Inhibition of Erythropoietin Activity by Cyanate

Abstract

Objective: Increased urea concentration is a measure of advanced renal failure and the adequacy of renal replacement therapy in end-stage renal disease (ESRD). Altered biologic activity due to changes in protein structure occurs when cyanate, formed spontaneously from urea, reacts with proteins. Carbamylation results in impaired erythropoietin (EPO) activity when high concentrations of cyanate react with EPO. In this study, the activity of carbamylated EPO (C-EPO), formed at a cyanate concentration which may occur in vivo, was studied in Sprague–Dawley rats. Material and Methods: The extent of carbamylation, causing loss of free amino groups, was monitored using trinitrobenzenesulfonic acid. Erythrocyte, hemoglobin, hematocrit and leukocyte levels were measured after either EPO, incubated EPO, C-EPO, physiologic saline or cyanate (1.5μM; 0.2ml) were injected subcutaneous twice weekly for 3 weeks in rats. Results: In vitro carbamylation of EPO was time- and concentration-dependent. C-EPO concentration increased as the duration of exposure to cyanate increased from 6 to 72 h, or as cyanate concentration increased from 15nM to 1.5μM. Injections of EPO caused significant increases in vivo in all erythropoietic measures. In contrast, injections of C-EPO, physiologic saline or 1.5μM cyanate caused no change from baseline. Conclusions: These results demonstrated diminished biologic activity in healthy rats by C-EPO formed in vitro at cyanate concentrations that may be found in vivo. C-EPO and high urea-derived cyanate levels may contribute to suboptimal erythropoietic responses to EPO therapy for chronic renal failure and ESRD, and may provide another measurement indicating inadequate dialysis.