AIMS:Cardiovascular events remain the leading cause of death in Western world. Atherosclerosis is the most common underlying complication driven by low-density lipoproteins (LDL) disturbing vascular integrity. Carbamylation of lysine residues, occurring primarily in the presence of chronic kidney disease (CKD), may affect functional properties of lipoproteins; however, its effect on endothelial function is unknown.METHODS AND RESULTS:Low-density lipoprotein from healthy donors was isolated and carbamylated. Vascular reactivity after treatment with native LDL (nLDL) or carbamylated LDL (cLDL) was examined in organ chambers for isometric tension recording using aortic rings of wild-type or lectin-like-oxidized LDL receptor-1 (LOX-1) transgenic mice. Reactive oxygen species (ROS) and nitric oxide (NO) production were determined using electron spin resonance spectroscopy. The effect of LDL-carbamyl-lysine levels on cardiovascular outcomes was determined in patients with CKD during a median follow-up of 4.7 years. Carbamylated LDL impaired endothelium-dependent relaxation to acetylcholine or calcium-ionophore A23187, but not endothelium-independent relaxation to sodium nitroprusside. In contrast, nLDL had no effect. Carbamylated LDL enhanced aortic ROS production by activating NADPH-oxidase. Carbamylated LDL stimulated endothelial NO synthase (eNOS) uncoupling at least partially by promoting S-glutathionylation of eNOS. Carbamylated LDL-induced endothelial dysfunction was enhanced in LOX-1 transgenic mice. In patients with CKD, LDL-carbamyl-lysine levels were significant predictors for cardiovascular events and all-cause mortality.CONCLUSIONS:Carbamylation of LDL induces endothelial dysfunction via LOX-1 activation and increased ROS production leading to eNOS uncoupling. This indicates a novel mechanism in the pathogenesis of atherosclerotic disease which may be pathogenic and prognostic in patients with CKD and high plasma levels of cLDL.

Introduction and Aims: According to the modern tendencies, the important formation factors of cardiovascular calcification in chronic kidney disease (CKD) are oxidative stress (OS) and chronic inflammation. However, the role of the indicated processes in mechanisms of cardiac valve calcification (CVC) in patients under the predialysis period of CKD were not asserted enough. In this conditions, it is also reasonable to research the functional activity of endothelium, especially of nitric oxide (NO) system, since endothelial dysfunction is one of the key mechanisms, which mediated inflammatory effects on the cardiac valve apparatus. The purpose of the current study was (1) to determine the role of activation of free radical oxidation of lipids and systemic inflammation in mechanisms of valve calcification in predialysis CKD patients, and (2) to establish in CVC group the relationship between the inflammatory markers and NO system state. Methods: We enrolled 167 (male/female, 78/89; age, 48.7 ± 13.2 years; eGFR-MDRD, 51.0 ± 28.2 ml/min per 1.73 m) predialysis (stage I: 8.4%, stage II: 28.1%, stage III: 38.9%, stage IV: 18.0%, stage V: 6.6%) CKD patients. All subjects underwent echocardiographical examination for detection of valve calcification. Plasma content of malondialdehyde (MDA), superoxide dismutase (SOD) and catalase (CAT) activities, ceruloplasmin (CP) and glutathione (GSH) concentrations as indices of pro/antioxidant system were determined using the standard methods. Inflammatory markers were performed by evaluating the serum levels of C-reactive protein (CRP) (immunoturbidimetric method), fibrinogen (gravimetric analysis) and circulating immune complexes (CICs) (polyethylene glycol precipitation test). Plasma content of nitrites (NO2-) (Green L.C. et al., 1982) was measured as marker of NO system activity. Data are presented as mean ± SD. Used nonparametric statistics methods: Mann-Whitney Utest in order to compare indices in two groups, Spearman’s rank R correlations for establishment of presence and strength of the relationship between the research parameters. Results: CVC was detected in 28.7% of predialysis CKD patients: isolated calcification of mitral valve – in 6.0%, aortal valve – in 7.2%, both valves – in 15.6%. In CVC group indices of MDA (p < 0.001), CP (p < 0.001), CRP (p < 0.001), fibrinogen (p = 0.005), CICs (p = 0.010) were higher, and SOD (p < 0.001), CAT (p = 0.061), GSH (p = 0.005) – lower compared to the group without calcification. Predialysis CKD patients with valve calcification had lower content of NO2(0.053 ± 0.016 vs. 0.070 ± 0.016 mmol/L, Z = 4.999, p < 0.001) than subjects without calcification. For the first time it was established that in CVC group (n = p;48) NO2level was correlated with CRP (R = −0.565, p < 0.001), fibrinogen (R = −0.415, p = 0.003) and CICs (R =− 0.512, p < 0.001) concentrations. Conclusions: (1) CVC in predialysis? CKD patients is combined with the development of OS and systemic inflammation. (2) Valve calcification under the predialysis period of CKD is characterized by the reduced content of stable metabolite of NO – NO2-, which in turn is closely related with the inflammatory parameters. (3) Availability of complex of metabolic disturbances in predialysis patients with CVC points towards the necessity of the search of means with normalizing influence on the level of lipid peroxidation, antioxidant defense, inflammatory processes and NO system state for prevention and treatment of the indicated cardiac damages.