Regulation Of Pericyte Metabolic Reprogramming Restricts The AKI To CKD Transition

Background:Acute kidney injury(AKI)is a common critical clinical state in which renal function declines rapidly in a short time.AKI is associated with high rates of hospitalization and mortality.Approximately 29%of hospitalized patients with AKI develop chronic kidney disease(CKD)and end-stage renal disease.Renal interstitial fibrosis is the hallmark of AKI to CKD progression4 and is characterized by increased myofibroblasts and excessive extracellular matrix deposition.However,current therapies targeting renal fibrosis are still lacking.The AKI to CKD transition has become a severe public health problem and has caused a substantial social and economic burden.Extensive studies have indicated that pericytes are the major source of scar-derived myofibroblasts in kidney fibrosis.Pericytes,source of mesenchymal cells,expressed platelet-derived growth factor receptor-β(PDGFR-β)and chondroitin sulfate proteoglycan 4(NG2)and melanoma cell adhesion molecule(CD146).Recent evidence suggests that inhibition of pericyte-myofibroblast transition(PMT)by genetic or pharmacological methods halts kidney fibrosis progression.Thus,delaying or preventing PMT might be a promising therapeutic strategy to prevent kidney fibrosis.Metabolic reprogramming,refers to the physiological or pathological condition,in order to meet the demand of power supply,the energy source has changed.Cell differentiation is often accompanied by changes in energy metabolism.Zhao et al.found that the homeostasis between Fatty acid oxidation(FAO)and glycolysis determines the fate change of fibroblasts into myofibroblasts for transdifferentiation.Activation of FAO and inhibition of glycolysis can reduce fibroblast transdifferentiation.In addition,AMP-activated protein kinase(AMPK)is an important sensor of cellular energy,which is involved in the regulation of glycolysis and fatty acid oxidation.At present,there are no studies on the metabolic changes in pericytes in CKD during AKI progression.Therefore,in this study,we first observed pericytes transdifferentiation and metabolic changes of pericytes during AKI to CKD progression,and then observed the effects of regulating metabolic reprogramming on pericyte transdifferentiation,and further explored the upstream molecular mechanisms.Objective:1.To observe PMT and metabolic change of pericytes during AKI to CKD transition.2.To investigate whether regulating metabolic reprogramming can inhibit PMT,and delay AKI to CKD transition.3.To further explore the underlying mechanism of regulating metabolic reprogramming during PMT.Methods:One:Animal experiments1.u IRI was used as the AKI-CKD animals model in this study,male C57BL/6 mice aged 6-8 weeks were randomly numbered.The u IRI model was established by unilateral clamping of the renal vessels for 30min,followed by removal of the contralateral kidney one day before sampling.The mice were sacrificed at 2,7,and 14 days after the operation,and blood and kidneys were collected.Blood urea nitrogen and creatinine were measured with Quanti Chrom Urea and Creatinine Assay kits.Periodic acid-Schiff(PAS)to observe renal injury,masson staining and sirius red staining were used to observe the deposition of collagen in the kidney,immunofluorescence was used to observe the expression ofα-SMA,Fibronectin,and Collagen I in the kidney.2.Observe the transdifferentiation and metabolic changes of pericytes:co-immunofluorescence was used to observe whether PDGFR-β~+pericytes,CD146~+pericytes and NG2~+pericytes transdifferentiate intoα-SMA~+myofibroblasts in the AKI to CKD progression.And to explore the mechanism of pericyte transdifferentiation,we sort pericytes by using PDGFR-βmagnetic beads at different time points after u IRI.Transcriptome sequencing was used to analyze the FAO gene(Peroxisome proliferator-activated receptor gamma coactivator-1α(PGC1α)and Carnitine palmitoyltransferase 1A(CPT1A),and glycolysis-related genes expression(Hexokinase 2(HK2),Pyruvate Kinase M 2(PKM2),Lactate Dehydrogenase A(LDHA)in PDGFR-β~+pericytes.q PCR and co-immunofluorescence were used to verify the expression of FAO and glycolysis-related genes in pericytes after u IRI.3.Administration drugs to regulate the metabolic changes of pericytes:PGC1α activator to activate FAO or HK2 inhibitor to inhibit glycolysis,and detect pericyte transdifferentiation and renal fibrosis.4.Treatment with AMPK activator,observe the changes of FAO and glycolysis in pericytes,pericyte transdifferentiation,and renal fibrosis.Two:Cell experiments:Because of the lack of cell lines for pericytes in the kidney,C3H/10T1/2 is commonly used to study the biological functions of renal pericytes,also known as pericytes.TGF-βis involved in regulating metabolic reprogramming,and TGF-βexpression and secreted significantly increased in the u IRI model,we used TGF-βtreated pericyte-like cells mimicking the vivo environment.1.TGF-βinduced pericytes transdifferentiation:Western blot and immunofluorescence detected the expressions ofα-SMA and Fibronectin after 5ng/m L TGF-βtreated for72 hours.2.The expressions of FAO and glycolysis-related genes were detected by q PCR and Western blot.Seahorse 96XFe detected the oxygen consumption rate and the extracellular acidification rate in pericyte-like cells.3.Administration of drugs to regulate the metabolic changes of pericytes:pericyte transdifferentiation was observed by administration of PGC1αactivator to activate FAO or HK2 inhibitor to inhibit glycolysis.4.Treatment with AMPK signaling pathway activator/inhibitor,observed the changes of FAO,glycolysis and transdifferentiation of pericytes.Results:One:animal experiments1.Co-immunofluorescence showed thatα-SMA was rarely expressed in the sham control group,and PDGFR-β~+α-SMA~+,CD146+α-SMA~+,NG2~+α-SMA~+began to increase at 2 days after u IRI,and increased markedly at 7 and 14 days after u IRI.2.GSEA analysis showed that mitochondrial fatty acid beta-oxidation was significantly down-regulated;q PCR and co-immunofluorescence showed that results showed that PPARgamma coactivator-1α(PGC1α),which are related to fatty acid metabolism,and their target genes carnitine palmitoyltransferase 1A(CPT1A)were expressed at markedly lower levels;Heatmap showed that compared with the Sham group,the expression of glycolysis related genes were significantly increased.q PCR and co-immunofluorescence showed pericytes increased HK2 expression along the AKI-CKD continuum.3.In the u IRI mice model,PGC1αactivator ZLN-005 upregulated PGC1αexpression and its downstream target gene CPT1A in pericytes,enhanced pericytes FAO,reduced lipid deposition;HK2 inhibitor 2-DG significantly reduced the expression level of HK2 and the enzymatic activity of HK2,resulting in decreased lactate production in pericytes;Activation of FAO or inhibition of glycolysis can reduced the expression of NG2~+α-SMA~+and PDGFR-β~+α-SMA~+and prevent the AKI to CKD progression.4.Mechanismly,AICAR activates PGC1αand CPT1A expression in pericytes,increases FAO levels in pericytes,inhibits the expression of HIF1αand HK2 in pericytes and reduces the glycolysis level of pericytes.AMPK promotes the metabolic switch from glycolysis to FAO and inhibits pericyte transdifferentiation.In addition,AICAR inhibits the pericytes transdifferentiation and reduces renal fibrosis.Two:Cell experiments1.TGF-βinduced the expression ofα-SMA and Fibronectin in the pericytes,suggesting that TGF-βsignificantly induces pericytes transdifferentiation.2.TGF-βinduced the down-regulation of PGC1αand CPT1A,impaired FAO level.TGF-βinduced the upregulation of glycolytic enzymes HK2,PKM2,and LDHA,increasing the glycolysis level of pericytes.3.PGC1αactivator ZLN-005 significantly reduced TGF-β-induced upregulation of PGC1αand CPT1A in m RNA and protein levels,enhanced pericytes FAO,and reduced TGF-β-induced lipid deposition in pericyte-like cells.HK2 inhibitor 2-DG significantly inhibited the TGF-β-induced increase in the m RNA and protein levels of HK2,reduced the extracellular acidification rate,and decreased glycolysis in pericyte-like cells.4.AMPK activator AICAR promoted the expression of PGC1α/CPT1A,and increased pericyte oxygen consumption rate,and ATP production,reducing TGF-β-induced lipid deposition.At the same time,AICAR significantly inhibited TGF-β-induced increase in HIF1α/HK2 expression and reduced the rate of glycolysis,and lactic acid production.In addition,AMPK inhibitor Compound C decreased the expression of PGC1α/CPT1A and increased in HIF1α/HK2 expression,Therefore,AMPK promoted pericyte glycolysis switch to FAO.Conclusions:1.Pericytes transdifferentiate into myofibroblasts in AKI-CKD transition.2.Decreased FAO and increased glycolysis control the fate of pericyte transdifferentiation during AKI-CKD progression.3.Enhancement of FAO or inhibiting glycolysis can effectively reduce pericyte transdifferentiation,and delay AKI to CKD transition.4.AMPK is an upstream molecule that promotes the metabolic switch from glycolysis to FAO.Specifically,AMPK enhances FAO by activating PGC1α/CPT1A signaling pathway and inhibits glycolysis by inhibiting HIF1α/HK2 signaling pathway.