| ObjectiveFibrosis is a key factor which results in the reduction of survival time of renal allograft.At present,effective therapy strategy for the intervention of fibrosis in renal allografts remain lacking.Our study focused on the function of fatty acid oxidation(FAO)pathways.We attempted to determine whether abnormal expression of the ratelimiting enzymes of FAO pathways affect the progression of fibrosis in renal allograft,ascertain molecular mechanism,and verify the inhibition of fibrosis through targeting abnormal lipid metabolism in renal transplantation model of rats.Based on our results,we will provide theoretical foundation and experimental evidence for the therapy of fibrosis in renal allograft in clinic.Methods1.To determine the association between abnormal lipid metabolism and fibrosis of renal allografts(1)Body mass index(BMI),triacylglycerol(TG),total cholesterol(TC),lowdensity lipoprotein cholesterol(LDL-C),and high-density lipoprotein cholesterol(HDL-C)of kidney transplant patients were detected.These results were further analyzed correlations with fibrosis,total interstitial inflammation,tubulitis,glomerulitis,C4 d deposition,vascular fibrous intimal thickening,and arteriolar hyaline thickening.(2)Differential genes were detected among each grade of chronic allograft nephropathy(CAN)in the online database;Analyzed the enrichment pathways of differential genes compared serious fibrosis group(CAN Ⅲ)with control group.2.To investigate the influence of rate-limiting enzymes in FAO pathways on fibrosis of renal allograft(1)Analyzed the association between carnitine palmitoyl transferase(CPT1),a rate-limiting enzyme in mitochondrial FAO pathway,and progression of fibrosis of renal allograft through immunohistochemistry(IHC),Masson stain,and the online X database results.(2)Analyzed the association between Acyl-Co A Oxidase 1(ACOX1),a ratelimiting enzyme in peroxisomal FAO pathway,and progression of fibrosis of renal allograft through immunohistochemistry(IHC),Masson stain,and the online database results.(3)Knocked down ACOX1 in HK-2 cell,and conducted RNA-sequence(RNAseq)detection in shACOX1-HK-2 and sh Ctrl-HK-2 cells.Differential genes were carried out Deis Ge Net analysis.3.To define ACOX1 deficiency facilitates fibrosis of renal allografts through inducing epithelial-mesenchymal transition(1)Analyzed enrichment pathways related to EMT in renal transplant database.(2)Ascertained that EMT facilitates fibrosis of renal allografts in tissue samples and online database.(3)Spatial transcriptome detection confirmed EMT exists in ACOX1-deficient cluster.(4)Determined correlation between EMT and ACOX1 and CPT1 in transplanted kidney biopsy samples through IHC stain.(5)Detected phenotypic alteration related to EMT in shACOX1-HK-2 cells and rat renal tubular epithelial cell NRK and human embryonic kidney cells HEK-293 cells exposed in ACOX1 inhibitor.(6)Kidney-specific ACOX1 heterozygous deletion mice(ACOX1-KD)were constructed to detect the expression level of ACOX1 and the localization of EMT molecules with ACOX1 in the kidney;(7)Detected the expression levels of EMT molecules in mice treated with ACOX1 inhibitor.4.To define ACOX1 deficiency facilitates fibrosis of renal allografts through inducing extracellular matrix reorganization(1)Analyzed enrichment pathways related to ECM in renal transplant database.(2)Spatial transcriptome detection confirmed ECM organization exists in ACOX1-deficient cluster.(3)Analyzed enrichment pathways related to ECM in shACOX1-HK-2 cells.(4)Analyzed association between ACOX1 deficiency induced upregulation of ECM genes and fibrosis progress in renal allograft.(5)Detection of ECM molecular expression levels in a rat kidney transplant model;(6)Correlation between ACOX1 and ECM analyzed expression was verified in online database human tissue samples;(7)Validation of the promotion of ECM molecule expression after silencing ACOX1 or ACOX1 inhibitor treatment in in vitro cellular experiments;(8)Detection of the expression levels of ECM molecules in the kidney in ACOX1inhibitor-treated mice and ACOX1-KD mice.5.Determine the inhibitory effect of NFκB activation on ACOX1 expression in transplanted kidneys.(1)To validate the effects of hypoxia and oxidative stress on ACOX1 expression in the online database,cellular experiments,and transplanted kidney samples;(2)To verify the inhibitory effect of NFκB activation on ACOX1 expression in the online database,transplanted kidney tissue samples,rat kidney transplantation model,lipopolysaccharide-treated mouse model,and in vitro cell model;(3)Screening for epimerase modification mediating NFκB regulation of ACOX1 by correlation analysis in an online database;(4)Confirmation of DNMT1 expression and its relationship to EMT in human transplanted kidney samples and rat kidney transplant model samples;(5)Validate NFκB inhibition of ACOX1 expression through upregulation of DNMT1 levels in in vitro cellular assays and in a lipopolysaccharide-treated mouse model;6.Determination of the role of lipid accumulation in mediating EMT by knockdown of ACOX1(1)Detection of lipid droplet-encapsulated protein PLIN1 in human transplanted kidney samples in relation to fibrosis progression in transplanted kidneys and XII localization to ACOX1 expression;(2)Nile Red detection of lipid accumulation levels in shACOX1 HK-2 cells;(3)ABCD1 knockdown in HK-2 cells and correlation between lipid accumulation,EMT phenotype,ECM gene expression and online database analysis of ABCD1 and progression of transplanted kidney fibrosis;(4)Cultured HK-2 cells in medium supplemented with oleic acid and palmitic acid to detect the expression of EMT marker molecules and ECM remodeling genes.7.Determine the role of altered lipid fractions in mediating ACOX1-induced ECM knockdown.(1)The non-targeted lipidome was examined for changes in lipid fractions induced by ACOX1 knockdown;(2)Culture medium was supplemented with linoleic acid,linolenic acid and DHA to detect ECM-related molecular expression;(3)ACOX1 inhibitor-treated mice and kidney-transplanted rats were given PUFAs by gavage to detect renal function,fibrosis level,and ECM molecule expression levels.8.Determination of endoplasmic reticulum stress-mediated ECM remodeling induced by reduction of PUFAs(1)Immunoblotting and flow cytometry to determine the effect of ACOX1 inhibition on mitochondrial function;(2)On-line database analysis of the correlation between DECR1,the rate-limiting enzyme for oxidation of PUFAs in mitochondria,and the expression of EMT and ECM molecules;(3)The upregulation of DECR1 by ACOX1 silencing was determined in in vitro cellular assays;(4)Silencing of DECR1 in shACOX1 cells to determine the expression levels of ECM molecules;(5)Analysis of the correlation between DECR1 and the expression of endoplasmic reticulum stress molecules based on RNA-seq results and online data;(6)In vitro cellular experiments verified that ACOX1 knockdown or ACOX1 inhibitor treatment induced ER stress;(7)Endoplasmic reticulum stress inhibitor treatment of shACOX1 cells and detection of ECM molecule expression levels;(8)Endoplasmic reticulum stress molecule expression was detected after PUFA supplementation in shACOX1 cells;9.To determine the role of targeting ACOX1 signaling in delaying transplant-related kidney fibrosis.(1)Validate the delaying effect of targeting NFκB and DNMT1 on transplanted kidney fibrosis in a rat kidney transplantation model.(2)Treatment of a rat model with the peroxisome proliferator-activated receptor α(PPARα)activator fenofibrate and Determine ACOX1 levels and fibrosis levels in transplanted kidneys.(3)The correlation between PUFA levels and fibrosis progression was verified in blood samples from kidney transplant patients.(4)Establish a rat unilateral kidney transplantation model and feed rats with the same total fat content and different n-3/n-6 PUFA content.Observe the large transplanted kidneys and compare the differences;Masson’s stain to detect the degree of fibrosis;hematoxylin-eosin stain to detect whether each organ is diseased;IHC to detect the expression level of ECM-related molecules in the transplanted kidneys.(5)The delaying effect of ER stress inhibitor treatment on transplanted kidney fibrosis was verified in a rat kidney transplantation model.Results1.To determine the association between abnormal lipid metabolism and fibrosis of renal allografts(1)BMI was not associated with fibrosis in renal allografts.In cases that had undergone transplantation operations more than 5 years prior,the percentage of overweight patients(BMI>25)displayed an increasing trend along with fibrosis progression.The amounts of triacylglycerol(TG)in cases with ci-scores of 2 and 3were significantly higher than those in cases with ci-scores 0 or 1.Cases with XIV percentages of high TG(>1.75 mmol/L)make up 52.00% of ci0 cases,67.57% of ci1 cases,and 85.71% of ci2/3 cases in patients who had undergone transplantation operations more than 5 years prior(2)We screened 5474 differentially expressed genes from the comparison of CAN Ⅲ vs.control in the Saint-Mezard Transplant Kidney Dataset.Pathways related to lipid metabolism enriched in downregulated genes from the comparison of CAN Ⅲ vs.control.FAO pathway accounted for the main position in these enriched pathways.2.To investigate the influence of rate-limiting enzymes in FAO pathways on fibrosis of renal allograft(1)CPT1 expression was not associated with fibrosis in renal allograft.(2)ACOX1 was deficient in tissues of renal allograft with serious fibrosis.(3)Differential genes induced by knocking down ACOX1 enrich in fibrosis pathway.3.To define ACOX1 deficiency facilitates fibrosis of renal allografts through inducing epithelial-mesenchymal transition(1)EMT pathway enriched in tissues with serious fibrosis.(2)Molecules related to mesenchymal phenotype increased along with fibrosis progression.(3)Molecules related to EMT increased in cluster 9 which ACOX1 displays lowest expression.(4)CPT1 expression was not associated with fibrosis development,and ACOX1 expression showed negative correlation with labeled molecules of mesenchymal phenotype in biopsies of renal allografts.(5)ACOX1 deficiency and inhibitor treatment induced EMT.(6)EMT molecules in the kidneys of ACOX1-KD mice were negatively correlated with ACOX1 expression;(7)Treatment of mice with ACOX1 inhibitors resulted in upregulation of EMT molecule expression.4.To define ACOX1 deficiency facilitates fibrosis of renal allografts through inducing extracellular matrix reorganization(1)EMT pathway enriched in tissues with serious fibrosis.(2)ECM pathway enriched in cluster 9 which ACOX1 displays lowest expression.(3)Upregulated genes induced by ACOX1 deficiency enriched in ECM pathway.(4)Upregulated genes induced by ACOX1 deficiency which related to ECM organization associated with fibrosis progression in renal allograft.(5)Increased expression of ECM molecules in transplanted kidneys compared to in situ kidneys in a rat kidney transplantation model;(6)ACOX1 was negatively correlated with ECM expression analyzed in human tissue samples from the online database;(7)ACOX1 knockdown or ACOX1 inhibitor treatment promotes the expression of ECM molecules;(8)Increased expression of ECM molecules in the kidneys of ACOX1 inhibitortreated mice and ACOX1 KD mice.5.To determine the inhibitory effect of NFκB activation on ACOX1 expression in transplanted kidneys.(1)Hypoxia and oxidative stress promote the expression of ACOX1;(2)NFκB activation inhibits ACOX1 expression in the online database,transplanted kidney tissue samples,rat kidney transplantation model,lipopolysaccharide-treated mouse model,and in vitro cell model;(3)DNMT1 was negatively correlated with ACOX1 expression in the online database;(4)DNMT1 expression was positively correlated with progression of transplanted kidney fibrosis in human transplanted kidney samples and rat kidney transplant model samples;(5)NFκB inhibited ACOX1 expression by upregulating DNMT1 levels in in vitro cellular assays and lipopolysaccharide-treated mouse models;6.To define lipid accumulation induced by ACOX1 deficiency mediates EMT(1)PLIN1 expression in human transplanted kidney correlated with ACOX1 XVI expression;(2)ACOX1 deficiency induced lipid accumulation.(3)ABCD1 deficiency induced lipid accumulation,EMT,and ECM reorganization;ABCD1 expression showed low significant correlation with fibrosis progression in renal allografts.(4)Oleic acid or palmitic acid treatment induced EMT,but did not affect ECM organization.7.To define alteration of lipid constitution induced by ACOX1 deficiency mediates ECM organization.(1)ACOX1 deficiency induced decreased PUFAs.(2)The common treatment of LA,α-LA,and DHA inhibited the expression of genes related to ECM reorganization.(3)Gavage of PUFAs reverses downregulation of renal function,increased levels of fibrosis and expression of ECM molecules in ACOX1 inhibitor-treated or renal transplantation models;8.Determination of Endoplasmic Reticulum Stress Mediating PUFA ReductionInduced ECM Remodeling(1)Inhibition of ACOX1 upregulated mitochondrial function;(2)DECR1 expression in transplanted kidneys did not correlate with EMT molecules and correlated positively with the expression of ECM molecules;(3)Knockdown of ACOX1 upregulates DECR1 levels;(4)Silencing of DECR1 in shACOX1 cells resulted in decreased expression of ECM molecules;(5)DECR1 is significantly positively correlated with the expression of endoplasmic reticulum stress molecules;(6)ACOX1 knockdown or ACOX1 inhibitor treatment induced endoplasmic reticulum stress;(7)Endoplasmic reticulum stress inhibitor treatment of shACOX1 cells suppresses ECM molecule expression;(8)Supplementation of shACOX1 cells with PUFAs inhibited the expression of endoplasmic reticulum stress molecules;9.To determine the role of targeting ACOX1 signaling in delaying transplant-related kidney fibrosis.(6)Pharmacological targeting of NFκB and DNMT1 in a rat kidney transplantation model delayed the progression of transplanted renal fibrosis.(1)Fenofibrate treatment upregulates ACOX1 expression levels and delays the degree of fibrosis in transplanted kidneys.(4)Reduction of PUFAs in blood samples from kidney transplant patients correlates with fibrosis progression;(5)Rats fed a diet enriched in n-3 PUFAs had reduced levels of fibrosis in their transplanted kidneys,and the expression of ECM molecules was reduced.(9)Treatment with an endoplasmic reticulum stress inhibitor in a rat kidney transplantation model significantly delayed transplant kidney fibrosis.Conclusions1.FAO pathway was suppressed in fibrotic renal allograft;ACOX1 deficiency facilitated fibrosis progression in renal allograft through inducing EMT and ECM reorganization.2.TLR4-NFκB signaling activation inhibits ACOX1 expression in a DNMT1-dependent manner;Lipid accumulation in renal tubular epithelial cells and overconsumption of PUFAs-induced endoplasmic reticulum stress due to ACOX1 deficiency promotes EMT and ECM remodeling respectively;3.Pharmacological targeting of NFκB,DNMT1,PPARα,and ER stress signaling... |
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