1. Volume 80, Issue 4, Pages 378-388 (August 2011)
Adenosine A2A receptor activation prevents progressive kidney fibrosis in a model of immune-associated chronic inflammation 
Gabriela E. Garcia, Luan D. Truong, Jiang-Fan Chen, Richard J. Johnson, Lili Feng 
Kidney International 
Volume 80, Issue 4, Pages (August 2011)
DOI: /ki
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2. Figure 1
Adenosine 2A receptor (A2AR) activation at day 14 after injection of anti-glomerular basement membrane (GBM) antibody (Ab) attenuates glomerular injury and macrophage infiltration: periodic acid-Schiff (PAS) staining and immunohistochemistry stained for ED1+ monocytes/macrophages of kidney sections of rats with anti-GBM glomerulonephritis (GN; n=6 in each group). In control rats there was severe glomerular hypercellularity, necrotizing lesion, crescent formation, and prominent macrophage infiltration. In rats treated with A2AR agonist, glomerular injury and macrophage infiltration were significantly attenuated. Worsening of glomerular injury with increased crescent formation was observed in rats treated with A2AR antagonist.
Kidney International  , DOI: ( /ki )
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3. Figure 2
Effects of adenosine A2 receptor (A2AR) activation and inactivation on urinary protein excretion and serum creatinine. (a) Determination of proteinuria (mg per 24h) from Wistar-Kyoto (WKY) rats with anti-glomerular basement membrane (GBM) glomerulonephritis (GN) after treatment with vehicle, adenosine 2A receptor (A2AR) agonist, or A2AR antagonist (antag). A2AR agonist blocked and A2AR antagonist increased proteinuria. *P<0.01 vs control, **P<0.05 vs control, δP<0.01 vs A2AR antagonist. (b) Serum creatinine (SCr; mg/dl). The SCr levels in A2AR-agonist-treated group were not different from those of normal rats. *P<0.05 vs normal, **P<0.03 vs control, δP<0.01 vs A2AR antagonist, &P<0.01 vs normal.
Kidney International  , DOI: ( /ki )
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4. Figure 3
Detection of apoptotic cells by TdT-mediated dUTP nick end labeling (TUNEL) assay and determination of macrophage-derived chemokine/chemokine (C-C motif) ligand 22 (MDC/CCL22) expression by RNase protection assay. (a, b) Immunostaining of macrophages and TUNEL assay demonstrated increased apoptosis in the glomeruli and tubulointerstitium in the control group. (c) MDC/CCL22 mRNA expression was suppressed by adenosine 2A receptor (A2AR) agonist. Probes contain polylinker regions and are longer than the protected bands. Rat ribosomal L-32 gene was used as a housekeeping gene.
Kidney International  , DOI: ( /ki )
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5. Figure 4
Effects of adenosine 2A receptor (A2AR) activation and inactivation on collagen (Col) I, Col III, Col IV, E-cadherin, and α-smooth muscle actin (α-SMA) deposition. Normal controls (a, f, k, p, u), control group at day 14 (D14) after the injection of anti-glomerular basement membrane (GBM) antibody (Ab; b, g, l, q, v), A2AR-agonist-treated group (c, h, m, r, w) at day 20 (D20), control group at day 20 after the induction of anti-GBM glomerulonephritis (GN; d, i, n, s, x), and A2AR-antagonist-treated group at day 20 (e, j, o, t, y). At day 20, A2AR agonist treatment reduced Col IV, Col I, and Col III deposition (c, h, m). A2AR agonist reversed Col IV and Col III deposition as Col IV and Col III expression in A2AR-agonist-treated group was significantly lower at day 20 than in the control group at day 14 (b, c, l, m). A2AR antagonist significantly enhanced Col IV, Col I, and Col III deposition (e, j, o). At day 20, A2AR agonist treatment restored E-cadherin expression (r, s) and A2AR antagonist enhanced E-cadherin loss compared with control group (s, t). A2AR agonist reduced appearance of α-SMA-positive cells in the kidney at day 20 (w, x). Tubular α-SMA was significantly reversed by A2AR agonist (v, w, x). In contrast, A2AR antagonist increased the expression of α-SMA in both glomeruli and tubules (x, y).
Kidney International  , DOI: ( /ki )
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6. Figure 5
Morphometric analysis of collagen (Col) IV, Col I, Col III, E-cadherin, and α-smooth muscle actin (α-SMA) expression in the kidneys by computer-assisted image quantification (Optimas 6.5, Optimas, Bothell, WA). Results from normal kidneys, glomerulonephritic kidneys from day 14 (D14) and day 20 (D20) after the injection of anti-glomerular basement membrane (GBM) antibody (Ab), kidneys from rats with anti-GBM glomerulonephritis (GN) treated with adenosine 2A receptor (A2AR) agonist (CGS) or A2AR antagonist (ZM) are shown. Twenty fields were analyzed at magnifications of × 400 (collagens) or × 200 (E-cadherin and α-SMA). Data were expressed as mean±s.d. percent (%) area (n=6 in each group).
Kidney International  , DOI: ( /ki )
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7. Figure 6
RNase protection assay for transforming growth factor-β1 (TGF-β), tissue inhibitor of matrix metalloproteinase-1 (TIMP-1), thrombospondin-1 (TSP-1), and osteopontin-1 (OPN-1). Macrophages isolated from nephritic glomeruli at day 20 (D20) express TGF-β, TIMP-1, TSP-1, and OPN-1 (a, b). In established glomerulonephritis (GN), profibrotic molecules TSP-1, OPN-1, and TGF-β were highly induced in whole kidneys. Adenosine 2A receptor (A2AR) agonist treatment prevented the induction of TSP-1 and significantly reduced the expression of OPN-1 and TGF-β in the kidney (c, d). Probes contain polylinker regions and are longer than the protected bands. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GM-CSF, granulocyte macrophage-colony stimulating factor; IFNβ, interferon β; IFNγ, interferon γ; Ltβ, lymphotoxin β; L32, ribosomal protein L32; MIF, macrophage migration inhibitory factor; TGFβ1, transforming growth factor-β1; TGFβ2, transforming growth factor-β2; TGFβ3, transforming growth factor-β3; TNFα, tumor necrosis factor α; TNFβ, tumor necrosis factor β.
Kidney International  , DOI: ( /ki )
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8. Figure 7
Selective activation of adenosine 2A receptor (A2AR) modulates genes that affect matrix turnover as determined by RNase protection assay. (a) In anti-glomerular basement membrane (GBM) glomerulonephritis (GN), matrix metalloproteinase-2 (MMP-2) was expressed and was unaffected by A2AR agonist or A2AR antagonist (antag). (b) Increased MMP-2 mRNA expression was associated with an induction in the expression of tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) in control rats and in A2AR-antagonist-treated rats. In contrast, a significant reduction of TIMP-1 expression was observed in A2AR-agonist-treated rats. *P<0.01 vs control. Probes contain polylinker regions and are longer than the protected bands.
Kidney International  , DOI: ( /ki )
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9. Figure 8
Phenotype of macrophages infiltrating nephritic kidneys. In control rats there was prominent glomerular and tubulointerstitial accumulation of macrophages (ED1+ cells), expressing both CD169 and CD163 markers. In rats treated with adenosine 2A receptor (A2AR) agonist, glomerular and interstitial accumulation of macrophages was markedly reduced, and <50% expressed CD163. In rats treated with A2AR antagonist, glomerular and interstitial macrophages expressed predominantly CD169 (91 and 71.94%, respectively).
Kidney International  , DOI: ( /ki )
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10. Figure 9
Expression of JunD determined by RNase protection assay. JunD mRNA expression was attenuated by adenosine 2A receptor (A2AR) agonist and restored by A2AR antagonist (antag) in the nephritic glomeruli. In activated peritoneal macrophages, A2AR activation downregulated and A2AR inactivation upregulated JunD mRNA expression.
Kidney International  , DOI: ( /ki )
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11. Figure 10
Systemic antigen-specific immune response and glomerular immunoglobulin G (IgG) deposition. Circulating titers of rat anti-rabbit IgG at serum dilution of (a) 1:20 or (b) 1:100 were not different among the nephritic groups. *P<0.01 vs normal. (c) Immunofluorescence staining of rabbit IgG and rat IgG. Immunofluorescence staining revealed rabbit IgG and rat IgG binding along the capillary walls of glomeruli in a linear pattern, with no discernible difference in the intensity among the groups.
Kidney International  , DOI: ( /ki )
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