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). Membeers of this family have been reported to have the AmtB fold (Ferrada and Superti-Furga 2022). A sensitive wlectrochemiluminescence urea sensor for dynamic monitoring of urea transport in living cells has been described (Feng et al. 2023).

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 JK^a/JK^b 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). Mammalian UTs are members of the Kidd blood group family (PMID 40198053).  The Kidd blood group system consists of three antigens present on RBCs—Jk^a, Jk^b, and Jk3. These antigens are not found on granulocytes, lymphocytes, monocytes, and platelets. Similar to other Kidd antigens, Kidd antigens are resistant to ficin, papain, trypsin, chymotrypsin, and pronase.

Elevin cryo-EM structures of four UT members in resting states, urea transport states, or inactive states bound with synthetic competitive, uncompetitive or noncompetitive inhibitor have been described (Huang et al. 2024). The results indicated that the binding of urea via a conserved urea recognition motif (URM) and urea transport via H-bond transfer along the Q^Pb-T^5b-T^5a-Q^Pa motif among different UT members. Distinct binding modes of the competitive inhibitors 25a and ATB3, the uncompetitive inhibitor CF11 and the noncompetitive inhibitor HQA2 provide different mechanisms for blocking urea transport and achieving selectivity through L-P pocket, UCBP region and SCG pocket, respectively. In summary, this study allows structural understanding of urea transport via UTs but also affords a structural landscape of hUT-A2 inhibition by competitive, uncompetitive and noncompetitive inhibitors, which may facilitate developing selective human UT-A inhibitors as a new class of salt-sparing diuretics (Huang et al. 2024).