Beneficial effects of the activation of the angiotensin-(1-7) MAS receptor in a murine model of adriamycin-induced nephropathy

Abstract

Angiotensin-(1-7) [Ang-(1-7)] is a biologically active heptapeptide that may counterbalance the physiological actions of angiotensin II (Ang II) within the renin-angiotensin system (RAS). Here, we evaluated whether activation of the Mas receptor with the oral agonist, AVE 0991, would have renoprotective effects in a model of adriamycin (ADR)-induced nephropathy. We also evaluated whether the Mas receptor contributed for the protective effects of treatment with AT1 receptor blockers. ADR (10 mg/kg) induced significant renal injury and dysfunction that was maximal at day 14 after injection. Treatment with the Mas receptor agonist AVE 0991 improved renal function parameters, reduced urinary protein loss and attenuated histological changes. Renoprotection was associated with reduction in urinary levels of TGF-β. Similar renoprotection was observed after treatment with the AT1 receptor antagonist, Losartan. AT1 and Mas receptor mRNA levels dropped after ADR administration and treatment with losartan reestablished the expression of Mas receptor and increased the expression of ACE2. ADR-induced nephropathy was similar in wild type (Mas(+/+) ) and Mas knockout (Mas (-/-)) mice, suggesting there was no endogenous role for Mas receptor activation. However, treatment with Losartan was able to reduce renal injury only in Mas(+/+) , but not in Mas (-/-) mice. Therefore, these findings suggest that exogenous activation of the Mas receptor protects from ADR-induced nephropathy and contributes to the beneficial effects of AT1 receptor blockade. Medications which target specifically the ACE2/Ang-(1-7)/Mas axis may offer new therapeutic opportunities to treat human nephropathies.

Figure 1. Adriamycin-induced morphological changes in glomerular and tubular regions of the kidney.

Representative photographs of PAS-stained glomerular and tubular regions noticed in control mice (Sham, A, B), and 7 (C, D), 14 (E, F) and 21 (G, H) days after injection of adriamycin (10 mg/kg). Glomerular damage (large arrows), tubule-interstitial changes, atrophy of tubular epithelial cells (arrowheads) and tubular enlargement (thin arrows, insert) increased in the renal cortex from day 7 (D) to day 14 (F, insert, arrow). Global sclerosis was observed in many glomeruli (E, asterisks). Resorption droplets were present in tubular cells at day 14 (panel F, thin arrows). Histological changes stabilized on day 21 (G, H). Original magnification 40× objective.

Figure 2. Effects of the treatment with the Mas receptor agonist, AVE 0991, and the AT1 receptor blocker, Losartan, on adriamycin-induced renal injury.

Adriamycin (ADR, 10 mg/kg) was injected in the tail vein of Balb/c mice. Animals were treated daily with vehicle (VE, filtered water), AVE0991 (AVE, 3 mg/kg) or Losartan (LOS, 10 mg/kg) by gavage from days 7 to day 14 day after ADR injection. The sham group received a single injection of NaCl 0.9% in the tail vein and was treated with filtered water. Microalbuminuria (A) and serum albumin levels (B) were evaluated on day 14 in n = 6–10 mice per group. The panel also shows urinary levels of TGF-β (C) in n = 4–5 mice per group. (*) for P<0.05 when compared to VE group and (#) for P<0.05 when compared to sham group.

Figure 3. Histological changes and index of tubulointertitial and glomerular injury of ADR-induced nephrosis at day 14.

Adriamycin (10 mg/kg) was injected in tail vein of Balb/c mice, on day 0 in vehicle-treated, VE (filter water), AVE0991-treated, AVE, (AVE 0991, 3 mg/kg) and Losartan-treated, LOS groups (losartan, 10 mg/kg). Representative photographs of PAS stained of glomerular and tubular regions were obtained at 14^th day. All mice were treated by gavage, daily, 7^th to 14^th day after ADR-induction. Glomerular (large arrow) and tubular injuries (thin arrows) observed in vehicle-treated mice (A–B) were attenuated by treatment with AVE 0991 (C–D) or Losartan (E–F). Original magnification 4× objective (A, C) and 40× (B, D). The indexes of tubulointerstitial (G) and glomerular (H) injuries were graded in a blind manner, as described in methods section. Symbols represent results in single animals and the trace is median value for 5–8 animals. (*) for P<0.05 when compared to 14^th day after ADR-induction group and (#) for P<0.05 when compared to sham group.

Figure 4. mRNA expression of Mas, AT1 and ACE2 in the kidneys after injection of adriamycin.

In A, Mas and AT1 mRNA levels were evaluated in the kidney before (Sham) and 7, 14 and 21 days after injection of adriamycin (ADR, 10 mg/kg). In B, Mas and AT1 mRNA levels at day 14 in kidneys of mice injected with adriamycin that were treated with vehicle (VE) or losartan (LOS, 10 mg/kg). In C, ACE2 mRNA levels at day 14 in kidneys of mice injected with adriamycin that were treated with vehicle (VE) or losartan (LOS, 10 mg/kg). The dotted line across the graphs represents levels in control animals. Renal mRNA levels of receptors and ACE2 were estimated by real time PCR. Results are mean ± SEM of n = 5 mice per group. (*) for P<0.05 when compared to sham group.

Figure 5. Treatment with losartan protects from adriamycin-induced renal damage in wild type (Mas^+/+) but not Mas deficient (Mas^−/−) mice.

Adriamycin (10 mg/kg) was injected in the tail vein of Mas^+/+ (A–D) and Mas^−/− (E–H) FVBN mice. Animals were treated with water (VE, A–B and E–F) or Losartan (LOS, 10 mg/kg, C–D and G–H) and morpohological changes evaluated at day 14 after adriamycin injection. Glomerular (large arrow) and tubular injuries (thin arrows) are showed. Indices of tubulointerstitial (I) and glomerular (J) injuries were graded in a blind manner, as described in the Methods section. PAS-stained sections and magnification 10× (A, C, E, G) and 40× (B, D, F, H). Symbols represent results in single animals and the trace is median value. (*) for P<0.05 when compared to VE-treated group and (#) for P<0.05 when compared to sham group.