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

Abstract Angiotensin‐converting enzyme‐2 (ACE2) and Mas receptor are the major components of the ACE2/Ang 1‐7/Mas axis and have been shown to play a protective role in hypertension and hypertensive nephropathy individually. However, the effects of dual deficiency of ACE2 and Mas (ACE2/Mas) on Ang II‐induced hypertensive nephropathy remain unexplored, which was investigated in this study in a mouse model of hypertension induced in either ACE2 knockout (KO) or Mas KO mice and in double ACE2/Mas KO mice by subcutaneously chronic infusion of Ang II. Compared with wild‐type (WT) animals, mice lacking either ACE2 or Mas significantly increased blood pressure over 7‐28 days following a chronic Ang II infusion (P < .001), which was further exacerbated in double ACE2/Mas KO mice (P < .001). Furthermore, compared to a single ACE2 or Mas KO mice, mice lacking ACE2/Mas developed more severe renal injury including higher levels of serum creatinine and a further reduction in creatinine clearance, and progressive renal inflammation and fibrosis. Mechanistically, worsen hypertensive nephropathy in double ACE2/Mas KO mice was associated with markedly enhanced AT1‐ERK1/2‐Smad3 and NF‐κB signalling, thereby promoting renal fibrosis and renal inflammation in the hypertensive kidney. In conclusion, ACE2 and Mas play an additive protective role in Ang II‐induced hypertension and hypertensive nephropathy. Thus, restoring the ACE2/Ang1‐7/Mas axis may represent a novel therapy for hypertension and hypertensive nephropathy.

phropathy remain unexplored, which was investigated in this study in a mouse model of hypertension induced in either ACE2 knockout (KO) or Mas KO mice and in double ACE2/Mas KO mice by subcutaneously chronic infusion of Ang II. Compared with wild-type (WT) animals, mice lacking either ACE2 or Mas significantly increased blood pressure over 7-28 days following a chronic Ang II infusion (P < .001), which was further exacerbated in double ACE2/Mas KO mice (P < .001). Furthermore, compared to a single ACE2 or Mas KO mice, mice lacking ACE2/Mas developed more severe renal injury including higher levels of serum creatinine and a further reduction in creatinine clearance, and progressive renal inflammation and fibrosis. Mechanistically, worsen hypertensive nephropathy in double ACE2/Mas KO mice was associated with markedly enhanced AT1-ERK1/2-Smad3 and NF-κB signalling, thereby promoting renal fibrosis and renal inflammation in the hypertensive kidney. In conclusion, ACE2 and Mas play an additive protective role in Ang II-induced hypertension and hypertensive nephropathy. Thus, restoring the ACE2/Ang1-7/Mas axis may represent a novel therapy for hypertension and hypertensive nephropathy.

| INTRODUC TI ON
Hypertensive nephropathy is a major complication of hypertension and is the main cause of chronic kidney disease (CKD) characterized by progressive renal fibrosis and inflammation. 1 Angiotensin II (Ang II), the major functional peptide of the renin-angiotensin system (RAS), has been long recognized as a key mediator in CKD, particularly in hypertension-associated nephropathy. 1 It is well accepted that Ang II can be positively regulated by the classical Ang I-converting enzyme (ACE)/Ang II/Ang II type 1 receptor (AT1) axis, a critical pathway leading to end-stage renal disease. 2,3 Accumulating evidence has shown that the pathogenic actions of the ACE/Ang II/AT1 axis can be countered by the angiotensin-converting enzyme 2 (ACE2)/Ang 1-7/Mas receptor (Mas) axis.
As a homolog of ACE, the monocarboxypeptidase ACE2 can oppose ACE activity via conversion of Ang II to Ang 1-7, which binds to the Mas receptor to exert opposite effects on Ang II. [4][5][6] is highly expressed in the normal kidney, mainly located in the proximal tubule, 7 and plays an essential role in cardiovascular and kidney diseases. 8 ACE2 deficiency is associated with exaggerated kidney injury in mouse models of diabetes, Ang II-induced hypertension and obstructive nephropathy, [9][10][11][12][13] whereas treatment with human recombinant ACE2 (hrACE2) can slow the progression of diabetic and hypertensive kidney injury. 14,15 Ang 1-7 is an endogenous ligand for the G protein-coupled receptor Mas. 16 When Mas is deleted (Mas -/-) in mice with the FVB/N genetic background, they have higher blood pressure than their wild-type (WT) counterpart (Mas +/+ ). 17 However, a difference in the mean arterial pressure was not observed in Mas +/+ and Mas -/mice with the C57BL/6 background. 18 Nevertheless, the genetic deletion of Mas leads to glomerular hyperfiltration and microalbuminuria. 19

| Proteinuria and renal function analysis
Twenty-four-hour urine was collected at day 28 after Ang II infu-

| Real-time PCR analysis
Total RNA was extracted from kidney tissues using TRIzol ® , and mRNA levels of collagen I, α-SMA, IL-1β, renin, angiotensinogen, aminopeptidase A (APA) and aminopeptidase N (APN) were detected by real-time PCR as described previously. The PCR primer sequences were listed in Table 1. The housekeeping genes GAPDH were used as internal controls. The ratio of the mRNA examined to GAPDH was calculated and is expressed as mean ± SEM.

| Statistical analysis
Data obtained from this study were expressed as mean ± SEM.
Statistical analyses were performed using one-way ANOVA using Prism 5.0 (GraphPad Software, San Diego, CA, USA).  Figure 1C) and creatinine clearance (Ccr, Figure 1D). All mice receiving chronic Ang II infusion developed hypertension (Figure 2A

| Double deletion of ACE2/Mas genes promotes Ang II-induced renal fibrosis and inflammation
Chronic Ang II infusion resulted in a moderate increase in the expres-

| D ISCUSS I ON
WT + Ang II; $ P < .05, $$$ P < .001 vs. ACE2 KO + Ang II and Mas KO + Ang II. Scale bar, 50 μm production to counteract the effect of Ang II in WT mice, which was reduced in mice lacking ACE2 or ACE2/Mas, but not in Mas KO mice, we failed to obtain meaningful levels of Ang 1-7 in serum and intrarenal tissues due to its low concentrations. Thus, changes in urinary levels of Ang 1-7 remain unexplained and may require further investigation.
In summary, ACE2 and Mas may function additively to protect against Ang II-mediated hypertension and hypertensive nephropathy as mice with double deletion of ACE2 and Mas developed more severe hypertension and hypertensive nephropathy when compared to either ACE2 or Mas KO mice. Enhanced AT1-ERK1/2-Smad3 signalling may be a key mechanism by which the dual deletion of ACE2 and Mas further promoted hypertension and hypertensive kidney disease. Results from this study reveal that the ACE2/Ang 1-7/Mas axis represents opportunities for the future development of therapeutics against hypertensive disorders. Research foundation (DFG SFB1365).

CO N FLI C T O F I NTE R E S T
The authors declare that there are no competing interests associated with the manuscript.