| Background/Aims:In the past decades,chronic kidney disease(CKD)has become a global public health problem due to its high prevalence,deleterious effects,and tremendous health care expenditures.Cardiovascular disease is the main complication of patients with chronic kidney disease and remains the leading cause of morbidity and mortality in chronic kidney disease,especially in end stage renal disease(ESRD).However,the high prevalence and incidence of cardiovascular disease in patients with CKD cannot be adequately explained by traditional risk factors,therefore other non-traditional risk factors such as endothelial dysfunction have aroused both nephrologist and cardiologist's great interest.Previous studies have shown that endothelial dysfunction is an initiator of cardiovascular events,and it's closely related to the occurrence and development of chronic kidney disease.Therefore,early and accurate detection of vascular endothelial function in patients with CKD is very important for the diagnosis,treatment and prognosis of CKD.Leptin is a pleiotropic hormone mainly produced in adipose tissue and plays vital roles in regulating food intake and energy expenditure.In addition,leptin has been shown to be involved in many other important physiological processes,including neuroendocrine,angiogenesis,reproductive development,immune regulation and wound repair.Recent clinical studies have revealed positive correlations between leptin and cardiovascular parameters,such as myocardial infarction,arterial rigidity and coronary atherosclerosis.And animal studies have also reported that leptin administration can induce high blood pressure and proteinuria.Leptin is cleared from the circulation by the kidney through glomerular filtration and metabolic degradation in renal tubules and many studies have reported elevation of serum leptin levels in CKD patients.However,previous researches offered suggestive but limited evidence for a pathogenic role of leptin in CKD.Especially,data about the influence of leptin on the endothelium status in adults with CKD are scarce.Hence,the present study was done to evaluate the serum concentration changes of leptin in CKD patients compared with healthy subjects,analyze the putative association between leptin and endothelial function in CKD adults and then explore how leptin contributes to endothelial dysfunction via in vitro experiments,which aims at finding out a new way to understand the high morbidity of cardiovascular events and a potential protective mechanism for endothelium in patients with CKD.MethodsThis study includes two parts:?clinical cross-sectional study,which aimed at evaluating the serum concentration changes of leptin in CKD patients compared with healthy subjects and analyzing the association between leptin and endothelial function in CKD adults;?in vitro experiments,which were done to explore the effect of leptin on endothelial function and the possible signals involved in leptin-induced endothelial dysfunction.1 Clinical cross-sectional studyA total of 140 non-diabetic,clinical stable CKD patients and 140 healthy subjects who were age-and gender-matched were enrolled in this study.We collected the basic information,including sex,age,height,weight and blood pressure,and blood lipid,blood glucose,liver and kidney function,high sensitive C reactive protein and fasting blood samples.After collecting all samples,we measured serum sICAM-1,sVCAM-1 and leptin levels with commercially available ELISA kits according to the manufacturer's specifications.What's more,we further detected brachial artery flow-mediated dilatation(FMD)in 46 patients and 30 controls to evaluate their endothelial function.Data were analyzed with SPSS 17.0 software.The correlation between continuous variables was assessed by Spearman's rho.P<0.05 was considered statistically significant.2 In vitro experiments2.1 The effect of leptin on adhesion molecule production of HUVECs:we cultured HUVECs in vitro with leptin(100ng/ml)for different time,and then examined sICAM-1,sVCAM-1 levels in the supernatants by ELISA according to the manufacturer's instructions.We also examined total ICAM-1 and VCAM-1 expression of HUVECs under leptin stimulation,at both the mRNA and protein levels,using real-time PCR and western blot.2.2 The effect of leptin on endothelial cell migration:we performed a scrap-wound-healing assay on a monolayer of HUVECs and the modified Transwell(polycarbonate membrane inserts for 24-well plate with pore diameter size:8 ?m)migration assay simultaneously to test the motility of cultured HUVECs subjected to leptin.2.3 The effect of leptin on endothelial monolayer permeability:HUVECs were plated in the upper compartments of chamber(clear polyester membrane inserts for 12-well plate with pore diameter size:0.4 ?m)and incubated overnight to allow formation of monolayers,and then paracellular permeability of endothelial monolayer was studied in a Trans well system by measuring the flux of 40kDa FITC-dextran across the monolayer.2.4 The effect of leptin on cytoskeleton rearrangement of HUVECs:cytoskeleton changes of HUVECs under different conditions were captured using a confocal microscope after we stained F-actin and vinculin.2.5 The signal pathways involved in leptin-induced endothelial dysfunction:western blot was used to explored the the impact of leptin on AKT/GSK3? and P-catenin signals,including the protein expression of AKT,p-AKT,GSK3P and p-GSK3?;and changes of P-catenin at total and nuclear protein level.To further ascertain the role of AKT and ?-catenin signaling pathway in leptin-induced endothelial dysfunction,we constructed AKT shRNA plasmid and ?-catenin shRNA lentivirus to knock down the gene expression of AKT or ?-catenin.After transfecting HUVECs with shRNA vectors,we examined adhesion molecules production,endothelial cell migration and permeability changes,as well as cytoskeleton alteration.Results1 Clinical cross-sectional study:1.1 Leptin levels are significantly raised in CKD patients than healthy controls.1.2 Serum adhesion molecule levels are increased and FMD is decreased in CKD patients than controls.1.3 Spearman's correlation shows that leptin expression significantly positively relates to sICAM-1 and sVCAM-1 in CKD patients.In addition,elevated leptin is significantly associated with the reduction of FMD in 46 patients with CKD.2 In vitro experiments:2.1 Leptin induces adhesion molecule production of HUVECs:when we cultured HUVECs with leptin for 6,12,24,and 48 hours,significant increase of both sICAM-1 and sVCAM-1 levels in the supernatants could be observed following 12 hours' incubation with leptin,with the greatest effect after 24 hours' exposure.After24 hours' incubation with leptin,we also observed a dramatic increase of total ICAM-1 and VCAM-1 expression at both mRNA and protein levels.2.2 Leptin promotes endothelial cell migration:Compared with control cells,leptin enhanced endothelial cell migration,resulting in accelerating closure speed of the uniform-width scratch wound and increasing cell migration through Transwell membranes with pores.2.3 Leptin increases endothelial monolayer permeability:Compared with control cells,FITC-dextran fluorescent intensity in the media from abluminal chamber stimulated by leptin was stronger.The difference was statistically significant.2.4 Leptin promotes cytoskeleton rearrangement of HUVECs:Compared with untreated cells,remarkable reorganization of the microfilament network could be observed,which was characterized by the formation of stress fibers that spanned the cell body.The stress fibers became dramatically thick and robust.What's more,vinculin,as a necessary protein for cross-linking of actin filaments,highly expressed after leptin treatment.2.5 The signal pathways involved in leptin-induced endothelial dysfunction:2.5.1 AKT/GSK3? pathway is required in leptin-induced endothelial dysfunction:Leptin induced a significant upregulation of p-AKT accompanying with hyper-phosphorylation of GSK3?.Otherwise,leptin made no influence on total protein expression of AKT or GSK3?.After AKT knockdown,adhesion molecular overexpression,endothelial motility change,monolayer permeability increase and cytoskeleton disorganization caused by leptin stimulation,could be partially mimicked.P-GSK3? overexpression could also be abrogated by AKT knockdown.2.5.2 Nuclear accumulation of ?-catenin is involved in leptin-induced endothelial dysfunction:Compared with control group,leptin did not alter total ?-catenin protein but upregulated nuclear expression of ?-catenin.?-catenin knockdown could partially but obviously inhibit leptin-induced increase in adhesion molecules synthesis and disruption of F-actin cytoskeleton.Meanwhile,the endothelial cell motility was weakened;permeability was ameliorated.We also determined the impact of AKT knockdown on ?-catenin translocation under leptin stimulation and discovered that pretreatment with AKT shRNA vectors demonstrated a notable abrogation of ?-catenin translocation.Conclusion1.In patients with CKD,elevated leptin level is associated with endothelial dysfunction.2.Leptin can induce endothelial dysfunction in vitro,which is charactered by adhesion molecular overexpression,endothelial migration enhancement and monolayer permeability increase.3.Leptin may contribute to endothelial dysfunction by disarrangement of F-actin cytoskeleton via a mechanism involving the AKT/GSK3? and ?-catenin pathway. |
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