Fig. 1: Expression and functional characterization of the urea transporters.

a The dendrogram clustering representation of the evolutionary relationship of UTs from different species, including human, bovine, mouse, rat, zebrafish and Desulfovibrio vulgaris (dv_UT). Five UT members are expressed in human, containing UT-B and four UT-As. The squares and the circles represent UTs encoded by gene Slc14a1 and Slc14a2, respectively. The UTs whose structure were determined in our paper are highlighted with red color. dv, Desulfovibrio vulgaris. b Tissue distribution of UT expressed in the human kidneys. UT-A1 and UT-A3 are expressed in the principal cells of the inner medullary collecting duct (IMCD), UT-A2 is expressed in the thin descending limb (TDL) of the loop of Henle, and UT-B is expressed in the renal descending vasa recta (DVR). c The membrane expression levels of the human UT-A1-A3, UT-B and zf-UT, which were overexpressed in SF9 cells using a Bac-to-Bac baculovirus expression system. Data are represented as mean ± SEM from 3 independent experiments (n = 3). d The size-exclusion chromatography elution profiles of the purified hUT-A2 on Superose 6 Increase 10/300 column. The peak of hUT-A2 was approximately 14.5 ml. e Representative SDS-PAGE results of the purified hUT-A2 (n = 2). After cross-linking with glutaraldehyde, the hUT-A2 showed at a size corresponding to trimer form displayed on the SDS-PAGE. f Schematic diagram of the ¹⁴C-labeled urea transportation assay. Each cell well was incubated with 300 μl of DMEM supplemented with 10 mmol/L urea and 1 μCi of ¹⁴C-urea for 30 min. After incubation, the cells were washed with cold 1xPBS. Then, the cells were lysed and transferred into 5 ml of scintillation fluid in a scintillation vial for measured by using a scintillation counter. The detailed experimental procedures are shown in the Methods section. g Urea permeability shown by the detection of remaining ¹⁴C-labeled urea. Columns of cells treated with vehicle or 100 μM inhibitor 25a are colored gray or orange respectively. Data are represented as mean ± SEM from 3 independent experiments (n = 3). ***P < 0.001, statistical differences were determined by the two-sided unpaired Student’s t-test (compared with vehicle).