Effects of Ang-(1–7) on Serum Levels of Ang II and SOD in Mice

To evaluate the short-term systemic effects of Ang-(1–7), we measured serum levels of Ang II and SOD at days 1 and 7 after treatment. As depicted in Fig. 1, the levels of Ang II were significantly elevated in the model group compared to the sham group, whereas the SOD levels were significantly reduced (P < 0.01). Treatment with Ang-(1–7) for one day markedly reduced Ang II levels in a dose-dependent manner, with the low and medium doses showing significant decreases compared to the model group (P < 0.05). By day 7, Ang II levels partially recovered in all treatment groups but remained lower than the model. As shown in Fig. 1B, serum SOD activity was significantly reduced in the model group relative to sham (P < 0.01). Treatment with Ang-(1–7) for one day significantly restored SOD activity in the low and medium dose groups (P < 0.05). By day 7, the high dose group showed the most pronounced increase in SOD activity compared to the model group (P < 0.01), indicating a sustained antioxidant effect.

These findings suggested that Ang-(1–7) rapidly attenuated Ang II elevation and enhanced antioxidant defense in a dose- and time-dependent manner.

Fig. 1

Effects of Ang-(1–7) on serum Ang II (A) and SOD (B) levels in mice following I/R injury. Mice were treated with low (144 µg/kg), medium (288 µg/kg), or high (576 µg/kg) doses of Ang-(1–7) for 1 or 7 days. Data were represented as mean ± SD (n = 6). **P < 0.01 vs. Sham group; #P < 0.05, ##P < 0.01 vs. Model group. Ang-(1–7): Angiotensin-(1–7); Ang II: Angiotensin II; SOD: Superoxide Dismutase; I/R: Ischemia-Reperfusion

Immunohistochemical Analysis of Collagen I and TGF-β1 Expression in Renal Tissue Following Ang-(1–7) Treatment

To assess the anti-fibrotic effects of Ang-(1–7), we performed immunohistochemical staining of Collagen I and TGF-β1 in mouse kidney tissue on days 1 and 7 after treatment. As shown in Fig. 2, both Collagen I and TGF-β1 were significantly upregulated in the I/R model group compared to the sham group (P < 0.01), indicating fibrotic activation following renal injury. However, treatment with Ang-(1–7) resulted in a significant reduction in TGF-β1 expression levels relative to the model group (P < 0.05 or P < 0.01). Furthermore, the expression levels of Collagen I were notably decreased in the medium and high-dose groups. The most pronounced reduction in both Collagen I and TGF-β1 expression was observed in the high-dose Ang-(1–7) group after seven days of treatment. Subsequently, the high dose of Ang-(1–7) was selected for further experiments.

Fig. 2

Immunohistochemical analysis of Collagen I (A) and TGF-β1 (B) expression in renal tissues of mice treated with different doses of Ang-(1–7): low (144 µg/kg), medium (288 µg/kg), or high (576 µg/kg), for 1 or 7 days (400 × magnification). Data were represented as mean ± SD (n = 3). **P < 0.01 vs. Sham group; #P < 0.05, ##P < 0.01 vs. Model group. Ang-(1–7): Angiotensin-(1–7); TGF-β1: Transforming Growth Factor-β1

Effects of Ang-(1–7) on Body Weight and Blood Pressure in Mice

To evaluate the systemic effects of Ang-(1–7) in the setting of renal I/R injury, we monitored changes in body weight and blood pressure 7, 14, and 28 days. As shown in Fig. 3A, all I/R-injured groups exhibited a significant reduction in body weight compared to their pre-surgery levels (P < 0.05 or P < 0.01), most notably at day 7, with partial recovery observed by day 14 and day 28. The sham group maintained stable body weight across all time points. In terms of blood pressure (Fig. 3B), the model group showed a significant decrease in both SBP and DBP at day 14 compared to the sham group (P < 0.01), suggesting impaired cardiovascular regulation following I/R injury. Interestingly, treatment with Ang-(1–7) significantly restored both SBP and DBP at day 14 compared to the model group (P < 0.05 or P < 0.01), indicating a protective role in maintaining hemodynamic stability. Losartan treatment showed a similar trend.

These findings demonstrated that while I/R injury induced transient weight loss and hypotension, Ang-(1–7) helped reverse blood pressure decline, especially during the subacute phase.

Fig. 3

Effects of Ang-(1–7) on body weight and blood pressure following renal I/R injury in mice. (A) Body weight measured before and after surgery at 7, 14, and 28 days in Sham, Model, Losartan (10 mg/kg/day), and Ang-(1–7) (576 µg/kg) groups (**P < 0.01 vs. Before). (B) Systolic blood pressure (SBP) and diastolic blood pressure (DBP) measured by tail-cuff plethysmography at corresponding time points (**P < 0.01 vs. Sham group; #P < 0.05, ##P < 0.01 vs. Model group). Data were expressed as mean ± SD (n = 6 ~ 9). Ang-(1–7): Angiotensin-(1–7); SBP: Systolic Blood Pressure; DBP: Diastolic Blood Pressure; I/R: Ischemia-Reperfusion

Effects of Ang-(1–7) on Renal Function, RAS Activation, Inflammatory and Oxidative Biomarkers in Mice

To comprehensively evaluate the renoprotective effects of Ang-(1–7), we assessed multiple biochemical parameters related to renal function, injury, inflammation, and oxidative stress at days 7, 14, and 28 following I/R injury.

As depicted in Fig. 4A-C, the model group exhibited a significant increase in serum creatinine, urea, and Ang II levels at 7, 14, and 28 days compared to the sham group (P < 0.05), indicating sustained renal dysfunction and systemic RAS activation. Treatment with Ang-(1–7) and losartan significantly reduced these markers at corresponding time points (P < 0.05), suggesting a protective effect in preserving renal function and modulating RAS balance.

Urinary biomarkers were also analyzed. As shown in Fig. 4D-F, urinary AGT, Kim-1, and NGAL levels were markedly elevated in the model group across time points (P < 0.05). Ang-(1–7) treatment significantly lowered AGT and Kim-1 at day 14 (P < 0.05), supporting its role in alleviating tubular injury and inflammation.

In renal tissue, Fig. 4G-H showed that the SOD activity was significantly suppressed in the model group at day 7 (P < 0.01), while Ang II tissue levels remained unchanged. Notably, Ang-(1–7) administration significantly increased renal SOD levels at day 7 compared to the model group (P < 0.05), indicating an antioxidant effect at the tissue level.

Together, these results suggest that Ang-(1–7) confers renoprotective effects by modulating systemic and local RAS activity, reducing inflammatory markers, and enhancing antioxidant defenses.

Fig. 4

Effects of Ang-(1–7) on renal function, RAS activation, inflammatory and oxidative biomarkers in mice subjected to renal I/R injury. Mice were treated with Ang-(1–7) (576 µg/kg) or Losartan (10 mg/kg), and parameters were assessed at 7, 14, and 28 days post-injury. (A-C) Serum levels of Creatinine (A), Urea (B), Ang II (C). (D-F) Urinary levels of AGT (D), Kim-1 (E), and NGAL (F); (G-H) Renal tissue levels of Ang II (G) and SOD (H). Data were expressed as mean ± SD (n = 6). *P < 0.05, **P < 0.01 vs. Sham group; #P < 0.05, ##P < 0.01 vs. Model group. Ang-(1–7): Angiotensin-(1–7); RAS: Renin-Angiotensin System; I/R: Ischemia-Reperfusion; Ang II: Angiotensin II; AGT: Angiotensinogen; Kim-1: Kidney Injury Molecule-1; NGAL: Neutrophil Gelatinase-Associated Lipocalin; SOD: Superoxide Dismutase

Effects of Ang-(1–7) on Renal Fibrosis in Mice

To assess the anti-fibrotic effects of Ang-(1–7), we assessed the renal expression of fibrotic markers including Fibronectin, Collagen I, and TGF-β in renal tissues by immunohistochemistry and qRT-PCR at 7, 14, or 28 days after I/R injury (Fig. 5A-F). Immunohistochemical staining analysis showed that the model group exhibited significantly increased positive expression of all three markers at all time points compared to the sham group (P < 0.01), indicating progressive renal fibrosis following injury. Semi-quantitative image analysis at day 28 revealed significant attenuation of all three markers in mice treated with Ang-(1–7) (P < 0.01), with levels comparable to or lower than those observed in the Losartan group. To corroborate the histological findings at the transcript level, mRNA expression of Fibronectin, Collagen I, and TGF-β was measured by qRT-PCR, showing a parallel reduction in Ang-(1–7)-treated mice (P < 0.01).

These findings suggested that Ang-(1–7) exerted sustained anti-fibrotic effects through the downregulation of extracellular matrix proteins and pro-fibrotic signaling molecules, highlighting its potential therapeutic value in preventing CKD progression following AKI.

Fig. 5

Effects of Ang-(1–7) on renal fibrosis markers following I/R injury. Mice were treated with Ang-(1–7) (576 µg/kg) or Losartan (10 mg/kg) and sacrificed at 7, 14, or 28 days post-injury (400× magnification). (A) Immunohistochemical staining analysis of FN at day 28; (B) Relative mRNA expression of FN at day 28; (C) Immunohistochemical staining analysis of Collagen I at day 28; (D) Relative mRNA expression of Collagen I at day 28; (E) Immunohistochemical staining analysis of TGF-β at day 28; (F) Relative mRNA expression of TGF-β at day 28. Data were expressed as mean ± SD (n = 3). **P < 0.01 vs. Sham group; ##P < 0.01 vs. Model group. Ang-(1–7): Angiotensin-(1–7); FN: Fibronectin; TGF-β: Transforming Growth Factor-β

Effects of Ang-(1–7) on RAS Components and Neuroinflammatory Markers in the Paraventricular Nucleus

To explore the central regulatory effects of Ang-(1–7), we assessed the expression of key renin-angiotensin system (RAS) components and neuroinflammatory markers in the paraventricular nucleus (PVN) of the hypothalamus at 7, 14, and 28 days post-surgery.

As depicted in Figs. 6, the model group exhibited significantly increased expression of AGT, AT1R, ACE, GFAP, PRR, and IBA-1, along with a reduction in ACE2 compared to the sham group (P < 0.01), indicating central RAS activation and glial inflammation following renal injury. Treatment with Ang-(1–7) significantly reversed these alterations at multiple time points. Specifically, Ang-(1–7) reduced AGT, AT1R, ACE, PRR, GFAP, and IBA-1 expression, while restoring ACE2 expression at day 28 (P < 0.01).

These results suggested that Ang-(1–7) exerted neuroprotective and anti-inflammatory effects in the PVN, potentially through modulation of central RAS balance and glial activation, which might contribute to its systemic renoprotective actions.

Fig. 6

Effects of Ang-(1–7) on RAS components and neuroinflammatory markers in the paraventricular nucleus (400 × magnification). (A-G) Immunohistochemical analysis of AGT (A), AT1R (B), GFAP (C), ACE2 (D), ACE (E), PRR (F), and IBA-1 (G) expression. Data were expressed as mean ± SD (n = 3). **P < 0.01 vs. Sham group; ##P < 0.01 vs. Model group. Ang-(1–7): Angiotensin-(1–7); FN: Fibronectin; RAS: Renin-Angiotensin System; AGT: Angiotensinogen; AT1R: Angiotensin II Type 1 Receptor; GFAP: Glial Fibrillary Acidic Protein; ACE: Angiotensin-Converting Enzyme; PRR: Prorenin Receptor; IBA-1: Ionized Calcium Binding Adapter Molecule 1

Effects of Ang-(1–7) on the Expression of RAS-related, Inflammatory, and Oxidative Stress Proteins in Renal Tissue

To elucidate the molecular mechanisms underlying the renoprotective effects of Ang-(1–7), Western blot analysis was performed to evaluate the expression of key proteins in renal tissues at 7, 14, and 28 days after I/R injury.

At day 7 (Fig. 7A), the model group exhibited significant upregulation of AGT, ACE, ACE2, MCP-1, NOX2, p-Akt, and p-ERK1/2 compared to the sham group (P < 0.05), indicating early activation of both the classical RAS axis and inflammatory signaling. Ang-(1–7) treatment significantly reversed these changes (P < 0.05 or P < 0.01).

At day 14 (Fig. 7B), ACE, NOX2, and MCP-1 remained significantly elevated in the model group, whereas ACE2 and p-Akt were downregulated, suggesting ongoing inflammation and dysregulation of the protective RAS arm (P < 0.05 or P < 0.01). Ang-(1–7) effectively mitigated these abnormalities, restoring ACE2 and p-Akt while suppressing NOX2 and MCP-1 expression (P < 0.05 or P < 0.01).

By day 28 (Fig. 7C), the model group showed sustained upregulation of AGT, MCP-1, NOX2, p-Akt, and p-ERK1/2, while ACE and ACE2 levels were not significantly different from the sham group (P < 0.05 or P < 0.01). Ang-(1–7) administration continued to exert beneficial effects by reducing these pathological markers, particularly MCP-1, NOX2, p-Akt, and p-ERK1/2 (P < 0.01).

These findings indicated that Ang-(1–7) confered renoprotection through suppression of pro-inflammatory and oxidative pathways and restoration of RAS homeostasis.

Fig. 7

Effects of Ang-(1–7) on RAS-related, inflammatory, and oxidative stress markers in renal tissues. A. Western blot analysis of protein (AGT, ACE, ACE2, MCP-1, NOX2, p-Akt, p-ERK1/2) expression in renal tissues after 7 days of treatment. B. Western blot analysis of protein (AGT, ACE, ACE2, MCP-1, NOX2, p-Akt, p-ERK1/2) expression in renal tissues after 14 days of treatment. C. Western blot analysis of protein (AGT, ACE, ACE2, MCP-1, NOX2, p-Akt, p-ERK1/2) expression in renal tissues after 28 days of treatment. Data were expressed as mean ± SD (n = 3). *P < 0.05, **P < 0.01 vs. Sham group; #P < 0.05, ##P < 0.01 vs. Model group. Ang-(1–7): Angiotensin-(1–7); RAS: Renin-Angiotensin System; AGT: Angiotensinogen; ACE: Angiotensin-Converting Enzyme; MCP-1: Monocyte Chemoattractant Protein-1; NOX2: NADPH Oxidase 2