At physiological expression levels the Kidd blood group/urea transporter protein is not a water channel.

The Kidd (JK) blood group locus encodes a urea transporter that is expressed on human red cells and on endothelial cells of the vasa recta in the kidney. Here, we report the identification in human erythroblasts of a novel cDNA, designated HUT11A, which encodes a protein identical to the previously reported erythroid HUT11 urea transporter, except for a Lys(44) --> Glu substitution and a Val-Gly dipeptide deletion after proline 227, which leads to a polypeptide of 389 residues versus 391 in HUT11. Genomic typing by polymerase chain reaction and transcript analysis by ribonuclease protection assay demonstrated that HUT11A encodes the true Kidd blood group/urea transporter protein, which carries only 2 Val-Gly motifs. Upon expression at high levels in Xenopus oocytes, the physiological Kidd/urea transporter HUT11A conferred a rapid transfer of urea (which was insensitive to p-chloromercuribenzene sulfonate or phloretin), a high water permeability, and a selective uptake of small solutes including amides and diols, but not glycerol and meso-erythritol. However, at plasma membrane expression levels close to the level observed in the red cell membrane, HUT11A-mediated water transport and small solutes uptake were absent and the urea transport was poorly inhibited by p-chloromercuribenzene sulfonate, but strongly inhibited by phloretin. These findings show that, at physiological expression levels, the HUT11A transporter confers urea permeability but not water permeability, and that the observed water permeability is a feature of the red cell urea transporter when expressed at unphysiological high levels.

Characterization of the gene encoding the human Kidd blood group/urea transporter protein. Evidence for splice site mutations in Jknull individuals.

The Kidd (JK) blood group is carried by an integral membrane glycoprotein which transports urea through the red cell membrane and is also present on endothelial cells of the vasa recta in the kidney. The exon-intron structure of the human blood group Kidd/urea transporter gene has been determined. It is organized into 11 exons distributed over 30 kilobase pairs. The mature protein is encoded by exons 4-11. The transcription initiation site was identified by 5'-rapid amplification of cDNA ends-polymerase chain reaction at 335 base pairs upstream of the translation start point located in exon 4. The 5'-flanking region, from nucleotide -837 to -336, contains TATA and inverted CAAT boxes as well as GATA-1/SP1 erythroid-specific cis-acting regulatory elements. Analysis of the 3'-untranslated region reveals that the two equally abundant erythroid transcripts of 4.4 and 2.0 kilobase pairs arise from usage of different alternative polyadenylation signals. No obvious abnormality of the Kidd/urea transporter gene, including the 5'- and 3'-untranslated regions, has been detected by Southern blot analysis of the blood of two unrelated Jknull individuals (B.S. and L.P.), which lacks all Jk antigens and Jk proteins on red cells, but was genotyped as homozygous for a "silent" Jkb allele. Further analysis indicated that different splice site mutations occurred in each variant. The first mutation affected the invariant G residue of the 3'-acceptor splice site of intron 5 (variant B.S.), while the second mutation affected the invariant G residue of the 5'-donor splice site of intron 7 (variant L.P.). These mutations caused the skipping of exon 6 and 7, respectively, as seen by sequence analysis of the Jk transcripts present in reticulocytes. Expression studies in Xenopus oocytes demonstrated that the truncated proteins encoded by the spliced transcripts did not mediate a facilitated urea transport compared with the wild type Kidd/urea transporter protein and were not expressed on the oocyte's plasma membrane. These findings provide a rational explanation for the lack of Kidd/urea transporter protein and defect in urea transport of Jknull cells.

The molecular basis of the Kidd blood group polymorphism and its lack of association with type 1 diabetes susceptibility.

The Kidd blood group locus encodes a urea transporter which is expressed on human red cells and in the kidney. This gene is located on chromosome 18q12, and evidence for linkage and association with type 1 diabetes mellitus has been reported. To investigate this further, the genetic basis for the blood group Jk(a)/Jk(b) polymorphism was first determined by sequencing reverse-transcribed reticulocyte RNAs from Jk(a+b-) and Jk(a-b+) donors. The Jk(a)/Jk(b) polymorphism was caused by a transition (G838A), resulting in a Asp280Asn amino acid substitution and an MnlI restriction fragment length polymorphism (RFLP). Using the MnlI RFLP, we found that the Jk(a)/Jk(b) polymorphism was not in linkage disequilibrium with type 1 diabetes in 228 multiplex UK and US families tested.

Endothelial cells of the kidney vasa recta express the urea transporter HUT11.

The cell specific expression of the human urea transporter HUT11 in human and rat kidneys was investigated by immunochemistry and in situ hybridization. Using specific rabbit polyclonal antibodies directed against the N-terminal part and the C-terminal part of HUT11, we found that endothelial cells of medullary vasa recta (outer and inner medulla) express HUT11. In addition, an HUT11-related protein expressed by smooth muscle cells of cortical arterioles can be detected by the anti-HUT11 N-terminal peptide antibody but not the anti-HUT11 C-terminal peptide antibody. The endothelial expression of HUT11 was confirmed by double labeling with anti-CD31 and anti-von Willebrand factor antibodies. No HUT11-positive cells expressed alpha smooth muscle actin, Tamm Horsfall protein, cytokeratin 18, or CAM-L1, a marker of the principal cells of collecting ducts. Medullary vasa recta of developing and mature rat kidneys were also stained with the anti-HUT11 C-terminal peptide antibody. By in situ hybridization using a specific HUT11 35S-cDNA probe on human kidney sections, medullary vasa recta but not other renal structures were found to express HUT11 mRNA. We conclude that endothelial cells of medullary vasa recta express HUT11, a specific urea transporter, which may play a role in urea recycling within the kidney and in the mechanisms of urinary concentration and urea excretion.

Functional differentiation of the human red blood cell and kidney urea transporters.

The recent cloning of two urea transporters will allow to better understand their role in the urinary concentrating mechanism. This physiological approach needs to be sustained by a knowledge of their functional characteristics. We compared the pharmacological properties of the human red blood cell and kidney urea transporters (HUT11 and HUT2) in the Xenopus oocyte expression system. Both proteins allow the rapid transfer of urea but not of water. Both are inhibited by phloretin, although with different half-maximal inhibitory concentrations (IC50; 75 microM, for HUT11 and 230 microM for HUT2). Whereas para-chloromercuribenzene sulfonate inhibits HUT11 with an IC50 of 150 microM, it does not inhibit HUT2, whatever the concentration used. We demonstrate that thiourea diffuses through HUT11 with a Michaelis constant (Km) of 40 mM, but not through HUT2. In contrast, it inhibits urea transport through both proteins. This identification of a substrate binding site independent from the transport activity is the first step in the understanding of the molecular events underlying urea transport.

Molecular characterization of a new urea transporter in the human kidney.

A cDNA clone (HUT2) sharing 61.1% and 89.9% sequence identity with the human erythroid (HUT11) and the rabbit (UT2) urea transporters, respectively, was isolated by homology cloning from a human kidney library. HUT2 transcripts were restricted to the kidney and the HUT2 polypeptide was not immunoprecipitated with blood group Kidd-related antibodies (anti-Jk3) in coupled transcription-translation assays. Functional expression studies in Xenopus oocytes demonstrated that HUT2-mediated urea transport was not inhibited by p-chloromercuribenzene sulfonate (pCMBS) which, however, inhibited the urea flux mediated by HUT11. These findings demonstrate that at least two distinct urea transporters are present in human tissues. By in situ hybridization, the gene encoding HUT2 has been assigned to chromosome 18q12.1-q21-1, as found previously for the Kidd/urea transporter HUT11, suggesting that both genes evolved from duplication of a common ancestor.

Kidd blood group and urea transport function of human erythrocytes are carried by the same protein.

The gene encoding the urea transporter of human erythrocytes (HUT11 clone) has been cloned recently (Olives, B., Neau, P., Bailly, P., Hediger, M. A., Rousselet, G., Cartron, J. P., and Ripoche, P. (1994) J. Biol. Chem. 269, 31649-31652). Now, this gene has been assigned to chromosome 18q12-q21 by in situ hybridization, as also found for the Kidd (Jk) blood group locus. In coupled transcription-translation assays, the HUT11 cDNA directed the synthesis of a 36-kDa protein which was immunoprecipitated by a human anti-Jk3 antibody produced by immunized Jk(a-b-) donors whose red cells lack Kidd antigens. The anti-Jk3 antibody also immunoprecipitated a protein material of 46-60 kDa from all red cell membranes, except those from Jk(a-b-) cells. After N-glycanase digestion the 46-60-kDa component was reduced to 36 kDa. A rabbit antibody raised against the predicted NH2-terminal amino-acids of the HUT11 protein reacted on immunoblots with a 46-60-kDa component present in all human erythrocytes except those from Jk(a-b-) individuals. Jk(a-b-) red cells lack the Kidd/urea transport protein and have a selective defect of the urea transport capacity, but a normal water permeability and aquaporin-associated Colton blood group antigens. These findings indicate that the erythrocyte urea transporter is encoded by the Kidd locus and may have implications for the biology of urea transporters and their tissue-specific regulation.

Cloning and functional expression of a urea transporter from human bone marrow cells.

A rapid passive urea transport has been previously described in the mammalian renal inner medullary collecting duct epithelial cells and in mammalian erythrocytes. Recently, a vasopressin-regulated urea transporter (UT2) has been cloned from a rabbit kidney medullary cDNA library (You, G., Smith, C. P., Kanai, Y., Lee, W. S., Stelzner, M., and Hediger, M. A. (1993) Nature 365, 844-847). We now report the cloning and characterization of a complementary DNA (HUT11) encoding an urea transporter isolated from a human bone marrow library. It encodes a 43,000-Da polypeptide of 391 amino acids that exhibited 63% sequence identity with the rabbit urea transporter and a similar membrane topology. HUT11 carries 2 putative glycosylation sites and 10 cysteines, of which only 7 are conserved at an equivalent position in UT2. HUT11 transcripts have been identified in human erythroid and renal tissues. Expression studies in Xenopus oocytes demonstrated that HUT11 mediates a facilitated urea transport that was inhibited, as described in mammalian erythrocytes, by very low concentrations of phloretin, p-chloromercuribenzene sulfonate, and urea analogues. No unidirectional movements of charged molecules, glycerol, or water were associated with HUT11 expression in oocytes. These findings suggest that HUT11 is most likely responsible for the facilitated urea transport in human red blood cells.