The Mechanism Of Adverse Restoration Of Acute Kidney Injury Mediated Through HIF-1α/p66Shc Pathway By Ischemia Reperfusion Injury

Background:Acute kidney injury(AKI)is a common clinical syndrome,and ischemia reperfusion injury(IRI)is a common clinical cause of AKI.In organisms,more than 90% of oxygen molecules are consumed in mitochondria.On the one hand,the process participates in the production of ATP to maintain cell physiological functions,while on the other hand,oxygen free radicals are produced and cause cell damage and diseases.Recent studies have shown that mitochondrial dysfunction,oxidative stress,autophagy,inflammation,etc.are closely related to the occurrence and development of AKI.After the occurrence of AKI in the kidney,the mitochondrial damage of renal tubular epithelial cells leads to adverse restoration and progressive fibrosis.The adaptor protein p66 shc plays an important role in mitochondrial oxidative stress damage and cell apoptosis,mediating mitochondrial dysfunction in renal proximal tubular epithelial cells.In addition,our previous studies have found that p66 Shc is highly expressed in the kidney tissue of IRI-induced rats with AKI.It is speculated that p66 shc may play a role in AKI-induced kidney injury,but the specific mechanism remains unclear.This article aims to clarify a possible mechanism in which ischemia-hypoxia damage involves in adverse restoration and progressive fibrosis by causing renal tubular epithelial cell mitochondrial damage,and then leading to tubule apoptosis and increased expression of fibrotic factors.The research discusses the function of HIF-1α/p66 Shc pathway in acute kidney injury and provides a theoretical basis for the clinical search for corresponding prevention and treatment methods.Methods:In vitro experiments,HK-2 cells were plasmid transfected in normoxia and acute hypoxia,and the expression of p66 Shc,RCAN1 and related factors were detected by WB,RT-q PCR and ELISA methods.In vivo experiments,lentivirus vectors carrying p66Shc-si RNA were intraperitoneally injected into sham-operated and IRI rats.The WB and immunohistochemistrical tests of kidney tissues of rats were performed to detect the expression of p66 Shc,RCAN1 and its downstream renal injury-related factors.Statistical method:Spss26.0 was used for statistical analysis,the measurement data was expressed as Mean±SD.The t test was used when the variances between different groups were homogeneous and in line with the normal division,otherwise,the nonparametric rank sum test was used.One-way analysis of variance(ANOVA)was used for comparison of more than three groups,SNK-Q or Tukey test was used when pair comparison was needed,Kruskal-Wallis test was used for comparison of variances,and Dunn’s Post Hoc test was conducted at the same time.Chi-square test or Fisher exact test was used to compare the counting data.P<0.05 indicated a statistically significant difference.Results:1.Both vivo and vitro experiments confirmed that compared with HK-2 cells and rats in the control group,HK-2 cells in acute hypoxia and rats with ischemia reperfusion injury shows significant difference(p< 0.05)in terms of p66 Shc and its phosphorylated p Ser36 level;the expression of mitochondrial damage factor Fis1 increased significantly,and the expression of Mfn1 decreased significantly(p<0.05);the expression of renal tubular damage factor Kim1 and NAGL increased significantly(p<0.05);the expression of apoptosis Caspase3 and Bax increased significantly,while the expression of Bcl-2 decreased significantly(p<0.05);the expression of fibrosis factors Collagen I,Collagen IV,TGF-β,and ɑ-SMA increased significantly(p<0.05);cellular inflammatory factors IL-6,TNF-α and MCP-1 expression increased significantly(p<0.05).The vivo experiment found that ischemia reperfusion injury induced renal tubule dilation.The tubule cells swelled and their striated borders disappeared.The glomeruli became sclerotic,and protein casts appeared.2.Vitro experiments have shown that under normoxic conditions,HIF1-ɑ induced an increase in the level of p66 Shc protein and phosphorylation,which further increased the expression of kidney injury factors Kim-1 and NAGL(p<0.05),the expression level of mitochondrial injury factor Fis1 increased significantly,and the expression level of Mfn1 decreased significantly(p<0.05).Under hypoxic conditions,compared with the control group,HIF1-α knockdown resulted in a decrease of expression and phosphorylation level of p66 Shc,a significant decrease in the expression of mitochondrial damage factor Fis1,a significant increase in the expression of Mfn1,and a significant decrease in the expression of renal damage factors(p<0.05).Further dual-luciferase reporter assay confirmed that HIF1-ɑ has a direct transcriptional regulation effect on p66 Shc.3.Vitro experiments proved that under normoxic conditions,overexpression of p66 Shc promoted a significant increase in RCAN1 expression,an increase in the expression of mitochondrial damage factor Fis1,and a decrease in Mfn1 expression,accompanied by an increase in the expressions of kidney damage factors Kim1 and NAGL(p<0.05).In hypoxic environment,p66 Shc silence plays a role in kidney injury protection by reducing the expression of RCAN1.The experimental results showed that the renal injury factor was significantly increased(p<0.05)in the group with RCAN1 non-intervention by p66 Shc overexpression,renal injury factors Fis1,Mfn1,mitochondrial injury factors Kim1 and NAGL did not show significant changes in their expression,suggesting that RCAN1 plays a role in the participation of overexpressed p66 Shc in kidney injury by damaging mitochondria and causing cell apoptosis.4.Vivo experiments proved that compared with the control group,the p66 Shc deficient mice showed a decrease in the expression of RCAN1,a decrease in the expression of mitochondrial damage factor Fis1,an increase in Mfn1 expression,and a decrease in the expression of kidney injury factors Kim1 and NAGL.The inflammatory factor and the level of its fibrosis factors also decreased significantly(p<0.05).The degree of renal fibrosis was improved,inflammatory cell infiltration reduced,and cell apoptosis also reduced.Conclusion:In kidney injury caused by ischemia reperfusion injury,the expression of HIF1-αincreases,and p66 shc and its ser36 phosphorylation level increases.HIF1-ɑ can also participate in the pathogenesis of kidney injury by directly regulating p66 Shc.P66shc mediates its effect on kidney injury through RCAN1.The reduce of p66 shc in IRI mice can significantly improve mitochondrial damage and kidney damage.