Main

The renin–angiotensin system has been recognized for many years as a critical pathway leading to chronic kidney disease. Renin promotes the production of angiotensin I (Ang I), which is converted to Ang II by Ang I-converting enzyme (ACE) and also possibly by other enzymes.1 Ang II, as an important active peptide, exerts a variety of biological and pathological effects on several target tissues and organs, including blood vessels, kidney, and heart under normal and disease conditions. ACE 2 is the homolog of ACE but counterbalances the ACE activity via promoting Ang II degradation to the vasodilator peptide Ang 1–7. Ang 1–7 is a biologically active peptide and acts on the Mas receptor to exert the opposite effect on Ang II.1 Ace2 is highly expressed in the normal kidney, largely by tubular epithelial cells.2 Increasing evidence shows that Ace2 has an essential role in the cardiovascular and kidney diseases.1, 3, 4 Ace2 inhibition also leads to the development of albuminuria in a mouse model of diabetes.5, 6

Direct evidence for ACE2 in the development of hypertensive kidney disease comes from the Ace2 gene knockout mice. Loss of Ace2 leads to the late development of glomerulosclerosis by 12 months of the age, and accelerates kidney injury in mouse models of diabetes and Ang II infusion.7, 8, 9 Recent finding that administration of human recombinant ACE2 inhibits progression of diabetic kidney disease clearly demonstrated a renoprotective role for ACE2 in the progression of chronic kidney diseases.10 All these studies suggest a negative regulatory role for ACE2 in blood pressure and Ang II-mediated hypertensive and diabetic nephropathy. The function of ACE2 in non-hypertension and diabetic condition was not yet clear. Furthermore, the mechanisms of ACE2 in protection against Ang II-mediated renal injury remain largely unclear. Thus, the present study aimed to investigate the potential role of Ace2 in chronic kidney disease independent of hypertension and diabetes and explored the renoprotective mechanisms of Ace2 in progressive renal fibrosis and inflammation.

As a mouse model of UUO is a well-established chronic kidney disease model without underlying hypertensive and diabetic conditions, we thus induced the UUO nephropathy in Ace2−/y mice and examined the hypothesis that loss of Ace2 may promote intrarenal Ang II-mediated renal fibrosis and inflammation independent of blood pressure. In addition, the mechanisms by which loss of Ace2 enhances renal fibrosis and inflammation were investigated.

MATERIALS AND METHODS

Obstructive Kidney Disease Model

Ace2−/y mice (C57BL/6 background) were generated as described previously.11 A mouse model of UUO nephropathy was induced in the littermate male Ace2+/y and Ace2–/y mice (20 g body weight, 8 weeks of age) by the left ureteral ligation as described previously.12 To investigate the role of Ace2 in the early and late stages of UUO, groups of eight Ace2+/y or Ace2–/y mice were killed at day 3 and day 7 after UUO. In addition, six age-matched Ace2+/y or Ace2–/y mice were received sham operation as control. The experimental procedures were approved by the Animal Experimental Committee at the Chinese University of Hong Kong. Kidney tissue samples were collected at day 3 and day 7 after UUO for histology, immunohistochemistry, western blot, and real-time PCR analyses.

Histology and Immunohistochemistry

Changes in renal morphology were examined in methyl Carnoy's fixed, paraffin-embedded tissue sections (4 μm) using the Masson trichrome staining. Immunohistochemistry was performed on paraffin sections using a microwave-based antigen retrieval technique.12, 13 Antibodies used in this study included rabbit antibodies recognized mouse phospho-Smad2/3, MCP-1, IL-1β, TNF-α, TGF-β1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), collagen I (Southern Tech, Birmingham, AL, USA), α-SMA (Sigma, St Louis, MO, USA), and rat anti-mouse monoclonal antibody to macrophages (F4/80) (Serotec, Oxford, UK) and rabbit polyclonal antibodies to CD3+ T cells (Abcam, Cambridge, UK). All slides (except α-SMA and phospho-Smad2/3 stained sections) were counterstained with hematoxylin.

Quantitation of immunostaining was carried on coded slides as previously described.12, 13, 14 IL-1β, TNF-α, and MCP-1 in the entire cortical tubulointerstitium (a cross-section of the renal cortex) were determined by the quantitative Image-Pro plus software (Media Cybernetics, Bethesda, MD, USA) as previously described.12, 13, 14 The number of F4/80+ cells in the tubulointerstitium was counted under high-power fields ( × 40 objective) by means of a 0.0625-mm2 graticule fitted in the eyepiece of the microscope and expressed as cells per millimeters squared (mm2).

Measurement of Blood Pressure and Ang II and Ang 1–7

Blood pressure was measured by tail-cuff method using the CODA non-invasive blood pressure system (Kent Scientific, Torrington, CT, USA) in conscious mice according to the manufacturer’s instruction. Both plasma and intrarenal Ang II and Ang 1–7 were measured using commercially available enzyme immunoassay kits (Peninsula Laboratories, San Carlos, CA, USA). Briefly, the kidney tissue was weighed, homogenized in 1 ml methanol on ice. Then, the samples were centrifuged at 12 000 g at 4°C for 10 min and the supernatant was collected and dried by evaporation. The dried samples were then reconstituted with EIA buffer. Blood plasma was collected from mice, and an equal amount of buffer A was added. The mixture was centrifuged at 17 000 g for 20 min at 4°C and the supernatant was passed through the pre-treated C-18 SEP-COLUMN. The elute peptide was freezer dried and was dissolved in EIA buffer. Concentrations of Ang II and Ang 1–7 were measured by the ELISA kits following the manufacturer's instructions.

Western Blot Analysis

Renal tissues were collected by carefully removing the renal pelvis and medullar tissues, and were frozen at −80°C freezer for western blot analysis as previously described.12, 13, 14 Briefly, after the protein was transferred onto a nitrocellulose membrane, the membrane was incubated overnight with primary antibodies against phospho-Smad3, phospho-IκBα (ser32), phospho-NF-κB/p65 (ser276), ERK1/2 (Cell Signaling Technology, MA, USA); IκBα, NF-κB/p65, phospho-ERK1/2, Smad2/3, Smad7, ACE2, Smurf2, Ang II receptor-1 (AT1) (Santa Cruz Biotech); α-SMA (Sigma), collagen I (Southern Tech), and GAPDH (Chemicon, Temecula, CA, USA) followed by the LI-COR IRDye 800-labeled secondary antibodies (Rockland Immunochemicals, Gilbertsville, PA, USA) in dark for 1 h at room temperature. Signals were scanned and visualized by Odyssey Infrared Imaging System (LiCor, Lincoln, NE, USA). The ratio of the protein interested was subjected to GAPDH and was densitometrically analyzed by the Image J software (NIH, Bethesda, MD, USA).

Real-Time PCR

Renal mRNA expression was quantitatively analyzed by real-time PCR with primers against mouse mRNA of IL-1β, TNF-α, MCP-1, TGF-β1, Smad7, collagen I, α-SMA, and GAPDH as described previously,12, 13, 14 whereas the primers for AT1 and Ace2 were described below: AT1: forward 5′-TGACTTTGGCCACCAGCAT-3′, reverse 5′-CCATTGTCCACCCGATGAA-3′; Ace2: forward 5′-ACCCTTCTTACATCAGCCCTACTG -3′, reverse 5′-TGTCCAAAACCTACCCCACATAT-3′. The reaction specificity was confirmed by melting curve analysis. The housekeeping gene GAPDH was used as an internal standard and the ratio of the mRNA examined to the GAPDH was calculated.

Statistical Analyses

Data obtained from this study were expressed as the mean±s.e.m. Statistical analyses were performed using one-way ANOVA, followed by Newman–Keuls Post Test from Prism 5.0 GraphPad Software (San Diego, CA, USA).

RESULTS

Ace2–/y Mice Promote Renal Fibrosis and Inflammation Independent of Blood Pressure in the UUO Nephropathy

Western blot analysis showed that Ace2 protein was not detectable in Ace2−/y mice, but was increased in the diseased kidney of Ace2+/y mice (Figure 1a and c). Both Ace2+/y and Ace2−/y mice had similar levels of blood pressure under sham-control (126±0.5 mm Hg in Ace2+/y mice vs 122±5.8 mm Hg in Ace2−/y mice) or UUO disease conditions over 7 days (125.8±2 mm Hg in Ace2+/y mice vs 121±5.5 mm Hg in Ace2−/y mice). Histologically, Masson trichrome staining showed no detectable abnormalities in normal or sham-operation Ace2−/y mice compared with Ace2+/y mice (Figure 1b and d). However, 3 days after UUO, Ace2−/y mice developed more intensive tubulointerstitial damage, including tubular atrophy, interstitial extracellular matrix accumulation, which became much more profound at day 7 after UUO (Figure 1b and d). Immunohistochemically, compared to the Ace2+/y mice, tubulointerstitial fibrosis was enhanced in Ace2−/y mice as demonstrated by more abundant collagen I (Figure 1e) and α-SMA (Figure 1i) accumulation in the fibrotic area of tubulointerstitium. Quantitative real-time PCR and western blot analyses further confirmed these findings that disrupted Ace2 largely enhanced collagen I and α-SMA mRNA and protein expression in the UUO kidney when compared with Ace2+/y mice (Figure 1f–h and j–l).

Figure 1
figure 1

Mice lacking Ace2 (KO) are promoted histological damage and renal fibrosis in the UUO nephropathy. (a) Western blot analysis of Ace2 expression in the kidney. (b) Masson's trichrome staining. (c) Semi-quantitative analysis of Ace2 protein expression detected by western blotting. (d) Semi-quantitative analysis of matrix protein accumulation detected by Masson's trichrome staining. (e) Immunohistochemical staining of collagen I. (f) Renal collagen I expression detected by western blot. (g) Semi-quantitative analysis of collagen I detected by western blotting. (h) Collagen I mRNA expression detected by real-time PCR. (i) Immunohistochemical staining of α-SMA expression. (j) α-SMA expression detected by western blot. (k) Semi-quantitative analysis of α-SMA protein expression by western blotting. (l) α-SMA mRNA expression detected by real-time PCR. Each bar represents mean±s.e.m. for at least six mice. *P<0.05, ***P<0.001 compared with sham-operation mice. #P<0.05, ##P<0.01, ###P<0.001 when compared with time-matched Ace2+/y (WT) mice with UUO. Magnification × 200.

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As inflammation is a critical process in the development of UUO, we next examined whether disruption of Ace2 gene influences renal inflammation in the UUO kidney. As shown in Figure 2, immunohistochemistry and real-time PCR revealed that compared with Ace2+/y mice, mice deficient for Ace2 exhibited a substantial increase in renal inflammation as demonstrated by a remarkable upregulation of pro-inflammatory cytokines (TNF-α, IL-1β) and chemokine MCP-1. Enhanced expression of renal TNF-α, IL-1β, and MCP-1 in Ace2–/y mice was accompanied by a significant increase in CD3+ T cells and F4/80+ cells infiltrating the tubulointerstitium (Figure 3).

Figure 2
figure 2figure 2

Mice lacking Ace2 (KO) are promoted renal inflammation in the UUO nephropathy. (a) Immunohistochemical staining of TNF-α expression. (b) Semi-quantitative analysis of TNF-α immunostaining. (c) TNF-α mRNA expression detected by real-time PCR. (d) Immunohistochemical staining of IL-1β. (e) Semi-quantitative analysis of IL-1β immunostaining. (f) IL-1β mRNA expression detected by real-time PCR. (g) Immunohistochemical staining of MCP-1 expression; (h) Semi-quantitative analysis of MCP-1 immunostaining. (i) MCP-1 mRNA expression detected by real-time PCR. Each bar represents mean±s.e.m. for at least six mice. **P<0.01, ***P<0.001 compared with sham-operation mice. #P<0.05, ##P<0.01, when compared with time-matched Ace2+/y (WT) mice with UUO. Magnification × 200.

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Figure 3
figure 3

Mice lacking Ace2 (KO) are promoted CD3+ T-cell and F4/80+ cell infiltration in the UUO nephropathy. (a) Immunohistochemical staining of CD3+ T cells. (b) Immunohistochemical staining of F4/80+ cell. (c) Quantitative analysis of CD3+ T cells. (d) Immunohistochemical staining of F4/80+ cell. CD3+ T-cell and F4/80+ cell infiltration in the tubulointerstitium are significantly increased in Ace2 −/y (KO) mice at day 3 after UUO, which becomes maximal at day 7. Each bar represents mean±s.e.m. for at least six mice. ***P<0.001 compared with sham-operation mice. ###P<0.001 when compared with time-matched Ace2+/y (WT) mice with UUO. Magnification × 200.

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Enhanced Ang II, TGF-β/Smad, and NF-κB Signaling Pathways Are Key Mechanisms by which Ace2−/y Mice Promote Renal Fibrosis and Inflammation in the UUO Nephropathy

It has been shown that Ang II is capable of activating the Smads to mediate fibrosis via both AT1-MAP kinase-crosstalk and TGF-β1-dependent pathways.15, 16, 17 We thus investigated whether enhanced renal fibrosis in the UUO kidney of Ace2−/y mice is associated with an increase in Ang II-mediated TGF-β/Smad pathway. As shown in Figure 4a and b, significant higher levels of intrarenal Ang II and Ang 1–7 were detected in Ace2+/y mice after UUO. Deletion of Ace2 resulted in doubling the intrarenal Ang II in the UUO kidney, while levels of intrarenal Ang 1–7 remained low (Figure 4a and b). Interestingly, the imbalance between Ang II generation and degradation occurred only locally in the UUO kidney, as plasma levels of Ang II and Ang 1–7 remained normal in Ace2+/y and Ace2−/y mice throughout the 7-day disease course (Figure 4c and d). Western blot and real-time PCR analyses detected that increased intrarenal Ang II in Ace2−/y mice was associated with a marked activation of the intrarenal Ang II signaling pathway, including upregulation of AT1 and activation of ERK1/2 mitogen-activated protein kinase (Figure 4e–h). Further study revealed that enhanced Ang II signaling in the UUO kidney of Ace2−/y mice was accompanied by a marked increase in renal TGF-β1 expression (Figure 5a, c and d) and higher levels of Smad2/3 phosphorylation and phospho-Smad2/3 nuclear translocation when compared with Ace2+/y mice (Figure 5b and e–g).

Figure 4
figure 4

Mice lacking Ace2 (KO) are promoted intrarenal Ang II and Ang II signaling in the UUO nephropathy. (a, b) Intrarenal Ang II and Ang 1–7 concentrations measured by ELISA. (c, d) Plasma levels of Ang II and Ang 1–7 measured by ELISA. (e) Western blot analysis of AT1 and phospho-ERK1/2. (f, g) Semi-quantitative analysis of AT1 protein and phospho-ERK1/2 protein by western blots. (h) AT1 mRNA expression detected by real-time PCR. Results show that deletion of Ace2 largely enhances intrarenal, but not circulating, Ang II concentrations, which results in the enhancement of AT1-dependent intrarenal Ang II signaling. Each bar represents mean±s.e.m. for at least six mice. *P<0.05, ***P<0.001 compared with sham-operation mice. #P<0.05, ##P<0.01, ###P<0.001 when compared with time-matched Ace2+/y (WT) mice with UUO.

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Figure 5
figure 5

Disrupted Ace2 enhances TGF-β/Smad signaling in the UUO nephropathy. (a) Immunohistochemical staining of TGF-β1 expression. (b) Immunohistochemical staining of phospho-Smad2/3 nuclear translocation. (c) Semi-quantitative analysis of TGF-β1 immunostaining. (d) TGF-β1 mRNA expression detected by real-time PCR. (e) Semi-quantitative analysis of phospho-Smad2/3 nuclear translocation. (f) Phosphorylation of Smad2/3 (P-Smad2/3) detected by western blots. (g) Semi-quantitative analysis of phosphorylation of Smad2/3 (P-Smad2/3) by western blotting. Results show that, compared to Ace2+/y (WT) mice, disrupted Ace2 (KO) largely enhances TGF-β1 mRNA expression and protein, resulting in a further increase in Smad2/3 signaling as demonstrated by its nuclear translocation and phosphorylation. Each bar represents mean±s.e.m. for at least six mice. ***P<0.001 compared with sham-operation mice. #P<0.05, ##P<0.01 when compared with time-matched Ace2+/y (WT) mice with UUO. Magnification × 200.

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We then determined the mechanism by which deletion of Ace2 promotes renal inflammation in the UUO kidney by examining the NF-κB signaling pathway. As shown in Figure 6, western blot analysis revealed that compared to Ace2+/y mice, enhanced activation of the NF-κB pathway such as an increase in the levels of phospho-IκBα and phospho-NF-κB/p65 was accompanied by degradation of IκBα and NF-κB/p65 in the UUO kidney of Ace2−/y mice (Figure 6).

Figure 6
figure 6

Disrupted Ace2 enhances NF-κB signaling in the UUO nephropathy. (a) Representative western blots. (b) Semi-quantitative analysis of phospho-IκBα. (c) Semi-quantitative analysis of phospho-NF-κB/p65. Results show that compared to Ace2+/y (WT) mice, deletion of Ace2 (KO) largely enhances phosphorylation of both IκBα and NF-κB/p65. Each bar represents mean±s.e.m. for at least six mice. ***P<0.001 compared with sham-operation mice. #P<0.05, ##P<0.01, ###P<0.001 when compared with time-matched Ace2+/y (WT) mice with UUO.

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Loss of Renal Smad7 Is an Underlying Mechanism Required for the Promotion of TGF-β/Smad-Mediated Renal Fibrosis and NF-κB-Driven Renal Inflammation in the UUO Nephropathy in Ace2−/y Mice

We next examined whether enhanced TGF-β/Smad and NF-κB signaling in Ace2−/y mice is associated with a loss of renal Smad7 via the Smurf2-ubiquitin degradation mechanism. As shown in Figure 7, western blot analysis detected that Smurf2 expression was significantly increased in the UUO kidney of Ace2+/y mice, which was further increased in the Ace2−/y mice (Figure 7a and b). Importantly, an increase in Smurf2 expression in the UUO kidney was associated with a reduction of renal Smad7 protein in Ace2+/y mice, which became more profound in mice lacking Ace2 (Figure 7a and c). Interestingly, in contrast to Smad7 protein, real-time PCR detected that Smad7 mRNA expression was significantly increased in the UUO kidney of Ace2+/y mice, which became higher in Ace2−/y mice (Figure 7d). An increase in Smad7 mRNA but loss of Smad7 protein along with upregulation of Smurf2 suggests that the Smurf2-dependent ubiquitin degradation of renal Smad7 occurs within the UUO kidney.

Figure 7
figure 7

Disrupted Ace2 enhances Smurf2-mediated degradation of renal Smad7 protein in the UUO nephropathy. (a) Representative western blots for renal Smurf2 and Smad7 expression. (b) Semi-quantitative analysis of Smurf2 protein. (c) Semi-quantitative analysis of Smad7 protein. (d) Real-time PCR detection of renal Smad7 mRNA. Results show that compared to Ace2+/y (WT), disrupted Ace2 (KO) largely enhances Smurf2 expression, which results in renal Smad7 protein degradation. Note that renal Smad7 mRNA is upregulated in the UUO nephropathy in both Ace2+/y and Ace2−/y mice. Each bar represents mean±s.e.m. for at least six mice. **P<0.01, ***P<0.001 compared with sham-operation mice. #P<0.05, ##P<0.01, ###P<0.001 when compared with time-matched Ace2+/y (WT) mice with UUO.

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DISCUSSION

The present study provided the evidence that loss of Ace2 enhanced renal fibrosis and inflammation in a mouse model of UUO nephropathy. Results from this study also delineated a critical role of Ace2 in negatively regulating endogenous Ang II-mediated progressive renal injury independent of systemic hypertension. Thus, Ace2 is an essential regulator in maintaining the balance between Ang II generation and Ang II degradation locally within the kidney regardless of systemic disease conditions. Furthermore, the present study also found that enhanced TGF-β/Smad and NF-κB signaling pathways were mechanisms by which Ace2−/y mice promote renal fibrosis and inflammation, which was attributed to a loss of renal Smad7 mediated by a mechanism of Ang II-induced Smurf2-dependent ubiquitin-degradation pathway.

Many studies have shown that the expression of Ace2 within the kidney varies with disease conditions. In hypertensive nephropathy, we found that renal ACE2 is largely reduced along with a marked upregulation of ACE and AT1 receptor in patients with hypertension.2 This is consistent with the findings in animal models of hypertension,18, 19 although renal ACE2 expression and activity is unaltered during established hypertension in adult SHRSP and TGR(mREN2)27 rats.20 Similarly, in diabetic nephropathy, while upregulation of Ace2 is noted in the diabetic kidney in mice8, 10, 21, 22 and in humans,23 downregulation of Aces is also reported.24, 25, 26 The functional importance of Ace2 is now established by recent findings that loss of Ace2 promotes but administration of recombinant human ACE2 inhibits exogenous Ang II-induced hypertensive nephropathy and streptozotocin-induced diabetic nephropathy.8, 9, 10, 11, 27 All these findings imply that Ace2 may protect against progressive renal injury in diseases associated with hypertension and diabetes. In the present study, we found that disruption of Ace2 largely enhanced intrarenal Ang II levels (a twofold increase), which was accompanied by much more severe renal fibrosis and inflammation, demonstrating a renoprotective role for Ace2 in obstructive nephropathy.

The finding of Ace2 as a critical regulator in maintaining the balance between endogenous Ang II generation and degradation locally in a mouse model of UUO nephropathy is clinically relevant. It implies that Ace2 not only functions to systemically regulate hypertensive renal injury but also acts as a local regulator for renal protection against the endogenous Ang II-mediated injury independent of hypertension. Indeed, the present study revealed that the local, but not systemic, Ang II generation and degradation was critically regulated by Ace2 as evidenced by the finding that disruption of Ace2 impaired the ACE2/Ang 1–7 axis but enhanced largely the ACE/Ang II/AT1-dependent Ang II generation in Ace2−/y mice (Ang II vs Ang 1–7=4:1). This finding was consistent with the observation in both animals and patients with diabetes or IgA nephropathy.21, 22, 23, 24, 25, 26, 28 Thus, Ace2 is an important counter-regulator for systemic hypertension and locally in determining the disease progression by balancing the intrarenal Ang II generation and degradation pathways.

Although ACE2 is a critical enzyme to degrade Ang I and Ang II to Ang 1–7, a number of alternative enzymes, including neprilysin, prolyl-endopeptidase, and thimet oligopeptidase, as well as ACE, are also capable of converting the Ang I and Ang II to Ang 1–7.4, 29, 30 Thus, the regulation of Ang 1–7 generation is under control by the multiple enzymes systems. It is possible that these Ang 1–7-generating enzymes may compensate each other to maintain the steady-state levels of Ang II and Ang 1–7 both locally and systemically, despite Ace2 as a major pathway for Ang 1–7 generation. This compensatory mechanism may explain our finding that the Ace2−/y and Ace+/y mice have similar levels of plasma Ang II and Ang 1–7 in both normal and the UUO conditions. It could be also true that because obstructive nephropathy was induced in the left kidney only, this leaves the opposite right kidney normal and enables to overcome the functional loss from the left nephropathy. In contrast, the balance between Ang II and Ang 1–7 was altered locally within the UUO kidney, which was much more profound in Ace2−/y mice, resulting in a fourfold increase in the ratio of intrarenal Ang II/Ang 1–7. This finding suggested that the compensatory mechanism locally in the UUO kidney was lost when the Ace2 pathway is disrupted. Therefore, the local vs systemically regulatory networking for both Ang II and Ang 1–7-generating pathways within the body may be attributed to the discrepancy in the levels of Ang II and Ang 1–7 between the plasma and the intrarenal tissue, although the precise mechanisms remain unknown.

A significant finding in the present study was the identification that enhanced TGF-β/Smad-mediated renal fibrosis and NF-κB-driven renal inflammation were key mechanisms by which loss of Ace2 enhanced progressive renal injury in a mouse model of UUO nephropathy. We and other investigators have previously demonstrated that Ang II is able to activate the TGF-β/Smad signaling via both TGF-β-dependent and -independent (through the ERK/p38 mitogen-activated protein kinase crosstalk pathway) mechanisms in vascular and renal cells.15, 16, 17 In the present study, we also found that loss of Ace2 largely increased intrarenal Ang II signaling via the AT1-mediated activation of ERK1/2 mitogen-activated protein kinase pathway. This was associated with enhanced TGF-β/Smad signaling (Smad2/3 phosphorylation and nuclear translocation) and progressive tubulointerstitial fibrosis in Ace2−/y mice when compared with Ace2+/y mice. Furthermore, in the context of renal inflammation, we also found that deletion of Ace2 promoted Ang II-stimulated NF-κB-dependent renal inflammation such as upregulation of IL-1β, TNF-α, MCP-1, and increased macrophage and T-cell infiltration in the UUO nephropathy. This was consistent with the known role of Ang II to activate NF-κB-mediated renal inflammation.31, 32 It is also possible that the development of intrarenal hypoxia within the UUO kidney may also be attributed to the development of renal inflammation.33 Prevention of Ang II-mediated renal oxidative stress, inflammation, and fibrosis by ACE2 supports the notion that loss of Ace2 promoted renal inflammation via the oxidative stress mechanism.9

More importantly, we also found that increased Ang II-induced Smurf2-dependent ubiquitin degradation of renal Smad7 may be the underlying mechanism required for promotion of TGF-β/Smad-mediated renal fibrosis and NF-κB-driven renal inflammation in the UUO nephropathy in Ace2−/y mice. We have previously shown that Smad7 is an integrated regulator that negatively regulates TGF-β/Smad-mediated renal fibrosis via its negative feedback-loop and NF-κB-dependent renal inflammation by the induction of IκBα, an inhibitor of NF-κB.34 Indeed, Smad7 acts as an adaptor protein that binds and recruits Smurf2, an E3 ubiquitin ligase, to the TGF-β1 receptor complex to promote its degradation through the proteasomal pathway.35 At the same time, ubiquitin-degradation of Smad7 occurs simultaneously.35 We have recently shown that Ang II can induce Smurf2 to degrade renal Smad7 in tubular epithelial cells in vitro.36 Once Smad7 is degraded, activation of Smad2/3 and renal fibrosis are enhanced. This is clearly demonstrated by the findings that Ang II activates Smad3 to induce epithelial-myofibroblast transition and renal fibrosis via Smurf2-dependent ubiquitin degradation of Smad7,36 and that upregulation of renal Smurf2 degrades renal Smad7 to promote TGF-β/Smad-dependent renal fibrosis in the UUO nephropathy.37 In addition, we also found that Smad7 is a negative regulator of NF-κB signaling.34 Overexpression of Smad7 is capable of inducing IκBα expression and preventing IκBα from phosphorylation and activation of NF-κB signaling, thereby inhibiting renal inflammation in vivo and in vitro.38, 39 In contrast, deletion of Smad7 promotes NF-κB-dependent renal inflammation in a mouse model of UUO.12 Taken together, loss of Smad7 promotes, while overexpression of Smad7 inhibits, TGF-β/Smad-mediated progressive renal fibrosis and NF-κB-driven renal inflammation as evidenced in a number of kidney disease models including UUO nephropathy,12, 37, 40 hypertension-associated remnant kidney disease,38, 41 diabetic nephropathy,14 and immunologically-mediated glomerulonephritis.42 Therefore, loss of Ace2 promoted Ang II-induced Smurf2-dependent ubiquitin degradation of renal Smad7 may be another essential mechanism by which Ace2−/y mice were promoted TGF-β/Smad-mediated renal fibrosis and NF-κB-driven renal inflammation in a mouse model of UUO nephropathy.