Absence of nicotinic acetylcholine receptor α7 subunit amplifies inflammation and accelerates onset of fibrosis: an inflammatory kidney model

Abstract

Inflammation is regulated by endogenous mechanisms, including anti-inflammatory cytokines, adenosine, and the nicotinic acetylcholine receptor α7 subunit (α7nAChR). We investigated the role of α7nAChR in protection against the progression of tissue injury in a model of severe, macrophage-mediated, cytokine-dependent anti-glomerular basement membrane (GBM) glomerulonephritis (GN), in α7nAChR-deficient (α7^−/−) mice . At d 7 after the injection of anti-GBM antibody, kidneys from α7^−/− mice displayed severe glomeruli (P < 0.0001) and tubulointerstitial lesions (P < 0.001) compared to kidneys from WT mice. An important finding was the presence of severe glomerulosclerosis in α7^−/− mice in this early phase of the disease. Kidneys of α7^−/− mice showed greater accumulation of inflammatory cells and higher expression of chemokines and cytokines than did those of WT mice. In addition, in α7^−/− fibrotic kidneys, the expression of fibrin, collagen, TGF-β, and tissue inhibitor of metalloproteinase (TIMP)-2 increased, and the expression of TIMP3 declined. The increase in counterregulatory responses to inflammation in α7^−/− nephritic kidneys did not compensate for the lack of α7nAChR. These findings indicate that α7nAChR plays a key role in regulating the inflammatory response in anti-GBM GN and that disruption of the endogenous protective α7nAChR amplifies inflammation to accelerate kidney damage and fibrosis.—Truong, L. D., Trostel. J., Garcia, G. E. Absence of nicotinic acetylcholine receptor α7 subunit amplifies inflammation and accelerates onset of fibrosis: an inflammatory kidney model.

There is increasing evidence of bidirectional communication between the immune and neuroendocrine systems. Cytokines, peptide hormones, and neurotransmitters, as well as their receptors, are present in the brain and in the endocrine and immune systems (1–3). Several immunoregulatory cytokines, including IL-1, IL-2, IL-6, IFN-γ, and TNF are produced in the immune system and the neuroendocrine system. Additionally, the synthesizing enzyme choline acetyltransferase, the signaling molecule acetylcholine (Ach), and the respective receptors (nicotinic and muscarinic) are expressed in nonneuronal cells. As an example, ACh, the principle vagal neurotransmitter, is produced by epithelial cells, T lymphocytes, and endothelial cells (4–6). Moreover, it significantly attenuates the release of TNF-α, IL-1β, IL-6, IL-18, and high-mobility group box 1 (HMGB1), but not the anti-inflammatory cytokine IL-10 in endotoxin-stimulated human macrophages in vitro (7). A recent study showed that stimulation of the vagus nerve activates adrenergic splenic neurons to release norepinephrine, which stimulates ACh synthesis in splenic memory T cells in a β2-adrenoreceptor-dependent manner. ACh released by these splenic T cells binds to the α7 nicotinic acetylcholine receptor (α7nAChR) expressed on macrophages in the red pulp and marginal zone, to suppress the synthesis and release of cytokines (8). In α7^−/− mice, inflammation is markedly increased in endotoxemia and adjuvant arthritis (9, 10). In contrast, stimulation of α7nAchR attenuates experimental arthritis and pancreatitis and also protects kidneys from renal ischemia-reperfusion injury (11–14).

Inflammation is beneficial in repairing injuries; however, it is detrimental when it proceeds in an uncontrolled manner or persists, leading to progressive fibrosis with loss of function (15). During inflammation, there is a release of proinflammatory cytokines and chemokines and recruitment of inflammatory cells that are major sources of profibrotic factors. Macrophages are key cellular mediators of inflammation, and the observation that macrophages ablation markedly attenuates fibrosis in various conditions suggests that these cells are among the main products of profibrotic molecules (16, 17).

Chronic and uncontrolled inflammation causes an excessive accumulation of extracellular matrix (ECM) components, such as collagen, which contributes to the formation of fibrosis. The amount of collagen and other ECM proteins is regulated by the balance between matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs). An imbalance of MMPs and TIMPs may play a pivotal role in fibrogenesis (18, 19).

There is growing evidence that inflammation plays a critical role in the development and progression of heart disease, cancer, stroke, diabetes, kidney disease, sepsis, and several fibroproliferative disorders. Consequently, understanding the mechanisms that regulate inflammation may offer therapeutic targets for inhibiting the progression of several diseases (20–22).

Anti-glomerular basement membrane (GBM) antibody-associated GN is analogous to human crescentic GN. It is characterized by the induction of several cytokines and chemokines and the infiltration of macrophages, which contribute to kidney damage. In this study, we investigated the role of α7nAChR in the protection from progression of kidney injury. We found that in α7nAChR knockout (KO) (α7^−/−) mice, injection of anti-GBM Ab induced extensive kidney injury and robust levels of proinflammatory cytokines. In addition, the profound inflammatory response led to early kidney fibrosis.

MATERIALS AND METHODS

Induction of anti-GBM GN in mice

C57BL/6J mice (The Jackson Laboratory, Bar Harbor, ME, USA) were preimmunized with rabbit IgG 5 d before injection of anti-GBM Ab (30 µl i.v.). Preparation of the anti-GBM Ab has been described (23, 24). Mice were euthanized at d 7, 8, 10, 14, and 21 after the induction of the disease, to determine the expression of α7nAChR in nephritic kidneys. Mice deficient for the α7nAChR were purchased from The Jackson Laboratory (B6.129S7-Chrna7^tm1Bay/J; stock number 003232). Anti-GBM GN was induced in 8-wk-old male and female mice and their age- and sex-matched wild-type (WT) littermate controls. Mice were euthanized at d 7 after the injection of anti-GBM Ab, and kidney tissue and blood were collected. Anti-GBM GN was induced in both heterozygous and homozygous mice, to determine the phenotypic consequences of disruption of α7nAChR in heterozygotes and homozygotes.

Pharmacological activation of α7nAChR in C57BL/6 mice was performed with the selective α7nAChR agonist GTS-21 (4 mg/kg i.p. daily; Sigma-Aldrich, St. Louis, MO, USA) for a period of 7 d (13). Treatment was started 1 d after the induction of the disease; control mice received the vehicle.

Serum creatinine was determined with the Cobas Integra Creatinine Plus system (Roche Diagnostics, Indianapolis, IN, USA). All experiments were performed in protocols approved by the Institutional Animal Care and Use Committees at Baylor College of Medicine and The University of Colorado Denver.

mRNA expression of cytokines and chemokines

Total RNA was isolated from kidneys with TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Five micrograms of total RNA from each sample was used in an RNase protection assay (RPA). mCK2b, mCK3b, mCK5c, and MMP1 multiprobe template sets (BD Pharmingen, San Jose, CA, USA) were used to investigate cytokine, MMP, and TIMP expression. A mouse suppressor of cytokine signaling (SOCS)3 322 bp probe (nt 369–691, as defined in GenBank accession number NM_007707) was generated by RT-PCR of spleen tissue. An RPA was performed with the Torrey Pines Biolabs kit (East Orange, NJ, USA) (24–27). Phosphoimage quantitation of the blots was performed with the PhosphorImager SI scanning instrument and ImageQuaNT software (Molecular Dynamics, Sunnyvale, CA, USA) (24, 28, 29). L-32 was used as the housekeeping gene. Final values were expressed as a ratio of the counts per minute (cpm) for the specific mRNA to L-32 mRNA, to ensure a constant quantity of RNA in each sample.

ELISA

The protein levels of IFN-γ, IL-1β, IL-6, IL-10, IL-12p70, TNFα, and chemokine (C-X-C motif) ligand (CXCL)-1/[macrophage inflammatory protein-2 (MIP-2)] in kidney tissue were assayed with the sandwich immunoassay Multispot Cytokine Assay (Meso Scale Discovery, Rockville, MD, USA), according to the manufacturer’s protocol. The lowest detectable limit was 0.08 pg/ml for IFN-γ, 0.11 pg/ml for IL-1β, 0.84 pg/ml for IL-6, 0.51 pg/ml for IL-10, 30.4 pg/ml for IL-12p70, 0.17 pg/ml for TNF-α, and 0.36 pg/ml for CXCL1/MIP-2.

Western blot analysis

Protein was obtained with a protein extraction reagent (T-PER; ThermoFisher Scientific, Inc., Waltham, MA, USA) containing protease inhibitors. Kidney expression of α7nAChR was detected with an anti-α7nAChR antibody (Abcam, Cambridge, MA, USA). Western blot analysis for detection of TIMP3 was performed on kidneys of α7^−/− and WT mice with GN, using anti-TIMP3 antibody (Cell Signaling Technology, Danvers, MA, USA). Anti-β-actin antibody (BD Biosciences, San Jose, CA, USA) was used as a loading control.

Detection of total and active TGF-β

TGF-β bioactivity was measured in kidneys by a quantitative solid-phase enzyme immunoassay, according to the instructions of the manufacturer (TGF-β1 E_max ImmunoAssay System; Promega, Madison, WI, USA). Total TGF-β1 was assayed by acidifying samples for 15 min at room temperature, followed by neutralization with 1 N NaOH. TGF-β1 levels were expressed in picograms per milligram protein. The minimum detectable level of biologically active TGF-β1 was 32 pg/ml with <3% cross-reactivity with TGF-β2 and -β3 at 10 ng/ml.

α-Bungarotoxin staining

Detection of α7nAChR with α-bungarotoxin (α-Bgt) was performed (9, 30) by incubating peritoneal macrophages and bone marrow–derived macrophages (BMDMs) with Alexa Fluor 594-labeled α-Bgt (Life Technologies, Grand Island, NY, USA) in the cell culture medium at 37°C for 45 min. Nonspecific binding was assessed by incubating the cells in nicotine (500 µM) 10 min before and during the labeling. The cells were washed 3 times with cell culture medium and then fixed for 15 min at room temperature in 4% paraformaldehyde-PBS solution, washed with PBS, and mounted for viewing by fluorescence confocal microscopy (FV1000; Olympus America, Center Valley, PA, USA). Kidney tissue was incubated with Alexa Fluor 594-labeled α-Bgt (1 µg/ml) at 4°C overnight in a dark, humid chamber.

Morphologic analysis, immunohistochemical phenotyping, and quantitation of leukocytes

Kidney samples fixed in formalin and methanol-Carnoy fixative solution were embedded in paraffin. Sections (3–4 μm) were stained with periodic acid-Schiff (PAS) reagent, to assess glomerular hypercellularity, sclerotic glomeruli, and tubulointerstitial (TIN) injury. Infiltrating macrophages in frozen tissue sections were immunohistochemically stained with mAb anti-CD68 (AbD Serotec, Raleigh, NC, USA) (23, 31, 32). Staining of frozen sections with the pan-T-cell marker CD3 (BD Biosciences) was also performed. Bound antibodies were detected with a horseradish peroxidase–based detection system. Positively stained cells per 60 glomeruli were counted and expressed per glomerular section. All quantitative morphologic analyses were performed in a blinded fashion.

Immunohistochemistry of collagen

Paraffin-embedded sections of methanol-Carnoy fixed tissue were stained for collagens. The primary antibodies used were polyclonal goat anti-type I, goat anti-type III, and goat anti-type IV collagen (Southern Biotech, Birmingham, AL, USA). The secondary antibodies consisted of peroxidase-coupled rabbit anti-goat IgG (Dako North America, Inc., Carpinteria, CA, USA). Histologic morphometry for collagen I, III, and IV was performed with a ScanScope digital scanner (Aperio Technologies, Inc., Vista, CA, USA). The results are expressed as the mean percentage of area ± sem.

Fibrin deposition

For detection of fibrin in glomeruli, sections were stained with monospecific FITC-labeled rabbit anti-human fibrinogen cross-reacting with mouse fibrinogen (Dako North America, Inc.). Labeled tissues were visualized with the confocal microscope (FV1000; Olympus) and analyzed by ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Peritoneal and BMDM isolation

Peritoneal macrophages were isolated from WT and α7^−/− mice by washing the peritoneal cavity twice with 10 ml PBS (33, 34). The macrophages were cultured in RPMI 1640 medium for 2 h at 37°C in a humidified 5% CO₂ incubator. Nonadherent cells were removed by gentle washing with PBS. Macrophages were maintained in RPMI 1640 medium with 3% fetal bovine serum (FBS) and 1% penicillin/streptomycin and were stimulated with LPS (5 ng/ml) for 4 and 24 h or with ACh chloride (AChCl) (100 µM; Sigma-Aldrich) in the presence of the ACh esterase inhibitor pyridostigmine bromide (1 mM; Sigma-Aldrich) for 24 h. BMDMs were isolated (35) by first harvesting bone marrow cells from femurs and tibias of C57BL/6 mice. The cells were incubated in DMEM containing 10% FBS, 1% penicillin/streptomycin, and 15% L929 cell-conditioned medium as a source of macrophage colony-stimulating factor at 37°C. After 18 h, nonadherent cells were transferred to a new dish and incubated for an additional 7 d.

TNF-α ELISA

TNF-α levels in sera were measured with an ELISA kit (R&D Systems, Minneapolis, MN, USA), according the manufacturer’s instructions.

Circulating antibody and glomerular IgG deposition

Mouse anti-rabbit IgG titers were measured by ELISA of sera that were collected at d 7 after the injection of anti-GBM Ab (36, 37). Bound mouse IgG was detected with peroxidase-conjugated anti-mouse IgG (Dako North America, Inc.) at 1:1000 dilution and 450 nm absorbance. Normal sera served as a negative control. IgG deposition was determined in kidney frozen sections by using FITC-labeled anti-rabbit and anti-mouse IgG (Dako North America, Inc.) (33, 38).

Statistics

Results for experiments are reported as means ± sem. Student’s t test and ANOVA followed by multiple comparisons (Bonferroni) were used to determine statistical differences between experimental groups. Values of P < 0.05 were considered statistically significant.

RESULTS

Expression of α7nAChR is induced in anti-GBM GN

Normal kidney had very little protein expression of α7nAChR; however, in anti-GBM GN the expression of α7nAChR was highly induced. Expression was most prominent at d 8 and 10 after the injection of the anti-GBM antibody and started to subside by d 14 (Fig. 1A).

Figure 1.

α7nAChR is increased in nephritic kidneys. A) Western blot analysis of α7nAChR protein expression. Representative Western blot showing induction of α7nAChR in anti-GBM GN. A band of approximately 50 kDa was detected as reported (39–41). B) Kidney tissue was stained for α7nAChR with Alexa Fluor 594-labeled α-Bgt. Nephritic kidney from d 8 after the induction of the disease was positive for α-Bgt; in contrast, a weak α-Bgt signal was observed in normal (Nl) kidney and in nephritic kidney at d 21. C) Peritoneal macrophages and BMDMs were stained with Alexa Fluor 594-labeled α-Bgt in the absence or presence of nicotine. Binding of α-Bgt was observed on macrophage surfaces. Treatment with nicotine reduced the intensity of binding.

To confirm this finding, kidney tissues were stained with fluorescently labeled α-Bgt. Low expression of α7nAChR was observed in normal kidney; however, α7nAChR expression increased in nephritic glomeruli on d 8 and decreased at d 21 (Fig. 1B). The immunofluorescence staining pattern did not correspond to endothelial, epithelial, or mesangial cells, suggesting that infiltrating cells express α7nAChR. To identify whether macrophages (the main cell recruited in anti-GBM GN) express α7nAChR, we stained the macrophages with fluorescently labeled α-Bgt. As demonstrated in Fig. 1C, peritoneal macrophages and BMDMs expressed α7nAChR. These data suggest that increased α7nAChR expression is a counterregulatory response to inflammation.

Absence of α7nAChR increases inflammation and kidney damage in anti-GBM GN

To investigate whether α7nAChR is essential in protection against kidney inflammation, we induced anti-GBM GN in α7^−/− mice. The mice developed normally and showed no gross anatomic defects, as reported in another study (9). We also examined the kidney phenotype of α7^−/− mice under normal conditions. Two-mo-old α7^−/− mice and WT mice did not show histologic differences in the glomeruli and the tubulointerstitium (Fig. 2A). At d 7 after the induction of GN, severe glomerular cell proliferation was observed in 92% of the glomeruli in the α7^−/− mice, in contrast to 35% in the WT mice. Of particular note, high degrees of glomerular sclerotic lesions and fibrinoid necrosis were observed in 50% of the α7^−/− mice, but in none of the WT mice. In addition to histologic changes in the glomeruli, the absence of α7nAChR resulted in extensive TIN injury (TIN index, 1.25 ± 0.6 vs. 3.4 ± 0.26, P < 0.001; Fig. 2A, C).

Figure 2.

Absence of α7nAChR increased kidney injury and macrophage infiltration in anti-GBM GN. A) PAS staining of kidney sections of WT and α7^−/− mice. Nephritic kidneys of α7^−/− mice had more severe lesions than those of WT mice. Nl, normal. B) Immunohistochemistry staining for CD68⁺ monocytes/macrophages of kidney sections of WT and α7^−/− mice. C) Quantitation of kidney injury and CD68⁺ cell infiltration from WT and α7^−/− mice with anti-GBM GN. Each data point represents sections from kidneys of 5 (WT) or 8 (α7^−/−) mice and is expressed as the mean ± sem. *P < 0.05; **P < 0.001; ***P < 0.0001.

Anti-GBM GN was induced in heterozygous and homozygous α7^−/− mice. No difference in the severity of GN was observed between heterozygous and homozygous mice (Fig. 3). Glomerular proliferation was observed in 39.5, 88.9, and 81.8% of the glomeruli in the WT, heterozygous, and homozygous groups, respectively. In addition, the index of TIN injury was similar between the heterozygous and homozygous groups (0 in WT, 3.25 ± 0.414 in heterozygous, and 3.7 ± 0.27 in homozygous; n = 6 in each group; P < 0.01 WT vs. heterozygous and P < 0.001 WT vs. homozygous). These data indicate that mutation in 1 copy of the gene is sufficient to disrupt the protective effect of this receptor.

Figure 3.

The severity of GN was similar in heterozygous and homozygous α7 KO mice. PAS staining of kidney sections from WT and heterozygous and homozygous α7 KO mice. Kidney injury was more severe in α7 KO than in WT controls, but the lesions were comparable between heterozygous and homozygous α7 KO mice.

Severity of GN in α7^−/− mice is associated with increased macrophage infiltration

To understand the mechanism of the increased kidney injury, we examined the infiltration of the kidneys by macrophages. More macrophages accumulated in glomeruli of the α7^−/− mice than in those of the WT mice (P < 0.0001, Fig. 2B). Immunohistochemical CD3 staining revealed no difference in T-cell infiltration between the WT and α7^−/− mice (Supplemental Fig. 1).

Cytokine expression increases in kidneys of α7^−/− mice during anti-GBM GN

Macrophage α7nAChR is essential for inhibiting cytokine synthesis. Accordingly, a potential mechanism by which the absence of α7nAChR could increase inflammatory damage and macrophage infiltration is through higher expression of cytokines and chemokines. In normal kidneys, there was sparse expression of cytokines and chemokines (Supplemental Fig. 2). However, in nephritic kidneys from α7^−/− mice, we found higher expression of the mRNA of the proinflammatory cytokines IL-1β, TNF-α, and IL-6 compared with levels in WT mice. Increased expression of the IL-1R antagonist (IL-1Ra) was also observed in kidneys of α7^−/− mice. Enhanced expression of the profibrotic cytokine TGF-β was present in α7^−/− mice. In addition, we found that mRNA expression of CXCL1/MIP-2, CXCL10/IFN-γ-induced protein (IP-10), CCL2/monocyte chemotactic protein (MCP)-1, and chemokine (C-C motif) ligand (CCL)-1/T-cell activator (TCA)-3 was highly induced in the nephritic kidneys of α7^−/− mice compared with expression in kidneys of WT mice (Figs. 4 and 5). ELISA analysis of IL-1β, IL-6, TNF-α, and CXCL1/MIP-2 confirmed that the protein level of these cytokines correlated with their mRNA levels in the kidney (Fig. 6). In addition, increased levels of IFN-γ were detected in nephritic kidneys compared to those of normal controls; however, the levels of IFN-γ were higher in the kidneys of α7^−/− mice. IL-12p70 was not detected in the kidney and, concordant with the mRNA expression, the protein levels of IL-10 were not different among the groups.

Figure 4.

Robust expression of cytokines and chemokines was induced in kidneys from α7^−/− mice compared with WT mice. A–C) RPA of cytokines and chemokines expressed in whole kidney. Cytokines and chemokines were highly induced in kidneys in the absence of α7nAchR. Each lane represents a single mouse. Probes contain polylinker regions and are longer than the protected bands. GAPDH and L-32 were used as housekeeping genes. CCL5/RANTES (RAN), regulated on activation of normal T cells expressed and secreted; MIF, macrophage migration inhibitory factor.

Figure 5.

Densitometric analysis of blots from RPAs of cytokines and chemokines expressed in the kidneys of α7^−/− mice and WT mice with anti-GBM GN. The data are expressed as the ratio of the cpm for the specific mRNA to L-32 mRNA, to ensure a constant quantity of RNA in each sample. Results were sampled from 5 (WT) or 8 (α7^−/−) mice per group and are expressed as means ± SD. *P < 0.05.

Figure 6.

Increased protein levels of cytokines and chemokines correlate with their mRNA levels in the kidney. ELISAs of cytokine and chemokine protein levels. Results are expressed as means ± sem *P < 0.05 vs. normal (Nl) and WT; **P < 0.005 vs. Nl and WT; ***P < 0.0005 vs. Nl and WT.

Active and total TGF-β1 are enhanced in kidneys from α7^−/− mice

In kidneys from α7^−/− mice, the active TGF-β1 fraction and total TGF-β1 increased significantly vs. levels in normal controls and WT mice. In contrast, no difference in active TGF-β1 and total TGF-β1 was observed in nephritic kidneys of WT mice compared to normal controls (Fig. 7).

Figure 7.

Increased active and total TGF-β1 in nephritic kidneys from α7^−/− mice. ELISAs of (A) active and (B) total TGF-β1. Both, active and total TGF-β1 were significantly increased in α7^−/− kidneys compared with those of normal (Nl) controls and WT. Results are expresses as means ± sem (n = 3 Nl and WT, n = 5 α7^−/−) *P < 0.05 vs. Nl and WT.

Increased expression of modulators of cytokine function does not compensate for the absence of α7receptor

Increased mRNA expression of IL-1Ra, the natural inhibitor of IL-1, was observed in nephritic kidneys of α7^−/− mice. In addition, the expression of SOCS3 mRNA was enhanced in the kidneys of α7^−/− mice (Figs. 4 and 8A). In contrast, the expression of SOCS3 in macrophages of α7^−/− mice was 4- and 21-fold less than that of the WT macrophages after its activation with LPS at 4 and 24 h, respectively. The expression of IL-1Ra was 1.7-fold less in α7^−/− macrophages than in WT macrophages, at both 4 and 24 h after LPS stimulation (Fig. 8B, C).

Figure 8.

Differential mRNA expression of IL-1Ra and SOCS3 in nephritic kidneys and macrophages. A) RPAs of SOCS3 mRNA expression in whole kidneys and (B, C) representative results of 3 assays to determine SOCS3 and IL-1Ra mRNA expression in macrophages. Kidneys from α7^−/− mice showed stronger expression of SOCS3 than did the WT kidneys (A). Conversely, there was a striking decrease in the expression of (B) SOCS3 and (C) IL-1Ra in activated macrophages from α7^−/− mice compared with those from WT mice. Each lane represents a single mouse. Probes contain polylinker regions and are longer than the protected bands. GAPDH and L-32 were used as housekeeping genes. The data are presented as the ratio of the cpm for the specific mRNA to L-32 mRNA, to ensure a constant quantity of RNA in each sample. Results are expressed as means ± sem. *P < 0.05 vs. normal (Nl); **P < 0.01 vs. WT; and ***P < 0.0001 vs. Nl.

Collagen and fibrin deposition are enhanced in kidneys from α7^−/− mice with anti-GBM GN

Type I and III collagens are normally present in the renal interstitium and blood vessels, and their expression is significantly increased in kidney diseases with fibrosclerotic lesions (42). In α7^−/− mice, increased depositions of collagen I and III were observed. In contrast, collagen I and III expression in WT mice was no different than in normal kidneys. In addition, high deposition of collagen IV, an important component of glomerular ECM, was found in the glomeruli of α7^−/− mice; however, collagen IV expression in WT mice was not different when compared with that in normal kidney controls (Fig. 9). Collagen IV deposition was also increased in the tubular basement membrane in kidneys from α7^−/− mice but not in WT mice.

Figure 9.

Absence of α7nAChR accelerates kidney fibrosis. Collagen I and III expression was increased in the interstitium, and collagen IV deposition was enhanced in the glomeruli from nephritic kidneys in α7^−/− mice. Results are expressed as means ± sem (n = 5 WT, n = 8 α7^−/− mice). *P < 0.05 vs. normal (Nl); ^&P < 0.05 vs. WT; **P < 0.01 vs. WT; ***P < 0.0001 vs. Nl and WT.

Anti-GBM GN is characterized by glomerular fibrin deposition, and functional studies have shown that fibrin is an important mediator of kidney injury. Immunofluorescence staining showed extensive fibrin deposition in most glomeruli of the α7^−/− mice, mainly in the glomeruli tuft. In contrast, scarce expression of fibrin was observed in the glomeruli of the WT mice (Fig. 10).

Figure 10.

Fibrin deposition is increased in nephritic kidneys from α7^−/−mice. Immunofluorescence micrographs of kidney tissue demonstrating significant fibrin deposition in the glomerular tuft of α7^−/− mice (n = 3 normal, n = 5 WT, n = 8 α7^−/−). Magnification, ×600. **P < 0.001 vs. WT; ^δP < 0.001 vs. normal (Nl); ***P < 0.0001 vs. Nl.

Kidney expression of TIMPs is differentially regulated in nephritic α7^−/− mice

We examined mRNA expression of mediators that could be responsible for the deposition and degradation of ECM. MMP-2 transcript levels were no different between WT mice and α7^−/− mice (Fig. 11). However, TIMP2 transcript was significantly increased in α7^−/− mice. In addition, mRNA expression of TIMP3, an MMP inhibitor that regulates inflammation and fibrosis, was significantly less in nephritic kidneys of α7^−/− mice. Western blot analysis of TIMP3 confirmed that the protein levels of this TIMP correlate with its mRNA levels in the kidneys of α7^−/− mice (Fig. 12A).

Figure 11.

Densitometric analysis of blots from RPAs of MMPs and TIMPs expressed in the kidneys of anti-GBM GN in α7^−/− and WT mice. The data are expressed as the ratio of the cpm for the specific mRNA to L-32 mRNA, to ensure a constant quantity of RNA in each sample. Results are expressed as means ± sem (n = 5 WT, n = 8 α7^−/−). **P < 0.01 vs. WT, ***P < 0.001 vs. WT.

Figure 12.

TIMP3 protein levels and serum TNF-α. A) TIMP3 was significantly reduced in nephritic kidneys from α7^−/−mice. Results are expressed as means ± sem. *P < 0.05 vs. normal (Nl) and WT. Quantification by ELISA of TNF-α in serum from mice (B) with or (C) without GN. Recombinant TNF-α was used as a positive control. Data are expressed as means ± sem. *P < 0.05 vs. WT; **P < 0.01 vs. Nl. D) Representative Western blot showing increased expression of TIMP3 in macrophages stimulated with ACh.

Because TIMP3 deficiency promotes inflammation via increased TNF-α, we measured the levels of serum TNF-α in α7^−/− and WT mice. The α7^−/− mice with GN had high levels of serum TNF-α compared to that in WT mice with GN. A notable finding is that serum levels of TNF-α were undetectable in α7^−/− mice and WT mice that were not challenged with anti-GBM Ab (Fig. 12B, C). Activation of α7nAChR by ACh inhibits cytokine expression in macrophages. Macrophages were stimulated with ACh, to determine whether activation of this receptor modulates TIMP3 expression. ACh increased TIMP3 expression in macrophages; however, LPS did not affect its expression (Fig. 12D) (43).

Kidney function is more severely affected in α7^−/− mice with anti-GBM GN

Induction of anti-GBM GN leads to increased serum creatinine; however, as a result of the severity of inflammatory lesions in the kidney in α7^−/− mice, serum creatinine was significantly higher in α7^−/− mice than in WT mice (P < 0.0001; Fig. 13A).

Figure 13.

A) Kidney function, (B) humoral immune response, and (C) glomerular IgG deposition in α7^−/− mice and WT mice. A) Serum creatinine was significantly higher in α7^−/− than in WT mice. Results are expressed as the mean ± sem. **P < 0.01 vs. normal (Nl); ***P < 0.0001 vs. WT and Nl. B) Circulating titers of mouse anti-rabbit IgG. Results are expressed as means ± sem. ***P < 0.0001 vs. Nl. C) Immunofluorescence staining of rabbit (A, B, WT and α7^−/−) and mouse (C, D, WT and α7^−/−) IgG.

Absence of α7nAChR does not affect antigen-specific humoral immune response or glomerular IgG deposition

To determine whether the systemic humoral immune response is affected in α7^−/− mice, we measured antigen-specific total mouse anti-rabbit IgG serum by ELISA. Total mouse anti-rabbit globulin IgG levels were similar between WT and α7^−/− mice (Fig. 13B). In addition, rabbit and mouse IgG glomerular depositions were not different in α7^−/− mice from those in WT mice (Fig. 13C).

Activation of the α7nAChR attenuates anti-GBM GN

Because absence of the α7nAChR exacerbates GN, we next investigated whether pharmacological activation of this receptor protects against kidney injury. Activation of α7nAChR with the selective α7nAChR agonist GTS-21 reduced the severity of anti-GBM GN. Although the glomerular proliferation was not different between the GTS-21-treated group and the control group, the fibrinoid necrosis and TIN injury were significantly reduced in the GTS-21 treatment group (Fig. 14A, B). Treatment with GTS-21 did not affect the immune humoral response or the deposition of rabbit or mouse IgG (Fig. 14C, D).

Figure 14.

Pharmacological activation of α7nAChR attenuates anti-GBM GN. A) PAS staining of kidney sections of vehicle- or GTS-21-treated mice. B) Quantitation of kidney injury. Each data point represents sections sampled from 6 mice per group and is expressed as the mean ± sem *P < 0.05. C) Circulating titers of mouse anti-rabbit IgG. Results are expressed as means ± sem. **P < 0.005 vs. normal; ***P < 0.0005 vs. normal. D) Immunofluorescence staining of rabbit (A, B, control and GTS-21, respectively) and mouse (C, D, control and GTS-21) IgG.

DISCUSSION

Our results indicate that mice lacking α7nAChR develop severe anti-GBM GN with a prominent inflammatory response, enhanced macrophage infiltration, and high expression of proinflammatory cytokines. Increased expression of the major profibrotic cytokine TGF-β1, reduction of TIMP3, and accumulation of collagen were also observed in α7^−/− mice during the early phase of the disease. Accordingly, these studies demonstrate that α7nAChR is an essential regulator of inflammation in anti-GBM GN and that the absence of the endogenous protective α7nAChR amplifies inflammation that leads to an accelerated onset of kidney fibrosis.

The protective role of α7nAChR in anti-GBM GN is supported by attenuation of kidney injury in mice treated with the specific agonist of α7nAChR, GTS-21.

Anti-GBM GN is characterized by the production of cytokines such as IL-1β and TNF-α from macrophages and intrinsic kidney cells (44, 45). Chemokines also play a key role in the pathogenesis of this model of GN. The use of antibodies against macrophage-attracting chemokines can reduce glomerular injury in anti-GBM GN (23, 26, 46). In addition, macrophages produce chemokines that can recruit more macrophages to amplify the inflammatory response. α7nAChR signal transduction suppresses the synthesis and release of cytokines in macrophages (5, 8, 9, 47). As a consequence, the absence of α7nAChR in macrophages could explain, at least in part, the robust expression of cytokines and chemokines in nephritic α7^−/− mice.

In α7^−/− mice, a natural specific IL-1 inhibitor, IL-1Ra, and a key physiologic regulator of inflammation, SOCS3, were increased in the kidney compared to values in WT mice. IL-1Ra competes with IL-1 for binding to its receptors and, in doing so, blocks the activity of IL-1. It has been demonstrated that IL-1Ra can ameliorate anti-GBM GN (48). This anti-inflammatory molecule is produced by macrophages, neutrophils (only ∼5% of the observed in monocyte/macrophages), and intrinsic kidney cells, including podocytes, proximal tubular epithelial cells, and epithelial cells of distal tubules (44, 49). SOCS3 proteins are induced by cytokines and therefore act in a classic negative-feedback loop to inhibit cytokine signal transduction (50). SOCS3 is expressed by macrophages, dendritic cells, glomerular mesangial cells, podocytes, and proximal tubular cells (50, 51). IL-1Ra and SOCS3 induction in the kidney of α7^−/− mice with GN could be a compensatory response to inflammation. Nevertheless, the magnitude of the severity of kidney injury with robust inflammatory response in nephritic α7^−/− mice indicates that IL-1Ra and SOCS3 did not compensate for the lack of α7nAChR. In contrast to increased expression of IL-1Ra and SOCS3 in nephritic kidneys of α7^−/− mice, peritoneal macrophages isolated from α-7^−/− mice had significantly attenuated expression of SOCS3 and IL-1Ra in response to LPS activation. These data suggest that intrinsic kidney cells are mainly responsible for the increased SOCS3/IL-1Ra expression in nephritic kidneys from α7^−/− mice and that the decreased SOCS3/IL-1Ra expression on macrophages prevents the regulation of cytokine production by these inflammatory cells.

In α7^−/− mice, kidney fibrosis was observed at an early stage of the disease (d 7). An increased expression of the profibrotic cytokine TGF-β1, with enhanced deposition of collagen, was found at d 7 after the injection of anti-GBM Ab in nephritic kidneys of α7^−/− mice. In contrast, none of the WT mice developed kidney fibrosis. α7^−/− macrophages could be responsible for the increased TGF-β1 production. Moreover, it has been reported that macrophages are a major source of TGF-β1 in fibrotic organs. Macrophages produce several mediators that induce kidney damage and modulate the production of ECMs, including TGF-β1, MMPs, and TIMPs (17, 52, 53). Studies of the PU.1 null mice, which lack macrophages and have low levels of TGF-β1, have shown that skin wounds can be repaired without scar formation. Those studies suggest that TGF-β1 is not essential for repair but is a cause of fibrosis (54).

The turnover of collagen and other ECM proteins is controlled by various MMPs and their inhibitors the TIMPs. Shifts in these opposing mechanisms (synthesis vs. catabolism) regulate the net increase or decrease in collagen within a tissue. The expression of MMP2 was not different in the kidneys of α7^−/− mice and WT mice; however, TIMP2 expression was increased in α7^−/− kidneys. These data suggest that up-regulation of TIMP2 could prevent ECM degradation in kidneys of α7^−/− mice.

TIMP3 is the most highly expressed TIMP in the kidney; deficiency of this protein is associated with age-related kidney fibrosis and the onset and progression of diabetic kidney disease (55, 56). Moreover, in patients with diabetic nephropathy, a significant reduction in kidney TIMP3 was identified (55). TIMP3 is the only known physiologic inhibitor of ADAM17, an MMP that controls the bioactivity of several growth factors and cytokines such as TNF-α, VCAM, and HB-EGF, molecules that are implicated in the development of kidney inflammation and fibrotic lesions (28, 57–59). In addition, TIMP3 deficiency promotes persistent vascular inflammation via increased TNF-α (60). Fibrotic kidneys of α7^−/− mice had reduced expression of TIMP3, and serum and kidney TNF-α was significantly higher in α7^−/− mice with anti-GBM GN than in WT mice. TNF-α is essential in the development and progression of GN; systemic administration of TNF-α induces glomerular damage and exacerbates glomerular injury in rats with anti-GBM GN. In contrast, in a rat model of crescentic nephritis, neutralization of endogenous TNF-α was effective in preventing acute glomerular inflammation and crescent formation and in treating established disease (61, 62). Serum TNF-α is increased, not only in patients with GN, but also in patients with chronic kidney disease (15, 63).

TIMP3 overexpression in macrophages protects from inflammation. Our finding that ACh induces TIMP3 in macrophages could suggest that an additional mechanism by which α7nAChR protects from inflammation and tissue injury is through up-regulation of TIMP3 (64, 65).

Fibrin deposition in anti-GBM GN is an important mediator of injury and progression of kidney damage. Macrophages appear to be directly responsible for glomerular fibrin deposition because of their ability to express procoagulant activity. In addition, products secreted by activated macrophages, mainly TNF-α and IL-1β, increase the tissue factor–like procoagulant activity of endothelial cells (66, 67). In α7^−/− mice, increased macrophage infiltration and higher levels of TNF-α and IL-1β in the kidney could explain enhanced glomerular fibrin deposition compared with that in WT controls. Coagulation influences several key aspects of the wound-healing response, and an imbalance in favor of a procoagulant state has the potential to dysregulate inflammatory and tissue repair programs and culminate in fibrosis (68).

It has been reported that activation of α7nAChR modulates the release of proinflammatory cytokines by macrophages; however, because we used mice that were globally deficient in α7nAChR, we cannot definitively conclude that the lack of macrophage α7nAChR was responsible for the increased inflammation and kidney injury observed in α7^−/− mice.

In the rodent kidney the presence of several nAChRs, including α2, α3, and α7 receptor subunit proteins, has been reported. α7nAChR has been detected by immunohistochemistry in the endothelium of cortical peritubular capillaries, expression that was increased after kidney ischemia-reperfusion. In addition, the rat proximal tubular epithelial cell line NRK52E also expresses α2, α3, and α7 subunit proteins (14, 69). Using αBgt, which is selective for α7 receptors over α3β4 receptors (IC₅₀, 1.6 nM and >3 µM, respectively), we did not detect staining in peritubular capillaries or tubular cells (70). This result could be attributable to different staining techniques or the use of mice instead of rats. Although macrophage α7nAChR has been well characterized and demonstrated to mediate anti-inflammatory effects, in this study, we cannot exclude the possibility that α7nAChR expressed in other cells protects kidneys from injury in GN (9, 47).

Although α7 subunits predominantly form homopentameric α7nAChRs, it has been reported that other nAChR subunits can combine with α7 to form heteromeric nAChRs, such as α7β2nAChRs. Although, heteromeric α7nAChRs have functional and pharmacological properties different from those of homomeric α7nAChRs, we cannot rule out the possibility that heteromeric α7nAChRs play a protective role in GN (71). However, the severe kidney injury observed in the absence of α7nAChR suggests that this receptor is a key regulator of kidney inflammation in anti-GBM GN.

Other limitations of this study are that GTS-21 is a selective partial α7nAChR agonist, and because these data are derived from murine studies, further challenges include the translation from mouse to human.

In summary, our results indicate that α7nAChR is essential for inhibiting inflammation in anti-GBM GN and that the absence of this receptor increases kidney injury by amplifying inflammation to accelerate kidney damage and fibrosis.

Nearly 45% of all deaths in the developed world are attributable to some type of pathogenic fibrosis (18). Therefore, controlling excessive inflammation would be of great potential therapeutic benefit for inhibiting progressive fibrosis. Our findings suggest that α7nAChR is an essential regulator of progressive inflammation and that pharmacological activation of this receptor may serve as a therapeutic target to prevent progressive injury in kidney and other chronic inflammatory diseases in humans, such as pulmonary fibrosis, liver fibrosis, cardiovascular disease, diabetes, and cancer, which are leading causes of morbidity and mortality. Notably, targeting α7nAChR, an endogenous anti-inflammatory molecule, could cause fewer side effects than those associated with the nonspecific immunosuppressive drugs used to treat GN and other chronic inflammatory diseases.

Glossary

α7^−/−

α7nAChR-deficient

α7nAChR

nicotinic acetylcholine receptor α7 subunit

αBgt

α-bungarotoxin

ACh

acetylcholine

ADAM

A disintegrin and metalloproteinase domain 17

BMDM

bone marrow–derived macrophage

cpm

counts per minute

CXCL

chemokine C-X-C motif

ECM

extracellular matrix

FBS

fetal bovine serum

GBM

glomerular basement membrane

GN

glomerulonephritis

HB-EGF

heparin-binding epidermal growth factor-like growth factor

HMGB1

high-mobility group box 1

KO

knockout

IL-1Ra

IL-1R antagonist

MCP

monocyte chemotactic protein

MIP

macrophage inflammatory protein

MMP

metalloproteinase

PAS

periodic acid-Schiff

RPA

RNase protection assay

SOCS

suppressor of cytokine signaling

TIMP

tissue inhibitor of metalloproteinase

TIN

tubulointerstitial

WT

wild-type