The apparent IC50 values for DMTU inhibition of UT-A1 were approximately independent of urea concentration, both with 0 intracellular [urea] and different extracellular [urea] (Fig. experienced a sustained, reversible reduction in urine osmolality from 1800 to 600 mOsm, a 3-collapse increase in urine output, and mild hypokalemia. DMTU did not impair urinary concentrating function in rats on a low protein diet. Compared to furosemide-treated rats, the DMTU-treated rats experienced higher diuresis and reduced urinary salt loss. In a model of Syndrome of Inappropriate Antidiuretic Hormone secretion, DMTU treatment prevented hyponatremia and water retention produced by water-loading in dDAVP-treated rats. Therefore, our results establish a rat model of UT inhibition and demonstrate the diuretic effectiveness of UT inhibition. screening of these compounds for diuretic effectiveness in rats. Seven urea analogs were also tested for UT inhibition (Fig. 2A). Two compounds, methylacetamide and dimethylthiourea (DMTU), showed UT-A1 inhibition activity, while the additional compounds were inactive (Fig. 2B). Fig. 2C summarizes UT-A1 and UT-B inhibition of the urea analogs, showing IC50 2C3 mM for DMTU inhibition of both UT-A1 and UT-B. Relatively poor inhibition was found for methylacetamide. Open in a separate window MIF Number 2 UT inhibition by urea analogsA. Structure of urea analogs tested. B. UT-A1 inhibition curves for urea analogs. C. Percentage inhibition of UT-A1 and UT-B urea transport (mean S.E, n=3). Characterization of urea transport inhibition by DMTU Concentration-inhibition measurements for DMTU inhibition of rat UT-B were carried out by stopped-flow light scattering, the Febuxostat D9 gold-standard for assay of UT-B urea transport (Fig. 3A, remaining). Fig. 3A (right) shows related IC50 of 2C3 mM for DMTU inhibition of rat UT-A1 and UT-B urea transport. DMTU inhibition of urea transport was fully reversible, as expected (Fig. 3B). The apparent IC50 ideals for DMTU inhibition of UT-A1 were approximately self-employed of urea concentration, both with 0 intracellular [urea] and different extracellular [urea] (Fig. 3C, remaining), and different intracellular [urea] and a fixed, 1600 mM inward urea gradient. These results define a non-competitive mechanism for DMTU inhibition of UT-A1 urea transport. DMTU competition with urea for UT-B urea transport, as analyzed by stopped-flow light scattering in rat erythrocytes, showed similar IC50 ideals (~2 mM) with different urea gradients (Fig. 3D), assisting a non-competitive inhibition mechanism. Fig. 3E shows DMTU inhibition of UT-A1 urea transport by an independent assay involving measurement of transepithelial urea transport from your basolateral to the apical answer in cells cultured on a porous filter. With this model urea permeability was improved by forskolin and reduced by a high concentration (15 mM) of DMTU to that of phloretin-treated cells; 3 mM DMTU, a concentration near its IC50 identified in plate reader assays, produced slightly greater than 50% inhibition, consistent with results from the fluorescence plate reader assay. Open in a separate window Number 3 Characterization of UT inhibition by dimethylthioureaA. DMTU inhibition of rat UT-B urea transport measured in erythrocytes by stopped-flow light scattering (remaining). DMTU concentration-inhibition of rat UT-A1 and UT-B (mean S.E., n = 3). B. Reversibility of DMTU inhibition of UT-A1 demonstrated from measurements of UT-A1 urea transport before DMTU addition, after addition of 3 mM DMTU, and 15 min after washing with PBS (remaining). Reversibility of DMTU inhibition of UT-B transferred measured by rat erythrocyte lysis assay (right) (mean S.E., n=3). C. Urea concentration-dependence of DMTU inhibition of Febuxostat D9 UT-A1. Measurements carried out as with Fig. 1A, but with different urea concentrations (1st and 2nd panels). Apparent IC50 like a function of extracellular urea concentration, [urea]e, at zero initial intracellular urea concentration (3rd panel), and as a function of intracellular urea concentration, [urea]i, for fixed 1600 mM urea gradient (right panel). D. Apparent IC50 for DMTU inhibition of UT-B like a function of extracellular urea concentration measured from light scattering in rat erythrocytes. E. Transepithelial urea transport in UT-A1-expressing MDCK cells. Cells were treated with 10 M forskolin only, forskolin + phloretin (0.7 mM), or forskolin plus 3 or 15 mM DMTU (mean S.E., n=3). Molecular modeling and computational docking were done to identify putative binding sites and modes of binding of DMTU and nicotine to rat UT-A1. Docking was carried out to the full intracellular and extracellular surfaces of the UT-A1 protein. The lowest energy binding present for DMTU expected by docking was located deep in the UT-A1 cytoplasmic pore (Fig. 4A), though additional less energetically beneficial potential binding sites were also recognized, including one deep in the.In short-term experiments, rats were administered 500 mg/kg DMTU (50 mg/ml in saline, IP) at 0 time and 125 mg/kg DMTU (IP) at 12 hours. model of Syndrome of Inappropriate Antidiuretic Hormone secretion, DMTU treatment prevented hyponatremia and water retention produced by water-loading in dDAVP-treated rats. Therefore, our results establish a rat model of UT inhibition and demonstrate the diuretic effectiveness of UT inhibition. screening of these compounds for diuretic effectiveness in rats. Seven urea analogs were also tested for UT inhibition (Fig. 2A). Two compounds, methylacetamide and dimethylthiourea (DMTU), showed UT-A1 inhibition activity, while the additional compounds were inactive (Fig. 2B). Fig. 2C summarizes UT-A1 and UT-B inhibition of the urea analogs, showing IC50 2C3 mM for DMTU inhibition of both UT-A1 and UT-B. Relatively poor inhibition was found for methylacetamide. Open in a separate window Number 2 UT inhibition by urea analogsA. Structure of urea analogs tested. B. UT-A1 inhibition curves for urea analogs. C. Percentage inhibition of UT-A1 and UT-B urea transport (mean S.E, n=3). Characterization of urea transport inhibition by DMTU Concentration-inhibition measurements for DMTU inhibition of rat UT-B were carried out by stopped-flow light scattering, the gold-standard for assay of UT-B urea transport (Fig. 3A, remaining). Fig. 3A (right) shows related IC50 of 2C3 mM for DMTU inhibition of rat UT-A1 and UT-B urea transport. DMTU inhibition of urea transport was fully reversible, as expected (Fig. 3B). The apparent IC50 ideals for DMTU inhibition of UT-A1 were approximately self-employed of urea concentration, both with 0 intracellular [urea] and different extracellular [urea] (Fig. 3C, remaining), and different intracellular [urea] and a fixed, 1600 mM inward urea gradient. These results define a non-competitive mechanism for DMTU inhibition of UT-A1 urea transport. DMTU competition with urea for UT-B urea transport, as analyzed by stopped-flow light scattering in rat erythrocytes, showed similar IC50 ideals (~2 mM) with different urea gradients (Fig. 3D), assisting a non-competitive inhibition mechanism. Fig. 3E shows DMTU inhibition of UT-A1 urea transport by an independent assay involving measurement of transepithelial urea transport from your basolateral to the apical answer in cells cultured on a porous filter. With this model urea permeability was improved by forskolin and reduced by a high concentration (15 mM) of DMTU to that of phloretin-treated cells; 3 mM DMTU, a concentration near its IC50 identified in plate reader assays, produced slightly greater than 50% inhibition, consistent with results from the fluorescence dish reader assay. Open up in another window Body 3 Characterization of UT inhibition by dimethylthioureaA. DMTU inhibition of rat UT-B urea transportation assessed in erythrocytes by stopped-flow light scattering (still left). DMTU Febuxostat D9 concentration-inhibition of rat UT-A1 and UT-B (mean S.E., n = 3). B. Reversibility of DMTU inhibition of UT-A1 proven from measurements of UT-A1 urea transportation before DMTU addition, after Febuxostat D9 addition of 3 mM DMTU, and 15 min after cleaning with PBS (still left). Reversibility of DMTU inhibition of UT-B carried assessed by rat erythrocyte lysis assay (correct) (mean S.E., n=3). C. Urea concentration-dependence of DMTU inhibition of UT-A1. Measurements completed such as Fig. 1A, but with different urea concentrations (1st and 2nd sections). Obvious IC50 being a function of extracellular urea focus, [urea]e, at zero preliminary intracellular urea focus (3rd -panel), so that as a function of intracellular urea focus, [urea]i, for set 1600 mM urea gradient (correct -panel). D. Obvious IC50 for DMTU inhibition of UT-B being a function of extracellular urea focus assessed from light scattering in rat erythrocytes. E. Transepithelial urea transportation in UT-A1-expressing MDCK cells. Cells had been treated with 10 M forskolin by itself, forskolin + phloretin (0.7 mM), or forskolin plus 3 or 15 mM DMTU (mean S.E., n=3). Molecular modeling and computational docking had been done.