Age-, gender-, and weight-matched cd73^−/− mice or littermate controls (WT) were subjected to IP alone (IP) or IP followed by 45 min of ischemia (IP + I) using a hanging weight system for atraumatic occlusion of the left renal artery. The IP protocol consisted of four cycles of ischemia and reperfusion (4 min each).
Statistics: One-way ANOVA was used to perform comparisons among groups. The F-test results are F (5,22) = 210.34, p < 0.001. All statistically significant differences within groups were compared by Tukey's post-hoc test (p < 0.05) and were indicated with a bracket (*, p < 0.01; **, p < 0.001).

A2BAR^−/− or age-, weight-, and gender-matched littermate controls (WT) were subjected to right nephrectomy followed by 45 min of left renal artery ischemia with or without prior in situ IP (consisting of four cycles of 4 min ischemia followed by 4 min reperfusion).
Renal function tests and renal histology were obtained after 24 h of reperfusion. (A) Creatinine clearance.
(B) Urinary flow rate.
(C) Urinary sodium excretion.
(D) Urinary potassium excretion.
(E–I) Representative HE staining (400× and 800×). Tissue damage was characterized by loss of tubular cells, tubular cast, and tubular dilation (arrows). Slice thickness was 3 μm.
(J) Quantification of histological tissue damage assessed by Jablonski index.
Statistics: (A–D) One-way ANOVA with Tukey's post-hoc test was performed (*, p < 0.001 versus WT + IP; **, p < 0.001 versus the A2BAR^−/− mice with IP). The F-test results are (A) F (4,28) = 207.93, p < 0.001; (B) F (4,26) = 50.96, p < 0.001; (C) F (4,25) = 57.65, p < 0.001; (D) F (4,25) = 57.65, p < 0.001.
(J) Kruskal-Wallis nonparametric analysis (chi-square = 17.175, degree of freedom (df) = 4, p = 0.002) with follow-up pairwise comparisons by Wilcoxon-Mann-Whitney test was performed with *, p < 0.05 versus WT + IP and **, p < 0.05 versus the A2BAR^−/− mice with IP.

A2BAR^−/− mice and their respective age-, weight-, and gender-matched littermate controls (WT) were subjected to right nephrectomy followed by 45 min of left renal artery ischemia with or without prior in situ IP (consisting of four cycles of 4 min ischemia followed by 4 min reperfusion). (A) TNF-α, (B) IL-6, (C) KC (mouse ortholog of human IL-8), and (D) IL-10 were assessed by real-time RT-PCR from renal tissues. Data were calculated relative to β-actin and are expressed as fold change compared to sham-operated animals without IP treatment.
(E) NFκB and (F) IκBα levels were evaluated in renal tissue homogenates using a mouse enzyme-linked immunosorbent assay (ELISA).
Statistics: One-way ANOVA with Tukey's post-hoc test was performed (*, p < 0.001 versus WT + IP; #, p < 0.05 versus WT+IP; **, p < 0.001 versus the A2BAR^−/− mice with IP). The F-test results are (A) F (4,15) = 39.28, p < 0.001; (B) F (4,15) = 53.40, p < 0.001; (C) F (4,15) = 201.01, p < 0.001; (D) F (4,15) = 61.46, p < 0.001; (E) F (4,15)= 75.76, p < 0.001; and (F) F (4,15) = 56.18, p < 0.001.

A2BAR antagonist (PSB1115): Age-, weight-, and gender-matched C57BL/6J underwent right nephrectomy. Prior to the surgery, immediately before ischemia, 5 min or 2 h after ischemia, they received the selective A2BAR antagonist PSB1115 (5 mg/kg, intravenous) or treatment with a vehicle control. They were exposed to 45 min of left renal artery ischemia with or without prior in situ IP (consisting of four cycles of 4 min ischemia followed by 4 min of reperfusion). (A) Plasma creatinine and (B) GFR as assessed by inulin clearance were obtained after 24 h of reperfusion.
Statistics: One-way ANOVA with Tukey's post-hoc test was performed (*, p < 0.001 versus the respective group without IP; **, p < 0.001 versus WT mice with IP and with PSB115 treatment). The F-test results are (A) F (9,54) = 42.20, p < 0.001; (B) F (9,55) = 51.67, p < 0.001.

(A–D) Age-, gender-, and weight-matched C57BL/6J mice were subjected to right nephrectomy followed by in situ IP with a hanging weight system for atraumatic occlusion of the left renal artery. The IP protocol consisted of four cycles of ischemia/reperfusion (4 min each), followed by indicated times of reperfusion. Transcriptional responses of the A1AR, A2AAR, and A2BAR of the A3AR were assessed by real-time RT-PCR. Data were calculated relative to β-actin and are expressed as fold change compared to sham-operated animals without IP treatment.
(E) Western blotting for renal A2BAR following IP treatment. Kidneys were excised at indicated times, flash frozen, and lysed; proteins were resolved by SDS PAGE and transferred to nitrocellulose. Membranes were probed with anti-A2BAR antibody. The same blot was reprobed for β-actin as a control for protein loading. One representative experiment of three is shown.
(F–I) A previously described A2BAR reporter mouse was used (A2BAR-KO/β-gal–knock-in mice). Renal tissues were prepared and stained for β-galactosidase (β-Gal) as indicative of the A2BAR gene promoter, which drives the expression of this reporter gene. Procedures are as detailed under Methods. (F, G) β-Gal staining of kidney derived from littermate controls (WT) or A2BAR^−/− mice, and captured at the indicated magnifications (16 ×) with an Olympus IX70 microscope combined with Hamamatsu charge-coupled device camera C4742–95.
(H, I) HE-staining histological examination of renal sections derived from the kidney shown in upper panel. Arrows point to β-Gal stained blood vessels. Fold magnification of captured images are 600×. WT mice were subjected to similar staining showing no expression. β-Gal expression in kidney is clear and localized only in blood vessels.
Statistics: (A–D) One-way ANOVA with Dunn's multiple comparison test (**, p < 0.001 versus control WT mice without IP [C]). The F-test results are (A) F (3,17) = 19.82, p < 0.001.

A2BAR^+/+ (A2BWT) or A2BAR^−/− (A2BKO) mice underwent lethal radiation followed by bone marrow transplantation with marrow derived from A2AAR^+/+ (A2AWT) or A2AAR^−/− (A2AKO) mice. Experiments were performed 8 wk following transplantation. Mice underwent right nephrectomy and underwent 45 min of left renal artery ischemia with or without prior IP (consisting of four cycles of 4 min ischemia followed by 4 min reperfusion). Following 24 h of reperfusion time, (A) plasma creatinine and (B) GFRs were measured.
Statistics: One-way ANOVA with Tukey's post-hoc test was performed (**, p < 0.001 versus the respective group with IP; *, p < 0.001 versus A2AWT→A2BKO). The F-test results are (A) F (7,40) = 32.45, p < 0.001; (B) F (7,40) = 29.67, p < 0.001.

(A–D) Previously characterized A1AR^−/−, A2AAR^−/−, A2BAR^−/−, and A3AR^−/− mice or their respective age-, weight-, and gender-matched littermate controls (WT) were subjected to right nephrectomy followed by 45 min of left renal artery ischemia with or without prior in situ IP (consisting of four cycles of 4 min ischemia followed by 4 min reperfusion). Plasma creatinine and GFR (as measured by inulin clearance) were obtained after 24 h of reperfusion. Note: Renal protection conferred by IP is abrogated in A2BAR^−/− mice.
Statistics: One-way ANOVA with Tukey's post-hoc test was performed (*, p < 0.001 versus WT + IP; **, p < 0.001 versus the respective gene targeted mice with IP). The F-test results are for plasma creatinine: (A) F (3,21) = 41.2, p < 0.001; (B) F (3,23) = 60.66, p < 0.001; (C) F (3,22) = 67.87, p < 0.001; (D) F (3,25) = 48.45, p < 0.001; and for GFR (A) F (3,20) = 79.34, p < 0.001; (B) F (3,21) = 59.12, p < 0.001; (C) F (3,22) = 109.75, p < 0.001; (D) F (3,22) = 93.43, p < 0.001.

A2BAR^−/− mice and their respective age-, weight-, and gender-matched littermate controls (WT) were subjected to right nephrectomy followed by 45 min of left renal artery ischemia with or without prior in situ IP (consisting of four cycles of 4 min ischemia followed by 4 min reperfusion).
(A–E) Chloracetate esterase staining. Arrows indicate granulocytes (magnification 600×).
(F) Quantification of granulocyte accumulation.
(G) Quantification of neutrophil tissue accumulation by measurement of myeloperoxidase (MPO).
Statistics: (F) Kruskal-Wallis nonparametric analysis (chi-square = 17.86, df = 4, p = 0.001) with follow-up pairwise comparisons by Wilcoxon-Mann-Whitney test was performed with *, p < 0.01 versus WT + IP and **, p < 0.01 versus the A2BAR^−/− mice with IP.
(G) One-way ANOVA with Tukey's post-hoc test was performed (*, p < 0.001 versus WT + IP; **, p < 0.001 versus the A2BAR^−/− mice with IP). The F-test result is F (4,22) = 46.26, p < 0.001.

A2BAR^−/− mice and their respective age-, weight-, and gender-matched littermate controls (WT) were subjected to right nephrectomy followed by 45 min of left renal artery ischemia with or without prior in situ IP (consisting of four cycles of 4 min ischemia followed by 4 min reperfusion). (A) Plasma or (B) tissue concentrations of nitrite (NO₂) and nitrate (NO₃) were measured using colorimetric-assay kits.
(C) Tissue nitric oxide synthetase (NOS) activity.
(D–H) Immunohistochemical staining for iNOS.
(I) iNOS expression score.
Statistics: (A–C) One-way ANOVA with Tukey's post-hoc test was performed (*, p < 0.001 versus WT + IP; **, p < 0.001 versus the A2BAR^−/− mice with IP). The F-test results are (A) F (4,22) = 89.90, p < 0.001; (B) F (4,30) = 32.41, p < 0.001; (C) F (4,25) = 36.54, p < 0.001.
(I) Kruskal-Wallis nonparametric analysis (chi-square = 19.1, df = 4, p = 0.001) with follow-up pairwise comparisons by Wilcoxon-Mann-Whitney test was performed with *, p < 0.01 versus WT + IP and **, p < 0.05 versus the A2BAR^−/− mice with IP.

They underwent treatment with BAY 60–6583 (60 μg/kg intravenous) or vehicle control given before the surgery, immediately before ischemia, 5 min or 2h after ischemia.
(A) GFR (as measured by inulin clearance and (B) Jablonski index for histological quantification of ischemic injury were obtained after 24 h of reperfusion.
(C, D) Representative HE staining (400× and 800×) of renal histology following 45 min of ischemia with (+BAY) or without BAY 60–6583 (−BAY) given prior to ischemia are displayed.
Statistics: (A) One-way ANOVA with Tukey's post-hoc test was performed (*, p < 0.01 versus the WT mice with BAY treatment, **, p < 0.001 versus WT mice with BAY treatment). The F-test results are (A) F (11,69) = 54.84, p < 0.001.
(B) Kruskal-Wallis nonparametric analysis (chi-square = 61.08, df = 11, p < 0.001) with follow-up pairwise comparisons by Wilcoxon-Mann-Whitney test was performed with *, p < 0.01 versus WT mice with BAY treatment.

A2BAR bone marrow chimeric mice [A2BAR^+/+→A2BAR^+/+ [WT→WT], A2BAR^−/−→A2BAR^−/− [KO→KO], A2BAR^−/−→A2BAR^+/+ [KO→WT] and A2BAR^+/+→A2BAR^−/− [WT→KO]) were generated. Experiments were performed 8 wk following transplantation. Mice underwent right nephrectomy and underwent 45 min of left renal artery ischemia with or without prior IP (consisting of four cycles of 4 min ischemia followed by 4 min reperfusion). Following 24 h of reperfusion time, (A) plasma creatinine and (B) GFRs were measured.
Influence of A2BAR agonist (BAY 60–6583) on renal function in bone marrow chimeric mice exposed to ischemia. A2BAR bone marrow chimeric mice were subjected to 45 min of ischemia with or without treatment with A2BAR agonist BAY 60–6583 (60 μg/kg intravenous). Following 24 h of reperfusion, (C) plasma creatinine, (D) GFR, and (E) histological tissue injury (Jablonski index) were obtained.
Statistics: (A–D) One-way ANOVA with Tukey's post-hoc test was performed (**, p < 0.001 versus the respective group without IP. The F-test results are (A) F (7,40)= 24.82, p < 0.001; (B) F (7,40) = 48.04, p < 0.001; (C) F (3,20) = 15.54, p < 0.001; (D) F (3,20) = 20.88, p < 0.001.
(E) Kruskal-Wallis nonparametric analysis (chi-square = 12, df = 3, p = 0.007) with follow-up pairwise comparisons by Wilcoxon-Mann-Whitney test was performed with *, p < 0.05 versus WT mice with BAY treatment.