| Objective: Chronic kidney disease(CKD)refers to a group of chronic diseases caused by various primary kidney diseases and a variety of secondary causes such as diabetes and hypertension.CKD has the epidemiological characteristics of high incidence,high disability rate,high mortality rate,and high economic burden,which has caused a great health and economic burden to society.Vascular calcification(VC)is one of the common complications of CKD,and an independent risk factor for cardiovascular events and mortality in patients with CKD.However,the current clinical treatment of VC in patients with CKD is mainly to regulate calcium and phosphorus,control secondary hyperparathyroidism,etc.,which cannot effectively prevent and treat VC.Therefore,exploring the pathogenesis of VC and finding effective drugs and methods to prevent and treat VC has always been the focus of attention.The pathological features of vascular calcification in CKD are mainly manifested by the calcification of the arterial media.Vascular smooth muscle cells(VSMCs)are the main cells of the arterial membrane,and their phenotypic transformation is a key link in the calcification of the middle membrane.In CKD environment,vascular smooth muscle cells show osteoblast-like changes,and then the contractile function is weakened,the apoptosis rate is increased,and vascular calcification is induced.The expression of a-SMA,SM22 a and other contractile proteins is decreased,and the expression of osteogenic markers such as Col I and BMP2 is up-regulated,leading to the decrease of normal vascular tone.Therefore,preventing and treating the phenotypic transformation of vascular smooth muscle cells may be an important research direction to effectively delay the calcification of CKD vessels.KAP1,also known as TRIM28 or TIF1β,is a transcriptional cofactor named for its ability to bind to members of the zinc family proteins(ZFPs)containing the KRAB domain.It is mainly localized in the nucleus,interacts with transcription factors,and plays an important regulatory role in the regulation of tumorigenesis,cell differentiation and embryonic development.At present,the study found that the content of KAP1 in atherosclerotic plaques increased significantly compared with the control group,and in vitro cell experiments have confirmed that knockdown KAP1 can significantly reduce the proliferation and migration of platelet-derived growth factorinduced vascular smooth muscle cells,while overexpression of KAP1 will produce the opposite result.In conclusion,KAP1 plays an important regulatory role in the phenotypic transformation of vascular smooth muscle cells,and is expected to become a potential target for the prevention and treatment of vascular calcification.The ERK signaling pathway is the main signaling pathway for cell proliferation,differentiation,apoptosis,and stress response under normal and pathological conditions.Previous studies have shown that the ERK signaling pathway is also an important regulatory pathway for phenotypic transformation of vascular smooth muscle cells.The activation of ERK pathway can promote VC,while ERK inhibitor can reduce the expression of Runx2 and ALP activity,thereby inhibiting the osteogenic differentiation of vascular smooth muscle cells.In this study,we first examined the expression of KAP1 and vascular smooth muscle cell contractile proteins and osteogenesis-related markers in the vascular tissues of CKD patients and rats with vascular calcification.Further,bioinformatics analysis was performed by using the expression microarray of rat vascular smooth muscle cells in the Gene Expression Omnibus(GEO)database of the public data platform to verify the difference in the expression of KAP1 between the normal group and the high phosphorus group.Next,the expression of KAP1,vascular smooth muscle cell contractile protein and osteogenesis-related markers in vitro cultured vascular smooth muscle cells under high phosphorus environment were detected by cell experiments,and we further investigated the role of KAP1 in vascular calcification in CKD by plasmid transfection with KAP1 overexpression and KAP1 knockdown to investigate the phenotypic switching and calcification induced by high phosphorus in VSMCs.In addition,the mechanism of KAP1 promoting phenotypic transformation and calcification of VSMCs was explored,which provided a theoretical basis and experimental evidence for revealing that KAP1 promotes the occurrence of CKD vascular calcification.This study consists of three parts,as follows:Part one Expression and significance of KAP1 in vascular tissues of patients with chronic renal disease and rats with chronic renal diseaseObjective: The expressions of KAP1 and VSMCs contractile proteins and osteogenic indexes in the vascular tissues of patients with CKD vascular calcification and rats were determined by immunohistochemistry.Methods:1.Patients with chronic kidney disease who were admitted to the Department of Nephrology,Cangzhou People’s Hospital from June 2020 to January 2021 were selected.Cephalic vein vascular tissues of patients during autologous arteriovenous fistula and reconstruction were collected.According to the results of Von Kossa staining,patients were divided into non-calcified group(NC)and calcified group(VC).2.Male SD rats were selected and randomly divided into two groups:control group and vascular calcification group.A rat model of vascular calcification of chronic kidney disease was constructed by 5/6 nephrectomy combined with a high-phosphate diet,and rats were sacrificed 12 weeks later with thoracic aortic vascular tissue retained.3.Immunohistochemistry was used to detect the expression of KAP1,a-SMA and Col I in various groups of vascular tissues.Results: Immunohistochemical results showed that KAP1 was significantly expressed in the vascular tissues of the VC group,and the expression of KAP1 was significantly increased in the VC group compared with the control group.KAP1 was mainly distributed in the nucleus,and the expression of cytoplasm and interstitium was not obvious.The expression of Col I protein in the vascular tissue of VC group was high.Compared with the control group,it was significantly increased in VC group.COL I was expressed in the extracellular matrix.The immunohistochemical results of a-SMA showed that the expression of a-SMA protein was obvious in the kidney tissue of the control group,and compared with the control group,the expression of a-SMA in the VC group was significantly reduced,and the expression and distribution of a-SMA were mainly in the cytoplasm of vascular smooth muscle cells.The above indicators,expression and trends were consistent in vascular tissues of CKD patients and CKD rats with vascular calcification.Summary: In the vascular tissues of patients with CKD vascular calcification and rats with CKD vascular calcification,KAP1 is highly expressed,which is consistent with the expression trend of osteoblast-related proteins,but contrary to the expression trend of smooth muscle actin.KAP1 may be involved in the occurrence and development of vascular calcification in CKD.Part two The effect of KAP1 on calcification of vascular smooth muscle cells in high phosphorus environment was investigatedObjective: To investigate the effect of KAP1 on the calcification of vascular smooth muscle cells in a high-phosphorus environment.Methods:1.Bioinformatics analysis was carried out by using the expression microarray of rat vascular smooth muscle cells(VSMCs)in the Gene Expression Omnibus(GEO)of the public data platform to explore the difference in the expression of KAP1 in the normal group and the high phosphorus group.2.For cell experiment verification,VSMCs were divided into normal control group(N)and high phosphorus group(β-GP,10 m M β-GP).After the specified time stimulated,total protein and RNA were extracted.The expression of KAP1、Col I、a-SMA were detected by Western blot or real-time PCR.Alizarin red staining and calcium determination were used to evaluate the calcification of VSMCs.3.Transfection plasmids were transfected with Lipofectamine 3000 and P3000,VSMCs were transferred to a six-well plate and transfected with different proportions.Cells were collected after 48 h,and the transfection effect of KAP1 was detected by Western blot.In order to observe the effect of knockdown KAP1 on the phenotypic transformation and calcification of VSMCs in a high-phosphorus environment,cells were divided into normal control group(N),high-phosphorus group(β-GP,10 m M β-GP),high-phosphorus + no-load plasmid transfection control group(β-GP + Scramble,10 m M β-GP + Scramble),and high-phosphorus + transfected KAP1 lowexpression plasmid group(β-GP + sh KAP1,10 m M β-GP + sh KAP1).In order to observe the effect of overexpression of KAP1 on the phenotypic transformation and calcification of VSMCs under normal culture environment,cells were divided into normal control group(N),normal + no-load plasmid transfection control group(Empty,normal + Control),and normal + transfected KAP1 overexpression plasmid group(OE KAP1,N+pc DNA3.1-KAP1).After 48 h stimulation,the activity of alkaline phosphatase was detected by alkaline phosphatase kit,and KAP1,Col I and a-SMA proteins expressions were detected by Western blot.The calcification of VSMCs were evaluated by alizarin red staining and calcium content determination.4.In order to explore whether KAP1 participates in the osteogenic differentiation of VSMCs stimulated by high phosphorus by affecting the osteogenic transcription factor Runx2,we divided VSMCs into 6 groups:normal control group(N),normal + no-load plasmid transfection control group(Empty,normal + Control),normal + transfected KAP1 overexpression plasmid group(OE KAP1,N+ pc DNA3.1-KAP1),high phosphorus group(β-GP,10 m M β-GP),high phosphorus + The no-loaded plasmid transfection control group(β-GP + Scramble group,10 m M β-GP + Scramble),the high phosphorus + transfected KAP1 knockdown plasmid group(β-GP + sh KAP1,10 m M β-GP + sh KAP1).After 48 h stimulation,Runx2 protein expression was detected by immunofluorescence,Western blot and real-time PCR.Results:1.The expression of KAP1 in calcified VSMCs was higher than that in normal group by GEO database analysis.2.Immunofluorescence,Western blot and real-time PCR results showed that the expression of KAP1 was significantly increased after 7d under high phosphorus.The difference was statistically significant(P<0.001).The expression of KAP1 was consistent with the expression trend of COL I and opposite to the expression trend of a-SMA.3.Compared with high phosphorus + empty plasmid transfection control group(β-GP + Scramble),the expression of KAP1 in high phosphorus group transfected with sh KAP1 plasmid was significantly decreased,the amount of mineralized nodules and calcium content were significantly decreased,the protein expression of COL I and ALP activity were significantly decreased,while the protein expression of a-SMA was significantly increased.This indicated that inhibition of KAP1 could attenuate VSMCs calcification.In addition,the results were reversed after normal group cells were transfected with the pc DNA3.1-KAP1 plasmid.4.Western blot and Real-time PCR results showed that the expression of Runx2 in VSMCs was significantly increased after high phosphorus stimulation for 48 h compared with the N group,while the expression of Runx2 was decreased after plasmid transfection of KAP1 knockdown(P<0.05).Immunofluorescence showed that the green fluorescence of KAP1 and the red fluorescence of Runx2 were increased in β-GP group,which was decreased in high phosphorus environment after KAP1 knockdown.Conversely,the opposite trend was observed when KAP1 was overexpressed in normal cells.Summary: In VSMCs with calcification induced by high phosphorus,the KAP1 level is increased,and KAP1 knockdown can inhibit VSMCs calcification.KAP1 promotes vascular calcification by activating the expression of Runx2 and its downstream osteoblast proteins.Part three Mechanism of KAP1 in calcification of vascular smooth muscle cells in high phosphorus environmentObjective: Mechanistic study of KAP1 in vascular smooth muscle cell calcification under a high phosphorus environment.Methods:1.To explore whether ERK signaling pathway is related to vascular calcification in CKD,the expression of p-ERK in thoracic aorta of CKD rats was detected by immunohistochemistry,and the expression of p-ERK in rat VSMCs induced by high phosphorus was detected by Western blot.2.In order to explore whether the ERK signaling pathway participates in the phenotypic transformation and calcification of high-phosphate-stimulated VSMCs,the p-ERK inhibitor PD0325901 was added.After blocking the ERK pathway,the expression of VSMCs phenotypically transform-associated proteins under high phosphorus environment were observed.The cells were divided into normal control group(N),high phosphorus group(β-GP,10 m Mβ-GP),high phosphorus + solvent DMSO group(β-GP + DMSO,10 m Mβ-GP + DMSO),high phosphorus + p-ERK inhibitor group(β-GP + PD0325901,10 m M β-GP + 5u M PD0325901).All groups were stimulated for48 h.Western blot was used to detect the expression of total-ERK(ERK)and phospho-ERK(p-ERK),as well as the expression of Col I and a-SMA.3.To investigate whether KAP1 plays a role in VSMCs osteogenic phenotype transformation and calcification through ERK signaling pathway,we overexpressed KAP1 in VSMCs under normal culture conditions by plasmid transfection and observed the expression of p-ERK protein.VSMCs were divided into three groups: normal control group(N),normal + no-load plasmid transfection control group(Empty,N+Control),and normal + transfected KAP1 overexpression plasmid group(OE KAP1,N+ pc-DNA3.1-KAP1).The protein expressions of ERK,p-ERK and p38 were detected by Western blot after 48 hours of stimulation.4.In order to further explore whether KAP1 participates in the osteogenic differentiation of VSMCs stimulated by high phosphorus through the ERK/Runx2 pathway,the p-ERK inhibitor PD0325901 was added.After blocking the ERK pathway,the affection of KAP1 over-expression on osteogenic and phenotypic transformation-related proteins of VSMCs under normal culture conditions were observed.The cells were divided into normal control group(N),normal + transfected KAP1 overexpression plasmid group(OE KAP1,N+ KAP1),normal + transfected KAP1 overexpression plasmid +PD0325901 group(N+KAP1+PD0325901,N+ pc DNA3.1-KAP1+ 5u M PD0325901).All groups were stimulated for 48 h.Western blot was used to detect Runx2 and downstream phenotypic transforming proteins Col I and a-SMA.Alizarin red staining was performed in each group to evaluate the calcification of VSMCs.Results:1.p-ERK was significantly increased in vascular tissues of CKD rats and in VSMCs induced by high phosphorus(P<0.001).2.Treatment of VSMCs with p-ERK inhibitor PD0325901 significantly reduced the expression of p-ERK and inhibited VSMCs osteogenic phenotype transformation(P<0.05).3.Compared with the control group,after overexpression of KAP1,the total-ERK was almost not affected,but the p-ERK protein was significantly increased(P<0.05),and the level of p38 did not change.4.Western blot results showed that,the activity of overexpression KAP1 on ERK signaling pathway and the enhancement of downstream Runx2 and COL I proteins were significantly inhibited after the addition of PD0325901.Compared with N+KAP1 overexpression group,the expression of a-SMA increased significantly after adding PD0325901.The results of alizarin red staining showed that after overexpression of KAP1 and then blocking of ERK pathway,the mineralized nodules in cells were significantly reduced compared with the N+ KAP1 overexpression group.Summary : The ERK signaling pathway was activated in the calcification environment of chronic kidney disease.KAP1 promotes calcification of vascular smooth muscle cells through the ERK/Runx2 signaling pathway.Conclusions:1.The expression of KAP1 in vascular smooth muscle cells was significantly increased in patients with vascular calcification in CKD and in rats with vascular calcification in CKD.KAP1 may be involved in the process of vascular calcification in CKD.2.KAP1 expression was significantly increased in rat vascular smooth muscle cells stimulated by high phosphorus,and its expression level was consistent with the expression of osteogenic transcription factor Runx2.KAP1 inhibition by plasmid transfection significantly reduced the osteogenic phenotype and calcification of vascular smooth muscle cells under high phosphorus environment,suggesting that KAP1 promotes vascular calcification in chronic kidney disease.3.ERK signaling pathway is activated in vascular smooth muscle cells of rats with vascular calcification in CKD.KAP1 promotes VSMC calcification through ERK/Runx2 signaling pathway. |
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