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Ischemia-reperfusion (I/R) injury occurs as a result of restoring blood flow to previously hypoperfused vessels or after tissue transplantation and is characterized by inflammation and microvascular occlusion. We report here that 4-[3-[6-amino-9-(5-ethylcarbamoyl-3,4-dihydroxy-tetrahydro-furan-2-yl)-9H-purin-2-yl]-prop-2-ynyl]-cyclohexanecarboxylic acid methyl ester (ATL146e), a selective agonist of the A(2A) adenosine receptor (A(2A)AR), profoundly protects mouse liver from I/R injury when administered at the time of reperfusion, and protection is blocked by the antagonist ZM241385. ATL146e lowers liver damage by 90% as assessed by serum glutamyl pyruvic transaminase and reduces hepatic edema and MPO. Most protection remains if ATL146e treatment is delayed for 1 h but disappears when delayed for 4 h after the start of reperfusion. In mice lacking the A(2A)AR gene, protection by ATL1465e is lost and ischemic injury of short duration is exacerbated compared with wild-type mice, suggesting a protective role for endogenous adenosine. I/R injury causes induction of hepatic transcripts for IL-1alpha, IL-1beta, IL-1Ra, IL-6, IL-10, IL-18, INF-beta, INF-gamma, regulated on activation, normal T cell expressed, and presumably secreted (RANTES), major intrinsic protein (MIP)-1alpha, MIP-2, IFN-gamma-inducible protein (IP)-10, and monocyte chemotactic protein (MCP)-1 that are suppressed by administering ATL146e to wild-type but not to A(2A)AR knockout mice. RANTES, MCP-1, and IP-10 are notable as induced chemokines that are chemotactic to T lymphocytes. The induction of cytokines may contribute to transient lymphopenia and neutrophilia that occur after liver I/R injury. We conclude that most damage after hepatic ischemia occurs during reperfusion and can be blocked by A(2A)AR activation. We speculate that inhibition of chemokine and cytokine production limits inflammation and contributes to tissue protection by the A(2A)AR agonist ATL146e.

The mechanisms involved in renal ischemia-reperfusion injury (IRI) are complex and appear to involve the early participation of bone marrow-derived cells. T lymphocytes participate in the pathogenesis of IRI. Sphingosine 1-phosphate (S1P) induces peripheral T cell depletion. Therefore, we hypothesized that S1P1 receptor activation protects kidney from IRI. FTY-720, a non-receptor-selective sphingosine analog, was given intraperitoneally to C57BL/6 mice, and animals were subjected to ischemia for 32 min followed by reperfusion for 24 h. Plasma creatinine, blood count, myeloperoxidase (MPO) activity, and renal histology were determined. IRI led to a marked increase in plasma creatinine, MPO activity, leukocyte infiltration, and vascular permeability. FTY-720 significantly decreased plasma creatinine in a dose-response manner with a maximal reduction of ∼73 and ∼69% with doses of 240 and 48 μg/kg, respectively. MPO, leukocyte infiltration, vascular permeability, and peripheral blood lymphocyte counts were markedly decreased with FTY-720 treatment. The protective effect of FTY-720 was reversed with VPC-44116, a selective S1P1 receptor antagonist. Furthermore, SEW-2871, a selective S1P1 agonist, significantly decreased plasma creatinine in a dose-response manner with a maximal reduction of ∼70% with a dose of 10 mg/kg. Analysis of kidneys by light microscopy revealed minimal histological signs of ischemic injury with FTY-720 or SEW-2871 treatment compared with the vehicle group. Using RT-PCR, we found a time-dependent increase in the S1P1 mRNA expression following IRI that begins after 2 h with the maximum expression at ∼4 h. We conclude that the protective effect of FTY-720 is due primarily to activation of S1P1 receptors. The mechanism of protection is not known but may be related to peripheral lymphocyte depletion or direct effects on kidney cells expressing S1P1 receptor. [ABSTRACT FROM AUTHOR]

The role of monocytes/macrophages in the pathogenesis of ischemia-reperfusion injury ORB is unknown. We sought to determine whether activation of macrophage adenosine 2A (A2A) receptors (A2ARs) mediates tissue protection We subjected C57B1/6 mice infused with clodronate [dichloromethylene bisphosphonate (Cl2MBP)] to IRI (32 min of ischemia followed by 24 h of reperfusion) to deplete them of macrophages. IRI induced an elevation of plasma creatinine that was reduced with Cl2MBP (26% of control). Adoptive transfer of murine RAW 264.7 cells reconstituted injury, an effect blocked significantly by A2A agonists (27% of plasma creatinine from mice reconstituted with macrophages). Macrophages subjected to A2A knockout by small interfering RNA were adoptively transferred to macrophage-depleted mice and reconstituted injury (110% of control mice); however, the increase in plasma creatinine was blocked by A2A agonists (20% of vehicle treatment). Finally, the A2A agonist effect on IRI was blocked in macrophage-depleted A2A-knockout mice reconstituted with wild-type RAW 264.7 cells. RNase protection assays 24 h after IRI demonstrated that macrophages are required for IL-6 and TGF-β mRNA induction. However, A2A agonist-mediated tissue protection is independent of IL-6 and TGF-β mRNA. We conclude that the full extent of IRI requires macrophages and that A2A agonist-mediated tissue protection is independent of activation of macrophage A2ARs. [ABSTRACT FROM AUTHOR]

Enhanced protection from renal ischemia: Reperfusion injury with A2A -adenosine receptor activation and PDE 4 inhibition. Background. We previously demonstrated in rats and mice that agonists of A2A -adenosine receptors (A2A -ARs) reduce renal injury following ischemia-reperfusion. We now extend these studies and examine the effects of ATL-146e (formerly DWH-146e), an A2A -AR agonist, and rolipram, a type IV phosphodiesterase (PDE 4) inhibitor, on murine renal injury following ischemia-reperfusion. Methods. C57BL/6 mice were treated with rolipram, ATL-146e, or both compounds combined and were subjected to renal ischemia for 32 minutes and reperfusion for 24 to 48 hours. In vitro studies were performed on suspended and adhering human neutrophils. Results. Continuous delivery of rolipram or ATL-146e during reperfusion reduced renal injury in a dose-dependent manner. Maximal protection was observed when ATL-146e was infused for six hours during reperfusion. Elevated plasma creatinine and myeloperoxidase activity produced by ischemia-reperfusion were reduced by rolipram (0.1 ng/kg/min) and ATL-146e (10 ng/kg/min) by up to approximately 60% and 70%, respectively. Co-infusion of both compounds produced a maximum reduction of plasma creatinine of approximately 90% and myeloperoxidase activity. In vitro studies on suspended and adhering human neutrophils demonstrated that selective stimulation of A2A -ARs by ATL-146e increased cAMP accumulation, reduced oxidative activity of activated neutrophils, and decreased activated neutrophil adherence. These responses were potentiated by rolipram. Conclusions. We conclude that the combined infusion of ATL-146e and rolipram leads to enhanced renal tissue protection from ischemia-reperfusion by mechanisms that may include reduced neutrophil adherence/recruitment and release of reactive oxygen species. [ABSTRACT FROM AUTHOR]

Presents information on a study which examined the effect of selective activation of A... adenosine receptors on ischemia-reperfusion injury in rat kidney. Methods of the study; Results and discussion.

The IL-23/IL-17 and IL-12/IFN-gamma cytokine pathways have a role in chronic autoimmunity, which is considered mainly a dysfunction of adaptive immunity. The extent to which they contribute to innate immunity is, however, unknown. We used a mouse model of acute kidney ischemia-reperfusion injury (IRI) to test the hypothesis that early production of IL-23 and IL-12 following IRI activates downstream IL-17 and IFN-gamma signaling pathways and promotes kidney inflammation. Deficiency in IL-23, IL-17A, or IL-17 receptor (IL-17R) and mAb neutralization of CXCR2, the p19 subunit of IL-23, or IL-17A attenuated neutrophil infiltration in acute kidney IRI in mice. We further demonstrate that IL-17A produced by GR-1+ neutrophils was critical for kidney IRI in mice. Activation of the IL-12/IFN-gamma pathway and NKT cells by administering alpha-galactosylceramide-primed bone marrow-derived DCs increased IFN-gamma production following moderate IRI in WT mice but did not exacerbate injury or enhance IFN-gamma production in either Il17a-/- or Il17r-/- mice, which suggested that IL-17 signaling was proximal to IFN-gamma signaling. This was confirmed by the finding that IFN-gamma administration reversed the protection seen in Il17a-/- mice subjected to IRI, whereas IL-17A failed to reverse protection in Ifng-/- mice. These results demonstrate that the innate immune component of kidney IRI requires dual activation of the IL-12/IFN-gamma and IL-23/IL-17 signaling pathways and that neutrophil production of IL-17A is upstream of IL-12/IFN-gamma. These mechanisms might contribute to reperfusion injury in other organs. [ABSTRACT FROM AUTHOR]

Chemokines and their receptors such as CCR2 and CX3CR1 mediate leukocyte adhesion and migration into injured tissue. To further define mechanisms of monocyte trafficking during kidney injury we identified two groups of F4/80-positive cells (F4/80(low) and F4/80(high)) in the normal mouse kidney that phenotypically correspond to macrophages and dendritic cells, respectively. Following ischemia and 3 h of reperfusion, there was a large influx of F4/80(low) inflamed monocytes, but not dendritic cells, into the kidney. These monocytes produced TNF-alpha, IL-6, IL-1alpha and IL-12. Ischemic injury induced in CCR2(-/-) mice or in CCR2(+/+) mice, made chimeric with CCR2(-/-) bone marrow, resulted in lower plasma creatinine levels and their kidneys had fewer infiltrated F4/80(low) macrophages compared to control mice. CX3CR1 expression contributed to monocyte recruitment into inflamed kidneys, as ischemic injury in CX3CR1(-/-) mice was reduced, with fewer F4/80(low) macrophages than controls. Monocytes transferred from CCR2(+/+) or CX3CR1(+/-) mice migrated into reperfused kidneys better than monocytes from either CCR2(-/-) or CX3CR1(-/-) mice. Adoptive transfer of monocytes from CCR2(+/+) mice, but not CCR2(-/-) mice, reversed the protective effect in CCR2(-/-) mice following ischemia-reperfusion. Egress of CD11b(+)Ly6C(high) monocytes from blood into inflamed kidneys was CCR2- and CX3CR1-dependent. Our study shows that inflamed monocyte migration, through CCR2- and CX3CR1-dependent mechanisms, plays a critical role in kidney injury following ischemia reperfusion. [ABSTRACT FROM AUTHOR]