The Role Of PDCD4 In The Pathogenesis Of Kidney Ischemia Reperfusion Injury

BackgroundRenal ischemia reperfusion injury(IRI),with rapid onset and progression,is one of the important cause in acute kidney Injury(AKI),which is prone to become chronic kidney injury and has a high mortality rate,and the incidence has increased in recent years.The pathogenesis of IRI is complex and uncertain,and multiple mechanisms are involved in its occurrence and development which brings difficulties to its prevention and treatment.Currently the drugs for treatment are limited to symptomatic treatment and the improvement of renal function is limited.Therefore,finding the key molecules involved in IRI injury for the prevention and treatment of IRI has important theoretical significance and practical application value.Programmed cell death 4(PDCD4)is an important tumor suppressor gene newly discovered in recent years.A large number of studies have shown that PDCD4 can inhibit the development of many tumor-related diseases by binding to the coding region of target genes and blocking the translation process of downstream target proteins,thereby affecting cell proliferation,apoptosis,transformation,invasion,autophagy and other biological process.However,research on PDCD4 has focused on tumor-related diseases,but its role in renal ischemia reperfusion injury remains unclear.Recent studies have shown that PDCD4 plays an important regulatory role in glycolipid metabolism-related diseases,and PDCD4 is closely related to inflammation and apoptosis.At the same time,numerous studies have shown that the mutual promotion of renal tubular cell apoptosis and inflammation plays a key role in acute kidney injury,and whether PDCD4 plays an important role in acute kidney injury is need to be further discussed.Purpose of research1.The role of PDCD4 in renal ischemia reperfusion injury was determined by in vivo animal experiments and in vitro cell experiments.2.To elucidate the mechanism of PDCD4 and to find the key targets involved in the renal ischemia reperfusion injury.MethodsPart I:expression,distribution and role of PDCD4 in acute renal ischemia reperfusion injury 1.1 Expression changes of PDCD4 in acute ischemia reperfusion injury1.1.1 The expression patterns of PDCD4 in different organ tissues and renal cell linesProtein expression of PDCD4 in heart,liver,spleen,lung,kidney,brain,intestine,testis of C57BL/6J mice was detected by Western blot(WB),and protein expression of PDCD4 in rat mesangial cells(RMC),rat mesangial cells(GENC),human podocyte(HPC)and rat renal tubular epithelial cells(NRK-52E)was detected in different renal cell lines.1.1.2.The expression of PDCD4 in renal ischemia reperfusion injuryC57BL/6J male mice aged 12 weeks were selected to establish the acute renal ischemia reperfusion model.Real-time quantitative PCR(Real-time RT-PCR),WB and immunohistochemical staining(IHC)were applied for 45 minutes after ischemia to detect the expression changes of mRNA and protein levels of PDCD4 at different time points after ischemia reperfusion,namely 24h,48h and 72h.Meanwhile,the dual-label method of immunofluorescence(IF)was used to detect the expression distribution of PDCD4 in renal proximal convoluted tubules,distal convoluted tubules and collecting tubes,as well as the expression change in renal ischemia reperfusion injury(IRI).1.1.3 The expression of PDCD4 in the kidney of patients with acute tubular necrosisIHC detected the expression level and distribution of PDCD4 in clinical renal biopsy specimens compared with paracancer tissues.1.1.4 The expression of PDCD4 in different ischemic injury models in vitroThree ischemic injury models were induced in NRK-52E.1.Glucose and oxygen deprivation experiment(OGD).The cells were cultured in a sugar-free and serum-free medium under hypoxia for 2h,and then the cells were cultured in a normal medium for 24h,48h and 72h for detection.2.Cells were cultured for 60min in serum-free medium containing anti-mycin A and 2-hypoxic glucose(AA/2-DG),and then changed to normal medium for 24h,48h and 72h.3.The cells were cultured for 12h in complete medium containing 50 ?m,200 ? m and 500 ?m cobalt chloride.WB was used to detect the expression of PDCD4 protein under the three in vitro ischemia reperfusion injury conditions.1.2 The role and mechanism of PDCD4 in renal ischemia reperfusioninjury1.2.1 The role of PDCD4 in renal ischemia reperfusion injuryWT male mice and PDCD4-/-knockout mice were randomly assigned to establish the acute renal ischemia reperfusion model.At different reperfusion time after45 min of ischemia using high performance liquid chromatography(HPLC)detect the contents of serum creatinine and automatic biochemical analyzer test serum urea nitrogen.Using Hematoxylin-eosin staining(HE staining),terminal deoxynucleotidyl transferase-mediated digoxigenindeoxyuridine nick-end labeling staining(TUNEL)to evaluate the role of PDCD4 in renal IRI.1.2.2 The mechanisms of PDCD4 in renal ischemia reperfusion injuryReal-time RT-PCR was performed to detect the mRNA changes of the representative inflammatory cytokines IL-1?,TNF-?,IL-6 and MCP-1 in the kidney tissues of the WT and PDCD4-/-mice control and model groups.CD68 labeled macrophages and Ly-6B labeled neutrophils were used to detect the distribution and changes of macrophages and neutrophils in kidney of WT and PDCD4-/-mice control and model groups.Part ii:PDCD4 induces renal ischemia reperfusion injury by regulating the Fgr-Notchl signaling pathway2.1 PDCD4 was involved in renal ischemia reperfusion injury through Fgr2.1.1 PDCD4 regulate renal ischemia reperfusion injury by Fgr in vivoMicroarray analysis technology was used to analyze the differentially expressed genes between WT and PDCD4-/-mice during renal ischemia/reperfusion injury,and the heat map of the differentially expressed genes during renal ischemia reperfusion injury was drawn.Real-time RT-PCR,WB and IHC were used to detect Fgr mRNA and protein levels in kidney tissues of WT and PDCD4-/-mice control and model group.2.1.2 PDCD4 regulate renal ischemia reperfusion injury by Fgr in vitroReal-time RT-PCR and WB were used to detect the changes of Fgr at different time points after OGD.The expression level of cytokines were detected by Real-time RT-PCR,flow cytometry and caspse3 activity were detected to determine whether PDCD4 influence inflammation and apoptosis in vitro.2.2 PDCD4 regulated the Notchl signaling pathway through Fgr2.2.1 PDCD4 regulated Notchl signaling pathway by Fgr in vivo and in vitro Real-time RT-PCR,WB and IHC were used to detect the mRNA and protein levels of Notch1 in kidney tissues of WT and PDCD4-/-mice control and model groups.In vitro assay detected the effect of Fgr on the expression level of Notchl in NRK-52E by WB.2.2.2 Renal overexpression of Fgr counteracted the protective effect of PDCD4 knockout on IRIThe WT and PDCD4-/-mice were randomly assigned to inject Fgr adeno-associated virus(AAV)labeled with GFP,namely AAV-Fgr-GFP.Renal ischemia reperfusion injury was established one month later.The samples were taken at 48h of reperfusion after 45min ischemia.HPLC was used to detect serum creatinine content.Automatic biochemical analyzer was used to detect serum urea nitrogen content.HE staining,TUNEL staining and other methods to determine whether overexpression of Fgr could counteract the protective effect of PDCD4 deficiency in acute ischemia reperfusion injury.Real-time RT-PCR detected the expression of cytokines and WB detected the expression of downstream gene Notch1.ResultPart ?:expression,distribution and role of PDCD4 in acute renal ischemia reperfusion injury1.1 The expression of PDCD4 was significantly increased in renal ischemia reperfusion injury1.1.1 Changes of PDCD4 expression in different organs and tissues of WT mice and renal cell linesWB results showed that PDCD4 was relatively abundant in kidney of WT mice,and PDCD4 was relatively more abundant in NRK-52E than in RMC,GENC and HPC,suggesting that PDCD4 may play an important role in kidney.1.1.2 The expression of PDCD4 dramatically increased after renal ischemia reperfusion injury in miceReal-time RT-PCR results showed that PDCD4 was significantly increased at 48h and 72h after ischemia reperfusion injury which was further confirmed by WB and IHC results.IF results showed that the expression of PDCD4 relatively increased in proximal convoluted tubules and collecting tubes in renal ischemia reperfusion injury.1.1.3 The expression of PDCD4 was significantly increased in the kidney of patients with acute tubular necrosis.IHC results showed PDCD4 was significantly increased in renal tissue of patients with acute tubular necrosis compared with adjacent renal carcinoma.1.1.4 The expression of PDCD4 was significantly increased in the hypoxia model in vitroWB results showed that PDCD4 protein was significantly increased in OGD,AA/2-DG and CoCl2 in vitro.1.2 The role and mechanism of PDCD4 in renal ischemia reperfusion injury1.2.1 PDCD4 deficiency significantly reduced renal ischemia reperfusion injuryThe PDCD4-/-mice were phenotypically normal and had no appreciable defects in renal morphology and function.However,PDCD4 deficiency significantly ameliorated renal IRI in mice,as evidenced by the reduction of serum creatinine and BUN(Figure 2b),improved morphologic injury,and reduced cell death as demonstrated by terminal deoxynucleotidyl transferase-mediated digoxigenindeoxyuridine nick-end labeling staining.1.2.2 PDCD4 deficiency alleviated acute renal ischemia reperfusion injury through inflammationIn addition,PDCD4 deficiency also significantly reduced inflammatory responses by decreasing the levels of proinflammatory mediators,and macrophage and neutrophil infiltration in the kidney from mice with IRI.Part ii:PDCD4 induces renal ischemia reperfusion injury by regulating the Fgr-notchl signaling pathway2.1PDCD4 induces renal ischemia reperfusion injury through Fgr2.1.1 In vivo experiments showed that PDCD4 regulated renal ischemia reperfusion injury by FgrUsing microarray analysis,we found that IRI-induced Fgr expression was significantly abolished by PDCD4 deficiency in mice,which was further confirmed by mRNA,Western blot,and immunohistochemical staining analyses.Together,these results indicate that PDCD4 positively mediated Fgr expression.2.1.2 In vitro OGD experiments showed that PDCD4 regulated the inflammation and apoptosis of renal tubular cells by FgrWe further found that overexpression of PDCD4 aggravated OGD-enhanced the production of pro-inflammatory mediators,as well as cell apoptosis accompanied by increased caspase-3 activity,which were counteracted by Fgr knockdown.2.2PDCD4 regulates the Notchl signaling pathway through Fgr2.2.1 In vivo and in vitro experiments showed that PDCD4 regulated Notch1 signaling pathway by FgrReal-time RT-PCR,WB and IHC results showed that the absence of PDCD4 significantly inhibited the expression of Notch1 induced by renal ischemia reperfusion injury.In vitro experiments showed that mRNA and protein expression levels of Notchl were increased under ODG conditions,but the expression of Notchl was significantly decreased under siRNA-Fgr.In vivo and in vitro experiments showed that PDCD4 regulated Notchl signaling pathway by Fgr.2.2.2 Renal overexpression of Fgr in kidney reverse the protective effect of PDCD4 deficiency in renal ischemia reperfusion injuryWB results showed the overexpression of Fgr in AAV-Fgr-GFP group compared with control group.The results of serum creatinine,urea nitrogen,HE staining,TUNEL staining and inflammatory factor detection showed that overexpression of Fgr could reverse the protective effect of PDCD4 deficiency in acute ischemia reperfusion injury.Fgr counteracted kidney injury in PDCD4-/-ischemic mice as evidenced by increased serum creatinine and BUN,deteriorated morphologic injury(Figure 6c-d),enhanced cell death as demonstrated by terminal deoxynucleotidyl transferase-mediated digoxigenindeoxyuridine nick-end labeling staining and increased inflammatory responses by augmenting the levels of proinflammatory mediators.Conclusion and innovation1.The role of PDCD4 in renal ischemia reperfusion injury was first described.PDCD4 was significantly induced in renal IRI.PDCD4 deficiency could alleviate renal IRI by decreasing inflammatory responses and the apoptosis of tubular epithelial cells.2.In this study,we first discovered that PDCD4 may regulate renal IRI through the Fgr-Notchl signaling pathway which provided important theoretical and experimental support for finding the targets to prevent and treat acute kidney injury.