Assessment of thrombin in the urine of glomerulonephritic patients by enzyme-linked immunosorbent assay

Abstract

Background: Accumulating evidence suggests that blood clotting occurs in inflamed glomeruli, although its role in the pathophysiology of glomerulonephritis remains to be elucidated. To address this issue, a simple and reliable method for evaluating clotting in glomeruli is necessary. Here, we developed an enzyme-linked immunosorbent assay (ELISA) for thrombin in urine to evaluate the degree of clotting activation in diseased glomeruli. Methods: Monoclonal antibodies against human α-thrombin were raised and used for sandwich ELISA to measure thrombin. Thrombin was measured in urine samples from normal volunteers and from patients with glomerulonephritis or disseminated intravascular coagulation (DIC). Results: Thrombin antigen was not detected in the urine of healthy volunteers or of patients with DIC, but was detected in the urine from two-thirds of glomerulonephritic patients. The average concentration in positive samples was 3.79 µg/L. Urinary thrombin concentrations measured by ELISA correlated well with thrombin activities measured by hydrolysis of a synthetic substrate. Conclusion: We suggest that thrombin antigen in urine measured by ELISA is not affected by systemic thrombin production in the vessels, and reflects blood clotting activation in glomerulonephritic lesions. A close relationship between urinary thrombin and glomerulonephritis indicates a possible involvement of clotting in disease development, and measurement of urinary thrombin may provide a real-time marker for monitoring renal diseases.