| The prevalence of chronic kidney disease(CKD)has increased significantly in recent decades and the global prevalence has reached 13.4%.Cardiovascular disease is the main cause of death in patients with CKD,accounting for about 50%of the total mortality in patients with chronic kidney disease.CKD is an independent risk factor for cardiovascular disease,while with the decline of renal function,the incidence of cardiovascular diseases such as atherosclerosis increased significantly.Endothelial dysfunction is an early event of cardiovascular disease,and is related to poor prognosis.In patients with CKD,such as toxin accumulation,inflammation,calcium and phosphorus metabolism disorders,can disrupt endothelial homeostasis and cause endothelial dysfunction.In-depth research on the influencing factors,signal transduction pathways and a series of pathophysiological processes of endothelial dysfunction in CKD patients will help us better prevent and reduce cardiovascular disease complications.Long non-coding RNAs(lncRNAs)are a type of RNA that has more than 200 nucleotides and does not have protein-coding functions.Through DNA methylation and demethylation,RNA interference,histone modification,chromatin remodeling,and imprinting genes and other mechanisms,it can affect cell proliferation,metabolism,and metabolism at the epigenetic level,transcription level,and post-transcriptional level,thereby affecting the expression of coding genes.The antisense non-coding RNA in the INK4 locus(ANRIL)of the INK4 locus is a long non-coding RNA located in the antisense direction of the INK4B-ARF-INK4 A gene cluster on chromosome 9p21.The polymorphism(SNP)is associated with various atherosclerotic vascular diseases such as coronary atherosclerotic heart disease and myocardial infarction.ANRIL is widely expressed in human vascular endothelial cells.When stimulating factors exist,ANRIL participates in the regulation of inflammatory response,cell apoptosis and other processes.With the decline of renal function,the body has a persistently low level of systemic inflammation,and the polymorphism of ANRIL in hemodialysis patients is significantly related to the incidence of major adverse cardiovascular events,suggesting that ANRIL plays an important role in the occurrence and development of cardiovascular complications of chronic kidney disease.However,the role and mechanism of ANRIL in the vascular endothelial dysfunction of CKD are still unclear.In-depth study of the role and mechanism of ANRIL in endothelial injury is of great significance.LncRNAs also have secondary and three-dimentional structures which enable them to have both RNA-and protein-like functions.Studies have shown that ANRIL can recruit PRC2 complex to change chromatin structure and regulate gene expression.EZH2 is one of the most important components of the PRC2 complex,which can trimethylates histone 3 lysine 27(H3K27me3)and cause chromatin compaction and transcriptional silencing.Studies have confirmed that EZH2 is involved in the occurrence and development of atherosclerosis and other vascular diseases.In addition,studies have suggested that EZH2 expression is also increased in renal biopsy tissues of patients with CKD,suggesting that EZH2 may play an important role in the endothelial dysfunction of CKD.To date,limited EZH2 target genes have been identified in endothelial cells,Brain-derived neurotrophic factor(BDNF)is highly expressed in endothelial cells and plays an important role in vascular diseases by promoting vascular development and endothelial cell budding and inducing angiogenesis.Studies have shown that BDNF expression was significantly reduced in plasma of patients with coronary heart disease and was associated with high levels of vWF.Moreover,BDNF mimics 7,8-Dihydroxyflavone(7,8-DHF)can improve cardiac loss,reduce cell death and correct mitochondrial fission abnormality.However,the relevant mechanism still needs to be further explored.Therefore,in this study,we detected the expression level of clinical ANRIL and analyzed the relationship between ANRIL level and endothelial dysfunction in CKD And the gene knockout mice model was established to verify its role in endothelial dysfunction.Then,the inducible factors of high expression of ANRIL were explored through serum stimulation and uremic toxin stimulation,furthermore,the specific mechanism of ANRIL in endothelial cell injury was explored.This will provide a new insight for the occurrence and development of endothelial dysfunction in chronic renal disease.Part ? The expression of ANRIL in patients with CKD and its association with endothelial dysfunctionObjective1.To determine the expression level of ANRIL in patients with CKD and analyze its correlation with renal function.2.To investigate the relationship between ANRIL and endothelial dysfunction in patients with CKDMethodsThe study included 60 cases of CKD patients and 45 cases of gender and age-matched healthy adults.Basic data such as gender,age,blood pressure and laboratory indicators such as blood lipids and renal function were collected,and the right brachial artery blood flow-mediated vasodilation(FMD)was measured by non-invasive ultrasound to assess the vascular endothelial function of the participants;fasting blood samples were collected to detect the ANRIL expression level in plasma.Compare the ANRIL expression between CKD and the control group,then analyze the correlation between ANRIL expression level and kidney function or vascular endothelial function.Results1.Clinical data analysis:there was no difference in age or gender between CKD patients and the control group,and there was no statistical difference in blood lipid and blood glucose test results between the two groups;Patients with CKD had higher blood pressure than the control group,P<0.001,the difference was statistically significant.2.Plasma ANRIL levels:real-time PCR test showed that compared with the control group,plasma ANRIL levels in CKD patients were significantly higher(P=0.02).3.Correlation between ANRIL level and renal function:Spearman correlation analysis showed that ANRIL expression was negatively correlated with eGFR(r=-0.284,P=0.032).4.Relationship between ANRIL level and endothelial dysfunction in patients with CKD:Spearman correlation analysis showed that ANRIL expression was negatively correlated with FMD level(r=-0.464,P=0.002)Part ? The role of ANRIL in endothelial injury in CKD miceObjective1.Construction of wild-type and ANRIL gene knockout CKD mice models.2.Detect the expression of ANRIL in vascular endothelial cells of CKD mice,and analyze its relationship with endothelial cell injury in CKD mice.Methods1.Construction of ANRIL-/-mice model:The ANRIL-/-mice model was constructed using CRISPR/Cas9 gene knockout technology;and to observe the appearance,color,shape and other general changes.2.Construction of CKD mice model:wild-type C57BL/6J mice were randomly grouped:sham operation group(Sham):the fat capsule was removed,and the kidney was preserved;CKD group:5/6 nephrectomy was performed.ANRIL-/-mice were randomly divided into groups:ANRIL-/-group:the fat capsule was removed and the kidney was preserved;the CKD/ANRIL-/-group:5/6 nephrectomy was performed.3.Clinical indicators and renal pathological changes in CKD mice:monitor the blood pressure and body weight of the mice;detect the serum creatinine,triglyceride,and total cholesterol levels;kidney tissue with PAS staining to observe the pathological changes of the kidney tissue.4.ANRIL expression in vascular endothelium:real-time PCR was used to detect the expression level of ANRIL in abdominal aorta;The expression and distribution of ANRIL in vascular endothelial cells were detected by RNA fluorescence in situ hybridization(FISH)combined with CD31 fluorescence double staining5.Detection of vascular endothelial injury:(1)Detection of protein related to vascular endothelial function:The mRNA expression of eNOS,VCAM-1,vWF were detected by real-time PCR and the protein expression of VCAM-1 in vascular endothelial cells were detected by immunohistochemistry.(2)Detection of mitochondrial fusion-related proteins:The mRNA expression of DRP-1,MFN2 were detected by real-time PCR and the protein expression of MFN2 in vascular endothelial cells were detected by immunohistochemistry.Results1.Construction of ANRIL-/-mice model:Genotype identification by gel electrophoresis showed that the size of PCR product was 704bp,and compared with wild-type mice,ANRIL-/-mice showed no difference in appearance,color,body shape,blood pressure,creatinine,etc.2.Clinical indicators and renal pathological changes in CKD mice:the blood pressure was increased in CKD mice(P<0.05),but there was no significant difference in body weight;the blood pressure of CKD/ANRIL-/-group was slightly lower than that in CKD group,P=0.04.Serum creatinine was significantly higher in CKD mice than in the sham group(P<0.001),and there was no statistically significant difference in serum triglyceride total cholesterol levels,the serum creatinine level in CKD/ANRIL-/-group was slightly lower than that in CKD group(P=0.04).PAS staining of CKD mice showed that the renal glomerular mesangial cells proliferated,the matrix increased,and the capillary loops collapsed or blocked,part of the glomeruli showed segmental or nodular sclerosis,renal tubules were atrophy with a large number of protein casts;knock out ANRIL could alleviate renal pathological injury.3.ANRIL expression in vascular endothelium:real-time PCR assay showed that ANRIL expression in the abdominal aorta was increased in the CKD mice(P<0.05),and FISH assay showed that ANRIL expression was increased in the vascular endothelial cells of CKD mice,and it was distributed in both cytoplasm and nucleus.4.Detection of vascular endothelial injury:(1)Detection of protein related to vascular endothelial function:real-time PCR assay showed that eNOS expression decreased and VCAM-1 and VWF expression increased in abdominal aorta of CKD mice;immunohistochemistry showed that VC AM-1 expression was increased in the abdominal aorta endothelial cells of CKD mice;and the above expression abnormalities were improved after ANRIL knockout,with statistically significant differences.(2)Detection of mitochondrial fusion-related proteins:real-time PCR assay showed that MFN2 expression decreased and DRP-1 expression increased in abdominal aorta of CKD mice;immunohistochemistry showed that MFN2 expression was increased in the abdominal aorta endothelial cells of CKD mice;and the above expression abnormalities were improved after ANRIL knockout,with statistically significant differences.Part ? Role and mechanism of ANRIL in uremic toxin-induced endothelial injuryObjective1.To determine whether serum derived from CKD patients and uremia toxin induce abnormal expression of ANRIL and its relationship with endothelial injury.2.To regulate the expression of ANRIL and verify the role and specific mechanism of ANRIL in uremic toxin-induced vascular endothelial cell injury.Methods1.Role of serum derived from CKD patients and uremia toxins in endothelial cell injury.1.1 Serum stimulation of endothelial cells:Human umbilical vein endothelial cells(HUVEC)were cultured and stimulated with serum from patients with CKD to simulate the environment of CKD in vivo.(1)ANRIL expression:Real-time PCR and FISH fluorescence staining were used to detect the expression and distribution of ANRIL in endothelial cells.(2)Detection of protein related to vascular endothelial function:The mRNA expression of eNOS,VCAM-1,vWF were detected by real-time PCR,The protein expression of eNOS,VCAM-1,vWF were detected by western bolt.(3)Mitochondrial detection:Mitosox Red was used to detect mitochondrial ROS levels in endothelial cells;Mitotracker Red probe was used to label mitochondria,and the morphological changes of mitochondria were observed under confocal microscope;the mitochondrial fusion-related protein DRP-1,MFN2 were detected by western bolt.1.2 Uremia toxin stimulates endothelial cells:(1)ANRIL expression:Endothelial cells were stimulated with different concentrations of uremia toxin,included Indoxyl sulfate(IS),hemuric acid(HA),indole-3-acetic acid(IAA)and homocysteine(Hcy),and the expression level of ANRIL was detected by real-time PCR.(2)Detection of endothelial cell injury:The endothelial cells were stimulated with IS in gradient concentration,and the expression of vascular endothelial function-related protein like eNOS,VCAM-1,VWF and the expression of mitochondrial fusion-related protein DRP-1 and MFN2 were detected by Western Blot.2.The role of ANRIL in endothelial cell injury2.1 Construction of specific ANRIL shRNA and ANRIL overexpression lentiviral vector and identification of its endothelial cell transfection efficiency.2.2 Role of ANRIL in endothelial cell injury(1)IS induces endothelial cell injury,groups:control group(PBS stimulation),IS group(1mM IS stimulation),IS/Scramble group(1mM IS stimulation+shRNA empty vector transfection),IS/Sh-ANRIL group(1mM IS stimulation+Sh-ANRIL transfection).The expression of endothelial function-related protein eNOS,VCAM-1,VWF was detected by real-time PCR and Western Blot.The expression of mitochondrial fusion-related protein DRP-1 and MFN2 was detected by Western Blot.(2)Stable cell lines overexpressed with ANRIL were constructed and further double transfected with Sh-ANRIL,groups:null(empty vector transfection),ANRIL(ANRIL overexpression),ANRIL/Sh-ANRIL(ANRIL overexpression+Sh-ANRIL double transfection),ANRIL/Scramble(ANRIL overexpression+shRNA empty vector double transfection).The expression of endothelial function-related protein eNOS,VCAM-1,VWF was detected by real-time PCR and Western Blot.The expression of mitochondrial fusion-related protein DRP-1 and MFN2 was detected by Western Blot.3.ANRIL mediates endothelial cell injury through the recruitment of EZH23.1 Verify the binding and regulatory relationship between ANRIL and EZH2:(1)Binding of ANRIL with EZH2:RN A pull-down combined with Western Blot was used to verify whether ANRIL was binding to histone methyltransferase EZH2;RNA-binding protein immunoprecipitation(RIP)verified the binding of ANRIL to EZH2,and detected the changes of the binding of ANRIL and EZH2 after the regulation of ANRIL expression.(2)Regulation of EZH2 expression by ANRIL:stable cell lines overexpressing ANRIL were constructed,and Sh-ANRIL was further double-transfected,groups:null,ANRIL,ANRIL/Sh-ANRIL,ANRIL/Scramble.The protein expression of EZH2 was detected by Western Blot.3.2 To verify the role of EZH2 in uremia toxin-induced endothelial cell injury:(1)IS(1mM)stimulated endothelial cells to detect EZH2 expression.(2)EZH2 interference vector(Si-EZH2)was constructed and the cells were divided into groups:si-NC(si-RNA empty vector transfection),IS/si-NC(1mM IS stimulation+si-RNA empty vector transfection),IS/si-EZH2(1mM IS stimulation+si-EZH2 transfection).The expression of endothelial function-related protein eNOS,VCAM-1,VWF was detected by real-time PCR and Western Blot.The expression of mitochondrial fusion-related protein DRP-1 and MFN2 was detected by Western Blot.3.3 ANRIL-EZH2 mediated vascular endothelial injury and its specific mechanism:The expression of ANRIL and EZH2 was regulated,groups:null,ANRIL,ANRIL/si-EZH2(ANRIL overexpression+si-EZH2 transfection),ANRIL/si-NC(ANRIL overexpression+si-NC transfection).The levels of histone methylation(H3K27me3)and brain-derived neurotrophic factor(BDNF)expression in endothelial cells were detected by Western Blot.(2)Chromatin immunoprecipitation combined with real-time PCR was used to detect the methylation level of BDNF promoter region and the binding status of EZH2.(3)ANRIL-EZH2-BDNF mediate endothelial injury,groups:null,ANRIL,ANRIL/si-EZH2,ANRIL/BDNF(ANRIL overexpression+stimulation of recombinant BDNF factor).?The expression of endothelial function-related protein eNOS,vWF was detected by Western Blot and the expression of VCAM-1 was detected by immunofluorescence.?The expression of mitochondrial fusion-related protein DRP-1,MFN2 was detected by Western Blot,and the expression of MFN2 was detected by immunofluorescence.Results1.Role of serum derived from CKD patients and uremia toxins in endothelial cell injury.1.1 Serum stimulation of endothelial cells:(1)ANRIL expression:Compared with the control group,stimulation with serum derived from CKD patients showed that ANRIL expression was increased in endothelial cells(P<0.05),and FISH staining results showed that ANRIL was distributed in nuclear and cytoplasm,and the expression was increased,the difference was statistically significant.(2)Detection of protein related to vascular endothelial function:After stimulation with serum derived from CKD patients,the expression of eNOS in endothelial cells decreased and the expression of VCAM-1 and vWF increased(p<0.05).(3)Mitochondrial detection:After stimulation with serum derived from CKD patients,the level of mitochondrial ROS in endothelial cells increased;and Mitotracker Red staining showed that the mitochondria of the endothelial cells in the control group were long rods,while those in the serum stimulation group of CKD patients were punctate;besides,Western Blot results showed that the expression of DRP-1 was increased and the expression of MFN2 was decreased in endothelial cells,P<0.05,the difference was statistically significant.1.2 Uremia toxin stimulates endothelial cells:(1)ANRIL expression:The uremic toxin IS,HA,IAA and Hcy could increase the expression of ANRIL in endothelial cells in a concentration-dependent manner,and the differences were statistically significant.(2)Detection of endothelial cell injury:IS stimulation decreased eNOS expression and increased VCAM-1,vWF expression in a concentration dependent manner(P<0.05);and Western Blot results showed that IS stimulation resulted in a dose-dependent increase in the expression of DRP-1 and a decrease in the expression of MFN2(P<0.05).2.The role of ANRIL in endothelial cell injury2.1 The expression level of ANRIL was significantly reduced after Sh-ANRIL transfection(P<0.05),and ANRIL overexpressed vector could significantly upregulated ANRIL expression(P<0.05).2.2 Role of ANRIL in endothelial cell injury(1)IS induced decreased expression of endothelial cell function-related protein eNOS,increased expression of VC AM-1 and vWF,also increased expression of DRP-1,and decreased expression of MFN2(P<0.05);inhibition of the ANRIL expression could reversed the abnormal expression of the above proteins(P<0.05).(2)Compared with null group,overexpression of ANRIL decreased the eNOS expression,and increased VCAM-1 and vWF expression(P<0.05);meanwhile,DRP-1 expression was increased and the MFN2 expression was decreased(P<0.05);the inhibition of ANRIL expression could reverse the abnormal expression of these proteins(P<0.05).3.ANRIL mediates endothelial cell injury through the recruitment of EZH23.1 Verify the binding and regulatory relationship between ANRIL and EZH2:(1)Binding of ANRIL with EZH2:RNA pull-down results showed that ANRIL could bind EZH2,and RIP test further verified the interaction,and the overexpression of ANRIL could increase the interaction,P<0.05,the difference was statistically significant.(2)Regulation of EZH2 expression by ANRIL:the overexpression of ANRIL could mediate the increase of EZH2 expression,and the double transfection of Sh-ANRIL could reverse the EZH2 expression(P<0.05).3.2 To verify the role of EZH2 in uremia toxin-induced endothelial cell injury:(1)IS stimulation can induce the increase of EZH2 expression in endothelial cells(P<0.05).(2)si-EZH2 interfered the EZH2 expression in endothelial cells,and reversed the decrease of eNOS expression and the increase of VC AM-1,vWF expression induced by IS stimulation(P<0.05).Meanwhile,the expression of DRP-1 was down-regulated and the expression of MFN2 was increased after si-EZH2 transfection(P<0.05).3.3 ANRIL-EZH2 mediated vascular endothelial injury and its specific mechanism:(1)The overexpression of ANRIL could mediate the increase of H3K27me3 level,which can be reversed after transfection with si-EZH2(P<0.05).Meanwhile,the overexpression of ANRIL induced the downregulation of BDNF expression,while the interference of EZH2 expression could restore the expression of BDNF(P<0.05).(2)CHIP results showed that after ANRIL overexpression,the level of H3K27me3 in the BDNF promoter region was significantly increased,and the binding of EZH2 in this promoter region was significantly increased(P<0.05).(3)ANRIL-EZH2-BDNF mediate endothelial injury:The results showed that compared with ANRIL overexpression,EZH2 inhibition or BDNF stimulation could reverse the low expression of eNOS and MFN2,and reduce the expression levels of VWF and DRP-1(P<0.05);meanwhile,the level of mitochondrial ROS also decreased after EZH2 inhibition or BDNF stimulation(P<0.05).Conclusion1.In this study,we found that the expression of plasma ANRIL was increased in patients with CKD,and circulating ANRIL level was inversely correlated with vascular endothelial dysfunction in CKD.2.ANRIL deficiency alleviated endothelial dysfunction in CKD mice by affecting the expression of endothelial function-related proteins and mitochondrial function3.This study illustrated the specific mechanism of ANRIL mediating endothelial cell damage in chronic kidney disease,we confirmed that ANRIL could bind to EZH2,and mediate BDNF promoter methylation through recruitment of EZH2 to the BDNF promoter region,then regulate the expression of endothelial function-related proteins and mitochondrial fission related proteins,eventually resulting in endothelial dysfunction.This study provides new insights for the study of endothelial dysfunction in CKD. |
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