Volume 83, Issue 6, Pages 1076-1086 (June 2013) A novel small-molecule thienoquinolin urea transporter inhibitor acts as a potential diuretic  Fei Li, Tianluo Lei, Juanjuan Zhu, Weiling Wang, Yi Sun, Jihui Chen, Zixun Dong, Hong Zhou, Baoxue Yang  Kidney International  Volume 83, Issue 6, Pages 1076-1086 (June 2013) DOI: 10.1038/ki.2013.62 Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 1 Activity of PU-14 against UT-B in human, rabbit, rat, and mouse. (a) Chemical structure of PU-14. (b) Dose-dependent inhibition activity for PU-14, determined by the osmotic lysis assay in human erythrocytes. (c) Dose-dependent inhibition activity for PU-14 in rabbit erythrocytes. (d) Dose-dependent inhibition activity for PU-14 in rat erythrocytes. (e) Dose-dependent inhibition activity for PU-14 in erythrocytes from wild-type (+/+) or UT-B knockout (-/-) mice. Mean±s.e.m., n=3. PU-14, 1-(3-Amino-6-methylthieno [2,3-b] quinolin-2-yl) ethanone. Kidney International 2013 83, 1076-1086DOI: (10.1038/ki.2013.62) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 2 Characterization of UT-B inhibition mechanism. Urea permeability was measured by stopped-flow light scattering in rat erythrocytes. (a) Concentration-inhibition curves for the time course of scattered light intensity measured in rat erythrocytes in the absence (bottom) or presence of PU-14 at indicated concentrations for 10min at 10°C in response to a 250-mmol/l inwardly directed urea gradient. (b) Membrane sidedness of UT-B inhibition. Experiments performed as in a, except that PU-14 (double as indicated concentration) was added only to the urea-containing solution (500mmol/l urea+phosphate-buffered saline (PBS)). (c) Time-inhibition curves for experiments done as in a, in which erythrocytes were incubated with 4μmol/l PU-14 for the indicated time. (d) Reversibility of UT-B inhibition. Where indicated, 4μmol/l PU-14 was washed out after 10min of incubation before stopped-flow measurements. (e) Inhibition of UT-B-mediated urea influx or efflux. Rat erythrocytes were incubated with 4μmol/l PU-14 for 10min and then subjected to a 250-mmol/l inflow- or outflow-directed urea gradient. PU-14, 1-(3-Amino-6-methylthieno [2,3-b] quinolin-2-yl) ethanone. Kidney International 2013 83, 1076-1086DOI: (10.1038/ki.2013.62) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 3 Activity of PU-14 against rat UT-A1. (a) Time course of urea transport. Rat UT-A1 stably transfected MDCK (Madin-Darby canine kidney) cells were treated (filled circles, open triangles, and filled square) or not treated (open circles) with 10μmol/l forskolin (FSK). Where indicated, phloretin (0.7mmol/l, filled square) and PU-14 (4μmol/l, filled circles) were present. (b) Mean±s.e.m., n=3; ***P<0.001. PU-14, 1-(3-Amino-6-methylthieno [2,3-b] quinolin-2-yl) ethanone. Kidney International 2013 83, 1076-1086DOI: (10.1038/ki.2013.62) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 4 Cellular toxicity of PU-14. MDCK (Madin-Darby canine kidney) cells were cultured in the absence or presence of PU-14 at the indicated concentrations for 72h. Cell viability in control group was regarded as 100%. Mean±s.e.m., n=3. PU-14, 1-(3-Amino-6-methylthieno [2, 3-b] quinolin-2-yl) ethanone. Kidney International 2013 83, 1076-1086DOI: (10.1038/ki.2013.62) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 5 Diuretic activity of PU-14. Rats were subcutaneously injected with 0, 3.125, 12.5, 50, and 100mg/kg of PU-14 just after a 2-h urine collection (time 0). Urine samples were collected every 2h in metabolic cages. (a) Urine output. (b) Urinary osmolality (Uosm). (c) Urinary urea concentration (Uurea). Mean±s.e.m., n=6; *P<0.05 and **P<0.01 compared with control rats. PU-14, 1-(3-Amino-6-methylthieno [2,3-b] quinolin-2-yl) ethanone. Kidney International 2013 83, 1076-1086DOI: (10.1038/ki.2013.62) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 6 Effect of PU-14 or hydrochlorothiazide (HCTZ) on urinary concentrating activity and renal handling of urea in rats. (a) Urine output. (b) Urinary osmolality (Uosm). (c) Urine urea concentration (Uurea). (d) Excretion (Excr.) of osmoles. (e) Urea excretion. (f) Excretion of nonurea solutes. Mean±s.e.m., n=6; *P<0.05, **P<0.01, and ***P<0.001 compared with control rats. #P<0.05 compared with PU-14-treated rats. PU-14, 1-(3-Amino-6-methylthieno [2,3-b] quinolin-2-yl) ethanone. Kidney International 2013 83, 1076-1086DOI: (10.1038/ki.2013.62) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 7 Long-term effect of PU-14 on urinary concentrating activity and blood solutes in rats. Rats were subcutaneously treated without or with PU-14 at 50mg/kg for 7 days. (a) Urine output. (b) Urinary osmolality (Uosm). (c) Blood Na+ concentration. (d) Blood K+ concentration. (e) Blood Cl− concentration. (f) Blood urea concentration. Mean±s.e.m., n=5; *P<0.05, **P<0.01, and ***P<0.001 compared with control rats; #P<0.05 compared with PU-14-treated rats. PU-14, 1-(3-Amino-6-methylthieno [2,3-b] quinolin-2-yl) ethanone. Kidney International 2013 83, 1076-1086DOI: (10.1038/ki.2013.62) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 8 Urinary concentrating ability in rats without (control) or with PU-14 after 18-h water deprivation. (a) Urine osmolality (Uosm). (b) Urine urea concentration (Uurea). (c) Concentration of urine nonurea solutes (Unonurea solutes). Mean±s.e.m.; n=6; *P<0.05 and **P<0.01 compared with control rats. PU-14, 1-(3-Amino-6-methylthieno [2,3-b] quinolin-2-yl) ethanone. Kidney International 2013 83, 1076-1086DOI: (10.1038/ki.2013.62) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 9 Concentration of osmoles, urea, and nonurea solutes in the inner medulla of rats without (control) or with PU-14 or hydrochlorothiazide (HCTZ) treatment fed water ad libitum. (a) Urine osmolality (Uosm). (b) Urine urea concentration (Uurea). (c) Concentration of urine nonurea solutes (Unon-urea solutes). Mean±s.e.m.; n=6, *P<0.05 compared with control rats; #P<0.05 compared with PU-14-treated rats. PU-14, 1-(3-Amino-6-methylthieno [2,3-b] quinolin-2-yl) ethanone. Kidney International 2013 83, 1076-1086DOI: (10.1038/ki.2013.62) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 10 Expression of urea transporters (UTs) and aquaporins (AQPs) in the medulla of rats without (control) or with PU-14 treatment. (a) Relative mRNA levels determined by fluorescence-based real-time reverse transcription–PCR (RT–PCR). Mean±s.e.m.; n=3. (b) Relative protein levels determined by western blot analysis. (b, left) Representative western blots of renal medulla homogenates, 25μg of protein/lane. β-Actin was used for normalization. (b, right) The quantitative densitometry analysis. Mean±s.e.m.; n=3. PU-14, 1-(3-Amino-6-methylthieno [2,3-b] quinolin-2-yl) ethanone. Kidney International 2013 83, 1076-1086DOI: (10.1038/ki.2013.62) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 11 Kidney histology in hematoxylin and eosin–stained sections of cortex (left) and inner medulla (right). Arrows indicate dilatation of collecting ducts. HCTZ, hydrochlorothiazide; PU-14, 1-(3-Amino-6-methylthieno [2,3-b] quinolin-2-yl) ethanone. Kidney International 2013 83, 1076-1086DOI: (10.1038/ki.2013.62) Copyright © 2013 International Society of Nephrology Terms and Conditions