Although recent preclinical and clinical studies have demonstrated that RLX may have important therapeutic potential in chronic kidney diseases, such as renal fibrosis and salt-sensitive hypertension , its role in AKI has never been elucidated The present findings offer first experimental evidence that the therapeutic administration of rhRLX at reperfusion significantly reduces renal injury and dysfunction caused by I/R in rats, while it has no effect on normal renal function in sham-operated animals. The dose of rhRLX used in this study has been previously reported to protect other organs against I/R injury and to prevent the development of acute pancreatitis [3, 27, 28]. Notably, to simulate the clinical conditions, in our experimental model, rhRLX was only applied during reperfusion, indicating that this treatment strategy could be potentially employed in several situations known to result in AKI, including renal transplantation, aortic aneurysm surgery, or X-ray contrast tracer-induced nephropathy, to protect or even rescue a kidney previously challenged by ischaemia. Our results further corroborate and extend the previous findings demonstrating that RLX is capable of ameliorating renal hemodynamics by inducing selective renal vasodilation and hyperfiltration in both rats and humans [29, 30]. In our experimental model, the improvements in the outcome of renal damage by rhRLX administration were associated with a significant inhibition of both the inflammatory response and oxidative stress induced by I/R. Namely, rhRLX reduced leucocyte adhesion to ischaemic-reperfused vascular endothelium, as suggested by its ability to suppress the expression of the adhesion molecule ICAM-1 and the activity of MPO, selected as typical markers of leucocyte inflammatory recruitment, which were both drastically up-regulated by I/R. At the same time, rhRLX significantly decreased the production of TNF-α, IL-1β and IL-18 in the kidney of animals that underwent I/R injury. Interestingly, this effect was associated with increased level of the anti-inflammatory cytokine IL-10, suggesting that RLX may operate a shift from a pro-inflammatory to an anti-inflammatory status. These results are consistent with previous reports demonstrating the role of RLX as a potent inhibitory factor in early vascular inflammation with prominent inhibitory effects on the expression of cytokines and adhesion molecules [31-33]. The attenuated inflammatory response caused by rhRLX treatment may also account for the decrease in tissue markers of oxidative stress, thus supporting the notion that release of ROS from activated leucocytes provides a major contribution to peroxidation of lipid membranes and free radical-induced DNA damage in the kidney. Besides, a direct effect of RLX on oxidative stress has also been recently demonstrated by Dschietzig et al. , showing that RLX stimulates CuZnSOD expression in rat aortic rings, by increasing the CuZnSOD promoter activity at different time-points. Our findings are in keeping with previous studies from our and other research groups showing that RLX exerts beneficial effects against organ ischaemic damage by reducing local leucocyte recruitment and oxidative stress [3, 4, 6]. Accordingly, RLX has also been proposed as a protective substance in preservation solutions for lung and liver transplantation [5, 35, 36]. Despite these intriguing data and the evidence that the kidney is the organ of greatest uptake of exogenously administered RLX , the specific signal transduction pathway by which RLX exerts its effects in the kidney remains to be fully elucidated. Previous studies have demonstrated that several renal biological actions of RLX, including its potent antifibrotic effects, are mediated by functional activation of the relaxin receptor RXFP1, which is expressed by specific renal cells, such as mesangial cells and myofibroblasts [37, 38]. RXFP1 signalling is complex, involving multiple pathways depending on the cell type under investigation; however, recent evidence suggests a key role for the nitric oxide pathway in mediating major renovascular effects of RLX . For instance, Sasser et al.  have demonstrated that RLX was ineffective in preventing chronic renal injury during administration of the nitric oxide synthase inhibitor N(ω)-nitro-l-arginine methyl ester (L-NAME), suggesting that the renoprotective effects of RLX are dependent on a functional NOS system. Although the exact signalling mechanisms of RXFP1 were beyond the scope of this study, we could demonstrate an involvement of the nitric oxide pathway in the RLX-mediated effects reported here: in fact, RLX administration was associated with eNOS activation and induction of iNOS expression, resulting in enhanced formation of nitric oxide in the microcirculation. In conditions associated with I/R, the enhanced formation of nitric oxide is beneficial, as it can cause local vasodilation, inhibit adhesion of platelets and leucocytes and promote angiogenesis . There is good evidence that agents that release nitric oxide or enhance the formation of endogenous nitric oxide attenuate organ injury/dysfunction in AKI [42, 43]. By a nitric oxide-dependent mechanism, RLX has been shown to strongly inhibit neutrophil activation, thereby reducing free radical generation, chemotaxis and platelet aggregation [44, 45]. Therefore, the reduced oxidative stress status and leucocyte activation here reported may be explained, at least in part, by the ability of RLX to up-regulate the NOS/nitric oxide pathway. Previous studies in cultured human endothelial cells have shown that RLX can evoke eNOS activation by phosphorylation of specific serine residues in Akt . Akt is a member of the phosphoinositide 3-kinase signal transduction enzyme family which, upon phosphorylation by its upstream regulator, can modulate inflammatory responses and apoptosis . A reduction in the activation of this important survival pathway has been recently demonstrated to make the kidney more susceptible to I/R insult [48, 49]. Here, we show that RLX caused a robust increase in Akt phosphorylation. This indicates a significant Akt activation, which in turn could promote eNOS phosphorylation and renal protection. An additional contribution to the regulatory effects of RLX on nitric oxide pathway may rely on its ability to affect ERK1/2 MAPK pathway, which is another important signal for cell survival . ERK activation protects renal epithelial cells from oxidative injury  and, particularly relevant to this study, it leads to iNOS induction in renal epithelial cells , renal myofibrobalsts , vascular smooth muscle cells  and murine macrophages . As we documented increased ERK1/2 activation in the presence of RLX, we propose that MAPK activation by RLX is, at least in part, responsible for the RLX-mediated modulation of iNOS expression. However, it must be underlined that ERK1/2 and Akt activation by RLX was recorded at 6 hrs after reperfusion. As RLX has a short serum half-life in rodents , we cannot rule out the possibility that RLX evokes an early intracellular signalling cascade leading to late ERK and Akt activation, thus resulting in increased NOS activity/expression.