The Role And Molecular Mechanism Of SHP-1 In Regulation Of Renal Ischemia/Reperfusion Injury

Ischemia/reperfusion?I/R?injury is an inevitable pathophysiological phenomenon during renal transplantation.It is occurred throughout the procedure of organ procurement,organ transportation,transplantation surgery and postoperative recovery.Renal ischemia reperfusion injury is a kind of aseptic inflammatory reaction with multiple kinds of cells in the kidney involved.It is characterized by apoptosis or necrosis or regeneration of renal tubular epithelial cells.In clinical,renal I/R injury contributes to delayed recovery of the function or for the graft and may lead to acute or chronic rejection.Renal tubular epithelial cells are the main cell units in kidney and are sensitive to the hypoxic environment.Figure out the process of injury and repair during ischemia/reperfusion injury,as well as the regulation and mechanism of critical genes in this process,may be an effective way to attenuate renal I/R injury.Protein tyrosine phosphatase SHP-1 is a critical member of the tyrosine phosphatase family and is responsible for negative regulation of tyrosine kinase phosphorylation signaling pathways in hematopoietic cells.The balance of phosphorylation and dephosphorylation is vital for maintaining homeostasis.Once stimulated,the kinase cascades activation and the anti-stress genes expression are up-regulated to cope with environmental stress.The phosphatase is responsible for removing the phosphate group of the substrate by hydrolysis,ultimately allowing the body to maintain homeostasis.The function of SHP-1 was first elucidated in SHP-1 gene-deficient mice for their special phenotype.That is,after SHP-1 deletion,the excessive activation of inflammation was uncontrolled,and finally the mice died of autoimmune reaction.Subsequently,SHP-1 was shown to be closely related to the functional status of various immune cells,involving in regulating the signaling pathway of many kinds of immune cells.However,the role of SHP-1 in non-hematopoietic cells is still unknown.In this study,renal ischemia reperfusion injury model was conducted on SHP-1 gene deletion mice.Compared with the wild type littermates,it was found that apoptosis and necrosis of renal tubular epithelial cells after SHP-1 deletion were significantly serious than wild type.This suggests that the mechanism of SHP-1 may be a negative regulator of apoptotic signaling pathway.After further detection of other apoptotic indicators,it was clearer that SHP-1 deletion caused mice to face more severe renal I/R injury,which confirmed that SHP-1 might regulate apoptosis signaling pathway to protect renal I/R injury.However,until now,it is not clear which kind of cell was responsible for the serious phenotype after SHP-1 deletion.From the results,there were at least two possible reasons.One was that SHP-1 may inhibit the activation of immune cells such as macrophages,thus reducing inflammation and protecting I/R injury.The other was that SHP-1 may directly act on renal parenchymal cells,such as renal tubular epithelial cells,to inhibit their apoptosis.Accordingly,we designed a macrophage clearance assay to verify the function of macrophages in it.By injecting clodronate liposome and PBS liposome into wildtype and SHP-1 deletion mice,we found that kidney damage was alleviated after removal of macrophages both in wildtype and SHP-1 deletion mice.Proinflammatory effect of macrophages was confirmed in renal ischemia reperfusion injury.However,by further comparison,the protective effect of clearing macrophages is comparable in the two genotype mice.That means the protective effect of clearing macrophages is not related to whether the absence of SHP-1 or not.At the same time,we performed continuous section and immunohistochemical staining of the kidney tissue after I/R,and found that the location of SHP-1 positive cells were consistent with that of AQP-1 positive cells,a specific biomarker of tubular epithelial cells,rather than that of F4/80 positive cells,which indicated macrophages.So we are more confident that SHP-1 functioned in renal tubular epithelial cells.To disclose the mystery of how SHP-1 could play an anti-apoptotic role in renal tubular epithelial cells,we constructed an in vitro cell model to explore the mechanism of SHP-1.According to literatures,we used CoCl₂ to mimic cellular hypoxia injury.A tubular epithelial cell line?TCMK1?was stimulated by CoCl₂ to induce apoptosis.After time and concentration exploration,we found that TCMK1 was sensitive to CoCl₂,and this apoptosis effect is dependent on stimulation time and stimulation concentration.TCMK1was stimulated with appropriate concentration?200?mol/L?and time?24 h?,the level of apoptosis and the expression level of apoptosis-related genes were up-regulated after CoCl₂ stimulation.These results confirmed the validity and stability of the cell model.To illustrate the protective effects of SHP-1,we constructed a SHP-1 overexpression plasmid and packaged it into a lentivirus.By infecting normal cultured TCMK1 cells,and subsequent puromycin selection and expression validation,we obtained a TCMK1 cell line?abbreviated as TCMK1 OV?with the ability to stably overexpress SHP-1.We immediately performed the functional assay of TCMK1 OV,which meant to use CoCl₂ to stimulate TCMK1 OV and cells infected with empty control virusm to assess apoptosis levels,for the same stimulation concentration and stimulation time,respectively.The results showed that the incidence of apoptosis in TCMK1 OV was significantly lower than that of the control cells.In order to fully assess the level of apoptosis,we used flow cytometry,immunofluorescence staining,and immunoblotting.All results consistently demonstrated that overexpression of SHP-1 significantly reduced apoptosis of renal tubular epithelial cells against hypoxia-induced by CoCl₂.To fully demonstrate the anti-apoptotic effect of SHP-1 in renal tubular epithelial cells,we supplemented loss-of-function experiments after knocking out or knocking down SHP-1 in renal tubular epithelial cells.We found that after SHP-1 down-regulated,apoptosis rate was significantly increased after CoCl₂ stimulation,which was consistent with the results of in vivo experiments.Based on these,we concluded that SHP-1 protected renal tubular epithelial cells from I/R injury by inhibiting apoptosis.The above results from cellular gain-of function and loss-of function experiments were consistent with the results of animal experiments,indicating the protective effect of SHP-1 against apoptosis.Furthermore,we examined the cleavage activation of caspase 3,a promoter of apoptosis,in animals and cells.The results indicated that the anti-apoptotic effect of SHP-1 was achieved by regulating the cleavage activation of caspase 3.However,how SHP-1 regulated the cleavage activation of caspase 3 more specifically was not known at present.One of the reasons for this condition is that the subcellular localization of SHP-1 in renal tubular epithelial cells is currently unknown.It is possible to act as a conventional cytoplasmic phosphatase,or to function as a transcription factor after tranlocated to nucleus as described in a few literatures.For aiding this judgement,we used cellular immunofluorescence staining experiments to determine.From the results,it showed SHP-1 indeed acted as a cytoplasmic molecule in renal tubular epithelial cells.This conclusion would guide the direction for the next step of intracellular mechanism of SHP-1.For figuring out the target molecule of SHP-1 in cytoplasm,we screened for potential SHP-1 substrates in the apoptotic signaling pathway.As we all acknowledged that caspase3 is an executor for initiating apoptotic signaling pathway.When screening for possible targets of SHP-1,we also added the screen criteria for the ability of regulating caspase 3activation.MAPK signaling pathway is widely involved in biological processes such as proliferation,differentiation,inflammation and apoptosis.And it was reported in the literature that MAPK signaling pathway participate in the activation of caspase 3.MAPK is a classical three-tiered-cascade activation mode,namely MAPKKK/MAPKK/MAPK.Among them,JNK was closely related to apoptosis.ASK1 belongs to the MAPKKK family and is an upstream regulatory molecule of JNK.We found through the literature that there is a relationship between ASK1 and SHP-1.We examined the expression of ASK1 in different expression states of SHP-1.We found that the expression of ASK in mRNA or protein level was not associated with the expression of SHP-1.In the exclusion of SHP-1 regulating ASK1 expression,combined with the function of SHP-1 tyrosine phosphatase,the results showed that SHP-1 overexpression can significantly inhibit the tyrosine phosphorylation level of ASK1,which meant SHP-1 regulated the post-translational modification of ASK1.Furtherly,by immunoprecipitation experiments,we confirmed the protein-protein interaction relationship of SHP-1 and ASK1.In order to clarify the domains of SHP-1 and ASK1 interaction,we constructed different truncations according to their respective protein structures.We found that the carboxyl terminal phosphatase domain of SHP-1 was necessary for binding to ASK1 by co-immunoprecipitation experiments.The kinase domain of the middle segment of ASK1was necessary for binding to SHP-1.Finally,we returned to the validation of the apoptotic signaling pathway.We found that the phosphorylation level of JNK was significantly increased in SHP-1 knockout stable cell line after CoCl₂ stimulation,compared with the empty vector transducted control cell line.That is,SHP-1 directly inhibits the phosphorylation of ASK1,thereby indirectly inhibiting the activation of JNK and the subsequent cleavage activation of caspase 3,and ultimately attenuating the apoptotic response.To sum up,this study systematically explored the important function of SHP-1,not just functioned in hematopoietic cells,but in renal tubular epithelial cells,which represent a kind of non-hematopoietic cells.We clarified the anti-apoptotic role of SHP-1 in renal I/R injury.Mechanically,ASK1,a target molecule of SHP-1,also has a new post-translational modification type,that is tyrosine phosphorylation.It provides a theoretical basis for the future development of drugs to effectively cure renal I/R injury.