We investigated the relationship between Ang-(1–7) [angiotensin-(1–7)] action, sHTN (systolic hypertension), oxidative stress, kidney injury, ACE2 (angiotensin-converting enzyme-2) and MasR [Ang-(1–7) receptor] expression in Type 1 diabetic Akita mice. Ang-(1–7) was administered daily [500 μg/kg of BW (body weight) per day, subcutaneously] to male Akita mice from 14 weeks of age with or without co-administration of an antagonist of the MasR, A779 (10 mg/kg of BW per day). The animals were killed at 20 weeks of age. Age-matched WT (wild-type) mice served as controls. Ang-(1–7) administration prevented sHTN and attenuated kidney injury (reduced urinary albumin/creatinine ratio, glomerular hyperfiltration, renal hypertrophy and fibrosis, and tubular apoptosis) without affecting blood glucose levels in Akita mice. Ang-(1–7) also attenuated renal oxidative stress and the expression of oxidative stress-inducible proteins (NADPH oxidase 4, nuclear factor erythroid 2-related factor 2, haem oxygenase 1), pro-hypertensive proteins (angiotensinogen, angiotensin-converting enzyme, sodium/hydrogen exchanger 3) and profibrotic proteins (transforming growth factor-β1 and collagen IV), and increased the expression of anti-hypertensive proteins (ACE2 and MasR) in Akita mouse kidneys. These effects were reversed by A779. Our data suggest that Ang-(1–7) plays a protective role in sHTN and RPTC (renal proximal tubular cell) injury in diabetes, at least in part, through decreasing renal oxidative stress-mediated signalling and normalizing ACE2 and MasR expression.

CLINICAL PERSPECTIVES

  • Enhanced intrarenal Agt gene expression/RAS activation induces systemic hypertension and kidney injury in diabetes and its effects can be countered by Ang-(1–7); however, the molecular mechanism(s) underlying the beneficial actions of Ang-(1–7) on systemic hypertension and kidney injury are not fully understood.

  • In the present study in diabetic Akita mice, Ang-(1–7) administration normalized systemic hypertension and attenuated glomerular injury and tubulointerstitial fibrosis. Ang-(1–7) decreased oxidative stress and expression of pro-hypertensive genes, and normalized the expression of ACE2 and MasR in the kidneys. Co-administration of A779, an antagonist of MasR effectively reversed most of the effects of Ang-(1–7).

  • Our results indicate the potential of Ang-(1–7) as a therapeutic agent for treatment of systemic hypertension and kidney injury in diabetes.

INTRODUCTION

ACE2 (angiotensin-converting enzyme 2), an enzyme homologue of ACE, was identified by two independent groups in 2000 [1,2]. ACE2 is 42% homologous with ACE in the catalytic domain and specifically cleaves AngI (angiotensin I) and AngII (angiotensin II) into Ang-(1–9) and Ang-(1–7) respectively. However, the catalytic efficiency of ACE2 on AngII is 400-fold greater than on AngI, resulting in direct Ang-(1–7) formation [3,4]. Evidence now supports a counter-regulatory role for Ang-(1–7) via its own receptor, the Ang-(1–7) receptor or MasR, which oppose many AT1-R (AngII subtype 1 receptor)-mediated outcomes (for reviews, see [57]).

Earlier studies on the effects of Ang-(1–7) on hypertension, and cardiovascular and renal function in hypertensive rats with or without diabetes yielded contradictory results. For instance, Giani et al. [8] reported Ang-(1–7) attenuation of SBP (systolic blood pressure) and reduction in renal oxidative stress and inflammatory markers in Zucker rats. Benter's group [9,10] reported decreased SBP by Ang-(1–7) in SHRs (spontaneously hypertensive rats), but not in streptozotocin-induced diabetic SHRs, despite amelioration of albuminuria and renal vascular dysfunction. In contrast, Tan et al. [11] found no significant reductions in SBP in response to Ang-(1–7) in SHR and Wistar–Kyoto rats.

We have established that high glucose induces ROS (reactive oxygen species) generation and stimulates Agt (angiotensinogen) (the sole precursor of angiotensins) gene expression in RPTCs (renal proximal tubular cells) in vitro [12,13]. Tg (transgenic) mice specifically overexpressing rat Agt in their RPTCs develop hypertension, albuminuria and kidney injury [14,15]. Hyperglycaemia and Agt overexpression act in concert to elicit sHTN (systolic hypertension) and RPTC apoptosis in diabetic Agt-Tg mice [16]. More recently, we documented that RAS (renin–angiotensin system) blockade and Cat (catalase) overexpression in RPTCs prevent sHTN and tubular ROS generation, suppress RPTC apoptosis and normalize ACE2 expression in RPTCs of diabetic Akita Agt-Tg [17] and Akita Cat-Tg mice [18], supporting the view that enhanced ROS generation and RAS activation are pivotal in down-regulation of ACE2 expression and renal injury in diabetes.

In the present study, we investigated the impact of Ang-(1–7) on sHTN, oxidative stress and kidney injury in diabetic Akita mice. We report that Ang-(1–7) administration prevented sHTN and renal oxidative stress, attenuated glomerular hyperfiltration, albuminuria, renal hypertrophy, tubulointerstitial fibrosis and tubular apoptosis, and inhibited pro-fibrotic as well as pro-apoptotic gene expression. Furthermore, Ang-(1–7) through MasR suppressed Agt and ACE expression and normalized renal ACE2 and MasR expression in Akita mice. Finally, Ang-(1–7) suppressed the expression of renal TACE [TNFα (tumour necrosis factor α)-converting enzyme) and NHE-3 (sodium/hydrogen exchanger 3) in Akita mice.

MATERIALS AND METHODS

Chemicals and constructs

Ang-(1–7) and A779 [D-Ala7-Ang-(1–7)] were purchased from Bachem Americas. A rabbit polyclonal antibody against rat Agt was generated in our laboratory (by J.S.D.C.) [19]. It is specific to intact rat and mouse Agt (55–62 kDa) and does not cross-react with pituitary hormone preparations or other rat or mouse plasma proteins. Monoclonal anti-NHE-3 antibody was a gift from Dr Orson Moe (UT Southwestern Medical Center, Dallas, TX, U.S.A.). The sources of other antibodies were: polyclonal anti-(bovine Cat) antibody and anti-β-actin monoclonal antibody (Sigma–Aldrich Canada), polyclonal anti-HO-1 (haem oxygenase 1) antibody (Assay Designs), monoclonal anti-(collagen type IV) antibody (Chemicon International), polyclonal anti-TGF-β1 (transforming growth factor β1), anti-Nox4 (NADPH oxidase 4) and anti-ACE (Santa Cruz Biotechnology), anti-MasR (Novus Canada), and anti-TACE (Enzo Life Sciences). Polyclonal anti-Nrf2 (nuclear factor erythroid 2-related factor 2) antibody was obtained from BD Biosciences. Oligonucleotides were synthesized by Invitrogen. Restriction and modifying enzymes were purchased from Invitrogen, Roche Biochemicals and GE Healthcare Life Sciences.

Ethics statement

The present study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol, animal care and experimental procedures were approved by the CRCHUM (Centre de recherche du Centre hospitalier de l’Université de Montréal) Animal Care Committee. All surgery was performed under sodium pentobarbital anaesthesia. Mice were killed by sodium pentobarbital overdose [75 mg/kg of BW (body weight)], and efforts were made to minimize suffering.

Physiological studies

Male heterozygous Akita mice (C57BL/6-Ins2Akita/J) were obtained from Jackson Laboratories (http://jaxmice.jax.org). Akita mice, an autosomal dominant model of spontaneous Type 1 diabetes in which insulin gene 2 is mutated, have decreased numbers of pancreatic islet β-cells and develop hyperglycaemia at 3–4 weeks of age, manifesting impaired renal function with increased oxidative stress markers in their RPTs (renal proximal tubules) by 30 weeks of age [20,21] and closely resembling those observed in Type 1 diabetes patients. Adult Akita mice (14 weeks of age) were treated subcutaneously with Ang-(1–7) (500 μg/kg of BW per day) with or without A779 (10 mg/kg of BW per day) and killed at 20 weeks of age (eight mice per group), as described previously [18]. Untreated non-Akita WT (wild-type) mice served as controls. All animals were given ad libitum access to standard mouse chow and tap water.

SBP was monitored in the morning with a BP-2000 tail-cuff pressure monitor (Visitech Systems) at least two or three times each week per animal, for 10 weeks [1418,2226]. The mice were habituated to the procedure for at least 15–20 min per day for 5–7 days before the first SBP measurements. SBP values are expressed as means±S.E.M. All animals were housed individually in metabolic cages for 24 h before being killed at 20 weeks of age. Body weight was recorded. Urine samples were collected and assayed for albumin and creatinine by ELISAs (Albuwell and Creatinine Companion; Exocell) [1418,2226]. Immediately following killing, the kidneys were removed, decapsulated and weighed. The left kidneys were processed for histology and immunostaining, and the right kidneys were harvested for isolation of RPTs by Percoll gradient [1418,2226].

The GFR (glomerular filtration rate) was estimated as described by Qi et al. [27], as recommended by the Animal Models of Diabetic Complications Consortium (http://www.diacomp.org/) with slight modifications [18,22,28].

Urinary Ang-(1–7) and AngII measurement

Mouse urinary AngII and Ang-(1–7) levels were quantified by specific ELISAs after extraction in accordance with the manufacturer's protocol (Bachem Americas) and were normalized by urinary creatinine levels [17,18,22,25,26].

Histology

Four to five kidney sections (3–4 μm thick) per kidney from eight animals per group were stained with PAS (periodic acid–Schiff) or Masson's trichrome, and assessed by two independent blinded observers under light microscopy. The collected images were analysed and quantified by the NIH ImageJ software (http://rsb.info.nih.gov/ij/) [1418,2226].

Mean glomerular volume on 30 random glomerular sections per mouse was assessed by Weibel's method [29] with Motic Images Plus 2.0 analysis software (Motic Instruments) [17,18,22,26]. Tubular luminal areas were measured on renal sections (six animals/group, four to five sections per kidney, four random fields per section, ten tubules around the glomerulus per field) with Motic Images Plus 2.0 analysis software [17,18,22,26]. Outer cortical RPTs with similar cross-sectional views and clear nuclear structures were selected. Mean cell volume was estimated by nucleation [30], as described previously [17,18,22,26].

Immunohistochemical staining was performed using the standard avidin–biotin–peroxidase complex method (ABC Staining System, Santa Cruz Biotechnology) [1418,2226]. Immunostaining with non-immune normal rabbit serum in non-Akita mouse kidneys served as control, with no immunostaining being observed (results not shown). Oxidative stress in RPTs in vivo was assessed by staining of frozen kidney sections with DHE (dihydroethidium) (Sigma–Aldrich Canada) and carboxy-H2DCFDA (6-carboxy-2′,7′-dichlorodihydrofluorescein diacetate) (Life Technologies) [18,31]. In these assays, non-fluorescent DHE is oxidized to fluorescent ethidium by superoxide anion, whereas non-fluorescent carboxy-H2DCFDA is oxidized to fluorescent carboxydichlorofluorescein by intracellular ROS (all species). The results were confirmed by standard immunohistochemical staining for HO-1, an oxidative-stress-inducible gene that confers cellular oxidative stress in vivo [18,25,32], Nox4 (a constitutively expressed and predominant form of Nox in diabetic kidneys) [33,34] and Nrf2 (a master regulator of redox balance in cellular cytoprotective responses) [35]. The percentage of apoptotic RPTCs was estimated semi-quantitatively by TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP nick-end labelling) assay (Roche Biochemicals) [1518,2226].

RT-qPCR (real-time quantitative PCR) assays for gene expression

Expression of mRNAs for Agt, ACE, ACE2, MasR, collagen IV, TGF-β1, Nox1, Nox2, Nox4, HO-1, Nrf2, Bax, Bcl-xL, TACE, NHE-3 and β-actin in RPTs was quantified by RT-qPCR with forward and reverse primers as described in [1518,2226] (see Supplementary Table S1).

Western blotting for estimation of protein expression

Western blotting for Nox4, Nrf2, HO-1, Agt, ACE2, ACE, MasR, TACE and NHE-3 was performed on RPT lysates [1518,2226]. The membranes were first blotted with antibodies against Nox4, Nrf2, HO-1, Agt, ACE2, ACE, MasR, TACE and NHE-3 and then re-blotted with anti-β-actin monoclonal antibodies and developed with chemiluminescent developing reagent (Roche Biochemicals). The relative densities of Nox4, Nrf2, HO-1, Agt, ACE2, ACE, MasR, TACE, NHE-3 and β-actin bands were quantified by computerized laser densitometry (ImageQuant software version 5.1, Molecular Dynamics).

Statistical analysis

Statistically significant differences between the experimental groups were analysed by Student's t test or one-way ANOVA and Bonferroni correction as appropriate. Data are expressed as means±S.E.M. P<0.05 was considered to be statistically significant.

RESULTS

Physiological parameters in Akita mice with or without Ang-(1–7) administration

We have previously reported significant differences in SBP between Akita and non-Akita WT mice as early as 8 weeks of age [17,18,22,26]. These differences increased with age (from week 14 until week 20). Ang-(1–7) administration protected Akita mice against SBP elevation compared with non-Akita mice. This protective effect of Ang-(1–7) on SBP in Akita mice was reversed by A779 co-administration (Table 1).

Table 1
Physiological Measurements
WTAkitaAkita + Ang-(1–7)Akita + Ang-(1–7) + A779
(A) Systolic blood pressure (SBP) 107.00±0.79 136.50±2.83 117.6±1.09 133.9±2.47 
(B) Blood glucose (mmol/L) 9.51±0.75 35.47±0.72*** 35.19±0.52*** 35.28±0.65*** 
(C) BW (g) 30.62±0.70 24.4±0.45*** 23.5±0.40*** 24.43±0.44*** 
(D) Kidney weight (KW) (mg) 366.2±8.14 547.6±18.42*** 415.6±13.03††† 429±12.15††† 
(E) KW/BW ratio (mg/g) 11.85±0.29 20.28±0.37*** 18.2±0.64***,†† 17.26±0.24***,††† 
(F) GFR (μl/min)/BW (g) 10.20±0.79 22.00±1.96 *** 16.03±0.92 **,† 19.68±1.17 *** 
(G) ACR (μg/ml/mg/dl) 0.28±0.02 3.14±0.87*** 0.67±0.16††† 0.70±0.13††† 
(H) Glomerular tuft volume (× 103 μm3110.3±12.17 178.2±15.36** 125.2±11† 148.7±8.31 
(I) RPTC volume (× 103 μm36.12±0.26 10.77±0.39*** 6.76±0.22††† 10.92±0.30*** 
(J) Tubular laminar area (μm286.51±2.28 184.00±5.32 96.42±6.23††† 177.00±3.42*** 
WTAkitaAkita + Ang-(1–7)Akita + Ang-(1–7) + A779
(A) Systolic blood pressure (SBP) 107.00±0.79 136.50±2.83 117.6±1.09 133.9±2.47 
(B) Blood glucose (mmol/L) 9.51±0.75 35.47±0.72*** 35.19±0.52*** 35.28±0.65*** 
(C) BW (g) 30.62±0.70 24.4±0.45*** 23.5±0.40*** 24.43±0.44*** 
(D) Kidney weight (KW) (mg) 366.2±8.14 547.6±18.42*** 415.6±13.03††† 429±12.15††† 
(E) KW/BW ratio (mg/g) 11.85±0.29 20.28±0.37*** 18.2±0.64***,†† 17.26±0.24***,††† 
(F) GFR (μl/min)/BW (g) 10.20±0.79 22.00±1.96 *** 16.03±0.92 **,† 19.68±1.17 *** 
(G) ACR (μg/ml/mg/dl) 0.28±0.02 3.14±0.87*** 0.67±0.16††† 0.70±0.13††† 
(H) Glomerular tuft volume (× 103 μm3110.3±12.17 178.2±15.36** 125.2±11† 148.7±8.31 
(I) RPTC volume (× 103 μm36.12±0.26 10.77±0.39*** 6.76±0.22††† 10.92±0.30*** 
(J) Tubular laminar area (μm286.51±2.28 184.00±5.32 96.42±6.23††† 177.00±3.42*** 
*

P < 0.05, **P < 0.01 and ***P < 0.005 compared with WT; †P < 0.05, ††P < 0.01 and †††P < 0.005 compared with Akita mice.

At 20 weeks, Akita mice exhibited elevated kidney weight/BW and heart weight/BW ratios, higher urinary ACR (albumin/creatinine ratio) and GFR than non-Akita mice (Table 1). Ang-(1–7) markedly attenuated these parameters in Akita mice, and its effects (except for urinary ACR and GFR) were reversed by A779 co-administration. (Table 1). In contrast, blood glucose levels did not differ significantly in Akita mice with or without Ang-(1–7) treatment (Table 1).

Cat expression and oxidative stress in Akita mouse kidneys

We have reported that Cat expression was RPTC-specific and co-localized to aquaporin-1-positive RPTCs [18]. We detected lower Cat levels in RPTCs from Akita mice (Figure 1A, panel b) than in non-Akita WT mice (Figure 1A, panel a). Ang-(1–7) normalized Cat expression in Akita mice (Figure 1A, panel c), and its effect was reversed by A779 (Figure 1A, panel d). DHE and carboxy-H2DCFDA staining demonstrated higher levels of oxidative stress in RPTCs from Akita mice (Figures 1B and 1C, panel b) than in non-Akita WT mice (Figures 1B and 1C, panel a). Ang-(1–7) decreased oxidative stress in Akita mice (Figures 1B and 1C, panel c), which was reversed by A779 (Figures 1B and 1C, panel d). Quantification of Cat activity, DHE and carboxy-H2DCFDA staining confirmed these findings (Figures 1D, 1E and 1F respectively). These results confirmed down-regulation of Cat expression and activity in diabetic Akita mice. Ang-(1–7) enhanced Cat expression and activity and effectively attenuated ROS production and its effects were reversed by A779 co-administration.

Catalase expression, catalase activity and oxidative stress in mouse kidneys at 20 weeks of age

Figure 1
Catalase expression, catalase activity and oxidative stress in mouse kidneys at 20 weeks of age

Immunohistochemical staining for Cat (A), DHE (red) (B) and carboxy-H2DCFDA (green) (C) in male mouse kidneys at age 20 weeks. Magnification ×200. Quantification of Cat activity (D) and semi-quantification of DHE (E) and carboxy-H2DCFDA (F) fluorescence in mouse kidneys. Results are means±S.E.M. (n±8 per group). *P<0.05, ***P<0.005, N.S., not significant.

Figure 1
Catalase expression, catalase activity and oxidative stress in mouse kidneys at 20 weeks of age

Immunohistochemical staining for Cat (A), DHE (red) (B) and carboxy-H2DCFDA (green) (C) in male mouse kidneys at age 20 weeks. Magnification ×200. Quantification of Cat activity (D) and semi-quantification of DHE (E) and carboxy-H2DCFDA (F) fluorescence in mouse kidneys. Results are means±S.E.M. (n±8 per group). *P<0.05, ***P<0.005, N.S., not significant.

ROS generation (Figure 2A), NADPH oxidase activity (Figure 2B), Nox4 mRNA and protein expression (Figures 2C–2E) were significantly elevated in the kidneys of Akita than in non-Akita mice. Administration of Ang-(1–7) normalized these changes and these actions were reversed by A779. Interestingly, Nox1 and Nox2 mRNA expression did not differ significantly among different groups (Figure 2C).

ROS generation, NADPH oxidase activity, and Nox1, Nox2 and Nox4 expression in mouse RPTs at 20 weeks of age

Figure 2
ROS generation, NADPH oxidase activity, and Nox1, Nox2 and Nox4 expression in mouse RPTs at 20 weeks of age

ROS generation (A) and NADPH oxidase activity (B) were quantified by lucigenin assays. Nox1, Nox2 and Nox4 mRNA expression in mouse RPTs was quantified by RT-qPCR (C). Immunostaining (D) and Western blotting (E) for Nox4 in mouse RPTs. Membranes were blotted for Nox4, then re-blotted for β-actin. Nox4 levels were normalized by corresponding β-actin levels. Corresponding values in non-Akita control littermates were considered as 100%. Results are means±S.E.M. (n±8). *P<0.05, **P<0.01, ***P<0.005, N.S., not significant.

Figure 2
ROS generation, NADPH oxidase activity, and Nox1, Nox2 and Nox4 expression in mouse RPTs at 20 weeks of age

ROS generation (A) and NADPH oxidase activity (B) were quantified by lucigenin assays. Nox1, Nox2 and Nox4 mRNA expression in mouse RPTs was quantified by RT-qPCR (C). Immunostaining (D) and Western blotting (E) for Nox4 in mouse RPTs. Membranes were blotted for Nox4, then re-blotted for β-actin. Nox4 levels were normalized by corresponding β-actin levels. Corresponding values in non-Akita control littermates were considered as 100%. Results are means±S.E.M. (n±8). *P<0.05, **P<0.01, ***P<0.005, N.S., not significant.

Consistently, immunostaining for the oxidative-stress-inducible proteins Nrf2 and HO-1 was significantly higher in the kidneys of Akita mice (Figures 3A and 3B, panel b) than in non-Akita WT mice (Figures 3A and 3B, panel a) mice. Once again, Ang-(1–7) normalized Nrf2 and HO-1 immunostaining (Figures 3A and 3B, panel c) and these actions were reversed by A779 (Figures 3A and 3B, panel d). Western blotting for Nrf2 (Figure 3C) and HO-1 (Figure 3D) and quantifying mRNA expression for Nrf2 (Figure 3E) and HO-1 (Figure 3F) confirmed these findings. Collectively, these results document Ang-(1–7) attenuation of enhanced oxidative stress in diabetic Akita mice.

Nrf2 and HO-1 expression in mouse kidneys at 20 weeks of age

Figure 3
Nrf2 and HO-1 expression in mouse kidneys at 20 weeks of age

Nrf2 (A) and HO-1 (B) immunostaining. Magnification ×200. Semi-quantification of Western blotting of Nrf2 (C) and HO-1 (D) in mouse kidneys. Values in non-Akita control littermates were considered as 100%. Quantification of mRNA levels of Nrf2 (E) and HO-1 (F) by RT-qPCR in mouse kidneys. Values in non-Akita control littermates were considered as arbitrary unit 1. Results are means±S.E.M. (n=6). *P<0.05, **P<0.01, ***P<0.005, N.S., not significant.

Figure 3
Nrf2 and HO-1 expression in mouse kidneys at 20 weeks of age

Nrf2 (A) and HO-1 (B) immunostaining. Magnification ×200. Semi-quantification of Western blotting of Nrf2 (C) and HO-1 (D) in mouse kidneys. Values in non-Akita control littermates were considered as 100%. Quantification of mRNA levels of Nrf2 (E) and HO-1 (F) by RT-qPCR in mouse kidneys. Values in non-Akita control littermates were considered as arbitrary unit 1. Results are means±S.E.M. (n=6). *P<0.05, **P<0.01, ***P<0.005, N.S., not significant.

Effect of Ang-(1–7) on Agt, ACE2, ACE, MasR, TACE and NHE-3 expression in Akita kidneys

Consistent with previous observations [17,18,22,26], Agt immunostaining was increased significantly in Akita mice (Figure 4A, panel b) compared with WT controls (Figure 4A, panel a). Ang-(1–7) attenuated Agt expression in Akita mice (Figure 4A, panel c), and its effect was reversed by A779 (Figure 4A, panel d). Quantification of Agt protein and Agt mRNA expression by respective Western blotting and RT-qPCR confirmed these findings (Figures 4B and 4C respectively). Furthermore, urinary AngII levels were significantly higher in Akita mice than in non-Akita WT mice (Figure 4D). Ang-(1–7) normalized urinary AngII levels in Akita mice with reversal by A779. In contrast, urinary Ang-(1–7) levels were significantly lower in Akita mice than in non-Akita WT mice (Figure 4E). Ang-(1–7) administration normalized urinary Ang-(1–7) levels in Akita mice with partial, statistically non-significant, reversal by A779.

Agt expression in mouse kidneys at 20 weeks of age

Figure 4
Agt expression in mouse kidneys at 20 weeks of age

(A) Agt immunostaining. Magnification ×200. Western blotting of Agt protein (B) and RT-qPCR of its mRNA level (C) in mouse RPTs. Values in non-Akita control littermates were considered as 100% control or arbitrary unit 1. Results are means±S.E.M. (n=8). **P<0.01, ***P<0.005, N.S., not significant. Urinary AngII (D) and Ang-(1–7) (E) levels. Urinary AngII and Ang-(1–7) levels in non-Akita control littermates were expressed as controls. Results are means±S.E.M. (n=8). *P<0.05, **P<0.01, ***P<0.005, N.S., not significant.

Figure 4
Agt expression in mouse kidneys at 20 weeks of age

(A) Agt immunostaining. Magnification ×200. Western blotting of Agt protein (B) and RT-qPCR of its mRNA level (C) in mouse RPTs. Values in non-Akita control littermates were considered as 100% control or arbitrary unit 1. Results are means±S.E.M. (n=8). **P<0.01, ***P<0.005, N.S., not significant. Urinary AngII (D) and Ang-(1–7) (E) levels. Urinary AngII and Ang-(1–7) levels in non-Akita control littermates were expressed as controls. Results are means±S.E.M. (n=8). *P<0.05, **P<0.01, ***P<0.005, N.S., not significant.

The RPTCs of WT controls (Figure 5A, panel a) exhibited decreased ACE staining relative to Akita mice (Figure 5A, panel b). Ang-(1–7) normalized ACE immunostaining with reversal by A779 co-administration in RPTCs from Akita mice (Figure 5A, panels c and d respectively). In contrast, ACE2 and MasR expression in the RPTCs of non-Akita WT controls (Figures 5B and 5C, panel a respectively) was significantly higher than in Akita mice (Figure 5B and 5C, panel b respectively). Ang-(1–7) normalized ACE2 and MasR immunostaining in RPTCs of Akita mice (Figures 5B and 5C, panel c respectively), and its effects were reversed by A779 co-administration (Figures 5B and 5C, panel d respectively). We confirmed these findings by immunoblotting and RT-qPCR for ACE, ACE2 and MasR protein (Figure 5D) and their respective mRNA expression in isolated RPTs (Figure 5E)

ACE, ACE2 and MasR expression in mouse kidneys at 20 weeks of age

Figure 5
ACE, ACE2 and MasR expression in mouse kidneys at 20 weeks of age

Immunostaining for ACE (A), ACE2 (B) and MasR (C) expression in mouse kidneys at 20 weeks of age. Magnification ×200. Western blotting of ACE, ACE2 and MasR (D) in mouse RPTs. Membranes were re-blotted for β-actin. ACE, ACE2 and MasR levels were normalized by corresponding β-actin levels. Values in non-Akita control littermates were considered as 100% control. RT-qPCR of mRNA levels of ACE, ACE2 and MasR in mouse kidneys (E). Values in non-Akita control littermates were considered as arbitrary unit 1. Results are expressed means±S.E.M. (n=8). *P<0.05, ***P<0.005, N.S., not significant.

Figure 5
ACE, ACE2 and MasR expression in mouse kidneys at 20 weeks of age

Immunostaining for ACE (A), ACE2 (B) and MasR (C) expression in mouse kidneys at 20 weeks of age. Magnification ×200. Western blotting of ACE, ACE2 and MasR (D) in mouse RPTs. Membranes were re-blotted for β-actin. ACE, ACE2 and MasR levels were normalized by corresponding β-actin levels. Values in non-Akita control littermates were considered as 100% control. RT-qPCR of mRNA levels of ACE, ACE2 and MasR in mouse kidneys (E). Values in non-Akita control littermates were considered as arbitrary unit 1. Results are expressed means±S.E.M. (n=8). *P<0.05, ***P<0.005, N.S., not significant.

In contrast with ACE2 and MasR expression, TACE and NHE-3 expression were reduced in RPTCs of Akita mice (Figures 6A and 6B, panel b) compared with WT mice (Figures 6A and 6B, panel a). Ang-(1–7) administration normalized TACE and NHE-3 expression in Akita mice (Figures 6A and 6B, panel c). These actions were reversed by A779 (Figures 6A and 6B, panel d). These findings were confirmed by immunoblotting for TACE and NHE-3 protein (Figures 6C and 6D respectively) in freshly isolated RPTs. mRNA levels of TACE and NHE-3, however, did not differ among different groups (Figures 6E and 6F respectively), as quantified by RT-qPCR.

TACE and NHE-3 expression in mouse kidneys at 20 weeks of age

Figure 6
TACE and NHE-3 expression in mouse kidneys at 20 weeks of age

Immunostaining for TACE (A) and NHE-3 (B) in mouse kidneys at 20 weeks of age. Magnification ×200. Western blotting of TACE (C) and NHE-3 (D) in mouse RPTs. Membranes were re-blotted for β-actin. TACE and NHE-3 levels were normalized by corresponding β-actin levels. Values in non-Akita control littermates were considered as 100% (control). RT-qPCR of mRNA levels of TACE (E) and NHE-3 (F) in mouse RPTs. Values in non-Akita control littermates were considered as arbitrary unit 100%. Results are means±S.E.M. (n=8). *P<0.05, **P<0.01, N.S., not significant.

Figure 6
TACE and NHE-3 expression in mouse kidneys at 20 weeks of age

Immunostaining for TACE (A) and NHE-3 (B) in mouse kidneys at 20 weeks of age. Magnification ×200. Western blotting of TACE (C) and NHE-3 (D) in mouse RPTs. Membranes were re-blotted for β-actin. TACE and NHE-3 levels were normalized by corresponding β-actin levels. Values in non-Akita control littermates were considered as 100% (control). RT-qPCR of mRNA levels of TACE (E) and NHE-3 (F) in mouse RPTs. Values in non-Akita control littermates were considered as arbitrary unit 100%. Results are means±S.E.M. (n=8). *P<0.05, **P<0.01, N.S., not significant.

Effect of Ang-(1–7) on renal fibrosis and pro-fibrotic gene expression in kidneys of Akita mice

Unlike non-Akita WT mice (Figure 7A, panel a), Akita mice exhibited renal structural damage (Figure 7A, panel b). Histological findings included tubular luminal dilatation and accumulation of cell debris in the tubular lumen. Some RPTCs were flattened. Remarkably, Ang-(1–7) administration in Akita mice markedly suppressed, but never completely prevented, these abnormalities (Figure 7A, panel c). A779 co-administration partially reversed Ang-(1–7)'s effect (Figure 7A, panel d). We observed significantly increased glomerular tuft volume, tubular luminal area and RPTC volume in Akita mice compared with non-Akita WT mice (Table 1). Ang-(1–7) partially reduced tubular luminal area and glomerular tuft volume, and completely normalized RPTC volume in Akita mice. Again, co-A779 administration partially reversed the effect of Ang-(1–7) on these parameters (Table 1).

Tubulointerstitial fibrosis in mouse kidneys at 20 weeks of age

Figure 7
Tubulointerstitial fibrosis in mouse kidneys at 20 weeks of age

PAS staining (A) and Masson's trichrome staining (B) (magnification ×600) and immunostaining for collagen IV (C) and TGF-β1 (D) (magnification ×200) expression in mouse kidneys at 20 weeks of age. (E) Quantification of extracellular matrix component accumulation (Masson's trichrome staining). (F) Quantification of immunoreactive collagen IV deposition. (G and H) RT-qPCR of mRNA levels of collagen IV (G) and TGF-β1 (H) in mouse kidneys. Values in non-Akita control littermates were considered as 100% control. Results are means±S.E.M. (n=8). **P<0.01, ***P<0.005, N.S., not significant.

Figure 7
Tubulointerstitial fibrosis in mouse kidneys at 20 weeks of age

PAS staining (A) and Masson's trichrome staining (B) (magnification ×600) and immunostaining for collagen IV (C) and TGF-β1 (D) (magnification ×200) expression in mouse kidneys at 20 weeks of age. (E) Quantification of extracellular matrix component accumulation (Masson's trichrome staining). (F) Quantification of immunoreactive collagen IV deposition. (G and H) RT-qPCR of mRNA levels of collagen IV (G) and TGF-β1 (H) in mouse kidneys. Values in non-Akita control littermates were considered as 100% control. Results are means±S.E.M. (n=8). **P<0.01, ***P<0.005, N.S., not significant.

We assessed the expression of collagenous components with Masson's trichrome staining (Figure 7B) and immunostaining for collagen type IV (Figure 7C) and TGF-β1 (Figure 7D). Kidneys from non-Akita WT mice exhibited significantly lower collagenous contents, collagen type IV and TGF-β1 (Figures 7B, 7C and 7D, panels a, respectively) relative to Akita mice (Figures 7B, 7C and 7D, panel b respectively). Ang-(1–7) markedly reduced glomerulotubular fibrosis (Figures 7B, 7C and 7D, panel c respectively), and its effect was reversed by A779 co-administration (Figures 7B, 7C and 7D, panel d respectively). Quantitative analysis of Masson's trichrome staining (Figure 7E), collagen immunostaining (Figure 7F), collagen type IV mRNA (Figure 7G) and TGF-β1 mRNA (Figure 7H) expression confirmed these findings. Collectively, the data indicated that Ang-(1–7) effectively prevented renal fibrosis in Akita mice.

Effect of Ang-(1–7) on tubular apoptosis in Akita kidneys

Next, we investigated the impact of Ang-(1–7) administration on tubular apoptosis in Akita mice by TUNEL assay. The number of TUNEL-positive nuclei in RPTCs from non-Akita WT mice (Figure 8A, panel a) were significantly lower than in Akita mice (Figure 8A, panel b). Ang-(1–7) significantly reduced the number of TUNEL-positive cells in Akita mice (Figure 8A, panel c) and its effect was reversed by A779 (Figure 8A, panel d). Consistently, active (cleaved) caspase-3 expression was lower in RPTCs from non-Akita WT controls (Figure 8B, panel a), but higher in Akita mice (Figure 8B, panel b). Ang-(1–7) attenuated active caspase-3 expression in Akita mice (Figure 8B, panel c) with its reversal by A779 (Figure 8B, panel d). Quantification of TUNEL-positive cell numbers (Figure 8C) and caspase-3 activity assays in isolated RPTs (Figure 8D) confirmed these findings.

Apoptosis in mouse kidneys at 20 weeks of age

Figure 8
Apoptosis in mouse kidneys at 20 weeks of age

(A) TUNEL staining (green) and (B) immunostaining for cleaved (active) caspase-3. Magnification ×200. Semi-quantification of apoptotic RPTCs (C) and caspase-3 activity in isolated RPTs (D). RT-qPCR of mRNA levels of Bax (E) and Bcl-xL (F). Assays were run simultaneously for mRNAs of Bax, Bcl-xL and β-actin. Bax and Bcl-xL mRNA levels were normalized by corresponding β-actin mRNA levels. mRNA levels in non-Akita control littermates were considered as 100%. Results are means±S.E.M. (n=8). *P<0.05, **P<0.01, ***P<0.005, N.S., not significant.

Figure 8
Apoptosis in mouse kidneys at 20 weeks of age

(A) TUNEL staining (green) and (B) immunostaining for cleaved (active) caspase-3. Magnification ×200. Semi-quantification of apoptotic RPTCs (C) and caspase-3 activity in isolated RPTs (D). RT-qPCR of mRNA levels of Bax (E) and Bcl-xL (F). Assays were run simultaneously for mRNAs of Bax, Bcl-xL and β-actin. Bax and Bcl-xL mRNA levels were normalized by corresponding β-actin mRNA levels. mRNA levels in non-Akita control littermates were considered as 100%. Results are means±S.E.M. (n=8). *P<0.05, **P<0.01, ***P<0.005, N.S., not significant.

Akita mice exhibited increased Bax mRNA expression (Figure 8E) and decreased Bcl-xL mRNA expression (Figure 8F) compared with non-Akita WT mice. These changes were normalized in Akita mice by Ang-(1–7), and the actions of Ang-(1–7) were reversed by A779 (Figures 8E and 8F).

DISCUSSION

The present study demonstrates that Ang-(1–7) treatment in Akita mice effectively attenuates oxidative stress, normalizes ACE2 and MasR expression in RPTCs, and suppresses expression of pro-hypertensive, pro-fibrotic and pro-apoptotic proteins. These findings indicate an important role for Ang-(1–7) in regulating renal oxidative stress and subsequently modulating sHTN and kidney injury in vivo.

Expanding our previous findings that Cat overexpression in RPTCs normalizes ACE2 expression and prevents hypertension, reduces tubulointerstitial fibrosis and RPTC apoptosis in Akita Cat-Tg mice [18], we show in the present study that Ang-(1–7) attenuates sHTN, indicating that intrarenal Ang-(1–7) formation is critical for counteracting AngII's impact in Akita mice. These observations highlight an important role of intrarenal ACE2 expression and Ang-(1–7) formation in thwarting hypertension and nephropathy development in diabetic mice.

The mechanisms underlying SBP elevation in Akita mice are largely unknown. The possibility that down-regulation of ACE2 and MasR gene expression and consequently high AngII/Ang-(1–7) ratios facilitate the development of hypertension has received considerable attention [57]. Indeed, our findings demonstrate significantly lower RPTC ACE2, MasR and urinary Ang-(1–7) and higher RPTC ACE and urinary AngII levels in Akita mice than in non-Akita WT mice. Ang-(1–7) normalized these changes and A779 co-administration reversed the effects of Ang-(1–7). These observations are consistent with our previous findings of markedly elevated ACE and depressed ACE2 expression in the kidneys of Akita Agt-Tg mice [17] as well as with studies on normotensive Lewis rats, which showed that RAS blockade increases cortical ACE2 activity and urinary Ang-(1–7) excretion [36]. Normal human kidneys express low ACE and high ACE2 levels, and this ratio is reversed in the kidneys of hypertensive and diabetic patients [3739]. Furthermore, AngII has been observed to up-regulate ACE and down-regulate ACE2 expression in HK2 cells in vitro [37]. Taken together, our data lend support to the concept that intrarenal RAS activation up-regulates Agt and ACE expression and down-regulates ACE2 and MasR expression via enhanced oxidative stress in RPTCs, ultimately contributing to hypertension development.

The mechanisms underlying the antihypertensive action of Ang-(1–7) are not well defined. One possibility is that Ang-(1–7) prevents or attenuates the influence of AngII on glomerular arterioles, thereby lowering glomerular pressure (hyperfiltration), glomerular tuft volume and, eventually, SBP. This possibility is supported by the studies by the Benter group [9,10], which showed that Ang-(1–7) reduces renal vascular resistance in diabetic hypertensive rats. Furthermore, other studies have shown that recombinant human ACE2 attenuates AngII-dependent and pressure-overload-induced hypertension in ACE2-knockout mice [4042], supporting an important counter-regulatory role for Ang-(1–7) in AngII-mediated sHTN and renal abnormalities.

Another possibility is that Ang-(1–7) could affect TACE (an enzyme responsible for ACE2 shedding) and NHE-3 (a key transporter mediating sodium reabsorption in the RPTs). Indeed, our data show increased renal TACE and NHE-3 expression in the RPTCs of Akita mice compared with that in WT mice. Ang-(1–7) administration normalized these changes and the effects of Ang-(1–7) were reversed by A779. These findings suggest that the SBP-lowering action of Ang-(1–7) is mediated, at least in part, via down-regulation of TACE and NHE-3 expression in the RPTs in Akita mice.

Since glomerular hyperfiltration and microalbuminuria are early clinical markers of hypertension- or diabetes-induced nephropathy, we monitored the GFR and urinary albuminuria, and detected enhanced GFR and microalbuminuria in Akita mice at 20 weeks. Ang-(1–7) significantly reduced, but never completely prevented, these changes. Surprisingly, A779 co-administration could not completely overturn Ang-(1–7)'s effect on the GFR and albuminuria despite complete reversal of SBP in Akita mice. The exact reasons for such discordant inhibitory actions remain largely unknown. One possibility is that different subtype(s) of Ang-(1–7) receptor might be present in podocytes that are less sensitive to A779 inhibition. This notion is further supported by the studies of Silva et al. [43], which showed that the Ang-(1–7) receptor is sensitive to D-Pro7-Ang-(1–7), but not to A779, in the aorta of Sprague–Dawley rats. Clearly, additional studies are needed to address this issue in podocytes.

The mechanism by which oxidative stress leads to interstitial fibrosis in Akita mice is far from being fully understood. It is possible that augmented ROS generation via enhanced Agt/AngII expression would stimulate TGF-β1, subsequently heightening the expression of extracellular matrix proteins, collagen type IV, and pro-fibrotic and pro-apoptotic proteins in RPTCs, with resultant apoptosis and, ultimately, interstitial fibrosis [44]. Indeed, neutralizing TGF-β alleviates fibrosis and tubular cell apoptosis in animal models of diabetes [45]. The present study shows higher TGF-β1 and collagen IV expression in RPTs from Akita mice than from non-Akita mice. Ang-(1–7) mitigated these changes, thus linking intrarenal oxidative stress to interstitial fibrosis.

To investigate further the role of oxidative stress in mediating the underlying mechanism(s) of Ang-(1–7) action on SBP regulation, we treated Akita mice with the ROS scavenger tempol (4-hydroxytempo) or with tempol and Ang-(1–7). Confirming the observations of Fujita et al. [46], tempol had no effect on SBP, whereas it attenuated renal oxidative stress in Akita mice (Supplementary Figure S1). In contrast, co-administration of tempol and Ang-(1–7) effectively lowered SBP and reduced renal oxidative stress in Akita mice. The reasons tempol failed to lower SBP in Akita mice are not known at present.

Confirming our previous findings in Akita kidneys [17,18,22,26], we detected higher number of apoptotic RPTCs in Akita mice, as was evident by higher percentages of TUNEL-positive RPTCs in parallel with increased active caspase-3 immunostaining and Bax mRNA expression and decreased Bcl-xL mRNA expression. Elevated Bax/Bcl-xL ratios are consistent with tubular apoptosis in Akita mice. Once again, these changes were mitigated in Akita mice by Ang-(1–7), and the actions of Ang-(1–7) were reversed by A779.

Our results may have clinical implications for the assessment of progression in Type 1 diabetes. Tubulointerstitial fibrosis and tubular apoptosis occur in the kidney in human Type 1 diabetes [47], and tubular atrophy appears to be a better indicator of disease progression than glomerular pathology [48]. We postulate that tubulointerstitial fibrosis and RPTC apoptosis may be the initial events leading to tubular atrophy in diabetes. Oxidative-stress-mediated decreases of Cat, ACE2 and MasR expression could further accelerate this process.

In summary, our data indicate an important role for Ang-(1–7) in inhibiting intrarenal oxidative stress, normalizing ACE2 and MasR expression, and subsequent preventing the development of hypertension and renal injury in Akita mice.

Abbreviations

     
  • ACE

    angiotensin-converting enzyme

  •  
  • ACR

    albumin/creatinine ratio

  •  
  • Agt

    angiotensinogen

  •  
  • Ang

    angiotensin

  •  
  • BW

    body weight

  •  
  • carboxy-H2DCFDA

    6-carboxy-2′,7′-dichlorodihydrofluorescein diacetate

  •  
  • Cat

    catalase

  •  
  • DHE

    dihydroethidium

  •  
  • GFR

    glomerular filtration rate

  •  
  • HO-1

    haem oxygenase 1

  •  
  • NHE-3

    sodium/hydrogen exchanger 3

  •  
  • Nox4

    NADPH oxidase 4

  •  
  • Nrf2

    nuclear factor erythroid 2-related factor 2

  •  
  • PAS

    periodic acid–Schiff

  •  
  • RAS

    renin–angiotensin system

  •  
  • ROS

    reactive oxygen species

  •  
  • RPT

    renal proximal tubule

  •  
  • RPTC

    renal proximal tubular cell

  •  
  • RT-qPCR

    real-time quantitative PCR

  •  
  • SBP

    systolic blood pressure

  •  
  • SHR

    spontaneously hypertensive rat

  •  
  • sHTN

    systolic hypertension

  •  
  • TACE

    TNFα (tumour necrosis factor α)-converting enzyme

  •  
  • Tg

    transgenic

  •  
  • TGF-β1

    transforming growth factor β1

  •  
  • TUNEL

    terminal deoxynucleotidyltransferase-mediated dUTP nick-end labelling

  •  
  • WT

    wild-type

AUTHOR CONTRIBUTION

Yixuan Shi performed research and contributed to discussion. Chao-Sheng Lo performed research and contributed to discussion. Ranjit Padda performed research. Shaaban Abdo performed research. Isabelle Chenier performed research. Janos Filep contributed to discussion, and reviewed/edited the paper before submission. Julie Ingelfinger contributed to discussion, and reviewed/edited the paper before submission. Shao-Ling Zhang performed research, contributed to discussion and reviewed/edited the paper before submission. John Chan contributed to discussion, wrote the paper, and reviewed/edited the paper before submission. John Chan is the guarantor of this work and, as such, had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Some of the data reported in the present paper have been presented as a poster communication at the 43rd Annual Meeting of the American Society of Nephrology, Atlanta, GA, U.S.A., 5–10 November 2013. Editorial assistance before submission was provided by the CRCHUM Research Support Office and Ovid Da Silva.

FUNDING

This work was supported in part by the Kidney Foundation of Canada [grant number KFOC120008 (to J.S.D.C.)], the Canadian Institutes of Health Research [grant numbers MOP-93650 and MOP-106688 (to J.S.D.C.), MOP-86450 (to S.L.Z.) and MOP-97742 (to J.G.F.)], the National Institutes of Health (NIH) of the U.S.A. [grant number HL-48455 (to J.R.I.)] and the Foundation of the CHUM (Centre hospitalier de l’Université de Montréal).

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Author notes

1

These authors contributed equally to this work.

2

Joint senior authors.

Supplementary data