Effects And Mechanisms Of Cold-induced RNA-binding Proteins On The Kidney During Deep Hypothermic Circulatory Arres

Background Deep hypothermic circulatory arrest(DHCA)is widely adopted in adult aortic arch surgeries and infant complex congenital heart surgeries.However,the hypothermic ischemia-reperfusion(I/R)environment during DHCA in cardiovascular surgeries is one of the main causes of postoperative acute kidney injury(AKI),for which there is still a lack of effective prevention and treatment measures.Previous studies showed that the expression of cold-inducible RNA-binding protein(CIRP)would be induced under the condition of hypoxia and hypothermia,and CIRP may exert protective effect on multiple organs.The purpose of this study was to investigate whether CIRP has a protective effect on kidney during hypothermic I/R and its potential mechanism.Methods This team established DHCA cardiac surgery model using adult male wild-type and Cirp knockout(Cirp-/-)rats to simulate clinical scenario.Rats were divided into Sham group,DHCA group and DHCA+Cirp-/-group with 5 in each group.Through RNA transcriptome and proteome sequencing analysis of kidney samples,the differences of gene expression in renal cells of wild-type and Cirp-/-rats after DHCA were compared,and the possible biological functions and molecular pathways were screened.Different biochemical and molecular biological techniques were used to detect and compare the perioperative physiological index,blood-gas analysis,postoperative renal function indicators,levels of AKI specific markers,renal pathological phenotype,renal cell ultrastructural changes,renal cell apoptosis,serum inflammatory factors,reactive oxygen species(ROS),related mRNA and protein expression and so forth.Results This study discovered that DHCA could significantly induce the expression of CIRP in rat kidney and promote the translocation of CIRP from nucleus to cytoplasm.After DHCA,the expression of prolyl hydroxylase 3(PHD3)in the kidney of Cirp-/-rats,the key regulator of hypoxia inducible factor-1α(HIF-1α),was significantly higher than that of wild-type rats,thus inhibiting the expression of HIF-1α(P<0.05).In addition,Cirp gene deletion resulted in increased ROS accumulation,significant activation of transforming growth factor-β1(TGF-β1)/p38 mitogen-activated protein kinase(MAPK)inflammatory pathway(P<0.05),higher levels of serum inflammatory factors,increased mitochondrial damage and cytochrome C release into the cytoplasm(P<0.05),increased expression of cytochrome C-apoptotic protease activating factor-1(Apaf-1)-cysteinyl aspartate specific proteinase(Caspase)9 in mitochondrial apoptosis pathway(P<0.05)and Fas-associated death domain protein(FADD)-Caspase 8 in death receptor apoptosis pathway(P<0.05),and Caspase 3,the joint target of the two apoptosis pathways,and Bcl-2-associated x protein(Bax)(P<0.05).Whereas,the expression of anti-apoptotic factor Bcl-2 in rat kidney decreased significantly after DHCA when Cirp gene was knockout(P<0.05).As a result,excessive apoptosis in Cirp-/-rats during DHCA aggravated the damage on structure and function of renal cells.Conclusion The results of Part I indicated that CIRP can regulate the expression of HIF-1α by inhibiting PHD3 during DHCA.Meanwhile,CIRP can protect renal function through alleviating renal I/R injury during deep hypothermic cardiac surgery by inhibiting inflammatory reaction and apoptosis.Background Under the stimulation of hypoxia and hypothermia,the expression of CIRP is prone to increase in many organs to help the body adapt to the adverse circumstances.Studies from our center have found that rat cardiac and cerebral tissue can secrete CIRP during hypothermic cardiopulmonary bypass(CPB)to alleviate I/R injury.The first part of this study proved that the rat kidney can also help resist oxidative stress inflammation and apoptosis in DHCA environment by secreting CIRP and regulating its downstream molecules.However,it remains unclear whether CIRP has similar expression characteristics and mechanism between human kidney cells and rat kidney.Besides,the effect of CIRP on PHD3/HIF-1α axis and the causal relationship between the activation of TGF-β1/p38 MAPK inflammatory pathway have not been thoroughly investigated.Therefore,we would discuss these issues in this section.Methods We established a deep hypothermic oxygen-glucose deprivation/reoxygenation(hOGD/R)model using human kidney tubular epithelial cell line(HK-2).Briefly,the HK-2 cells were treated in the condition of 18℃,1%O2,fetal bovine serum(FBS)-free and glucose-free medium for 6 h,and then cultured with 10%FBS complete medium at 37℃ 21%O2 for 30 min.HK-2 cells with CIRP or PHD3 gene knockdown were constructed by small interference RNA transfection,and siNC was used as negative control.The cells were divided into five groups including Control group,hOGD/R group,hOGD/R+siNC group,hOGD/R+siCIRP group and hOGD/R+siPHD3 group with three replications in each group.The expression of CIRP/PHD3/HIF-1α axis,inflammatory and apoptotic pathway protein,ROS level,inflammatory factor level,cell viability and apoptosis degree,mitochondrial membrane potential and energy metabolism were detected in each group and compared between groups.Results After the intervention of hOGD/R,the expression of CIRP in HK-2 cells increased significantly and translocated from the nucleus to the cytoplasm.Meanwhile,the viability of HK-2 cells decreased and significant apoptosis occurred(P<0.05).When siRNA was used to knock down CIRP gene,the expression of PHD3 protein was significantly increased(P<0.05),which led to a large amount of degradation of HIF-1αprotein.However,when the PHD3 gene was knocked down,HIF-1α was stably expressed in HK-2 cells,and the degree of apoptosis was significantly alleviated under the condition of hOGD/R(P<0.05).siNC had no significant effect on cell viability and CIRP expression(P>0.05).In hOGD/R environment,CIRP gene knockdown indirectly inhibited the expression of HIF-la through upregulating PHD3 gene,resulting in the limited expression of thioredoxin 1(Trx-1)and replication protein A2(RPA2),which resulted in a significant increase of intracellular ROS and damaged DNA accumulation,respectively(P<0.05).Whereas after the inhibition towards HIF-1α was eliminated by knocking down PHD3 gene,the expression of Trx-1 and RPA2 in HK-2 cells was significantly increased,and the accumulation of ROS was significantly alleviated compared with untransfected cells(P<0.05).Additionally,in consistent with the results in rat kidney,HIF-la degradation caused by additional CIRP gene inhibition induced significant activation of TGF-β1/p38 MAPK inflammatory pathway and increased production of inflammatory cytokines(P<0.05),decreased mitochondrial membrane potential(P<0.05),decreased ATP production and increased extramitochondrial cytochrome C release(P<0.05)compared with single intervention of hypothermic I/R.In comparison with hOGD/R group,the expressions of the key targets of mitochondrial apoptosis pathway Apaf-1 and Caspase 9,and the key proteins of death receptor apoptosis pathway FADD and Caspase 8,and the common target of the two apoptosis pathways Caspase 3 were all significantly increased in hOGD/R+siCIRP group(P<0.05).On the contrary,after the expression of HIF-1α was stabilized by siPHD3 transfection,the above inflammatory and apoptotic pathway targets were significantly inhibited(P<0.05),the production of ATP was significantly promoted and the percentage of apoptotic cells was significantly decreased(P<0.05).Conclusion These results suggested that under the condition of deep hypothermia I/R,renal cells can regulate the expression of PHD3/HIF-1α axis by secreting CIRP to promote the clearance of ROS and repair of damaged DNA,reduce oxidative stress inflammation and inhibit the activation of apoptosis pathway,so as to avoid excessive apoptosis and maintain energy metabolism of renal cells to help the body adapt to hypothermia and hypoxia stress.These mechanisms exist in both human and rat kidney cells.Background HIF-1α is the main regulator of cellular anti-hypoxia defense mechanism,which can widely participate in the pathophysiological processes including metabolism in I/R injury,promotion of erythropoiesis,anti-inflammation and anti-apoptosis.In normoxia,the expression of HIF-1α is regulated by PHD hydroxylation and degradation.Drugs targeting HIF-1α hydroxylase have been used in the treatment of several hypoxic diseases and chronic nephrotic anemia.Among them,a new oral PHD specific inhibitor enarodustat(JTZ-951)has been found to have a protective effect on kidney in different animal models of renal injury.The purpose of this part was to explore the effect of exogenous administration of enarodustat on renal deep hypothermic I/R injury using rat DHCA model and HK-2 cell hOGD/R model on the basis of the first two parts of this study.Methods HK-2 cells transfected with siCIRP or siNC were treated with enarodustat or dimethyl sulfoxide(DMSO)as control.The hOGD/R model was established according to the second part of the study.HK-2 cells were divided into hOGD/R group,hOGD/R+siCIRP group,hOGD/R+enarodustat group and hOGD/R+siCIRP+enarodustat group.The expression of CIRP/PHD3/HIF-la axis,apoptotic pathway protein and apoptotic percentage were detected and compared between groups.After intragastric administration of enarodustat or solvent control for 5 days before operation,adult male wild-type or Cirp-/-rats were used to establish DHCA cardiac surgery model.The rats were divided into DHCA group,DHCA+Cirp-/-group and DHCA+Cir-/-+enarodustat group.The expression of CIRP/PHD3/HIF-1α axis,apoptotic pathway protein,renal pathological phenotype and renal function were detected and compared between groups.Results Enarodustat pretreatment had no significant effect on the expression of CIRP in HK-2 cells.Compared with single hOGD/R intervention,the expression of PHD3 protein in HK-2 cells undergoing the administration of hOGD/R+enarodustat was significantly inhibited(P<0.05),while the expression of HIF-1α protein was significantly increased(P<0.05),the expression of Bax was significantly decreased(P<0.05),the expression of Bcl-2 was significantly increased(P<0.05),and the activation of Caspase 9,FADD,Caspase 8 and Caspase 3 was remarkably inhibited(P<0.05).Additionally,the percentage of apoptosis was significantly decreased in the detection of flow cytometry(P<0.05).After pretreatment of enarodustat,the upregulation of Bax,Caspase 9,FADD,Caspase 8 and Caspase 3 and the increase of apoptosis induced by siCIRP were significantly inhibited(P<0.05).Enarodustat intervention partially reversed the apoptosis trend of HK-2 cells after hOGD/R induced by CIRP silencing through inhibiting the expression of PHD3 and stabilizing the expression of HIF-1α.Consistent with the results in vitro,after intragastric administration of enarodustat,the expression of PHD3 in renal tissue was inhibited and the expression of HIF-1α was significantly increased(P<0.05).The trend of renal pathological injury in DHCA aggravated by Cirp-/-was significantly alleviated(P<0.05)that the score of renal injury in DHCA+Cirp-/-+enarodustat group was significantly lower than that in DHCA+Cirp-/-group(P<0.05),but there was no significant difference between DHCA+Cirp-/-+enarodustat group and DHCA group(P>0.05).Compared with DHCA+Cirp-/-group,the activation of Bax,Caspase-9,FADD,Caspase-8 and Caspase-3 was significantly inhibited and the expression of anti-apoptotic Bcl-2 protein was significantly increased after the administration of additional enarodustat(P<0.05).The levels of SCr,BUN and serum uric acid in DHCA+Cirp-/group were significantly higher than those in DHCA group(P<0.05).After DHCA operation,the levels of SCr and BUN in DHCA+Cirp-/-+enarodustat group were significantly lower than those in DHCA+Cirp-/-group(P<0.05),but there was no significant difference in the levels of renal function between DHCA+Cirp-/-+enarodustat group and DHCA group(P>0.05),which suggested that enarodustat can significantly reverse the renal function damage induced by Cirp knockout.Conclusion Enarodustat can partially counteract the renal deep hypothermic I/R injury aggravated by the deletion of CIRP expression through inhibiting excessive apoptosis,which confirms that CIRP can alleviate AKI after DHCA cardiac surgery through PHD3/HIF-1α axis.The novel inhibitor of HIF-1α hydroxylase may serve as a potential intervention for the prevention and treatment for AKI after deep hypothermic I/R.