Urea-derived Cyanate Forms Epsilon-amino-carbamoyl-lysine (homocitrulline) in Leukocyte Proteins in Patients with End-stage Renal Disease on Peritoneal Dialysis

Carbamoylated proteins have been located by using a site-specific polyclonal antihomocitrulline antibody and a fluorescent secondary antibody in leukocytes from patients with end-stage renal disease who were undergoing maintenance continuous ambulatory peritoneal dialysis. A covalent reaction with urea-derived cyanate and the epsilon-amino group of lysine forms homocitrulline residues in carbamoylated proteins. Isocyanic acid, the reactive form of cyanate, is spontaneously formed from urea in aqueous solution at physiologic pH and temperature. In washed, fixed monolayers of cells, an intracellular fluorescent antigen-antibody complex was located throughout the cytoplasm of polymorphonuclear neutrophils (PMNs) and monocytes from 11 patients with blood urea nitrogen (BUN) levels ranging from 32 to 102 mg/dl who were undergoing dialysis for 2 to 135 months. A punctate fluorescence present in the cell surface proteins of living cells demonstrated that lysine residues in the external domain of proteins were carbamoylated, forming homocitrulline. In contrast, we found a perinuclear fluorescence in PMNs in normal subjects with no history of renal insufficiency and BUN levels of 6 to 19 mg/dl. This suggests that homocitrulline is located in carbamoylated proteins within the perinuclear membrane, a structural organelle continuous with the endoplasmic reticulum. It appears that continuous exposure to urea-derived cyanate in low levels results in increasing carbamoylation of stable proteins over the PMN's lifetime. When normal PMNs were exposed to 120 mmol/L cyanate ion in vitro for 10 to 30 minutes, the ability of PMNs to release microbicidal superoxide was strongly inhibited. Thus protein carbamoylation may provide a regulatory mechanism. The altered function of PMNs in renal disease may be due in part to the posttranslational modification of proteins by urea-derived cyanate.