Dual deficiency of angiotensin‐converting enzyme‐2 and Mas receptor enhances angiotensin II‐induced hypertension and hypertensive nephropathy

Figures

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  Figure 1. Mice with double deletion of angiotensin (Ang)‐converting enzyme‐2 (ACE2) and Mas genes develop normally with a normal range of blood pressure and renal function. A, Systolic blood pressure (SBP). B, Bodyweight. C, Serum creatinine (Scr). D, Creatinine clearance (Ccr). Data represent the mean ± SEM for groups of 3‐5 mice

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  Figure 2. Double deletion of angiotensin (Ang)‐converting enzyme‐2 (ACE2) and Mas receptor enhances Ang II‐induced hypertension and hypertensive renal injury. A, Systolic blood pressure (SBP) at weeks 1, 2, 3, and 4. B, 24‐hour proteinuria. C, Serum creatinine (Scr). D, Creatinine clearance rate (Ccr). E, Renal periodic acid Schiff (PAS) staining at week 4 after Ang II infusion. Glomerular mesangial matrix expansion was scored. Data represent the mean ± SEM for groups of 6‐15 mice. *P < .05, **P < .01, ***P < .001 vs. WT + saline (WT + SL); # P < .05, ## P < .01, ### P < .001 vs. WT + Ang II; $ P < .05, $$ P < .01, $$$ P < .001 vs. ACE2 KO + Ang II and Mas KO + Ang II. Scale bar, 50 μm. WT + SL represents wild‐type (WT) mice received saline infusion; WT + Ang II represents WT mice received Ang II infusion; ACE2 KO + Ang II represents ACE2 KO mice received Ang II infusion; Mas KO + Ang II represents Mas KO mice received Ang II infusion and A/M KO + Ang II represents mice with double deletion of ACE2 and Mas received Ang II infusion

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  Figure 3. Double deletion of ACE2 and Mas receptor enhances Ang II‐induced renal fibrosis. A, Real‐time PCR detects α‐smooth muscle actin (α‐SMA) and collagen I (Col. I) mRNAs. B, Western blot analysis of α‐SMA and Col. I. C, Col. I deposition detected by immunohistochemistry. Data represent the mean ± SEM for groups of 5‐12 mice. *P < .05, **P < .01, ***P < .001 vs. WT + SL; # P < .05, ## P < .01, ### P < .001 vs. WT + Ang II; $ P < .05, $$ P < .01, $$$ P < .001 vs. ACE2 KO + Ang II and Mas KO + Ang II. Scale bar, 100 μm

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  Figure 4. Double deletion of ACE2 and Mas receptor enhances Ang II‐induced renal inflammation. A, F4/80+ macrophages infiltrating the kidney detected by immunohistochemistry. B, Monocyte chemoattractant protein‐1 (MCP‐1) expression in the kidney detected by immunohistochemistry. C, Western blot and Real‐time PCR (D) analysis of Interleukin (IL)‐1β expression. Data represent the mean ± SEM for groups of 6‐12 mice. *P < .05, ***P < .001 vs. WT + SL; # P < .05, ## P < .01, ### P < .001 vs. WT + Ang II; $ P < .05, $$$ P < .001 vs. ACE2 KO + Ang II and Mas KO + Ang II. Scale bar, 50 μm (A) or 100 μm (B)

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  Figure 5. Double deletion of ACE2 and Mas receptor enhances Ang II‐induced activation of AT1, ERK1/2, and Smad3 signalling in the hypertensive kidney. A, Western blot analysis of Ang II receptor type 1 (AT1), phospho‐ERK1/2 (p‐ERK1/2), phospho‐Smad3 (p‐Smad3). B, Immunohistochemistry of p‐Smad3 nuclear translocation. Data represent the mean ± SEM for groups of 6‐12 mice. *P < .05, ***P < .001 vs. WT + SL; # P < .05, ## P < .01, ### P < .001 vs. WT + Ang II; $ P < .05, $$ P < .01, $$$ P < .001 vs. ACE2 KO + Ang II and/or Mas KO + Ang II. Scale bar, 50 μm

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  Figure 6. Double deficiency of ACE2 and Mas genes inhibits angiotensinogen but not Renin, Angiotensinogen, Aminopeptidase A mRNA expression in the hypertensive kidney. Renal mRNA expression of Renin, Angiotensinogen, Aminopeptidase A (APA) and Aminopeptidase N (APN) is detected by Real‐time PCR. Data represent the mean ± SEM for groups of 8 mice. # P < .05 vs. WT + Ang II; $ P < .05, $$ P < .01 vs. ACE2 KO + Ang II and/or Mas KO + Ang II

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  Figure 7. Double deletion of ACE2 and Mas receptor enhances Ang II‐induced activation of nuclear factor κB (NF‐κB) signalling in the hypertensive kidney. A, Western blot analysis of Smad ubiquitination regulatory factors 2 (Smurf2), Smad7, phospho‐IκBα (p‐IκBα), and phospho‐NF‐κB/p65 (p‐p65). B, Immunohistochemistry of NF‐κB/p‐p65 nuclear translocation. Data represent the mean ± SEM for groups of 6‐12 mice. *P < .05, ***P < .001 vs. WT + SL; # P < .05, ## P < .01, ### P < .001 vs. WT + Ang II; $ P < .05, $$$ P < .001 vs. ACE2 KO + Ang II and Mas KO + Ang II. Scale bar, 50 μm