Comparison of three methods to quantify urinary aquaporin-2 protein

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Comparison of three methods to quantify urinary aquaporin-2 protein Fuminori Umenishi, Sandra N. Summer, Melissa Cadnapaphornchai, Robert W. Schrier Kidney International Volume 62, Issue 6, Pages 2288-2293 (December 2002) DOI: 10.1046/j.1523-1755.2002.00686.x Copyright © 2002 International Society of Nephrology Terms and Conditions

Figure 1 Characterization of the radioimmunoassay (RIA) for the measurement of urinary aquaporin-2 (AQP2). (A) Standard curve of AQP2 RIA. The human AQP2 peptide was serially diluted for making standard curve. (B) Urine dilution curve. Urine samples were serially diluted with PBS. Kidney International 2002 62, 2288-2293DOI: (10.1046/j.1523-1755.2002.00686.x) Copyright © 2002 International Society of Nephrology Terms and Conditions

Figure 2 Characterization of the immunoblot (IB) analysis for the measurement of urinary AQP2. (A) Immunoblot of BSA-AQP2 and urinary AQP2 proteins. The known amounts of BSA-AQP2 (10, 20, 30, 50, 75, 100, 150, and 200 pg) were used as a standard and were immunoblotted with urine samples (2, 4, 6, 8, and 10 μL). (B) The band intensities at different concentration of BSA-AQP2 were quantitated by densitometry scanning and the standard curve was obtained to quantitate the amount of urinary AQP2. Kidney International 2002 62, 2288-2293DOI: (10.1046/j.1523-1755.2002.00686.x) Copyright © 2002 International Society of Nephrology Terms and Conditions

Figure 3 Characterization of the enzyme-linked immunosorbent assay (ELISA) for the measurement of urinary AQP2. (A) Calibration curve. The calibration curve for the assay of urinary AQP2 was obtained by using the AQP2 peptide at concentrations of 0 to 400 pg. (B) Effects of SDS on the absorption of urinary AQP2 to a 96-well microplate. Urine samples were treated with various concentrations of SDS from 0 to 0.5%. The ELISA was performed as described in the Methods section. All values are shown as means ± SEM (pg/mL). (C) Urine dilution curve. Urine samples were serially diluted with PBS. Kidney International 2002 62, 2288-2293DOI: (10.1046/j.1523-1755.2002.00686.x) Copyright © 2002 International Society of Nephrology Terms and Conditions

Figure 4 Comparison of the (A) RIA, (B) IB, and (C) ELISA methods for quantifying urinary AQP2. AQP2 was measured in the urine at 0, 1, 2, 3, and 4 hours after water oral loading. All values are shown as means ± SEM (fmol/mg creatinine). Kidney International 2002 62, 2288-2293DOI: (10.1046/j.1523-1755.2002.00686.x) Copyright © 2002 International Society of Nephrology Terms and Conditions

Figure 5 Correlations between values of urinary AQP2 determined by the RIA, IB, and ELISA methods. Correlations between values were evaluated by the Pearson's correlation test. (A) r = 0.91, P < 0.0001; (B) r = 0.75, P < 0.0001; (C) r = 0.67, P < 0.0002. Kidney International 2002 62, 2288-2293DOI: (10.1046/j.1523-1755.2002.00686.x) Copyright © 2002 International Society of Nephrology Terms and Conditions

Figure 6 Correlations between urinary AQP2 and urinary osmolality (UOsm). Correlations between values were evaluated by the Pearson's correlation test. (A) r = 0.73, P < 0.0001; (B) r = 0.81, P < 0.0001; (C) r = 0.77, P < 0.0001. Kidney International 2002 62, 2288-2293DOI: (10.1046/j.1523-1755.2002.00686.x) Copyright © 2002 International Society of Nephrology Terms and Conditions