Angiotensin-[1–7] attenuates kidney injury in experimental Alport syndrome

Figure 1. Effects of Ang-[1–7] on kidney fibrosis in Col4a3−/− mice kidneys. (A) Tissue morphology of kidney from WT, Col4a3−/−, and Col4a3−/−+Ang-[1–7] mice. Images from glomerulus (left) and tubulointerstitium (right) are presented. (B,C) Comparison of expression level for fibrosis markers determined by immunoblotting (B) and qPCR (C) from the kidney of WT, Col4a3−/−, and Col4a3−/−+Ang-[1–7] mice (n = 4 mice/group). Scale bars, 25 μm. H&E, hematoxylin & eosin staining; MT, Masson’s trichrome staining; α-SMA, alpha smooth muscle actin; FN, fibronectin.

Figure 2. Effects of Ang-[1–7] on transforming growth factor-beta (TGF-β)/Smad pathway in Col4a3−/− mice kidneys. (A) Protein expression of the TGF-β and Smad proteins was assessed in kidney of WT, Col4a3−/−, and Col4a3−/−+Ang-[1–7] mice (n = 4 mice/group). β-actin was used as the endogenous control. (B) Comparison of mRNA expression level TGFβ determined by qPCR from the kidney WT, Col4a3−/−, and Col4a3−/−+Ang-[1–7] mice (n = 4 mice/group). (C) Representative immunohistochemical staining of TGF-β in renal cortex of WT, Col4a3−/−, and Col4a3−/−+Ang- [1–7] mice. Each column represents mean ± SEM. *P < 0.05 vs. WT mice; #P < 0.05 vs. Col4a3−/− mice.

Figure 3. Effects of Ang-[1–7] on inflammation and apoptosis in Col4a3−/− mice kidneys. (A) Comparison of protein expression level for HO-1 and ED-1 determined by immunoblotting from the kidney of WT, Col4a3−/−, and Col4a3−/−+Ang-[1–7] mice (n = 4 mice/group). β-actin was used as the endogenous control. (B) Comparison of mRNA expression level for inflammatory markers determined by qPCR from the kidney of WT, Col4a3−/−, and Col4a3−/−+Ang-[1–7] mice (n = 4 mice/group). (C) Comparison of protein expression level for proteins related to apoptosis determined by immunoblotting from the kidney of WT, Col4a3−/−, and Col4a3−/−+Ang-[1–7] mice (n = 4 mice/group). β-actin was used as the endogenous control. (D) Representative images of immunohistochemical staining for F4/80 (upper) and TUNEL (lower) in the kidney of WT, Col4a3−/−, and Col4a3−/−+Ang-[1–7] mice. Scale bars, 25 μm. Each column represents mean ± SEM. *P < 0.05 vs. WT mice; #P < 0.05 vs. Col4a3−/− mice. MCP-1, monocyte chemoattractant protein-1; TNF-α tumor necrosis factor-α; ICAM-1, intercellular adhesion molecule-1; VCAM-1, vascular cell adhesion molecule-1.

Figure 4. Effects of Ang-[1–7] on renin angiotensin system (RAS) in Col4a3−/− mice kidneys. Comparison of protein expression level of RAS components determined by immunoblotting from the kidney of WT, Col4a3−/−, and Col4a3−/−+Ang-[1–7] mice (n = 4 mice/group). β-actin was used as the endogenous control. Each column represents mean ± SEM. *P < 0.05 vs. WT mice; #P < 0.05 vs. Col4a3−/− mice. ACE, angiotensin converting enzyme; TACE, TNF-α converting enzyme.

Figure 5. Effects of Ang-[1–7] on mitogen-activated protein kinase (MAPK) pathway in Col4a3−/− mice kidneys. Protein expression of the total ERK, phosphorylated ERK (p-ERK), total JNK, phosphorylated JNK (p-JNK), P38 and phosphorylated P38 (pP38) was assessed in the kidney of WT, Col4a3−/−, and Col4a3−/−+Ang-[1–7] mice (n = 4 mice/group). β-actin was used as the endogenous control. Each column represents mean ± SEM. *P < 0.05 vs. WT mice; #P < 0.05 vs. Col4a3−/− mice.

Figure 6. Effects of Ang-[1–7] on fibrosis, inflammation and apoptosis in TGF-β treated HK-2 cell. (A) Comparison of expression level for fibrosis markers and Smad proteins determined by immunoblotting in HK-2 after stimulation with vehicles or recombinant human TGF-β (rhTGFβ). (B) Comparison of expression level for inflammation and apoptosis proteins determined by immunoblotting in HK-2 after stimulation with vehicles or rhTGFβ. β-actin was used as the endogenous control. Each column represents mean ± SEM. *P < 0.05 vs. control (CON) group; #P < 0.05 vs. rhTGFβ group.

Figure 7. Effects of Ang-[1–7] on RAS and MAPK pathway in TGF-β treated HK-2 cell. Comparison of expression level for RAS (A) and MAPK pathway proteins (B) determined by immunoblotting in HK-2 after stimulation with vehicles or recombinant human TGF-β (rhTGFβ). β-actin was used as the endogenous control. Each column represents mean ± SEM. *P < 0.05 vs. control (CON) group; #P < 0.05 vs. rhTGFβ group.