Ang-(1–7) and AngII for 1 hour and p65 and p50 subunits were detected by an indirect immunostaining using a mouse FITC-labeled secondary antibody (green staining). Nuclei were stained with propidium iodure (in red). In the merge, the yellow staining shows nuclear localization of the NF-κB proteins after Ang-(1–7) stimulation. The results are representative of 3 independent observations done by confocal microscopy. (B) Growth arrested cells were incubated with 10⁻⁷ mol/L Ang-(1–7), AngII or TNFα for 1 hour. Thereafter, nuclear extracts were isolated and NF-κB activity was determined by binding assay with a labeled NF-κB oligonucleotide and analyzed by EMSA. Data are expressed as an n-fold increase vs. control cells, as mean±SEM of 4 experiments. *P<0.05 vs. control. (C) Growth arrested cells were stimulated with 10⁻⁷ mol/L Ang-(1–7) or TNFα for 18 and 24 hours. Gene expression of MCP-1 and IL-6 was analyzed by real-time PCR. Results are expressed as mean±SEM of 4 experiments. *P<0.05 vs. control.