Effect Of Trimethylamine-N-oxide On CKD Rat Vascular Calcification And The Mechanisms Exploration

BackgroundCardiovascular complications are the main clinical problems in patients with chronic renal disease(CKD).Vascular calcification is a common cardiovascular complication of CKD.The increase of vascular calcification indicates the decrease of overall survival rate in patients with CKD.The exact mechanism of vascular calcification in CKD has not been fully elucidated.Inflammation is closely related to the occurrence and development of vascular calcification.Chronic inflammation is considered to be an important cause of ectopic soft tissue calcification.Recently,trimethylamine oxide(TMAO),a metabolite of intestinal flora,has attracted extensive attention because of its potential to promote atherosclerosis,cardiovasc?lar disease(CVD)and renal disease.In CKD,the increase of TMAO concentration is related to systemic inflammatory markers.However,the mechanism between TMAO and vascular calcification is not clear.ObjectiveVascular calcification is highly prevalent in patients with CKD.Increased plasma TMAO concentrations are found in patients with CKD.However,The effect of TMAO on vascular calcification is not yet established.In this study,we investigate whether TMAO participates in the progression of vascular calcification and its mechanism using in vitro,ex vivo and in vivo models.Methods and results1.To investigate the relationship between plasma TMAO levels and vascular calcification in patients with CKD,we compared the plasma TMAO level between aortic non-calcification group and aortic arch calcification group in CKD patients.We found that plasma TMAO levels in patients with aortic arch calcification were higher than those without aortic arch calcification,suggesting that TMAO may play a potential role in the pathogenesis of vascular calcification in patients with CKD.2.Rat vascular smooth muscle cells(VSMCs)were treated with TMAO(100?M?300?M?600?M)for 7 days under the induction of calcification medium.The results of alizarin red staining showed that TMAO treatment promoted the calcium deposition in VSMCs,the calcium content in VSMCs was significantly higher than that in calcified medium,and the m RNA expression of osteogenic differentiation related genes Runx2 and BMP2 was significantly higher than that in calcified medium.Western blot analysis further confirmed the positive regulatory effect of TMAO on the expression of Runx2 and BMP2.TMAO promoted calcified medium-induced calcification of VSMCs.similarly,we found that TMAO promoted calcified medium-induced calcification of aortic vascular rings;Further in vivo studies showed that TMAO promoted aortic calcification in rats CKD.3.In addition,we found that TMAO activated NLRP3 inflammatory and NF-?B signaling pathways in the process of promoting osteogenic differentiation and calcification of VSMCs.In order to further determine whether NLRP3 inflammasome and NF-?B signals regulate TMAO-induced calcification of VSMCs,MCC950(a selective inhibitor of NLRP3)and PDTC(an inhibitor of NF-?B)respectively were used to treat VSMCs in the presence of calcifying medium together with TMAO.Alizarin red staining showed that both MCC950 and PDTC decreased the calcium deposition in VSMCs on the 7th day,calcium content decreased significantly and the mRNA expression of Runx2 and BMP2 decreased significantly.These results suggest that NLRP3 inflammasome and NF-?B sign.als are involved in TMAO-induced calcification of VSMCs.Furthermore,we found that NF-?B signal regulated NLRP3 inflammasome and vice versa.These results suggest the NF-?B/NLRP3 signaling loop participates in TMAO-induced VSMC calcification.ConclusionsThis study for the first tine demonstrates that TMAO promotes vascular calcification through activation of NLRP3 inflammasome and NF-?B signals.Reducing the levels of TMAO could become a potential treatment strategy for vascular calcification in CKD.