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Published as doi: 10.1096/fj.07-8430com.
 (The FASEB Journal. 2008;22:445-454.)
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(The FASEB Journal. 2008;22:445-454.)
© 2008 FASEB

Adenosine A2A receptor activation and macrophage-mediated experimental glomerulonephritis

Gabriela E. Garcia*,1, Luan D. Truong{dagger},{ddagger}, Ping Li*, Ping Zhang*, Jie Du*, Jiang-Fan Chen§ and Lili Feng*,2

* Department of Medicine, Section of Nephrology, and

{dagger} Department of Pathology, Baylor College of Medicine, Houston, Texas, USA;

{ddagger} The Methodist Hospital, Houston, Texas, USA; and

§ Molecular Neuropharmacology Laboratory, Department of Neurology, Boston University School of Medicine, Boston, Massachusetts, USA

1Correspondence: Department of Medicine, Nephrology Section, Alkek N520, One Baylor Plaza, Houston TX, 77030, USA. E-mail: geg{at}bcm.tmc.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In immune-induced inflammation, leukocytes are key mediators of tissue damage. Since A2A adenosine receptors (A2ARs) are endogenous suppressors of inflammation, we examined cellular and molecular mechanisms of kidney damage to determine if selective activation of A2AR would suppress inflammation in a rat model of glomerulonephritis. Activation of A2AR reduced the degree of kidney injury in both the acute inflammatory phase and the progressive phase of glomerulonephritis. This protection against acute and chronic inflammation was associated with suppression of the glomerular expression of the MDC/CCL22 chemokine and down-regulation of MIP-1{alpha}/CCL3, RANTES/CCL5, MIP-1β/CCL4, and MCP-1/CCL2 chemokines. The expression of anti-inflammatory cytokines, interluekin (IL)-4 and IL-10, also increased. The mechanism for these anti-inflammatory responses to the A2AR agonist was suppression of macrophages function. A2AR expression was increased in macrophages, macrophage-derived chemokines were reduced in response to the A2AR agonist, and chemokines not expressed in macrophages did not respond to A2AR activation. Thus, activation of the A2AR on macrophages inhibits immune-associated inflammation. In glomerulonephritis, A2AR activation modulates inflammation and tissue damage even in the progressive phase of glomerulonephritis. Accordingly, pharmacological activation of A2AR could be developed into a novel treatment for glomerulonephritis and other macrophage-related inflammatory diseases.—Garcia, G. E., Truong, L. D., Li, P., Zhang, P., Du, J., Chen, J. F., Feng, L. Adenosine A2A receptor activation and macrophage-mediated experimental glomerulonephritis.


Key Words: chemokines • anti-inflammatory cytokines • tissue injury protection


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
WE STILL TREAT IMMUNOLOGICALLY INDUCED inflammatory diseases with non-specific immunosuppressive drugs that can cause significant morbidity (1) . For this reason, defining the pathways of organ damage could lead to design of more specific therapies.

Crescentic glomerulonephritis is a rapidly progressive glomerular disease with a poor prognosis. Macrophages can play an important role in the induction and development of the disease, and both the magnitude of proteinuria and the percentage of crescentic glomeruli are correlated with the number of macrophages that infiltrate the glomerulus; this is not the case with the number of CD8+ cells (2 , 3) . Macrophages are constituents of glomerular crescents in progressive crescentic glomerulonephritis, and they probably play a major role in the irreversible scarring that leads to kidney failure (4 5 6 7 8) . In Wistar-Kyoto (WKY) rats, small doses of anti-glomerular basement membrane (GBM) antibody induce proliferative and necrotizing glomerulonephritis with crescent formation plus infiltration of CD8+ cells and macrophages into the glomeruli (9 10 11 12 13) . In this model, macrophages accumulate in glomeruli during later phases and contribute to the progressive decline in kidney function (3 , 14) .

Chemokines mediate selective attraction and activation of various subsets of leukocytes (6 , 15 16 17) . In the induction of anti-GBM glomerulonephritis in WKY rats, CC chemokines play an important role. We found that CC chemokines, MCP-1/CCL2, MIP-1β/CCL4, RANTES/CCL5, and MDC/CCL22, and the CX3C chemokine, fractalkines/CX3CL1, are induced during the disease and that the expression of multiple chemokines coincides with the influx of CD8+ cell and ED1+ macrophages into the glomeruli. In in vitro studies these chemokines induced strong migratory response in inflammatory cells prepared from the nephritic glomeruli (9 10 11) .

The degree of inflammation can be tightly regulated by endogenous anti-inflammation pathways, including PPAR{gamma}, adiponectin, nitric oxide (NO), and adenosine receptors (18 19 20 21 22 23 24) . Genetic and pharmacological evidence supports a nonredundant role for both endogenous adenosine and A2A receptors (A2ARs) in protecting against acute inflammatory damage in models of inflammatory injury and systemic inflammation (25) . The anti-inflammatory mechanism involves down-regulation of activated immune cells in vivo (25) . Thus, the A2AR could modulate inflammatory processes because it is expressed in most cells involved in inflammation, including a variety of hematopoietic, endothelial, and smooth muscle cells (19 , 20 , 25 , 26) . We have found that quiescent peritoneal macrophages do not express A2AR, but after LPS was added, these cells express abundant A2AR. In vitro studies indicate that A2ARs are present on polymorphonuclear (PMN) leukocytes and can inhibit the expression of β2-integrins and adhesion and can suppress oxygen radical production, degranulation, and production of tumor necrosis factor (TNF) -{alpha} (27 28 29 30 31) . In addition, expression and activation of A2AR on macrophages can inhibit the production of interleukin (IL) -12, TNF-{alpha}, and NO, and enhance the secretion of IL-10 in response to lipopolysaccharide (LPS; refs. 32 33 34 35 ). Adenosine can inhibit proliferation, activation, and production of inflammatory cytokines in peripheral T cells, it also can enhance the production of anti-inflammatory cytokines (36 , 37) .

Because of the potential for beneficial effects of pharmacological activation of A2AR as a therapeutic target, we tested the hypothesis that A2AR activation would protect against immune-mediated inflammation in experimental glomerulonephritis. Activation of A2AR significantly reduced leukocyte infiltration into the kidney and prevented kidney injury during the acute inflammatory phase and the progressive phase of glomerulonpehritis. Our results indicate that activation of A2AR represents a potential therapeutic strategy for glomerulonephritis and possibly other macrophage-mediated diseases.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Induction, treatment, and analysis of anti-GBM glomerulonephritis
Animal studies were approved by the institutional animal care and use committee at Baylor College of Medicine. Male WKY rats (Harlan Sprague Dawley, Indianapolis, IN, USA), weighing 180–200 g, received an intravenous injection of 25 µl/100 g body weight of anti-GBM Ab as described previously (9 , 10 , 12 , 38) . We activated A2AR with a selective A2A receptor agonist, CGS 21680 (Tocris Cookson, Ellisville, MO, USA, 1.5 mg/kg ip twice a day, n=6) and inactivated A2AR using a selective receptor antagonist, ZM241385 (Tocris, 2 mg/kg ip twice daily, n=6) for a period of 5 days, as described previously (25) . In initial experiments, we pharmacologically activated or inhibited A2AR during the acute inflammatory phase of the disease by starting treatment 8 h after the injection of anti-GBM antibody (maximum glomerular deposition of IgG occurs 1 h after injection of anti-GBM antibody; ref. 39 ). In the second set of experiments, we began treatment with A2AR agonist at day 6 after the anti-GBM antibody was injected (the progressive phase of crescentic glomerulonephritis). Urine protein was assayed using sulfosalicylic acid, and rats were euthanized at day 6 or 12 to collect blood and kidney tissues. During the first hour, after the A2AR agonist or the A2AR antagonist was injected, tail-cuff blood pressure was measured in conscious rats (Visitech Systems, Apex, NC, USA) as described previously (40) .

mRNA expression of A2AR, chemokines, cytokines, and adhesion molecules
Rat A2AR nucleotides 459 to 801 (GenBank sequence accession number NM_053294) were used to generate A2AR probe from brain tissue by reverse transcriptase-polymerase chain reaction (RT-PCR). MDC/CCL22 (400 bp), RANTES/CCL5 (246 bp), MIP-1{alpha}/CCL3 (284 bp), factalkine/CX3CL1 (420 bp), MIP-3β/CCL19 (380 bp), MIP-1β/CCL4 (210 bp), MCP-1/CCL2 (239 bp), VCAM-1 (371 bp), ICAM-1 (292 bp), and L-32 (92 bp) riboprobes were generated by PCR reaction using cDNA templates. rCK1 (BD Pharmingen, San Diego, CA, USA) was used to investigate cytokine expression. Glomeruli were prepared by sequential sieving, and total RNA was isolated from glomeruli (11 , 41) . Three micrograms of total RNA from each sample was used in an RNase protection assay using the Torrey Pines Biolabs kit (Houston, TX, USA) as described previously (11 , 16 , 42 , 43) . Phosphoimage quantitiation was performed using the PhosphorImager SI scanning instrument and ImageQuaNT software (Molecular Dynamics, Sunnyvale, CA, USA; refs. 16 , 44 , 45 ).

Morphological analysis, immunohistochemical phenotyping, and quantitation of leukocytes
Kidney samples fixed in formalin or methanol-Carnoy fixative solution were embedded in paraffin. Two to three micrometer sections were stained with periodic acid-Schiff reagent to assess glomerular hypercellularity, necrotizing lesions, and formation of glomerular crescents (crescentic glomeruli per 100 glomeruli were calculated and expressed as a percentage). Infiltrating leukocytes were immunohistochemically stained for CD8+ and ED1+, as described previously (9 , 11 , 12) . Positively stained cells per 100 glomeruli were counted and expressed per glomerular section. All quantitative morphological analyses were performed in a blinded fashion.

Immunohistochemistry of A2AR, ED1+ cells, CD8+ cells, mesangial cells, and podocytes
Paraffin sections of methanol-Carnoy fixed tissue were stained with goat polyclonal anti-A2AR antibody (1:100, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and monoclonal antibody ED-1 against rat macrophages (Chemicon, Temecula, CA, USA) or monoclonal OX-8 against rat CD8 (BD Biosciences Pharmingen). Two-color dual antigen immunostaining was obtained by serial avidin-biotin peroxidase and alkaline phosphatase staining reactions with final chromogenic substrates of diamibenzidine (brown color) and Fast Red (red color), respectively, as described previously (12 , 46 , 47) .

Kidney sections were also stained with {alpha}-SMA (Dako, Carpinteria, CA, USA), a marker of activated mesangial cells or {alpha}-actinin-4 (AXXORA, LLC, San Diego, CA, USA), a marker for podocytes (48 , 49) .


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
A2AR expression in nephritic glomeruli
Normal glomeruli do not express A2AR. In response to the anti-GBM antibody, however, there was strong expression of A2AR from day 3 onwards (Fig. 1 A). The glomerular level of A2AR mRNA was 6.2-fold higher in anti-GBM Ab-treated rats than in normal glomeruli on day 3. A2AR expression peaked at day 5 (6.8-fold increase) and started to decrease by day 7 (5.2-fold increase). Double immunohistochemical staining of ED1+ cells and A2AR demonstrated that A2AR was virtually confined to macrophages. There was some staining in the location of glomerular endothelial cells (Fig. 1B ), but A2AR was not expressed in either CD8+ cells (Fig. 1C ) or in monocytes located in extraglomerular vessels (Fig. 1D ). These results suggest that increased expression of A2AR in macrophages might yield an anti-inflammatory defense against kidney damage.


Figure 1
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  		Figure 1. Induction of A2AR in nephritic glomeruli. A) RNase protection analysis was performed to determine A2AR mRNA expression in glomeruli of anti-GBM glomerulonephritis in WKY rats. A2AR was induced from day 3 onwards during anti-GBM glomerulonephritis. Expression of A2AR peaked at day 5 and started to decrease by day 7. Rat ribosomal L-32 gene was used as a housekeeping gene. Probe contains polylinker regions and is longer than protected bands. Expression levels of A2AR mRNA are relative to mRNA levels of housekeeping gene L-32. Data shown are representative of three separate experiments. B) Double immunohistochemical staining of ED1+ cells (brown color) overlap with A2AR (red color); thus A2AR was mainly in M{phi}. Interestingly, A2AR (red color; C) was not expressed in CD8+ cells (brown color) or monocytes (red color; D) located in extraglomerular vessels. B, C) ED1+ or CD8+ (right panel) cell staining after removal of red color (A2AR staining) from B and C (left panel) to demonstrate overlap of A2AR expression in ED1+ but not in CD8+ cells. Sections for staining were sampled on day 7 after anti-GBM antibody injection.

A2AR activation prevents glomerulonephritis
In the acute (days 1–5) phase of crescentic glomerulonephritis, control rats exhibited severe glomerular hypercellularity, necrotizing lesions, and crescentic formation (Figs. 2 and 3 ). Increased expression of {alpha}-SMA, a marker for mesangial cells injury, was inversely correlated with reduced {alpha}-actinin-4, a marker for podocytes, indicating the degree of glomerular injury (Fig. 4 ). In rats treated with CGS21680, a specific A2AR agonist, however, there was only minimal damage to the kidneys. Notably, crescent formation, a major characteristic of this model, was abolished by CGS21680 treatment, and necrotizing lesions were not observed, whereas glomerular hypercellularity was markedly attenuated by ~70% (Figs. 2 and 3a ). Treatment with the A2AR agonist also decreased {alpha}-SMA expression and restored the expression of {alpha}-actinin- 4 (Fig. 4) . There also was no proteinuria (Fig. 5 a). In contrast, in rats with anti-GBM glomerulonephritis treated with a specific antagonist for A2AR, ZM241385, there was more severe glomerular hypercellularity, necrotizing lesions, and crescentic formation compared to control rats (Figs. 2 and 3a ). A2AR antagonist also enhanced the expression of {alpha}-SMA and increased the loss of {alpha}-actinin- 4 (Fig. 4) . Finally, proteinuria was also significantly higher in ZM241385-treated vs. control rats with glomerulonephritis (Fig. 5a ). Thus, selective activation of A2AR at acute phase (day 1–5) confers kidney protection from damage.


Figure 2
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  		Figure 2. A2AR attenuates inflammatory infiltrates and renal injury during acute inflammatory phase and progressive phase in anti-GBM glomerulonephritis. Immunohistochemistry stained for CD8+ cells or ED1+ monocytes/macrophages, and periodic acid-Schiff staining of kidney sections of control, CGS21680 (CGS), and ZM241385 (ZM) -treated rats with anti-GBM glomerulonephritis. In acute phase of disease (days 1–5), control group of glomerulonephritis rats display severe glomerular hypercellularity, necrotizing lesions, crescentic formation, and prominent accumulation of CD8+ cells and ED1+ macrophages. In CGS-treated rats, crescentic formation and necrotizing lesions were prevented, and CD8+ and ED1+ macrophage infiltrates attenuated. Worsening of glomerular injury was observed in rats treated with ZM. In progressive phase of disease (days 6–11) CGS markedly reduced glomerular lesion and ED1+ cell infiltrate.


Figure 3
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  		Figure 3. Quantitation of CD8+ and ED1+ cell infiltration, glomerular hypercellularity, necrotizing lesions, and crescent formation in glomeruli from WKY rats with anti-GBM glomerulonephritis that were treated with vehicle, CGS21680, or ZM241385 during days 1–5 and days 6–11; 100 glomeruli per section were counted. Each data point represents sections sampled from 6 rats and is expressed as mean ± SE. *P < 0.0001 vs. control, **P < 0.005 vs. control, ***P < 0.05 vs. control, {sigma}P < 0.0001 vs. CGS.


Figure 4
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  		Figure 4. A2AR activation decreases {alpha}-SMA expression and restores {alpha}-actinin-4 expression in anti-GBM GN. Immunohistochemistry stained for {alpha}-SMA and {alpha}-actinin-4 of kidney sections of normal, control, CGS21680, and ZM241385-treated rats with anti-GBM glomerulonephritis. In acute phase of disease (days 1–5), control group of glomerulonephritis rats display increased expression of {alpha}-SMA and loss of {alpha}-actinin-4. In CGS-treated rats, expression of {alpha}-SMA was reduced and expression of {alpha}-actinin-4 restored. Increased expression of {alpha}-SMA and enhanced loss of {alpha}-actinin-4 was observed in rats treated with ZM. In progressive phase of disease (days 6–11) CGS markedly reduced {alpha}-SMA and restored {alpha}-actinin-4.


Figure 5
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  		Figure 5. Determination of proteinuria (milligrams of urine protein per 24 h) in Wistar-Kyoto rats with anti-GBM glomerulonephritis that were treated with vehicle, CGS21680, and ZM241385. In acute phase of disease, CGS completely blocked proteinuria in anti-GBM antibody-injected rats. Proteinuria was significantly higher in ZM-treated rats than control group. In progressive phase of glomerulonephritis, CGS markedly reduced proteinuria. Results were sampled from 6 rats per group and expressed as mean ± SE. *P < 0.0001 vs. control, **P < 0.005 vs. control, ***P < 0.05 vs. control, {delta}P < 0.005 vs. ZM, &P < 0.0001 vs. ZM.

Increased or normal blood pressure has been reported in mice lacking A2AR. We determined if A2AR antagonism could increase blood pressure and contribute to kidney damage (46 , 50) . There was no significant difference in systolic blood pressure among the groups at days 4 or 6 after induction of anti-GBM GN (D4: 147.75±18.7, 141.0±14.2, 149.7±14.1; D6: 134.6±8.01, 131.0±9.43, 137.0±5.6 in control, A2AR agonist, andA2AR antagonist treated rats, respectively).

Next, we treated rats in the progressive phase of glomerulonephritis (days 6–11) using the A2AR agonist, CGS21680. As shown in Figs. 2 and 3b , glomerular hypercellularity, necrotizing lesions, and crescentic formation were markedly reduced at day 12 compared to the results in the control group (88, 90, and 74%, respectively). A2AR activation also decreased {alpha}-SMA expression and restored the expression of {alpha}-actinin-4vs. results in the control group (Fig. 4) . Finally, CGS21680 reduced proteinuria within 24 h of administration; the inhibitory effect was sustained during the treatment period (Fig. 5b ). Thus, selective activation of A2AR also reduces kidney injury and improves proteinuria during the progressive phase of glomerulonephritis.

Prevention of glomerulonephritis by A2AR activation reduces lymphocyte/macrophage infiltration
To understand the mechanism for the prevention of kidney injury, we examined the infiltration of leukocytes into the kidney. As shown in Figs. 2 and 3a , there was a prominent accumulation of CD8+cells and ED1+ macrophages in glomeruli of control rats during the acute (days 1–5) inflammatory phase of glomerulonephritis. Treatment with CGS21680 dramatically attenuated infiltration of CD8+ cells and ED1+ macrophages into glomeruli. Pharmacological activation of A2AR in the progressive phase (days 6–11) blocked macrophage infiltration to levels only 26% of the level in glomeruli of control rats (Figs. 2 and 3b ). This model of glomerulonephritis is characterized by an early infiltration of CD8+ cells with a maximum increase on day 3 after the injection of anti-GBM Ab (13) . Consequently, CD8+ cells were not observed in either control or CGS21680-treated rats.

A2AR activation modulates the expression of chemokines and cytokines in anti-GBM glomerulonephritis
A potential mechanism by which activation of A2AR protects from inflammatory damage and leukocyte infiltration could be via suppression of cytokines/chemokines. We found that pharmacological A2AR activation in the acute inflammatory phase (days 1–5) did not reduce glomerular expression of proinflammatory cytokines IL-1β or TNF-{alpha} mRNA. In contrast, it significantly increased the expression of anti-inflammatory cytokines IL-4 and IL-10 (69 and 66%, respectively), compared to levels in glomeruli of control rats (Figs. 6 a and 7 a). Activation of A2AR abolished the induction of MDC/CCL22 and significantly attenuated the expression of MIP-1{alpha}/CCL3, MIP-1β/CCL4, RANTES/CCL5, and MCP-1/CCL2 (Figs. 6b, c and 7b, c ) but did not affect expression of fractalkine/CX3CL1 (Figs. 6c and 7c ). Interestingly, inhibition of A2AR by ZM241385 did not significantly change the expression of these chemokines. Expression of anti-inflammatory cytokines seemed to be attenuated by ZM241385, but the changes were not statistically significant compared to the control results. When compared with levels in CGS21680-treated rats, IL-10 and IL-4 were significantly reduced in rats treated with ZM241385 (64.2 and 61%, respectively; Figs. 6a and 7a ).


Figure 6
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  		Figure 6. Selective A2AR activation alters anti-GBM glomerulonephritis-induced cytokine expression. RNase protection assay of cytokines and chemokines expressed in glomeruli of anti-GBM glomerulonephritis in WKY rats. CGS in acute inflammatory phase of disease (days 1–5) increased expression of IL-4 and IL-10 (a), abolished increase of expression of MDC/CCL22, and attenuated expression of MIP-1{alpha}/CCL3, RANTES/CCL5, MIP-1β/CCL4, and MCP-1/CCL2 (b, c). ZM reduced IL-4 and IL-10 expression compared with CGS group (a). In progressive phase of disease (days 6–11), CGS significantly enhanced expression of IL-4 (d) and suppressed increase of expression of MDC/CCL22 (e). {lambda} = undigested probe. Each lane represents a single rat.


Figure 7
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  		Figure 7. Selective A2AR activation alters anti-GBM glomerulonephritis-induced cytokine expression. Densitometric analysis of blots from RNase protection assay of cytokines and chemokines expressed in glomeruli of anti-GBM glomerulonephritis in WKY rats. Data are presented as a ratio of cpm for specific mRNA/L-32 mRNA to ensure a constant quantity of RNA in each sample. Results were sampled from 6 rats per group and expressed as mean ± SD. *P < 0.05 vs. nl, {delta}P < 0.005 vs. nl, {gamma}P < 0.0005 vs. nl, **P < 0.05 vs. ctrl, {theta}P < 0.005 vs. ctrl, {lambda}P < 0.0005 vs. ctrl, {kappa}P < 0.05 vs. CGS, &P < 0.005 vs. CGS. nl = normal rat.

We also investigated the effects of the A2AR agonist and antagonist on the expression of VCAM-I and ICAM-1 in anti-GBM glomerulonephritis. Increased expression of VCAM-1 and ICAM-1 was observed in anti-GBM glomerulonephritis, and A2AR activation or inactivation did not significantly modify their expression in the acute inflammatory phase (days 1–5) compared to control rats (Fig. 8 ).


Figure 8
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  		Figure 8. Expression of adhesion molecules is not modified by pharmacological activation of A2AR. Densitometric analysis of blots from RNase protection assay of VCAM-1 and ICAM-1 expressed in glomeruli in anti-GBM glomerulonephritis. Data are presented as a ratio of cpm for specific mRNA/L-32 mRNA to ensure a constant quantity of RNA in each sample. Results were sampled from 6 rats per group and expressed as mean ± SD. *P < 0.05 vs. nl, {delta}P < 0.005 vs. nl, {gamma}P < 0.0005 vs. nl.

Pharmacological activation of A2AR in rats with established glomerulonephritis (days 6–11) significantly enhanced the expression of the anti-inflammatory cytokine IL-4 (104%) but not of IL-10 (Figs. 6d and 7d ). The induction of MDC/CCL22 chemokine was suppressed, but the expression of other chemokines and VCAM-1 or ICAM-1 did not change. Fractalkine/CX3CL1 expression significantly increased (Figs. 6e, f ; 7e, f ; and 8 ).

These results suggest that A2AR pharmacological activation modulates renal injury by altering expression of inflammatory and anti-inflammatory cytokines in the kidney.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Our results indicate that activation of adenosine A2ARs prevents infiltration of leukocytes into the kidney, suppresses glomerular inflammation, and protects the kidney from inflammatory injuries induced by anti-GBM glomerulonephritis. These benefits were observed both in the acute inflammatory phase and during the progressive phase of glomerulonephritis. The protection can be linked to the ability of activated A2AR to suppress infiltration of leukocytes, the key mediators of kidney damage.

We found that A2AR activation alters the expression of chemokines. Specifically, pharmacological activation of A2AR suppresses MDC/CCL22 expression and markedly reduces the expression of other chemokines during the acute inflammatory phase of glomerulonephritis (Fig. 6) . These changes in chemokine expression can explain the decreased infiltration of CD8+ and macrophages (6 , 9 10 11) . During the progressive phase of glomerulonephritis (days 6–11), we found suppressed MDC/CCL22 induction, whereas the expression of other chemokines (except for fractalkine/CX3CL1) was not modified. These results are consistent with the report that MDC/CCL22 plays a critical role in stimulating the influx of macrophages into glomeruli in anti-GBM glomerulonephritis (11) . Notably, MDC/CCL22 expression in anti-GBM glomerulonephritis is confined to macrophages (11) . As shown in Fig. 1 , at day 7 after the anti-GBM antibody was injected, macrophages are the major cellular site of A2AR expression in nephritic glomeruli. When MDC/CCL22 expression is inhibited by the A2AR agonist, CGS21680, there also is a dramatic inhibition of ED1+ macrophage infiltration. These results raise the following possibilities: 1) Activation of macrophage-specific A2AR suppresses MDC/CCL22 expression resulting in decreased macrophage infiltration in the acute phase of glomerulonephritis; and 2) macrophage infiltration in later stages of glomerulonephritis depends on MDC/CCL22 because blocking expression of this chemokine ameliorates the degree of functional damage. Interestingly, A2AR activation in the progressive phase of glomerulonephritis did not alter the expression of other chemokines, except for fractalkine/CX3CL1. Possibly increased fractalkine/CX3CL1 expression occurs because of endothelial cell activation in the nephritic glomeruli. This interpretation is suggested because fractalkine/CX3CL1 is mainly expressed by endothelial cells and we have shown that fractalkine/CX3CL1 is induced in the endothelium of nephritic glomeruli (10) . In contrast, we have not found A2AR in cultured glomerular endothelial cells (quiescent or activated) or HUVEC (quiescent). Thus, a plausible explanation for the finding that A2AR occupancy during the progressive phase of glomerulonephritis does not alter the expression of other chemokines, is that the chemokines at days 6–11 could be produced by A2AR-negative kidney cells.

Our finding that activation of A2AR did not suppress the expression of the proinflammatory cytokines, IL-1β and TNF-{alpha} (Fig. 6a, d ) is consistent with the notion that macrophages are not the predominant source of IL-1β and TNF-{alpha} in this model of glomerulonephritis (51 , 52) . On the other hand, in vitro studies indicate that IL-1β and TNF-{alpha} can up-regulate A2AR in THP-1 cells, human dermal microvascular endothelial cells, and PC12 cells (35 , 53 , 54) . Our results, therefore, suggest that IL-1β and TNF-{alpha} could induce A2AR expression in other cells and contribute to the beneficial effect of A2AR agonists in preventing glomerular damage.

In addition, we found that the expression of the anti-inflammatory cytokines IL-10 and/or IL-4 was enhanced by the A2AR activation during anti-GBM glomerulonephritis. These cytokines might also contribute to the beneficial effects of A2AR activation on kidney injury (55 , 56) .

Increasing evidence suggests that A2AR tissue-protection requires activation of receptors expressed on bone marrow-derived cells (57 , 58) . Our data indicate that macrophages are the target cells mediating the benefits of A2AR activation in both the acute and progressive phases of glomerulonephritis. First, A2AR expression was increased in macrophages but not in CD8+ cells during anti-GBM GN (Figs. 1B, C ). Second, macrophage derived-chemokines such as MDC/CCL22, MCP-1/CCL2, RANTES/CCL5, and MIP-1{alpha}/CCL3 were decreased in response to A2AR agonist. Third, chemokines not expressed in macrophages (e.g., fractalkine/CX3CL1) were unaffected by the A2AR agonist. Finally, macrophages are present in the crescents and in progressive glomerulonephritis and are correlated with the degree of glomerular injury (5 , 6 , 8) . It has been found that depletion of macrophages in this and other models of glomerulonephritis suppresses glomerular crescent formation and reduces proteinuria (3 , 7 , 59) . Accordingly, suppression of the expression of MDC/CCL22 and reduced expression of other chemokines when macrophage-A2AR activation occurs could explain in part the beneficial effect of CGS21680 in both acute and progressive phases of anti-GBM glomerulonephritis.

Notably, we found that the A2AR agonist also was beneficial in the late phase of anti-GBM glomerulonephritis when macrophage influx and crescent formation were at their maximum (day 6; refs. 13 , 60 ). These responses were associated with a significant reduction in macrophage infiltration (74%) suggesting that activation of A2AR on macrophages was responsible fro the suppression of glomerular injury.

To examine mechanisms underlying the beneficial effects of A2AR we determined the expression of {alpha}-SMA, a marker for mesangial cell injury and activation (49) and {alpha}-actinin-4, a marker for podocytes, in glomeruli of rats with anti-GBM glomerulonephritis. Earlier reports have demonstrated that changes in podocytes contribute to the severity of anti-GBM glomerulonephritis (48 , 61 , 62) . We found that the expression of {alpha}-SMA is increased and that {alpha}-actinin- 4 expression is reduced in anti-GBM GN. Treatment with A2AR agonist significantly decreased {alpha}-SMA expression and restored the expression of {alpha}-actinin- 4. In contrast, the A2AR antagonist enhanced the expression of {alpha}-SMA and increased the loss of the podocyte marker, {alpha}-actinin-4, compared to the controls rats. These data suggest that A2AR agonist may attenuate kidney injury through effects on mesangial cells and podocytes. Future experiments will determine if A2AR activation prevents mesangial cells injury and restores {alpha}-actinin-4 by reducing inflammation or through a direct effect on mesangial cells and podocytes.

A2AR activation or inactivation did not modify blood pressure in rats with anti-GBM glomerulonephritis. Earlier reports in mice lacking A2AR provide inconsistent changes in blood pressure (46 , 50) . The inconsistent results could reflect differences in strains of mice, route of administration of A2AR agonists, conscious vs. anesthetized animals, etc. Regardless, the effects of the A2AR agonist and antagonist we found are not related to changes in blood pressure.

Prolonged exposure to adenosine agonists could produce desensitization of A2AR (63 64 65) . In certain reports, desensitization was dose-dependent and reversible; suggested mechanisms for desensitization have included down-regulation of the expression of A2AR or of Gs proteins involved in the cellular signaling or phosphorylation of the A2A, G protein-coupled receptor (GPCR) by GPCR kinases. There are also reports, however, providing evidence that prolonged stimulation of A2AR does not lead to loss of functional response to adenosine agonists (66 , 67) . In our experiments, treatment with adenosine agonists increased expression of A2AR in glomeruli of rats with anti-GBM glomerulonephritis, providing a mechanism that would prevent desensitization (see Supplemental Data). Interestingly, in rats treated with ZM241385, we found decreased expression of A2AR. This finding could eliminate responses to endogenous adenosine-induced activation of A2AR and lead to additional kidney damage.

In summary, we have demonstrated that pharmacological activation of A2AR attenuates leukocyte infiltration into the kidney during both the acute and progressive phases of anti-GBM glomerulonephritis. Subsequent responses include decreased expression of chemokines and up-regulation of anti-inflammatory cytokines with suppressed inflammation. These events are associated with marked protection against glomerular injury. Consequently, A2AR activation could be a novel therapeutic strategy for modifying macrophage-mediated glomerulonephritis or other diseases dependent on macrophages.


   ACKNOWLEDGMENTS
 
The untimely death of our dear colleague L. Feng deprived us of a superb scientist. She not only contributed substantially to our understanding of mechanisms of inflammation in the kidney but also influenced the training of young scientists. Her enthusiasm, knowledge, and beautiful spirit will be greatly missed. This work was supported in part by a National Institutes of Health George O’Brien Center grant (P50 DK064233, G.E.G, L.D.T., and L.F.). We thank W. Mitch for critical review of the manuscript.


   FOOTNOTES
 
2 Deceased.

Received for publication March 20, 2007. Accepted for publication August 16, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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