Comparison of three methods to quantify urinary aquaporin-2 protein

Abstract

Background. Urinary excretion of aquaporin-2 (AQP2) is measurable, and is regulated by renal vasopressin action in the principal cells of the collecting duct. To date, two methods [radioimmunoassay (RIA) and quantitative immunoblot analysis (IB)] have been used for quantitation of urinary AQP2 protein. However, the actual amount of urinary AQP2 measured has not been directly compared by the RIA and IB. Recently, we have established an enzyme-linked immunosorbent assay (ELISA) for quantitation of urinary AQP2. The purpose of our current study was to compare three different immunoassay methods for measurement of urinary AQP2. Methods. After overnight dehydration, five normal subjects ingested an oral water load (20 mL/kg). Urine was collected at 0, 1, 2, 3, and 4 hours after oral water loading. Urinary AQP2 protein was quantitated in each sample by using the RIA, IB, and ELISA, and the correlation coefficients were compared among three different methods. Results. Values of urinary AQP2 at 0, 1, 2, 3, and 4 hours after oral water loading for RIA, IB, and ELISA were, respectively (fmol/mg creatinine): 0 h: 266 ± 28, 405 ± 74, 294 ± 41; 1 h: 159 ± 59, 267 ± 147, 195 ± 95; 2 h: 48 ± 17, 19 ± 10, 38 ± 18; 3 h: 79 ± 18, 70 ± 24, 80 ± 15; 4 h: 147 ± 21, 161 ± 31, 136 ± 15. All values were shown as means ± SEM. There was a significant positive correlation between: the IB and ELISA (r = 0.91, P < 0.0001); the IB and RIA (r = 0.75, P < 0.0001); and the RIA and ELISA (r = 0.67, P = 0.0002). The correlation between the IB and ELISA was therefore the best. Also, urinary AQP2 was positively correlated with urine osmolality among all three methods. Conclusions. The results indicate that the newly developed IB and ELISA methods are useful for measurement of urinary AQP2 and have an excellent correlation.