Measurement of urinary albumin using newly developed enzyme immunoassay in the rat

Abstract

Background. Since it is well known that microalbuminuria predicts the late development of diabetic nephropathy, the measurement of urinary albumin (Ualb) is important in human diabetic nephropathy. Ualb also increases in diabetic rats and the measurement of Ualb in the rat is important to determine diabetic nephropathy. Many methods have been developed for measuring human Ualb. However, there are few methods for rat Ualb assay. Since specific antialbumin antibody for rat should be used, the aim of this study was to basically evaluate a new enzyme immunoassay for determining Ualb in rats.

Methods. 24-h urine samples from male Sprague-Dawley (SD) rats and diabetic spontaneously hypertensive rats (SHR) were collected and measured.

Results. (1) Assay sensitivity was 16 ng/ml. (2) The urinary pH and presence of coexistent substances such as bovine serum albumin, glucose, urea, bilirubin, and hemoglobin, did not influence the Ualb measurement. (3) No changes were observed in Ualb after 28-day storage at 4°C, −20°C, or −80°C. (4) The coefficient of variation between 2.2 and 8.2% was 3.3 ± 0.9%. (5) The entire assay, including sample preparation, reagent addition, incubation, and end-point determination, can be completed within 90 min. (6) Ualb increased in SHR induced by streptozotocin compared with findings in non-diabetic SHR.

Conclusions. These data demonstrated that this method for the measurement of rat Ualb, is rapid, accurate, and sensitive, and it should also be helpful in the field of research in experimental diabetic nephropathy.