Functional impairment of HDL may contribute to the excess cardiovascular mortality experienced by patients with renal disease, but the effect of advanced renal disease on the composition and function of HDL is not well understood. Here, we used mass spectrometry and biochemical analyses to study alterations in the proteome and lipid composition of HDL isolated from patients on maintenance hemodialysis. We identified a significant increase in the amount of acute phase protein serum amyloid A1, albumin, lipoprotein-associated phospholipase A2, and apoC-III composing uremic HDL. Furthermore, uremic HDL contained reduced phospholipid and increased triglyceride and lysophospholipid. With regard to function, these changes impaired the ability of uremic HDL to promote cholesterol efflux from macrophages. In summary, the altered composition of HDL in renal disease seems to inhibit its cardioprotective properties. Assessing HDL composition and function in renal disease may help identify patients at increased risk for cardiovascular disease.

MPO-mediated decomposition of SCN and/or urea might be a relevant mechanism for generating dysfunctional HDL in human disease.

Carbamylation (or carbamoylation) of lysine residues and protein N-termini is a ubiquitous, non-enzymatic post-translational modification. Carbamylation at sites of inflammation is due to cyanate formation during the neutrophil oxidative burst and may target lysine residues within the antimicrobial peptide LL-37, which is secreted by activated neutrophils. The bactericidal and immunomodulatory properties of LL-37 depend on its structure and cationic nature, which are conferred by arginine and lysine residues. Therefore, carbamylation may affect the biological functions of LL-37. This may be of great importance in the context of using LL-37 as a target for drug development. The present study examined the kinetics and pattern of LL-37 carbamylation to investigate how this modification affects the bactericidal, cytotoxic, and immunomodulatory function of the peptide. The results indicated that LL-37 undergoes rapid modification in the presence of physiological concentrations of cyanate, yielding a spectrum of diverse carbamylated peptides. Mass spectrometry analyses revealed that the N-terminal amino group of Leu-1 was highly reactive and was modified almost instantly by cyanate to generate the predominant form of the modified peptide, named LL37C1. This was followed by the sequential carbamylation of Lys-8, Lys-12, and Lys-15, to yield LL37C8, and LL37C12,15, respectively. Carbamylation had profound and diverse effects on the structure and biological properties of LL-37. In some cases, anti-inflammatory LL-37 was rapidly converted to pro-inflammatory LL-37. Thus, caution should be exercised when treating patients with severe inflammatory conditions, such as sepsis, with pro-inflammatory LL-37.

Collectively, our data raise the possibility that cyanate amplifies vascular inflammation, linking inflammation, smoking, and uremia.

SummaryCarbamylation is a non-enzymatic post-translational modification induced upon exposure of free amino groups to urea-derived cyanate leading to irreversible changes of protein charge, structure and function. Levels of carbamylated proteins increase significantly in chronic kidney disease and carbamylated albumin is considered as an important biomarker indicating mortality risk. High plasma concentrations and long half-life make fibrinogen a prime target for carbamylation. As aggregation and cross-linking of fibrin monomers rely on lysine residues, it is likely that carbamylation impacts fibrinogen processing. In this study we investigated carbamylation levels of fibrinogen from kidney disease patients as well as the impact of carbamylation on fibrinogen cleavage by thrombin, fibrin polymerisation and cross-linking in vitro. In conjunction, all these factors determine clot structure and stability and thus control biochemical and mechanical properties. LC-MS/MS analyses revealed significantly higher homocitrulline levels in patient fibrinogen than in fibrinogen isolated from control plasma. In our in vitro studies we found that although carbamylation does not affect thrombin cleavage per se, it alters fibrin polymerisation kinetics and impairs cross-linking and clot degradation. In addition, carbamylated fibrin clots had reduced fiber size and porosity associated with decreased mechanical stability. Using mass spectroscopy, we discovered that N-terminally carbamylated fibrinopeptide A was generated in this process and acted as a strong neutrophil chemoattractant potentially mediating recruitment of inflammatory cells to sites of fibrin(ogen) turnover. Taken together, carbamylation of fibrinogen seems to play a role in aberrant fibrin clot formation and might be involved in haemostatic disorders associated with chronic inflammatory diseases.