Deletion of angiotensin-converting enzyme 2 exacerbates renal inflammation and injury in apolipoprotein E-deficient mice through modulation of the nephrin and TNF-alpha-TNFRSF1A signaling

Abstract

Background: The renin-angiotensin system (RAS) has been implicated in atherosclerotic lesions and progression to chronic kidney diseases. We examined regulatory roles of angiotensin-converting enzyme 2 (ACE2) in the apolipoprotein E (ApoE) knockout (KO) kidneys.

Methods: The 3-month-old wild-type, ApoEKO, ACE2KO and ApoE/ACE2 double-KO (DKO) mice in a C57BL/6 background were used. The ApoEKO mice were randomized to daily deliver either Ang II (1.5 mg/kg) and/or human recombinant ACE2 (rhACE2; 2 mg/kg) for 2 weeks. We examined changes in pro-inflammatory cytokines, renal ultrastructure, and pathological signaling in mouse kidneys.

Results: Downregulation of ACE2 and nephrin levels was observed in ApoEKO kidneys. Genetic ACE2 deletion resulted in modest elevations in systolic blood pressure levels and Ang II type 1 receptor expression and reduced nephrin expression in kidneys of the ApoE/ACE2 DKO mice with a decrease in renal Ang-(1-7) levels. These changes were linked with marked increases in renal superoxide generation, NADPH oxidase (NOX) 4 and proinflammatory factors levels, including interleukin (IL)-1beta, IL-6, IL-17A, RANTES, ICAM-1, Tumor necrosis factor-alpha (TNF-alpha) and TNFRSF1A. Renal dysfunction and ultrastructure injury were aggravated in the ApoE/ACE2 DKO mice and Ang II-infused ApoEKO mice with increased plasma levels of creatinine, blood urea nitrogen and enhanced levels of Ang II in plasma and kidneys. The Ang II-mediated reductions of renal ACE2 and nephrin levels in ApoEKO mice were remarkably rescued by rhACE2 supplementation, along with augmentation of renal Ang-(1-7) levels. More importantly, rhACE2 treatment significantly reversed Ang II-induced renal inflammation, superoxide generation, kidney dysfunction and adverse renal injury in ApoEKO mice with suppression of the NOX4 and TNF-alpha-TNFRSF1A signaling. However, rhACE2 had no effect on renal NOX2 and TNFRSF1B expression and circulating lipid levels.

Conclusions: ACE2 deficiency exacerbates kidney inflammation, oxidative stress and adverse renal injury in the ApoE-mutant mice through modulation of the nephrin, NOX4 and TNF-alpha-TNFRSF1A signaling. While rhACE2 supplementation alleviates inflammation, renal dysfunction and glomerulus injury in the ApoE-mutant mice associated with upregulations of Ang-(1-7) levels and nephrin expression and suppression of the TNF-alpha-TNFRSF1A signaling. Strategies aimed at enhancing the ACE2/Ang-(1-7) actions may have important therapeutic potential for atherosclerotic renal injury and kidney diseases.

Figures

Fig. 1

Renal ACE2, Ang II and Ang-(1-7) levels in the mice. a Representative Western blot analysis showing ACE2 protein levels in the mice kidneys. β-actin was used as an endogenous control. b, c The Ang II and Ang-(1-7) levels (b) and the ratio of Ang-(1-7)/Ang II (c) in the kidney cortex of mice. AU arbitrary units, WT wildtype, ApoE apolipoprotein E, KO knockout, DKO the ApoE/ACE2 double knockout, AngII angiotensin II. n = 5–6. *P < 0.05;**P < 0.01 compared with WT group; #P < 0.05, compared with ApoEKO group.

Fig. 2

Loss of ACE2 resulted in downregulation of renal nephrin levels in the ApoE/ACE2 DKO mice. The real-time PCR and Western blot analyses for nephrin mRNA (a), nephrin protein (b) and representative nephrin immunofluorescence images (c, d) showing downregulation of nephrin levels in the ApoE/ACE2 DKO kidneys compared with the ApoEKO kidneys. n = 5–6; β-actin or GAPDH was used as an endogenous control. In the immunofluorescence images, the red color represents nephrin and blue color represents DAPI stained nuclei. RE relative expression, AU arbitrary units, WTC wild-type control, ApoE Apolipoprotein E, KO knockout, DKO the ApoE/ACE2 double knockout mice. *P < 0.05; **P < 0.01 compared with WT control group; #P < 0.05 compared with ApoEKO group.

Fig. 3

ACE2 deficiency resulted in elevated AT1 receptor levels and renal inflammation in ApoE/ACE2 DKO kidneys. a, b The Western blot analysis for AT1 receptor protein (a) and representative immunohistological staining images for AT1 receptor (b) showing upregulation of AT1 receptor levels in the ApoE/ACE2 DKO kidneys compared with the ApoEKO kidneys. n = 5; β-actin was used as an endogenous control. c, d Quantification of cytokines in kidney tissues of the ApoEKO and ApoE/ACE2 DKO mice by the mouse cytokine-specific antibody arrays (array C3: upper; array C4: bottom). n = 3 for each group. Please see the related description in Table 3. AU arbitrary units, ApoE apolipoprotein E, KO knockout, DKO the ApoE/ACE2 double knockout. **P < 0.01 compared with ApoEKO group.

Fig. 4

ACE2 deficiency led to increases in renal inflammation in the ApoE/ACE2 DKO mice. The real-time PCR (a–f) and Western blot analyses (g–j) revealed the mRNA or protein levels of the inflammatory cytokines in mice kidneys, including TNFα, TNFRSF1A, TNFRSF1B, IL-1β, IL-6, and IL-17A. n = 5–6. GAPDH or β-actin was used as an endogenous control. RE relative expression, WTC wild-type control, ApoE apolipoprotein E, KO knockout, DKO the ApoE/ACE2 double knockout, TNFα tumor necrosis factor-α, IL interleukin. Please see other abbreviations in Table 3. *P < 0.05; **P < 0.01 compared with WT control group; #P < 0.05 compared with ApoEKO group.

Fig. 5

ACE2 deficiency resulted in increases in oxidative stress levels in the ApoE/ACE2 DKO kidneys. a, b The real-time PCR analysis revealed mRNA expression of the NADPH oxidase subunits NOX2 (a) and NOX4 (b) in mice kidneys (n = 6). GAPDH was used as an endogenous control. c, d Representative dihydroethidium fluorescence images (c), relative fluorescence values and lucigenin-enhanced chemiluminescence assay (d) exhibited the superoxide generation and NADPH oxidase activity in mice kidneys. n = 5. WTC wild-type control, ApoE apolipoprotein E, ACE2 angiotensin-converting enzyme 2, KO knockout, DKO the ApoE/ACE2 double knockout. *P < 0.05; **P < 0.01 compared with WT control group; #P < 0.05 compared with ApoEKO group.

Fig. 6

Treatment with rhACE2 abolished Ang II-mediated reduction in renal nephrin expression in ApoEKO mice. a Representative Western blot analysis showing ACE2 protein levels in the mice kidneys. b, c The Ang II and Ang-(1-7) levels (b) and the ratio of Ang-(1-7)/Ang II (c) in the kidney cortex of the ApoEKO mice treated with Ang II and/or human recombinant ACE2 (rhACE2). d, e The real-time PCR (d) and Western blotting analyses (e) exhibited levels of nephrin in mice kidneys. β-actin or GAPDH was used as an endogenous control. n = 5–6; RE relative expression, AU arbitrary units; **P < 0.01 compared with ApoEKO control group; #P < 0.05; ##P < 0.01 compared with ApoEKO + Ang II group.

Fig. 7

Treatment with rhACE2 prevented Ang II-mediated renal inflammation in the ApoEKO mice. The real-time PCR (a–f) and Western blot analyses (g–i) demonstrated the renal mRNA or protein expression of inflammatory cytokines in the ApoEKO mice, including TNFα, TNFRSF1A, TNFRSF1B, IL-1β, IL-6, and IL-17A (n = 4–6). GAPDH or β-actin was used as an endogenous control. RE relative expression, rhACE2 human recombinant ACE2, Ang II angiotensin II. Please see other abbreviations in Table 3. *P < 0.05; **P < 0.01 compared with ApoEKO control group; #P < 0.05; ##P < 0.01 compared with ApoEKO + Ang II group.

Fig. 8

Effects of rhACE2 on renal oxidative stress levels in the Ang II-infused ApoEKO mice. a, b The real-time PCR analysis revealed mRNA expression of the NADPH oxidase subunits NOX2 (a) and NOX4 (b) in mice kidneys (n = 4–6). GAPDH was used as an endogenous control. c–e Representative dihydroethidium fluorescence images (c), relative fluorescence values (d) and lucigenin-enhanced chemiluminescence assay (e) exhibited the superoxide generation and NADPH oxidase activity in mice kidneys. n = 4–5. AU arbitrary units, ApoE apolipoprotein E, ACE2 angiotensin-converting enzyme 2, KO knockout, rhACE2 human recombinant ACE2, Ang II angiotensin II, PEG-SOD polyethylene glycol-conjugated superoxide dismutase, DPI diphenylene iodonium chloride (NADPH oxidase inhibitor). *P < 0.05; **P < 0.01 compared with the ApoEKO control group; ##P < 0.01, compared with the ApoEKO + Ang II group.

Fig. 9

Renal ultrastructure changes in mice. The renal glomerulus ultrastructural changes were observed in kidneys of mice by transmission electron microscope analysis (a–g ×4200 magnification; h–k ×7400 magnification). Compared with the ApoEKO mice, renal ultrastructure injury was aggravated in the ApoE/ACE2 DKO mice or the Ang II-infused ApoEKO mice. These ultrastructure changes were characterized with renal mesangial cell necrosis (green star), the immune-complex (IC) formation, podocyte depletion from the glomerular wall linked with the foot process effacement (red arrow) and the thickening and stiffening glomerular capillary basement membrane (red triangle). WTC wild-type control, ApoE apolipoprotein E, ACE2 angiotensin-converting enzyme 2, KO knockout, DKO double knockout, rhACE2 human recombinant ACE2, Ang II angiotensin II, IC immune-complex.

Fig. 10

Effects and potential mechanisms of ACE2 on renal inflammation and injury in ApoE-deficient mice. ACE2 serves as a key regulator of Ang II-mediated actions in kidneys. On one hand, ACE2 deficiency results in downregulation of nephrin levels and greater increases in ROS production and expression of proinflammatory cytokines TNFα, IL-1, IL-6, and IL-17A, contributing to renal inflammation, oxidative stress and structural injury in the ApoEKO mice. On the other hand, rhACE2 treatment promotes nephrin levels and ameliorates the Ang II-induced kidney inflammation and ROS generation, functioning as a negative regulator for kidney dysfunction and renal injury in the ApoEKO mice.