Currently the mechanism by which limb IPC induces upregulati

Currently, the mechanism by which limb IPC induces upregulation of renal renalase is incomplete clear. Given that remote IPC-activated humoral signaling pathways is involved in the remote organ protection (Gassanov et al., 2014), the circulating cytokines released from the preconditioned limb muscle may increase the resistance to hypoxic injury in distant organ (Bonventre, 2012). In this context, several studies have revealed that low dose of TNFα has protective biological properties against ischemic injury and preconditioning with TNFα can mimic classic IPC in a time and dose dependent manner (Lecour et al., 2002; Smith et al., 2002), suggesting that TNFα may serve as a key mediator for limb IPC to induce reno-protection. This hypothesis was supported by the following observations: 1) circulating TNFα increased 6h after limb IPC, which preceded the cox inhibitor of renalase in the kidney that occurred at 24h after limb IPC; 2) Renalase expression was up-regulated in the kidney when exogenous TNFα was administered in the animal; 3) low dose of TNFα treatment increased renalase expression in cultured renal epithelial cells, which is required for cell survival in response to oxidant injury; and 4) administration of Humira, a TNFα antagonist, blocked limb IPC induced renalase upregulation. TNFα may be released from ischemic limb into the systemic circulation and then promotes renalase synthesis in the remote kidney. In line with our observations, other studies have also indicated that low dose of TNFα can protect against I/R injury in heart, liver and neural system (Kleinbongard et al., 2011; Perry et al., 2011; Watters and O\'connor, 2011; Feng et al., 2013). Nevertheless, how TNFα mediates renal protection has not been well defined. It is well known that TNFα is a pleiotropic cytokine involved in the regulation of infection, inflammation, autoimmune, and apoptosis (Pasparakis et al., 1996; Karatzas et al., 2014), thus, this cytokine may play a detrimental role through its pro-inflammatory effects in certain tissues/organs after acute injury. However, since low dose of TNFα can induce activation of multiple signaling pathways that regulate expression/activation of different molecule with diverse biological functions, it is possible that muscular TNFα released during limb IPC may induce activation of a signaling pathway(s) that triggers expression of a protective molecule such as renalase. In this context, we observed that limb IPC-induced renalase upregulation was accompanied by activation of NF-κB signaling in the kidney and pretreatment with either TNFα antagonist (Humira), or NF-κB inhibitor (PDTC), blocked renalase expression. Therefore, the NF-κB signaling pathway may play an essential role in transmitting the renal protective signal from TNFα to expression of some molecules (i.e., renalase) required for renal protection. In support of this hypothesis, activation of the TNFα/NF-κB signaling pathway has been reported to mediate survival of several cell types including cardiomyocytes (Lecour et al., 2005), hepatocytes (Feng et al., 2013) and neuron (Watters et al., 2011) as well as renal tubular cells as indicated in this study. Interesting, it was been reported that low dose of TNFα increased cardiomyocytes\' tolerance to toxicity of “high TNFα” (Cacciapaglia et al., 2014). Although we can no rule out the possibility that limb IPC -induced activation of TNFα signaling pathway may also causes a deleterious effect, the protective effect of TNFα/NF-κB signaling observed in the current study suggests that, at least in the case of CIN, the beneficial effects mediated by this pathway overrides its deleterious effects to the kidney. Further investigations are needed to address this issue. Other molecules and signaling pathways may also be involved in the renal protection after limb IPC. Animal and human studies have revealed that transient limb IPC can lead to a rapid increase of serum EPO (Oba et al., 2015). Increased expression of endogenous EPO or administration of exogenous EPO has been reported to play a protective effect against renal injury induced by diverse stimuli. As such, remote IPC may also induced defense against ischemic injury in kidney through release of EPO from the kidney (Gardner et al., 2014; Diwan et al., 2008). Moreover, it is possible that catecholamines may also play a role in limb IPC-induced renalase upregulation. It has been reported that remote IPC can lead to increased catecholamines levels in myocardial tissue and catecholamines can evoke renalase synthesis (Wang et al., 2014a; Li et al., 2008). In addition, pretreatment with catecholamines can mimic the effect of preconditioning (Bankwala et al., 1994; De Zeeuw et al., 2001). Thus, it will be interesting to further examine, to what extent, various molecules/signaling pathways contribute to the reno-protection after limb IPC and whether they act coordinately to regulate this response.