Role Of Phosphatase SHP-1 In Kidney Ischemia Reperfusion Injury And The Underlying Immunological Mechanisms

Organ transplantation has become the most effective therapeaty for end stage organ failure, while the high quality of donor graft always being the first guarantee. Ischemia reperfusion (IR) injury is an inevitable consequence occurring in organ procurement and transplantation. It can cause primary none function or delayed graft function, and increase the incidence of both acute and chronic rejection. Althrough lots of studies reported approaches that can alleviate IR injury, the need for effective therapeutic modalities to prevent renal IR injury is still quite urgent. The pathophysiologic process of IR includes failure of mitochondria energy synthesis, calcium overload, free radical damage and cell apoptosis. However, more and more studies demonstrated that during the complex pathophysiologic process after IR, mammalian sentinel toll-like-receptor (TLRs) recognizes specific endogenous danger molecules that have been altered from their native state or accumulate in non-physiologic sites or amounts during tissue injury, such as highmobility group box 1 protein (HMGB1), heat-shock proteins, hyaluronan, and fibrinogen. Upon ligand recognition, TLRs are activated and initiate a proinflammatory response by the release of cytokines/chemokines and attraction of inflammatory cells. Except for TLR3, all TLRs control these innate immune responses through a conserved downstream signaling pathway, starting with the translocation of the adapter molecule MyD88 (myeloid differentiation factor 88) that ultimately leads to the early activation of NF-kB. Besides this pathway, TLR4 and TLR3 can use an alternative signaling cascade, the MyD88-independent route, which specifically involves the translocation of adapter molecule TRIF (TIR domain–containing adapter inducing IFN-β), in combination with the adapter protein TRAM (TRIF-related adapter molecule) that subsequently leads to the production of type I IFN and the expression of Interferon-inducible genes.SHP-1 (also named SHPTP-1, SHP, HCP and PTP1C) is an intracellular protein tyrosine phosphatase that contains two tandemly linked Src homology 2 domains at its amino termini, followed by a catalytic domain. The SHP-1 protein has been well studied in the immune system and primarily functions as a negative regulator of signal transduction. Our previous studies demonstrated that SHP-1 inhibited TLR-initiated production of proinflammatory cytokines by suppressing the activation of MAPKs and NF-κB, but it increased TLR- and RIG-I-activated type I interferon by directly binding to the kinase domain of IRAK1 and thus inhibiting IRAK1 activity. Our results identify SHP-1 as a unique regulatory molecule that balances the production of proinflammatory cytokines and type I interferon in innate immune responses and thus contributes to immune homeostasis.Since the mechanisms underlying renal IRI is much complex a pathophysiologic process, it's interesting to evaluate the specific role of SHP-1 in regulating TLR signaling in the setting of renal IRI. Furthermore, as reported by others, ROS generated by oxidation can regulate the enzymatic activity of SHP-1. IR can induce intense oxidative stress. Whether this oxidation has already played a role in the regulation of SHP-1, which in turn participates in the development of inflammatory response after IR? In other words, is it possible that SHP-1 has already participated in IR induced inflammatory resoponse as a dampened anti-inflammatory mechanism? To this end, we first investigated the SHP-1 expression after renal IR and the possible regulatory mechanism. After that, we studied the employed in vivo and in vitro IR model in SHP-1 defcient mice or primary cells to document the regulatory role of SHP-1 in the inflammatory response after renal IR. We further investigated the effect of SHP-1 in modulating TLR signaling after renal IR. Finally, we confirmed the results by over-expressing SHP-1 in mice before renal IR.Part I Renal IR regulated the expression and enzymatic activity of SHP-1 and the underling mechanismWe first assessed the expression SHP-1 during renal IR by employing a mice bilitary renal IR model together with an in vitro simulated IR model in primary cultured mouse renal tubular epithelial cells (TEC) and peritoneal macrophages (M(?)). IR significantly decreased the mRNA as well as the protein content of SHP-1 both in the kidney and in the primary cultured cells. The SHP-1 phosphatase activity in kidney homogenate also decreased after IR. A number of data emphasized the important role for ROS in fine-tuning tyrosine phosphorylation-dependent signaling pathways through transient oxidation and inactivation of members of the PTP family. In this study, we demonstrated that, other than transient inactivation, ROS generated from renal IR further decreased the expression and phosphotase activity of SHP-1 in kidney. We confirmed that this inhibition also existed in the SHP-1 over-expressing M(?) after H₂O₂ treatment. Importantly, the inhibition caused by oxidative stress is almost at the same time course as that caused by renal IR, which is known to generate lots of reactive oxygen species (ROS) within hours after reperfusion. Interestingly, pretreating mice with a superoxide dismutase agonist and antioxidant MnTMPyP almost entirely blunted the SHP-1 down-regulation in the kidney post renal IR. Together, these data suggest that renal IR down-regulates SHP-1 expression through a mechanism involving ROS.Part II The effect of SHP-1 deficiency in renal damage after renal IR and its regulatory mechanism in TLRs signalingWe next studied the effect of SHP-1 deficiency on renal function after IR injury. The phosphatase activity assay showed that mev/+ mice have significantly decreased SHP-1 phosphatase activity in renal tissue. We demonstrated that the SHP-1 deficient mev/+ mice are more susceptible to renal IR than the WT controls. Increased susceptibility in mev/+ mice was associated with enhanced inflammatory responses as evidenced by strikingly increased leucocyte accumulation and proinflammatory cytokine production in the kidney.Mev/+ mice displayed more robust up-regulation of TLR4/TLR2 and their endogenous ligands, as well as enhanced phosphorylation of MAPKs and NF-κB in comparison to the WT controls, indicating an over-reactive TLRs signaling pathway. Renal production of proinflammatory cytokines downstream of the TLRs signaling (e.g., TNF-α, IL-1βand IL-6) increased more prominently in mev/+ mice after IR. Cytokines produced in response to TLRs activation may damage renal cells via direct cytotoxicity (induction of dysfunction and/or apoptosis) as well as attracting more leucocytes to infiltrate and cause further damage indirectly. Some proinflammatory cytokines have interactions between each other. And some of them, like TNF-α, are capable of up-regulating their own expression. However, it is generally believed that TLRs signaling controls the production of most proinflammatory cytokines through the MAPK and NF-κB pathways. Consistently, our results also showed SHP-1 regulate TLR-triggered proinflammatory cytokine production mainly through MAPK and NF-κB pathways.A pro-apoptotic action in acute myocardial ischemia has been proposed for SHP-1 based on its binding to Fas-R, and increasing Akt phosphorylation in myocardial cells. In view that TNF-αis a well known cytokine which induces cell apoptosis, the similar extent of apoptosis observed between WT and mev/+ mice after renal IR in our model may reflect the increase of TNF-αproduction. Besides, enhanced JNK phosphorylation in mev/+ mice during renal IR might be another explanation, since activation of JNK reportedly triggered cell apoptosis.Partâ…¢Over-expression of SHP-1 dispalyed protective effect against renal damage after renal IR through inhibition of inflammatory responseFurther proof of a protective role for SHP-1 came from in vivo transfection experiments. SHP-1 was transiently over-expressed in the kidney via tail vein injection using an adenovirus vector. Over-expression of SHP-1 significantly increased the SHP-1 phosphatase activity by two folds at 48 hours post transfection in the kidney tissues. Transient SHP-1 over-expression in the kidney of WT and mev/+ mice resulted in decreased production of proinflammatory cytokines and preserved renal function after IR injury.In conclusion, the present study revealed a protective role of SHP-1 in renal IR, most likely due to inhibition of the TLR-triggered innate immune responses. These findings shed light on better understanding of the activation of innate immune system, and encourage targeting SHP-1 in the development of novel molecular therapies for renal IR injury.