1.A.28 The Urea Transporter (UT) Family
Members of the UT family are found in vertebrate animals and bacteria but not in other eukaryotes or in archaea (Minocha et al., 2003). In a single species (i.e., rat or human) there are at least seven isoforms. These isoforms are splice variants of two adjacent genes in humans and mice, Slc14a2, which encodes the type UT-A variants, and Slc14a1, which encodes the UT-B variants. UT-A1-4 are expressed mainly in the renal tubules while UT-A5 is expressed only in the testis. UT-B1 is expressed in red blood cells, in endothelial cells of the descending vasa recta irrigating renal medulla and in other tissues (Minocha et al., 2003). The physiology of UT family members is described by Sands (2003). UTs are targets of small molecule diuretics (Esteva-Font et al. 2015). A phenylphthalazine compound, PU1424, is a potent UT-B inhibitor, inhibiting human and mouse UT-B-mediated urea transport with IC50 values of 0.02 and 0.69 mumol/L, respectively, and exerted 100% UT-B inhibition at higher concentrations (Ran et al. 2016).
Urea transporters (UTs) include two UT subfamilies, UT-A and UT-B. The UT-A subfamily includes six members, UT-A1 to UT-A6, which are mainly expressed in kidney. The UT-B subfamily has only one member, UT-B1, that has a wide distribution in the body. UTs play important roles in urinary concentrations as determined by the phenotypic analysis of 6 UT selective knockout mouse models. UTs might be diuretic targets, and UT inhibitors might be developed as novel diuretics (Li and Yang 2018). UT-B1 is the Kidd (JK) blood group glycoprotein. The JKa/JKb antigenic polymorphism in human UT-B1 is due to an Asp280Asn substitution on the external loop separating TMSs 7 and 8, while the ABO blood group type is due to a glycan linked to Asn211 in the large, central, extracellular loop between TMSs 5 and 6 (Lucien et al., 2002).
Most of the UT proteins vary in size from 380-400 residues and exhibit 10 putative transmembrane helical spanners, but mammalian urea transporters such as UT-A1 of the rat are 920-930 residues long. They exhibit an internal duplication with a total of 20 TMSs (Minocha et al., 2003). This duplication is lacking in the other forms. Isoforms A2-A5 are splice variants of A1. B1 and B2 are of the same size as A2-A5. At least one of these proteins (UTB or UT3) can transport water as well as urea (Yang and Verkman, 2002). A channel-type mechanism is probable. UT1 and UT2 may be derived from a single gene by alternative splicing. A human protein (spQ15849) is 397 residues long, exhibits 10 putative TMSs and is internally duplicated.
Homologues of the mammalian UT family members have been identified in several bacteria. The gene encoding the Actinobacillus pleuropneumoniae homologue, Utp, is in the urea utilization gene cluster which also encodes a Ni2+-ABC transporter and urease (Bosse et al., 2001). Utp is 300 aas long and has ten putative TMSs. The first 129 residues of this bacterial protein are homologous to residues 55-187 and 220-349 of the frog protein, thus demonstrating the presence of a putative 5 TMS repeat element.
Urea is highly concentrated in the mammalian kidney to produce the osmotic gradient necessary for water re-absorption. Free diffusion of urea across cell membranes is slow owing to its high polarity, and specialized urea transporters have evolved to achieve rapid and selective urea permeation. Levin et al. (2009) presented a 2.3 A structure of a functional urea transporter from the bacterium Desulfovibrio vulgaris. The transporter is a homotrimer, and each subunit contains a continuous membrane-spanning pore formed by the two homologous halves of the protein. The pore contains a constricted selectivity filter that can accommodate several dehydrated urea molecules in single file. Backbone and side-chain oxygen atoms provide continuous coordination of urea as it progresses through the filter, and well-placed alpha-helix dipoles provide further compensation for dehydration energy. Thus, the urea transporter operates by a channel-like mechanism. The structure reveals the physical and chemical basis of urea selectivity (Levin et al., 2009).
Urea transporters (UTs) play an important role in urine concentration. UT-knockout mice exhibit a urea-selective urine-concentrating defect, without affecting electrolyte balance, suggesting that UT-B inhibitors have the potential to be developed as novel diuretics. Li et al. 2019 characterized the thienopyridine, 5-ethyl-2-methyl-3-amino-6-methylthieno[2,3-b]pyridine-2,5-dicarboxylate (CB-20) with UT inhibitory activity as a diuretic, inhibiting both UTB and UTA1. CB-20 exhibited good absorption and rapid clearance. (Li et al. 2019).
Frog urinary bladder ADH-regulated urea transporter
Urea transporter of Rana esculenta (O57609)
Kidney urea transporter, UT-A1 (mediates transepithelial urea transport in the inner medullary collecting duct for urinary concentration. Interacts with the C-terminus of Snapin (O95295) and SNARE-associated protein) (Mistry et al., 2007). Also transports formamide, acetamide, methylurea, methylformamide, ammonium carbamate, and acrylamide, and possibly dimethylurea and thiourea as well (Zhao et al., 2007). Mutation of the N-linked glycosylation sites reduces urea flux by reducing the UT-A1 half-life and decreasing its accumulation in the apical plasma membrane. The related erythrocyte urea transporter, UTB (UT-B; TC# 1.A.28.1.5) has been reviewed (Bagnasco, 2006). Mutation of the N-linked glycosylation sites reduces urea flux by reducing the UT-A1 half-life and decreasing its accumulation in the apical plasma membrane (Chen et al. 2006). In vivo, inner medullary collecting duct cells may thus regulate urea uptake by altering UT-A1 glycosylation in response to AVP stimulation.
UT-A1 of Rattus norvegicus
THe Urea transporter channel protein of 337 aas and 11 TMSs. The 3-d structure (2.3 Å resolution) is available (Levin et al., 2009). Urea binding and flux as well as dimethylurea (DMU) transport have been modeled (Zhang et al. 2017).
Urea channel of Desulfovibrio vulgaris (A1VEP3)
Urea transporter 1 or UT-B1 (Solute carrier family 14 member 1; Urea transporter of the erythrocyte) (Bagnasco 2006). A phenylphthalazine compound, PU1424, is a potent UT-B inhibitor, inhibiting human and mouse UT-B-mediated urea transport with IC50 values of 0.02 and 0.69 mumol/L, respectively, and exerted 100% UT-B inhibition at high concentrations (Ran et al. 2016). Another potent inhibitor is the thienopyridine, CB-20 (5-ethyl-2-methyl-3-amino-6-methylthieno[2,3-b]pyridine-2,5-dicarboxylate) (Li et al. 2019). UT-B catalyzes transmembrane water transport which can be ued as a reporter system (Schilling et al. 2016). Knocking out both UT1 and UT2 increases urine output 3.5-fold and lowers urine osmolarity (Jiang et al. 2016). The double knockout also lowered blood pressure and promoted maturation of the male reproductive system. Thus, functional deficiency of all UTs causes a urea-selective urine-concentrating defect with few physiological abnormalities in extrarenal organs (Jiang et al. 2016). UT-B may be related to the occurrence of melanoma and play a role in tumor development (Liu et al. 2018).
SLC14A1 of Homo sapiens
Urea transporter 2 (Solute carrier family 14 member 2) (Urea transporter, kidney). Knocking out both UT1 and UT2 increases urine output 3.5-fold and lowers urine osmolarity (Jiang et al. 2016). The double knockout also lowered blood pressure and promoted maturation of the male reproductive system. Thus, functional deficiency of all UTs causes a urea-selective urine-concentrating defect with few physiological abnormalities in extrarenal organs (Jiang et al. 2016). A potent inhibitor of both UT1 and UT2 is the thienopyridine, CB-20 (5-ethyl-2-methyl-3-amino-6-methylthieno[2,3-b]pyridine-2,5-dicarboxylate) (Li et al. 2019).
SLC14A2 of Homo sapiens
Putative urea transporter of 306 aas and 9 TMSs
UT of E. coli
Utp of Actinobacillus pleuropneumoniae