Mechanism Of Metabolic Reprogramming And PI3K-Akt-mTOR Pathway In Pericyte-myofibroblast Transition

Background:The poor regeneration or repair of acute renal injury can contribute to renal fibrosis,which gradually progresses to chronic kidney disease and end-stage renal disease.The histological feature of renal fibrosis is the massive deposition of extracellular matrix in renal interstitium,such as collagen and elastin.The extracellular matrix is mainly synthesized and secreted by myofibroblasts.Recent studies have found that pericytes are the main source of renal myofibroblasts.Under the stimulation of ischemia,hypoxia or inflammatory factors,renal tubular epithelial cells and vascular endothelial cells can secret transforming growth factor ?1(TGF?3)and induce pericyte-myofibroblast transition.Metabolic reprogramming was previously supposed as the conversion of the main metabolic phenotype from oxidative phosphorylation to glycolysis,also known as the Warburg effect.However,with the deepening of research,metabolic reprogramming is no longer limited to changes in the balance of glycolysis and oxidative phosphorylation,but extends to changes in the metabolism of virous nutrients such as fatty acids,amino acids and glutamine.Studies have found that metabolic reprogramming is closedly related to the transdifferentiation process of neurons,podocytes,adipose precursor cells,and so on.At present,there are few studies on the metabolism of renal pericytes,and the mechanism of metabolic reprogramming in pericyte-myofibroblast transition are still unclear.Objectives:To analyze the main transcriptomic changes in pericyte-myofibroblast transition,and to verify the significant changes in cell proliferation and cell metabolism pathways.Using a model of pericyte-myofibroblast transition in vitro to observe the specific changes of glycolysis and oxidative phosphorylation,and to in-depth study the mechanism of PI3K-Akt-mTOR pathway in pericyte-myofibroblast transition.Thus,providing a new theoretical basis for the intervention in renal fibrosis.Methods:1.Compared the gene expression profilies of pericytes and pericyte-derived myofibroblasts through bioinformatics analysis,and screened out differentially expressed genes(DEGs)related to pericyte-myofibroblast transition.The molecular pathways of DEGs were identified through KEGG(Kyoto encyclopedia of genes and genomes)enrichment.Then,we studied the significant changes in cell proliferation and metabolic pathways in vivo and in vitro.2.Studied the changes of cell proliferation pathway during pericyte-myofibroblast transition in vivo and in vitro.(1)In vivo experiment:The acute kidney injury to chronic kidney disease(AKI-CKD)model was established through unilateral renal ischemia-reperfusion injury in C57BL/6J mice.The mice were sacrificed at 2 days,7 days,14 days and 21 days after the surgery,and blood serum and kidney samples were collected.Serum creatinine and blood urea nitrogen were detected,and Masson staining was used to assess the levels of collagen deposition in renal interstitium.Immunohistochemical staining was used to detect the expression of platelet-derived growth factor receptor ?(PDGFR?),thus to observe the proliferation of pericytes.(2)In vitro experiments:Magnetic bead sorting(PDGFR?-positive)was used to extract primary pericytes,and the pericytes were identified by immunofluorescence staining.We used 5ng/mL TGF?1 to stimulate pericytes and induce the in vitro model of pericyte-myofibroblast transition.Used the immunofluorescence staining,Western Blot and RT-qPCR to detect the expression changes of alpha-smooth muscle actin(?-SMA),vimentin and desmin to evaluate the pericyte transdifferentition.RT-qPCR was used to detect the expression of genes related to cell proliferation.3.Used the in vitro model of pericyte-myofibroblast transition to observe the changes in the metabolic pathways:The Seahorse real-time ATP rate test kit was used to clarify the metabolic phenotype of pericytes.Relative quantitatification of metabolites were examined using cell-targeted metabolites detection.The change of glycolysis and oxidative phosphorylation levels during pericyte-myofibroblast transition was detected by the Seahorse Cell Energy Metabolism Analyzer.The expression levels of metabolic enzymes and other proteins were examined by Western Blot and RT-qPCR.The glucose level in the culture medium was detected by the Glucose and Sucrose Colorimetric/Fluorometric Assay Kit.The membrane potential of mitochondria was evaluated,and we observed the mitochondria by electron microscopy.4.We inhibited hexokinase using 2-DG and reduced the glycolysis level in pericyte-myofibroblast transition model in vitro.Then we evaluated the transition of pericyte by detecting the expression of ?-SMA,vimentin and desmin.The activation of PI3K-Akt-mTOR pathway during the pericyte-myofibroblast transition was detected through Western Blot.We used LY294002 and Rapamycin to inhibit PI3 K-Akt-mT OR pathway,and detected the glycolysis levels by Seahorse Glycolysis Pressure Kit.Using immunofluorescence,Western Blot and other methods to evaluated the expression of glycolysis enzymes and pericyte transition.Results:1.Metabolic pathways and cell proliferation changed significantly in the process of pericyte-myofibroblast transition.Through bioinformatics analysis,a total of 1485 DEGs(including 884 up-regulated genes and 601 down-regulated genes)were identified by comparing gene expression profiling of pericytes and myofibroblasts.Through the KEGG pathway enrichment,we found 58 pathways,including 14 pathways related to metabolism(metabolic pathway,carbon metabolism,PI3K-Akt signaling pathway,etc.)and 6 pathways related to cell proliferation(cell cycle and p53 signaling pathway,etc.)2.The activation of cell proliferation during the pericyte-myofibroblast transition in vivo and in vitro.We successfully established AKI-CKD model in mice:the renal function deteriorated rapidly on the 2nd day after the operation,and the renal function improved on the 7th day,14th and 21stday after the operation,but it was still higher than the sham operation group in those days.Masson staining showed that the renal fibrosis began to appeared on the 7th day after surgery,and large amounts of collagen were deposited in the renal interstitium after the 14th day.As the fibrosis worsened,the number of PDGFR?-positive pericytes also increased significantly.Successfully purified pericytes by magnetic beads sorting:cells were positive for PDGFR?,CD73,and negative for CD31,?-SMA,and E-cadherin.The positive rates of PDGFR? and CD73 were 85.7%and 75.3%,respectively.Stimulation with 5ng/mL TGF?1 for 48 hours could significantly induce the in vitro model of pericyte-myofibroblast transition:the expression of ?-SMA increase at 24h after sitimulation,and the expressions of ?-SMA,vimentin and desmin increase increased significantly at 48h.Compared with the control group,the expression of CyclinE1,CDK1,CDK2,CDK4,Ki67 and other cell proliferation-related genes increased significantly in the TGF?1-48h group.3.Both glycolysis and oxidative phosphorylation increased during the pericyte-myofibroblast transition.The ATP produced by pericyte mitochondrial oxidative phosphorylation is 2 to 7 times that produced by glycolysis under normal circumstances.Compared with the control group,31 metabolites increased significantly in the TGF?1-48h group.These metabolites were mainly concentrated in the pathways of carbon metabolism,glycolysis/gluconeogenesis,oxidative phosphorylation,etc.Compared with the control group,the expression of glucose transporter was increased and the glucose in the culture medium was dcreased in the TGF?1-48h group.The level of glycolysis,the expression of glycolytic enzymes and glycolytic metabolites also increased significantly.The expression of pyruvate dehydrogenase was higher in the TGF?1-48h group.Compared with the control group,the transcription level of mitochondrial transcription factor A increased,while the transcription level of motility-related protein 1 decreased in the TGF?1-48h group.The proportion of rod-shaped mitochondria increased,the mitochondrial membrane potential increased,and the level of oxidative phosphorylation increased in the TGF?1-48h group.There were a few differential metabolites in the the tricarboxylic acid cycle between two groups,However,when compared the ratio of glycolytic reserve and spare respiratory capacity between the two groups,we found that the the ratio of TGF?1-48h group was significantly higher than that of the control group.4.Inhibiting glycolysis reduced pericyte-myofibroblast transition.After inhibiting the activity of hexokinase by 2-DG,the glycolysis level in the model of pericyte-myofibroblast transition model was significantly reduced,and the expression of glycolysis enzymes downstream was also reduced.Compared with the control group,the expression levels of ?-SMA,vimentin and desmin in the 2-DG intervention group were significantly reduced.5.Reduced glycolysis and the pericyte-myofibroblast transition by inhibiting PI3K-Akt-mTOR pathway.Western Blot results suggested that the PI3K-Akt-mTOR pathway was activated during pericyte-myofibroblast transition,specifically manifested as the increased phosphorylation levels of PI3K,Akt and mTOR.When inhibited the PI3K-Akt-mTOR pathway using LY294002 or Rapamycin,the level of glycolysis was reduced,and the expression level of hexokinase 2 was also decreased.Meanwhile,the expression of ?-SMA,vimentin and desmin was significantly reduced after inhibiting the PI3K-Akt-mTOR pathway.Conclusions:1.Metabolic pathways and cell proliferation pathways are significantly changed during the pericyte-myofibroblast transition.2.Renal pericytes mainly rely on oxidative phosphorylation to produce ATP.Both the glycolysis and oxidative phosphorylation levels were increased during the pericyte-myofibroblast transition.3.Reducing the level of glycolysis can inhibit the pericyte-myofibroblast transition.4.The PI3K-Akt-mTOR pathway can regulate pericyte-myofibroblast transition by mediating glycolysis.