Prevention of Angiotensin II–Mediated Renal Oxidative Stress, Inflammation, and Fibrosis by Angiotensin-Converting Enzyme 2

The ongoing epidemic of chronic kidney disease is a health care problem worldwide,^1–3 and preventing the progression of chronic kidney disease to end-stage renal disease is one of the major goals of current nephrology practice. At the tissue level, kidney biopsy samples from patients with severe chronic kidney disease and end-stage renal disease exhibit prominent fibrosis, not only in the glomeruli and renal vasculature but also in the tubuolinterstituim. Fibrosis in the tubulointerstitial compartment is the best histological predictor of clinical outcome in chronic kidney disease.^4–8 Activation of the renin-angiotensin system and the subsequent generation of angiotensin (Ang) II (Ang II) is an important mediator of tubulointerstitial fibrosis and progression to end-stage renal disease.^9,10 Angiotensin-converting enzyme 2 (ACE2) is a pleitropic monocarboxypeptidase capable of metabolizing Ang II into Ang 1 to 7.^11–14 In the cardiovascular system, ACE2 suppresses Ang II–mediated myocardial hypertrophy and fibrosis and prevents cardiac dysfunction.^14,15 ACE2-deficient mice have age-dependent glomerulosclerosis develop¹⁶ and are more susceptible to diabetic renal injury,^17,18 suggesting that ACE2 is a key modulator of kidney diseases. In this study, we directly assessed the hypothesis that ACE2 is a negative regulator of Ang II-induced renal injury and tubulointerstitial fibrosis.

Materials and Methods

Detailed methods are available in the online Supplement (available online at http://hyper.ahajournals.org).

Experimental Animals and Protocols

Mutant mice were back-crossed into a pure C57BL/6 background for >10 generations, as previously described.^19,20 The 10-week-old male ACE2 knockout (KO) (Ace2^−/y) and their littermate wild-type (WT; Ace2^+/y) mice underwent in vivo Ang II infusion. An osmotic minipump (model 1002; Alza) was implanted subcutaneously at the dorsum of the neck to infuse Ang II (1.5 mg/kg⁻¹/d⁻¹) or saline (vehicle) for 14 days. In a separate experiment, Ang II-infused WT mice were treated with placebo or recombinant human ACE2 (rhACE2; 2 mg/kg⁻¹/d⁻¹, intraperitoneal). All experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health (NIH Publication 85-23, revised 1996), Institutional Guidelines, and the Canadian Council on Animal Care.

Histology and Immunohistochemistry

Kidneys were fixed with 10% buffered formalin and embedded in paraffin. Picrosirius red staining and immunostaining for α-smooth muscle actin and CD3-positive lymphocytes were performed as previously described.^21,22

TaqMan Real-Time Polymerase Chain Reaction and Western Blot Analysis

RNA expression levels of various genes were determined by TaqMan (Applied Systems Inc, Streetsville, Ontario, Canada) real-time polymerase chain reaction as described previously.^19,20,23 Expression analysis of the reported genes was performed by TaqMan reverse-transcription polymerase chain reaction using ABI 7900 sequence detection system; 18S rRNA was used as an endogenous control. The primers and probes for mRNA expression analysis by Taqman real-time polymerase chain reaction are listed in the online Supplemental Table I (available online at http://hyper.ahajournals.org). Western blot analysis was used to detect phosphorylated or total proteins or both as described previously.^14,20,23

Generation and Characterization of Human Recombinant ACE2

The extracellular domain of human ACE2 (amino acid residues 1–740; molecular weight=101 kDa)²⁴ was expressed recombinantly in Chinese hamster ovary cells under serum-free conditions in a chemically defined medium as previously described.^14,25 The enzymatic turnover of rhACE2 with Ang II substrate was 5.2±0.1 μmol/mg⁻¹/min⁻¹. The purity of the expression product was 99.99% measured by high-performance liquid chromatography.

Ang Peptide Measurement

The excised kidneys were retrograde-perfused with cold phosphate-buffered saline solution to expel any remaining blood and quickly snap-frozen in liquid nitrogen. Renal Ang II and Ang 1 to 7 levels were measured by radioimmunoassay in the Hypertension and Vascular Disease Centre Core Laboratory at Wake Forest University School of Medicine as previously described.^14,20

Dihydroethidium Fluorescence

Dihydroethidium, an oxidative fluorescent dye, was used to measure superoxide (O₂⁻) levels in kidney tissues from ACE2 KO and WT mice as previously described.^14,20

Lucigenin-Enhanced Chemiluminescence

The activities of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in kidney tissue were quantified by lucigenin-enhanced chemiluminescence as previously described.^19,20

Statistical Analysis

Results are shown as mean±SEM. Statistical analysis was performed using SPSS 11.5 software (SPSS) either by Student t test or by ANOVA, followed by multiple comparison testing (Student-Neuman-Keuls test) as appropriate. A value of P<0.05 was considered statistically significant.

Results

Loss of ACE2 Worsens Ang II-Induced Tubulointerstitial Fibrosis, Renal NADPH Oxidase Activation, and Inflammation

Ang II (and its pro-oxidant effects) is a well-known activator of increased tissue fibrosis, including renal fibrosis.^26–29 Loss of ACE2 enhanced the susceptibility to Ang II-induced tubulointerstitial fibrosis based on picrosirius red staining (Figure 1A). Activation of α-smooth muscle actin expression is a key marker of increased pathological fibrosis and immunohistochemical staining showed a mild elevation in WT kidneys, with a greater increase in ACE2 KO kidneys (Figure 1B) in response to Ang II. Expression analysis confirmed higher renal mRNA expression of α-smooth muscle actin (Figure 1C) and the fibrosis-associated genes, transforming growth factor-β (Figure 1D) and procollagen type Iα1 (Figure 1E), without a differential effect on procollagen type IIIα1 expression (Figure 1F) in Ang II-infused ACE2 KO compared to WT mice. Western blot analysis confirmed increased protein levels of collagen I (Figure 1G), whereas collagen III (Figure 1H) levels were increased similarly in Ang II-treated ACE2 KO vs WT kidneys. Clearly, ACE2 deficiency increases Ang II-induced tubulointerstitial fibrosis.

• Download figure
• Download PowerPoint

In WT mice, infusion of Ang II resulted in a marked reduction in renal ACE2 protein levels (Supplemental Figure SIA, available online at http://hyper.ahajournals.org). We subjected ACE2 KO (Ace2^−/y) and WT (Ace2^+/y) mice to a 14-day period of Ang II infusion (1.5 mg/kg⁻¹/d⁻¹). Renal Ang II levels were increased 4-fold in ACE2 KO kidneys compared with a 2-fold increase in WT kidneys without altering renal Ang 1-7 levels (Supplemental Figure SIB, C). Ang II–mediated NADPH oxidase activation and superoxide generation is a pivotal mechanism of Ang II–mediated renal injury.^30–33 In the absence of ACE2, Ang II-induced superoxide production and NADPH oxidase activation were greater in ACE2 KO mice compared with WT mice (Supplemental Figure SID-F). Real-time polymerase chain reaction analysis of the expression of NADPH oxidase subunits revealed that loss of ACE2 augments Ang II-induced mRNA expression of p47^phox (Supplemental Figure SIG). Increased inflammation is a key mediator of Ang II–mediated injury,^34–36 and as such we assessed for changes in expression of proinflammatory cytokines and chemokines. Expression of the cytokine interleukin (IL)-1β and the chemokine chemokine (C-C motif) ligand 5 (CCL5) were significantly greater in ACE2 KO compared with WT kidneys, whereas the expression levels of tumor necrosis factor-α and IL-6 did not show a differential response (Supplemental Figure SIIA-D, available online at http://hyper.ahajournals.org). The increased renal Ang II levels, NADPH oxidase activation, and proinflammatory state resulted in greater phosphorylation of extracellular-regulated kinase (ERK) ½ pathways with increased protein kinase C (PKC)-α protein levels in the absence of a differential effect on PKC-β1 levels (Supplemental Figure SIIE-G). Together, these data show that loss of ACE2 increased renal oxidative stress, inflammation, and activation of pathological signaling pathways in response to Ang II.

Treatment With rhACE2 Reverses Ang II-Induced Renal NADPH Oxidase Activation and Proinflammatory Changes

We next evaluated whether rhACE2 treatment (2 mg/kg⁻¹/d⁻¹, intraperitoneal) can prevent Ang II–mediated pathological remodeling in the kidneys. Two weeks of treatment with rhACE2 (2 mg/kg⁻¹/d⁻¹, intraperitoneal) results in marked and persistent elevation in plasma ACE2 activity,¹⁴ with the development of extremely low levels of anti-ACE2 IgG titers (14.7±3.5 ng/mL) in only 7 of the 20 mice screened. Renal Ang II levels increased 2-fold in response to 2 weeks of Ang II infusion, which was normalized by rhACE2 (Figure 2A), whereas renal Ang 1-7 levels were unchanged (Figure 2B). In this model, plasma Ang II level is drastically elevated from a basal value of 38.5±8.1 to 494±65.9 pg/mL in response to Ang II infusion and is partially reduced by rhACE2 treatment (212±51 pg/mL; P<0.01).¹⁴ Ang II infusion in WT mice for 2 weeks resulted in a significant increase in systolic blood pressure (168.2±3.7 mm Hg; n=12; P<0.01), which was partially reduced with rhACE2 treatment (136.2±3.3 mm Hg; n=12; P<0.01) compared to vehicle-infused WT mice receiving placebo (106.2±3.1 mm Hg) or rhACE2 (104.2± 4.1 mm Hg; n=8). Ang II-induced increase in superoxide generation in the kidney was largely prevented by rhACE2 (Figure 2C, D) because of a suppression of the Ang II-induced activation of NADPH oxidase (Figure 2E). The superoxide scavengers, polyethylene glycol-superoxide dismutase (650 U/mL; Figure 2C, D) and diphenylene iodonium (10 μmol/L; Figure 2E), were used to confirm the Ang II–mediated superoxide generation. Expression analysis confirmed a complete normalization of p47^phox (Figure 2F), NOX2 (Figure 2G), and NOX4 (Figure 2H) mRNA expression in WT mice receiving Ang II and treated with rhACE2.

• Download figure
• Download PowerPoint

Consistent with a reduction in renal Ang II levels and superoxide production, expression of the inflammatory cytokines, including IL-1β (Figure 3A), CCL5 (Figure 3B), tumor necrosis factor-α (Figure 3C), and IL-6 (Figure 3D), were restored to baseline values in mice infused with Ang II and treated with rhACE2. T-lymphocyte infiltration in the kidney markedly increased in response to Ang II, which was prevented by treatment with rhACE2 (Figure 3E; Supplemental Figure SIII, available online at http://hyper.ahajournals.org). Interestingly, rhACE2 completely normalized Ang II-induced increased phosphorylation of ERK½ (Figure 3F) and the increase in PKC-α (Figure 3G) and PKC-β1(Figure 3H) protein levels. These data illustrate the potency of rhACE2 to suppress Ang II-induced pathological remodeling and renal injury.

• Download figure
• Download PowerPoint

Treatment With rhACE2 Attenuates Ang II–Mediated Tubulointerstitial Fibrosis and Prevented Changes in ACE2 and AT1 Receptor Levels

Histological analysis confirmed that treatment with rhACE2 largely prevented the Ang II–mediated tubulointerstitial fibrosis, based on picrosirius red staining (Figure 4A) and immunostaining for α-smooth muscle actin (Figure 4B). The prevention of oxidative stress and the proinflammatory state suggest that rhACE2 also may have antifibrotic effects in the kidney. Treatment with rhACE2 reduced Ang II-induced mRNA expression of fibrosis-associated genes, including α-smooth muscle actin (Figure 4C), transforming growth factor-β (Figure 4D), procollagen type Iα1 (Figure 4E), and procollagen type IIIα1 (Figure 4F) in WT mice. In agreement with these changes at the mRNA level, Western blot analysis of collagen I (Figure 4G) and collagen III (Figure 4H) showed a marked increase in response to Ang II, which was suppressed by rhACE2 treatment. Ang II–mediated renal fibrosis was also associated with decreased membrane-fractionated E-cadherin protein levels consistent with epithelial-to-mesenchymal transition, which was prevented by rhACE2 (Supplemental Figure SIV, available online at http://hyper.ahajournals.org). These findings clearly indicate that treatment with rhACE2 minimizes the adverse effects of Ang II on the kidney, thereby preventing the development of tubulointerstitial fibrosis and damage. The complete normalization of renal Ang II levels and Ang II–mediated effects implies renal-specific changes were elicited by rhACE2. Western blot analysis revealed that rhACE2 prevented the Ang II–mediated loss of native ACE2 in the kidneys (Figure 5A) while normalizing the increased AT1 receptor levels (Figure 5B) without altering the level of the Mas receptor (Figure 5C).

• Download figure
• Download PowerPoint

• Download figure
• Download PowerPoint

Discussion

ACE2 is the first known homolog of human ACE and functions as a pleiotropic monocarboxypeptidase responsible for the degradation of a range of peptides with a high catalytic efficiency.^11–13 We showed that loss of ACE2 promotes the progression of kidney disease by augmenting Ang II-induced renal injury. In contrast, rhACE2 prevented the molecular, signaling, and histological correlates of Ang II–mediated injury in the kidney. Ang II–mediated loss of renal ACE2 protein may serve as a positive feedback mechanism whereby Ang II-induced injury is perpetuated. In an ACE2-deficient state, Ang II infusion results in a greater pressor response,³⁷ whereas rhACE2 can suppress Ang II-induced pressor response in conscious mice.^14,38 Ang II-induced pressor response is a key mediator of kidney damage,^33,39 and in our experimental model the Ang II-induced pressor response was reduced by rhACE2.

Regulation of renal Ang II levels, coupled with corresponding changes in plasma Ang II levels,^14,37,38 highlights a key role of ACE2 in the metabolism of Ang II. In our experimental system, it is possible that the parallel infusion of ACE2 and Ang II may have contributed to inactivation of Ang II before absorption. ACE2 had a minimal effect on renal Ang 1 to 7 levels in our model. These results suggest that the potent and high-capacity ability of neprilysin^40–42 and Ang II-activated ACE system to metabolize (and reduce) Ang 1 to 7^43,44 are likely the major determinants of steady-state renal Ang 1 to 7 levels. The lack of a change in renal Ang 1 to 7 levels also may have resulted from AT1-mediated intracellular sequestration of Ang II, thereby rendering it resistant to Ang 1 to 7–producing enzymes. We linked changes in Ang II levels with variation in NADPH oxidase activation and O₂⁻ production. Renal NADPH oxidase, which is stimulated by Ang II, increases renal O₂⁻ production and is linked to renal vasoconstriction, renal failure, and tubular dysfunction.^45,46 Ang II activates a plethora of signaling cascades, including ERK½ and PKC signaling pathways.^29,47,48 In this study, Ang II infusion resulted in elevated phosphorylation levels of ERK½ and increased protein levels of PKC-α and PKC-β1 in WT mice, which were significantly enhanced by loss of ACE2 and suppressed by treatment with rhACE2, respectively.

Our data also show a clear role of Ang II-induced proinflammatory state and its ability to be modulated by ACE2. Ang II–mediated inflammation and T-lymphocyte infiltration has emerged as a key player in renal injury and hypertension.^21,36,49 In particular, expression levels of IL-1β and CCL5 were positively regulated in the absence of ACE2, whereas rhACE2 completely prevented their upregulation in response to Ang II. Consistent with previous findings,²¹ our results show that Ang II induces T-lymphocyte infiltration to the kidney, which is a known mediator of IL-1β, IL-6, and the chemokine CCL5 production. Importantly, rhACE2 was able to completely suppress the infiltration of T lymphocytes. CCL5 is a member of the β-chemokine family that is chemotactic for monocytes/macrophages and lymphocytes, whereas IL-1β activates T lymphocytes and plays a pivotal role in the regulation of inflammatory cell infiltration in a variety of renal diseases.^50,51

Ang II directly activates renal fibroblasts through its local tissue actions and renal fibroblasts express AT1 receptors, resulting in increased production of collagen and transforming growth factor-β.^28,52 Loss of ACE2 augments Ang II–mediated mRNA expression of fibrosis-associated genes, including α-smooth muscle actin, transforming growth factor-β, and procollagen type Iα1, leading to increased tubulointerstitial fibrosis. Treatment with rhACE2 markedly decreased the expression of these fibrosis markers. Although treatment with rhACE2 resulted in a global normalization of expression pattern and signaling pathways, loss of ACE2 did not enhance Ang II-induced expression of tumor necrosis factor-α, IL-6, procollagen type IIIα1, and PKC-β1 levels. These differential responses suggest that Ang II infusion in WT mice reached a regulatory threshold level for the activation of these pathways such that the increase in renal Ang II levels in ACE2 KO mice resulted in no further increase. Recombinant human ACE2 also suppressed the Ang II–mediated increase in AT1 receptor and decrease in ACE2 protein levels, respectively, thereby eliciting renal-specific changes capable of further minimizing the detrimental effects of Ang II. The reduced AT1 receptor protein by rhACE2 may have decreased renal Ang II levels because of a reduction in the internalization of Ang II.

Inhibition of ACE2 function is associated with an age-dependent glomerulosclerosis,¹⁶ accelerated glomerular injury in Akita diabetic mice,¹⁷ and streptozocin-induced diabetes,^18,53 providing definitive evidence that ACE2 is renoprotective and that reduced ACE2 expression might contribute to the progression of kidney disease.^54–56 Moreover, rhACE2 slows the progression of diabetic nephropathy.²⁵ Taken together, these observations have given strong support to the concept that ACE2 plays an important role in negatively regulating renal injury. Enhancing ACE2 action by the use of rhACE2 or activator of endogenous ACE2⁵⁷ may minimize the progression of kidney disease.

Perspectives

In summary, loss of ACE2 enhances the susceptibility to Ang II-induced hypertension, renal oxidative stress, and pathological signaling, resulting in worsening tubulointerstitial fibrosis. In contrast, rhACE2 prevents Ang II-induced oxidative stress and pathological signaling. Ang II-induced pressor response is a key mediator of kidney damage and is controlled in part by ACE2. The exacerbation of Ang II-induced renal injury in ACE2 KO mice and the rescue by rhACE2 clearly is linked to an increase and decrease in renal Ang II levels, respectively. Given the upregulation of the renin-angiotensin system in kidney disease, loss of ACE2 function certainly can promote the progression of kidney disease by augmenting Ang II-induced renal injury. ACE2 may act as a protective mechanism in the kidney to limit the pathological activation of the systemic or local renin-angiotensin system or both.

Sources of Funding

J.C.Z. was supported by the National Natural Science Foundation of China . Financial support was received from the Canadian Institute for Health Research (G.Y.O., grant 86602 ; Z.K., grant 84279 ), Alberta Innovates–Health Solutions (G.Y.O. and Z.K.), and Canadian Diabetes Association (J.W.S. and G.Y.O., grant OG-3-08-2559-JS ).

Disclosure

G.Y.O. is a Clinician Investigator Scholar of Alberta Innovates–Health Solutions and a Distinguished Clinician Scientist of the Heart and Stroke Foundation of Canada and Canadian Institutes of Health Research. Z.K. is a New Investigator of the Heart and Stroke Foundation of Canada and a Scholar of Alberta Innovates–Health Solutions. M.S. is COO of Apeiron Biologics and owns stock in that company. H.L. is CEO of Apeiron Biologics and owns stocks in that company. J.M.P. is Founder of Apeiron Biologics and owns stock in that company.

Footnotes