Overexpressed angiotensin-converting enzyme in neutrophils suppresses glomerular damage in crescentic glomerulonephritis

Abstract

While angiotensin-converting enzyme (ACE) regulates blood pressure by producing angiotensin II as part of the renin-angiotensin system, we recently reported that elevated ACE in neutrophils promotes an effective immune response and increases resistance to infection. Here, we investigate if such neutrophils protect against renal injury in immune complex (IC)-mediated crescentic glomerulonephritis (GN) through complement. Nephrotoxic serum nephritis (NTN) was induced in wild-type and NeuACE mice that overexpress ACE in neutrophils. Glomerular injury of NTN in NeuACE mice was attenuated with much less proteinuria, milder histological injury, and reduced IC deposits, but presented with more glomerular neutrophils in the early stage of the disease. There were no significant defects in T and B cell functions in NeuACE mice. NeuACE neutrophils exhibited enhanced IC uptake with elevated surface expression of FcγRII/III and complement receptor CR1/2. IC uptake in neutrophils was enhanced by NeuACE serum containing elevated complement C3b. Given no significant complement activation by ACE, this suggests that neutrophil ACE indirectly preactivates C3 and that the C3b-CR1/2 axis and elevated FcγRII/III play a central role in IC elimination by neutrophils, resulting in reduced glomerular injury. The present study identified a novel renoprotective role of ACE in glomerulonephritis; elevated neutrophilic ACE promotes elimination of locally formed ICs in glomeruli via C3b-CR1/2 and FcγRII/III, ameliorating glomerular injury.NEW & NOTEWORTHY We studied immune complex (IC)-mediated crescentic glomerulonephritis in NeuACE mice that overexpress ACE only in neutrophils. Such mice show no significant defects in humoral immunity but strongly resist nephrotoxic serum nephritis (less proteinuria, milder histological damage, reduced IC deposits, and more glomerular neutrophils). NeuACE neutrophils enhanced IC uptake via increased surface expression of CR1/2 and FcgRII/III, as well as elevated serum complement C3b. These results suggest neutrophil ACE as a novel approach to reducing glomerulonephritis.

Keywords: C3; angiotensin-converting enzyme; complement; crescentic glomerulonephritis; immune complex; neutrophil.

Graphical abstract

Figure 1.

Overexpressed angiotensin-converting enzyme in neutrophils ameliorates glomerular injury in immune complex-mediated crescentic glomerulonephritis. A: proteinuria in the early phase of nephrotoxic serum nephritis (NTN), 24 h after nephrotoxic serum (NTS) injection. Each point represents data from one mouse [naïve wild-type (WT) mice: 2.8 ± 0.2, WT mice at 24 h: 4.6 ± 0.3, and NeuACE mice at 24 h: 4.2 ± 0.4]. *P < 0.05; ***P = 0.0009. NS, no significance. n = 15 for each group. B: proteinuria on day 7 of NTN (WT male mice: 32.1 ± 2.4, WT female mice: 35.6 ± 1.8, NeuACE male mice: 18.8 ± 2.4, and NeuACE female mice: 14.8 ± 3.5). **P < 0.005; ****P < 0.0001. n = 15 for each group. C–E: histological assessment of glomerular injury in NTN mice on day 7. n = 15 for each group. C: glomeruli with fibrinoid necrosis stained with periodic acid-methenamine silver (WT mice: 74.2 ± 8.2% and NeuACE mice: 7.3 ± 1.7%). ****P < 0.0001. D: the same assay was performed for crescents with periodic acid-Schiff stain (WT mice: 4.2 ± 1.1% and NeuACE mice: 0.2 ± 0.1%). **P < 0.003. E: representative image of glomeruli with or without fibrinoid necrosis and crescent formation. F: neutrophil infiltration into glomeruli from WT mice and NeuACE mice with NTN 2 or 24 h after NTS injection (0.35 ± 0.11 per glomerulus in the 2-h WT group vs. 0.80 ± 0.15 per glomerulus in the 2-h NeuACE group, ****P < 0.0001; 0.24 ± 0.08 per glomerulus in the 24-h WT group vs. 0.29 ± 0.03 per glomerulus in the 24-h NeuACE group, P = 0.93). n = 10 for each group. G: representative image of esterase-positive cells in the glomerulus 2 h after NTS injection. Arrows indicate neutrophils. H and I: interstitial neutrophils. H: esterase-positive cells in interstitial areas [50 high-power fields (HPF)/mouse, n = 10 mice for each group] were evaluated 2 h and 7 days after NTS injection. I: representative image of esterase-positive cells in the interstitium on day 7. Arrows indicate neutrophils. J and K: macrophage infiltration to glomeruli. J: Mac-2-positive cells in glomeruli from WT mice (n = 15) and NeuACE mice (n = 15) on day 7 (WT mice: 2.22 ± 0.21 and NeuACE mice: 0.97 ± 0.14). ***P = 0.0002. K: representative image of Mac-2-positive cells in the glomerulus. All scale bars = 25 µm.

Figure 2.

Lesser glomerular immune complex deposition in NeuACE mice. A: rabbit IgG and mouse IgG staining of kidney tissues from wild-type (WT) mice (n = 10) or NeuACE mice (n = 10) with nephrotoxic serum nephritis on day 7. Scale bars = 25 µm. B and C: staining of rabbit IgG (B) and mouse IgG (C) was evaluated semiquantitatively (rabbit IgG: 4 ± 0.0 in WT mice vs. 3.12 ± 0.24 in NeuACE mice, **P = 0.0031; mouse IgG: 3.65 ± 0.20 in WT mice vs. 2.40 ± 0.31 in NeuACE mice, **P = 0.0038). D and E: representative electron microscopy images. WT mice showed more abundant electron-dense immune complex deposits (*) in the glomerular capillary subendothelial space (D) and in the mesangial area (E) than NeuACE mice. Uranyl acetate and lead citrate stain was used. Original magnification: ×19,000. Scale bars = 2 µm. F: autologous murine IgG (anti-rabbit IgG) levels were determined by ELISA from serum samples from WT (n = 12) or NeuACE (n = 10) mice on day 7 of nephrotoxic serum nephritis. Titers in serial dilutions of serum are expressed in arbitrary units [optical density (OD) 450 nm] (×100 dilution: 0.82 ± 0.02 in WT mice vs. 0.85 ± 0.03 in NeuACE mice, P = 0.30; ×1,000 dilution: 0.57 ± 0.03 in WT mice vs. 0.63 ± 0.04 in NeuACE mice, P = 0.23; and ×10,000 dilution: 0.23 ± 0.02 in WT mice vs. 0.30 ± 0.03 in NeuACE mice, P = 0.12). G: recall assay to rabbit IgG in splenocytes. Splenocytes were isolated from WT mice (n = 6) or NeuACE mice (n = 6) after 10 days of immunization of rabbit IgG. Cells were incubated with or without rabbit IgG for 24 h. Supernatants were collected to measure interferon (IFN)-γ by ELISA (WT mice: 0.0 ± 0.0 pg/mL with no rabbit IgG vs. 314.2 ± 27.6 pg/mL with rabbit IgG, *P < 0.05; NeuACE mice: 0.0 ± 0.0 pg/mL with no rabbit IgG vs. 429.2 ± 27.8 pg/mL with rabbit IgG, ***P = 0.0007). H: the same assay as shown in G was done for IL-6 production (WT mice: 19.3 ± 0.75 pg/mL with no rabbit IgG vs. 320.7 ± 7.4 pg/mL with rabbit IgG, *P = 0.014; NeuACE mice: 23.0 ± 0.42 pg/mL with no rabbit IgG vs. 432.6 ± 16.1 pg/mL with rabbit IgG, *P = 0.0123).

Figure 3.

NeuACE neutrophils show enhanced immune complex (IC) uptake. A: neutrophil adhesion on IC-coated plates. Neutrophils were seeded on a plate precoated with ICs (mouse IgG and rabbit IgG) and incubated at 37°C for 30 min. Neutrophils were fixed with 4% paraformaldehyde. After being washed, cells were stained with Flash Phalloidin Green 488. The number of adherent neutrophils was counted under a microscope [without coating: 13.7 ± 0.8 cells/high-power field (hpf) in NeuACE mice and 12.7 ± 1.5 cells/hpf in wild-type (WT) mice, no significance (NS); with coating: 146.2 ± 5.1 cells/hpf in NeuACE mice and 101.2 ± 12.9 cells/hpf in WT mice, ***P = 0.0006]. n = 6 for each group. B: IC uptake assay in neutrophils. Neutrophils were incubated with preformed ICs (FITC-labeled mouse IgG and rabbit IgG) at 37°C for 30 min. After a wash, IC uptake by neutrophils (FITC-positive population in CD11b+Ly-6G+/hiLy-6Cdim/+gate) was detected by flow cytometry. IC-FITC mean fluorescent intensity (MFI) was calculated from the histogram (vehicle: 1.1 ± 0.1-fold MFI in NeuACE mice and 1.0 ± 0.0-fold MFI in WT mice, NS; with IC: 7.4 ± 0.2-fold MFI in NeuACE mice and 5.8 ± 0.1-fold MFI in WT mice, ****P < 0.0001). n = 8 for each group. C: live/dead assay in IC-treated neutrophils. Neutrophils were incubated with preformed ICs at 37°C for 0, 30, 60, and 120 min. The percentage of dead cells was detected in the neutrophil population by flow cytometry (naïve: 98.1 ± 0.7% in WT mice vs. 98.1 ± 0.5% in NeuACE mice, P = 0.98; 30 min: 97.8 ± 0.5% in WT mice vs. 98.2 ± 0.5% in NeuACE mice, P = 0.55; 60 min: 93.6 ± 1.7% in WT mice vs. 96.9 ± 0.5% in NeuACE mice, P = 0.09; 90 min: 63.9 ± 5.1% in WT mice vs. 83.3 ± 6.2% in NeuACE mice, *P = 0.04). n = at least 4 for each group. D: reactive oxygen species (ROS) production in IC-treated neutrophils. Neutrophils were treated with ICs at 37°C for 30 min. ROS production was detected by flow cytometry. ROS MFI was calculated from the histogram (vehicle: 0.6 ± 0.2-fold MFI in WT mice vs. 0.7 ± 0.2-fold MFI in NeuACE mice, NS; with IC: 7.4 ± 0.4-fold MFI in WT mice vs. 11.7 ± 0.5-fold MFI in NeuACE mice, ****P < 0.0001). n = 8 for each group. E: IL-1β production from IC-treated neutrophils (217.1 ± 11.0 pg/mL in WT mice vs. 265.4 ± 17.3 pg/mL in NeuACE mice, *P = 0.02). n = 21 for each group. F: TNF-α production from a similar experiment to that shown in E (78.3 ± 9.2 pg/mL in WT mice vs. 66.9 ± 7.5 pg/mL in NeuACE mice, NS). n = 15 for each group.

Figure 4.

Complement C3b-mediated removal of immune complexes (ICs) is upregulated in NeuACE mice. A: receptor expression profile (CR1/CR2, FcγRII/III, and FcγRI) on neutrophils by flow cytometry [CR1/2: 1.4 ± 0.0-fold mean fluorescent intensity (MFI) in NeuACE mice vs. 1.0 ± 0.3-fold MFI in wild-type (WT) mice, ****P < 0.0001; FcγRII/III: 1.1 ± 0.0-fold MFI in NeuACE mice vs. 1.0 ± 0.0-fold MFI in WT mice, *P = 0.0265; FcγRI: 1.0 ± 0.0-fold MFI in NeuACE mice vs. 1.0 ± 0.0-fold MFI in WT mice, no significance (NS)]. n = 5 for each group. B: effects of CR1/2 blockade and/or FcγRII/III blockade on IC uptake in neutrophils. Preincubated neutrophils with anti-CR1/2 blocking antibody and/or anti-FcγRII/III antibody at room temperature for 10 min were incubated with ICs at 37°C for 30 min. IC uptake by neutrophils was measured by flow cytometry. The results are expressed as fold MFIs against the condition of WT neutrophils with anti-CR1/2 antibody and anti-FcγRII/III antibody (no blocking antibodies: 5.7 ± 0.1-fold MFI in WT mice vs. 7.3 ± 0.3-fold MFI in NeuACE mice, ****P < 0.0001; anti-FcγRII/III antibody only: 2.9 ± 0.1-fold MFI in WT mice vs. 3.0 ± 0.1-fold MFI in NeuACE mice; anti-CR1/2 antibody only: 1.4 ± 0.1-fold MFI in WT mice vs. 1.0 ± 0.1-fold MFI in Neu ACE mice, NS; anti-FcγRII/III antibody and anti-CR1/2 antibody: 1.2 ± 0.1-fold MFI in WT mice vs. 1.2 ± 0.1-fold MFI in NeuACE mice, NS). n = 5 for each group. C: expression levels of chemokine (C-X-C) ligand (CXCL)1, CXCL2, and CXCL5 in kidneys with nephrotoxic serum nephritis (NTN; day 7) from WT mice (n = 3) and NeuACE mice (n = 3) by Western blot. D: serum levels of complement C3 from WT mice (n = 16) and NeuACE mice (n = 13) by ELISA (1,683.0 ± 64.3 µg/mL in WT mice vs. 1,951.0 ± 160.7 µg/mL in NeuACE mice, NS). E: serum levels of complement C3b from WT mice (n = 16) and NeuACE mice (n = 13) by ELISA (18.3 ± 3.2 ng/mL in WT mice vs. 45.2 ± 6.1 ng/mL in NeuACE mice, ***P = 0.0003). F: effect of serum from WT mice and NeuACE mice on IC uptake by neutrophils. Neutrophils from WT mice or NeuACE mice were incubated with ICs in culture media supplemented with 5% serum from WT mice or NeuACE mice at 37°C for 30 min. IC uptake by neutrophils was detected by flow cytometry, and IC-FITC MFI was calculated from the analysis. Lane 1, 1.0 ± 0.0-fold MFI; lane 2, 1.3 ± 0.1-fold MFI; lane 3, 3.2 ± 0.2-fold MFI; lane 4, 3.9 ± 0.1-fold MFI; lane 5, 4.0 ± 0.2-fold MFI; lane 6, 4.7 ± 0.2-fold MFI. Lanes 1 vs. 2, *P < 0.05; lanes 3 vs. 4, **P < 0.01; lanes 5 vs. 6, **P < 0.01; lanes 3 vs. 5, **P < 0.01; lanes 4 vs. 6, **P < 0.01. n = 5 for each group. G: effects of angiotensin-converting enzyme inhibitor or bradykinin 2 receptor antagonist on CR1/2 and FcγRII/II of neutrophils. NeuACE mice were treated with ramipril (5 mg/kg/day, every day, orally), HOE-140 (100 µg/kg/day, every day, subcutaneously), or vehicle only for 3 wk and euthanized to collect neutrophils to analyze by flow cytometry. Expression of CR1/2 and FcγRII/III was expressed as MFI (CR1/2: 1.0 ± 0.1-fold MFI with vehicle, 0.6 ± 0.1-fold MFI with ramipril, and 0.8 ± 0.1-fold MFI with HOE-140, *P < 0.05 for ramipril vs. vehicle; FcγRII/III: 1.0 ± 0.0-fold MFI with vehicle, 0.8 ± 0.1-fold MFI with ramipril, and 1.0 ± 0.0-fold MFI with HOE-140, *P < 0.05 for ramipril vs. vehicle). n = 5 for each group. H: effects of ramipril, an angiotensin-converting enzyme inhibitor, or losartan, an angiotensin receptor blocker, on NTN mice. WT NTN mice and NeuACE NTN mice were treated with ramipril (10 mg/kg/day) or losartan (20 mg/kg/day) from day 2 to day 7 every day by oral gavage. Proteinuria was measured on day 7 (WT mice with vehicle: 35.3 ± 3.0; WT mice with ramipril 25.0 ± 2.9; WT mice with losartan: 20.7 ± 2.6; NeuACE mice with vehicle: 14.4 ± 2.3; NeuACE mice with ramipril: 9.7 ± 2.6; NeuACE mice with losartan: 9.0 ± 1.4). *P < 0.05; ***P = 0.0008. n = 10 for each group.

Figure 5.

Role of neutrophilic angiotensin-converting enzyme (ACE) in immune complex (IC)-mediated crescentic glomerulonephritis. The model depicts the ameliorated glomerular injury in NeuACE mice. Mouse IgG against rabbit IgG is produced upon immunization. Neutrophilic ACE contributes preactivation of complement C3 to generate serum C3b in NeuACE mice. Once nephrotoxic serum is injected, mouse IgG-rabbit IgG ICs are formed. ICs are efficiently captured by NeuACE neutrophils via increased surface CR1/2 and FcγRII/III and eliminated in the reticuloendothelial system, leading to reduced glomerular damage.