The Mechanism Of PHF14 Regulating Glucose Metabolism Switch In Promoting Renal Tubular Epithelial Cells Repair After Acute Kidney Injury

Objective:This study aimed to explore the role of PHF14,a newly identified transcriptional regulatory protein,of which expression is regulated by hypoxia-inducible factor(HIF)-1α,on the regulation of glucose metabolism in renal tubular epithelial cells and renal repair after acute kidney injury induced by ischemia-reperfusion and related mechanisms.Methods:1.The IRI-induced-AKI mouse model and the control sham-operated mouse model were established by using 8-12-week-old male wild-type mice C57BL/6 by bilateral renal pedicle clipping for 25 minutes,and the mice were sacrificed at the corresponding time points after modeling,Then,serum samples were collected to detect serum urea nitrogen,kidney tissues were obtained for HE staining,and Western blot was used to detect the expression changes of HIF-1α,PHF14,and PKM2 in the process of AKI.2.The PHF14 gene in mice was inducibly knocked out using tamoxifen.PHF14-knockout mouse genotype: PHF14 flox/flox Cre+;control mouse genotype: PHF14 flox/flox Cre-,8-to 12-week-old male mice were subjected to 5 consecutive intraperitoneal injections of tamoxifen to induce knockout of PHF14.Experiments were performed to confirm whether the mouse PHF14 gene was knocked out.The IRI-induced-AKI mouse models constructed by clamping bilateral renal pedicles for 25 minutes and the control sham-operated mouse model were established and sacrificed 24 h after modeling,the serum samples were collected for the detection of urea nitrogen and NGAL,kidney tissue was obtained for the detection of lactic acid,HE staining and immunofluorescence were performed to detect PHF14,M2 pyruvate kinase(PKM2),c-Myc expression,Western blot and Real-time PCR were performed to detect the expression of HIF-1α,PHF14,c-Myc,PKM2.In addition,mice were sacrificed 28 days after modeling,and kidney tissue was obtained for Western blot to detect the expression of PHF14,Fn,α-sma and Masson staining.3.Utilizing the NCBI-GEO database to obtain the gene expression profile after silencing PHF14,and analyzing the differences of the related genes to explore the influence of PHF14 upon the expression of the key enzymes and enzymes that promote glycolysis,and analyzing the down-regulated genes and the regulatory mechanisms that may be related to them.PHF14-knockout NRK-52 E cells and Cas9 NRK-52 E cells as control constructed by the Crispr-cas9 system.After hypoxia treatment for 24 h with hypoxia culture bag,the morphological changes of cells were observed and the supernatant of medium was collected to determine glycolysis products(lactic acid),Western Blot were performed to detect the expression of HIF-1α,PHF14,c-Myc,and PKM2.Results:1.The expressions of HIF-1α,PHF14 and PKM2 were time-dependent during AKI in mice after bilateral renal clipping for 25 minutes.The serum urea nitrogen concentration in the tested mice surged at first and then decreased,and reached a peak at 24 hours after reperfusion.Compared with sham-operated mice,the renal tissue damage of the mice in model group was more obvious.2.After 25 minutes of ischemia and then 24 hours of reperfusion in bilateral kidneys,the serum urea nitrogen,serum NGAL and renal tissue lactate content of the model mice were significantly increased compared with those of the sham-operated mice.Compared with the control mice,the degree of kidney injury and the elevated levels of serum urea nitrogen and serum NGAL were more significant in the control mice,and the expressions of PKM2 and c-Myc and the determination of lactate in the kidney tissue were significantly lower than those in the control mice.After 25 minutes of bilateral renal ischemia and reperfusion for 28 days,the degree of renal tissue fibrosis in PHF14 knockout mice was increased comparing with that of control mice,and the expression levels of Fn and α-sma were significantly increased.3.The differentially expressed gene analysis of the gene expression profiles of the cell lines that silenced PHF14 revealed that the expression of key enzymes in glycolysis(PKM2,etc.),pyruvate dehydrogenase kinase(PDK),which inhibits oxidative phosphorylation and promotes glycolysis,and c-Myc were down-regulated.The enrichment analysis of KEGG signaling pathway was performed on the differentially expressed genes,and it was found that the down-regulated genes were enriched in the Wnt signaling pathway.After hypoxia treatment for 24 hours,the expression levels of PHF14,c-Myc,and PKM2 in PHF14-knockout NRK-52 E cells and the level of lactate determination in the culture medium were significantly decreased compared with the control Cas9 cells.Conclusions:In vivo and in vitro experiments proved that PHF14 could up-regulate the level of glycolysis of renal tubular epithelial cells under hypoxic conditions to promote renal tubular repair.Ischemia and hypoxia of renal tissue,in the renal tubular epithelial cells,which increased the expression level of HIF-1α,further upregulated the expression level of PHF14.The up-regulated PHF14 further regulated the expression of c-Myc,which promoted the expression of PKM2 and then upgraded the level of glycolysis and improved energy supply of cells and promoted the repair of renal tubular epithelial cells and reduced the chronic transformation rate of AKI.