Mechanism Of Wnt1/?-catenin Pathway In Leptin Induced Endothelial Dysfunction In Chronic Kidney Disease

Backgroud/Obj ectiveChronic kidney disease(CKD)arises from many heterogeneous disease pathways that alter the function and structure of the kidney irreversibly,over months or years.Studies have shown that cardiovascular disease(CVD)is associated with CKD,and the mortality in CKD is significantly higher than that in patients with normal kidney,which is the cause of death in more than half of CKD patients.Atherosclerosis originates from endothelial cells.Endothelial dysfunction(ED)is the basis of atherosclerosis,which is obvious in the early stage of CKD patients.With the development of the disease to end stage kidney disease(ESKD),the incidence of ED is gradually increasing,and the risk of CVD death is also increasing.At present,the mechanism of ED in CKD is not clear.Many pathways including inflammation,oxidative stress,low vitamin D,hyperphosphatemia,and accumulation of endogenous inhibitors upstream of NO synthase are involved in it.Leptin is a 16 kDa protein synthesized and secreted by adipose tissue.Recent studies have shown that leptin not only participates in energy metabolism,but also promotes oxidative stress,inflammation and lipid disorder,thus increasing the risk of CVD.Leptin is also related to angiogenesis,immune regulation and hemostatic factors.Leptin is a biomarker of coronary heart disease/acute coronary syndrome risk.In addition,leptin is eliminated by the kidney through glomerular filtration and metabolic degradation of the renal tubules.Nevertheless,few researchers have studied the relationship between plasma leptin levels and CKD.Therefore,existing research results suggest that leptin may be the missing link between inflammation and ED in CKD.Wnt pathway regulates cell proliferation and survival by controlling the expression of differentiation related genes.So far,three Wnt pathways have been found:one ?-catenin dependent pathway and two ?-catenin independent pathways.The Wnt/?-catenin signaling pathway is an evolutionarily conserved signaling cascade for cell adhesion,embryonic development,damage repair and homeostasis of tissues and organs.Metastasis associated protein 1(MTA1)is a component of nucleosome remodeling and histone deacetylation complex.It is widely expressed in a variety of human cell lines and cancer tissues,and plays an important role in cell survival,diffusion,migration and invasion.A large number of studies suggest that MTA1 is an important regulatory molecule of Wntl pathway.In the study of non-small cell lung cancer cells,the down-regulation of MTA1 expression can inhibit Wnt/?-catenin pathway,and up-regulation of MTA1 expression can activate Wnt/?-catenin pathway.In recent years,studies have confirmed that Wnt/?-catenin pathway plays an important role in endothelial cell injury.Simvastatin can promote ED by activating Wnt/?-catenin pathway under oxidative stress.Therefore,we speculate that MTA1/Wnt/?-catenin pathway may play an important role in leptin induced ED in CKD patients.Therefore,we collected and compared the clinical data and serum samples of CKD patients and healthy controls(HCs),detected the levels of leptin and ED related serum markers,and further detected the flow mediated dilation(FMD)in this study;nevertheless,we also investigated the pathogenesis of leptin and ED through human umbilical vein endothelial cells(HUVECs)cultured in vitro.Our study will provide new ideas and therapeutic targets for the further prevention and treatment of ED in CKD patients.Materials and methodsThis study was divided into two parts.The first part was a clinical study to determine the relationship between leptin and ed in CKD patients.The second part is in vitro experiment to explore the effect of leptin on endothelial cells and its mechanism.1.Clinical study1.1 ParticipantsPatients attending the Department of Nephrology,Shandong Provincial Hospital Affiliated with Shandong University from January 2014 to December 2019 who met the inclusion and exclusion criteria were assessed for this study.HCs,whose age and gender matched the patients,were also enrolled.1.2 Clinical data collectionThe age,gender,smoking history,onset time and past histories of the patients were recorded in detail by questionnaire,and anthropometry was completed according to the standard measurement method,including blood pressure,height and weight.The measurement of height and weight need to be completed at the same time,and then the body mass index(BMI)is calculated.1.3 Blood sample collection and detection1.3.1 Routine laboratory testingSerum creatinine,triglycerides,total cholesterol,low density lipoprotein cholesterol,high density lipoprotein cholesterol,fast blood glucose,serum insulin and hypersensitive C-reactive protein were detected.1.3.2 Enzyme-linked immunosorbent assays(ELISA)The levels of serum leptin,interleukin-6(IL-6),monocyte chemoattractant protein-1(MCP-1)and endothelin-1(ET-1)were detected.1.3.3 Homeostasis model assessment of insulin resistance calculation 1.4 Flow-mediated dilatation(FMD)was detected to evaluate endothelial function.2.Vitro experimentHUVECs were used in this study.2.1 Effects of leptin on the synthesis of inflammatory factors in HUVECs HUVECs were cultured in vitro and stimulated with leptin(100 ng/ml)for different time(0 h,6 h,12 h,24 h).The levels of IL-6,MCP-1 and ET-1 in the supernatant of HUVECs were detected by ELISA.The gene and protein levels of IL-6,MCP-1 and ET-1 in HUVECs were measured by real-time polymerase chain reaction(RT-PCR)and Western blot(WB).2.2 Effect of leptin on migration of HUVECs2.2.1 Scratch healing testWhen HUVECs covered 80%of the surface with layers of cells,a sterilized pipette tip was scratched into a line with the same width.The initial scratch width of HUVECs was recorded as A;the scratch width of HUVECs in intervention group and control group was recorded as B after 12 h.Scratch healing rate=(A-B)/A×100%.2.2.2 Transwell cell migration assayHUVECs single cell suspension of 100 UL in control group and intervention group were added into Transwell chamber and cultured for 7 h to make cells migrate.2.3 Effect of leptin on HUVECs monolayer permeability HUVECs were cultured in Transwell upper chamber(membrane aperture:0.4 ?m)to form endothelial monolayer.The permeability of endothelial monolayer to FITC dextan was detected to explore the effect of leptin on HUVECs monolayer permeability.2.4 The effect of leptin on the skeleton rearrangement of HUVECs FITC phalloidin was used to stain the F-actin of ECS under different intervention conditions to explore whether leptin can induce cyto skeleton reorganization.The changes of vinculin were detected by laser confocal microscopy immunofluorescence.2.5 Molecular mechanism of leptin induced ED After 24 h of leptin treatment,the protein levels of MTA1,Wnt1,?-catenin and phosphorylated ?-catenin(Y654)were measured by WB.To verify the role of MTA1 and Wnt1/?-catenin pathway in leptin induced ED,we further constructed MTA1 shRNA and Wnt shRNA lentivirus and transfected HUVECs respectively.After MTA1 or Wntl knockout,the expression of inflammatory factors,cell migration,permeability,cytoskeleton rearrangement and phosphorylation of ?-catenin in HUVECs were detected.Results1.Clinical study1.1 The serum leptin concentrations were significantly increased in the CKD patients compared with the healthy controls,and the difference was statistically significant(7.89(3.79-9.33)ng/ml vs 5.49(5.49-7.73)ng/ml;P=0.039)?1.2 The serum levels of IL-6,MCP-1 and ET-1 in CKD patients were significantly higher compared with HCs,(IL-6:6.18(4.21-7.25)pg/ml vs 4.89(3.89-5.91)pg/ml,P=0.002;MCP-1:213.48(158.33-269.34)pg/ml vs 168.72(117.30-214.62)pg/ml,P<0.001;ET-1:2.27(1.41-3.06)pg/ml vs 1.34(0.97-1.77)pg/ml,P<0.001).1.3 These CKD patients had a lower FMD than the controls(4.49(2.83-5.74)in patients vs.8.29(5.52-9.07)in controls,P<0.05).1.4 Serum leptin levels were positively correlated with IL-6,MCP-1 and ET-1 levels in CKD patients(IL-6:r=0.119,P=0.034;MCP-1:r=0.115,P=0.039;ET-1:r=0.144,P=0.010);serum leptin levels were negatively correlated with FMD(r=-0.294,P=0.006).2.Vitro study2.1 Effect of leptin on infammatory factor expression in HUVECs To determine the direct effect of leptin on ED,we examined the expression of HUVECs proinflammatory cytokines(IL-6,ET-1 and MCP-1)after leptin(100 ng/ml)stimulation at 0,6,12,24 and 48 h by ELISA.After 12 h of leptin incubation,the levels of IL-6,ET-1,and MCP-1 increased significantly,with the greatest effect after 24 h of exposure.The results of RT-PCR showed that compared with those of the control group,the mRNA levels of IL-6,ET-1 and MCP-1 in the HUVECs increased after 24 h of leptin exposure.WB analysis showed that 100 ng/ml leptin induced the expression of inflammatory factors at the protein level.2.2 Leptin promotes endothelial cell migration In the scratch assay,100 ng/ml leptin increased endothelial cell migration compared with the control(P<0.01).We also performed a transwell migration assay using the same cell set and found that leptin resulted in strong upregulation of migration compared with the control(P<0.01).2.3 Leptin increases the monolayer permeability of HUVECs Compared with the control group,the fluorescence intensity of FITC dextran in Transwell inferior ventricle in leptin stimulated group was significantly increased(P<0.01)2.4 Leptin induces the remodeling of the F-actin cytoskeleton and vinculin After leptin stimulation,the actin skeleton distribution at the cell edge was significantly decreased,and F-actin was significantly disordered,thickened and clustered into bundles to form stress fibers across the cell body,accompanied by an obvious increase in vinculin recruitment.2.5 Molecular mechanism of leptin induced ED2.5.1 Leptin induces ED by activating MTAl-Wntl pathway WB showed that Wnt1 and MTA1 were significantly increased in leptin treated HUVECs for 24 hours(P<0.01).In order to further determine whether MTA1 and Wntl were involved in leptin induced ED,we used lentivirus to transfect MTA1 shRNA and Wnt1 shRNA to reduce their expression,and verified the knockout efficiency(P<0.01).In addition,after transfecting HUVECs with MTA1 shRNA,the increase of Wntl expression induced by leptin stimulation was significantly inhibited(P<0.05),while Wnt shRNA did not affect the expression of MTA1 induced by leptin(P>0.05).WB and RT-PCR showed that knockdown of MTA1 and Wnt1 significantly reduced the increased expression of IL-6,MCP-1 and ET-1 mRNA and protein induced by leptin stimulation(P<0.05);wound healing test and Transwell test showed that knockdown of MTA1 and Wntl could significantly improve the migration ability of HUVECs stimulated by leptin(P<0.05);meanwhile,FITC phalloidin staining showed that knockdown of MTA1 and Wnt1 knockdown could reduce the cytoskeleton damage caused by leptin.2.5.2 Leptin promote ED by inducing nuclear translocation and phosphorylation of ?-CateninWB showed that after leptin stimulation,the total protein of ?-catenin in HUVECs had no significant change compared with the control group(P>0.05),but the levels of nuclear ?-catenin and phosphorylated ?-catenin(Y654)were significantly increased(P<0.01).The expression of total ?-catenin protein in MTA1 or Wnt1 knockdown HUVECs stimulated by leptin was not significantly different from that in wild-type HUVECs stimulated by leptin(P>0.05),but the levels of nuclear ?-catenin and phosphorylated ?-catenin(Y654)were significantly lower than those in wild-type HUVECs stimulated by leptin(P<0.05)Conclusion1.Leptin levels in CKD patients were elevated,and the serum leptin concentration was associated with endothelial dysfunction.2.Leptin can mediate endothelial dysfunction by inducing the synthesis of inflammatory factors,leading to the rearrangement of F-actin skeleton and the enhancement of cell migration and monolayer permeability.3.Leptin induces nuclear migration and phosphorylation of ?-catenin by activating MTA1/Wnt1,and then mediates endothelial dysfunction.