Localization of the urea transporter UT-B protein in human and rat erythrocytes and tissues

Richard T. Timmer1,*, Janet D. Klein2,*, Serena M. Bagnasco3, John J. Doran2, Jill W. Verlander4, Robert B. Gunn1, and Jeff M. Sands2

Departments of 1 Physiology, 2 Medicine (Renal Division), and 3 Pathology, Emory University School of Medicine, Atlanta, Georgia 30322; and 4 Department of Medicine, College of Medicine Electron Microscopy Core Facility, University of Florida College of Medicine, Gainesville, Florida 32610


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

A new polyclonal antibody to the human erythrocyte urea transporter UT-B detects a broad band between 45 and 65 kDa in human erythrocytes and between 37 and 51 kDa in rat erythrocytes. In human erythrocytes, the UT-B protein is the Kidd (Jk) antigen, and Jk(a+b+) erythrocytes express the 45- to 65-kDa band. However, in Jk null erythrocytes [Jk(a-b-)], only a faint band at 55 kDa is detected. In kidney medulla, a broad band between 41 and 54 kDa, as well as a larger band at 98 kDa, is detected. Human and rat kidney show UT-B staining in nonfenestrated endothelial cells in descending vasa recta. UT-B protein and mRNA are detected in rat brain, colon, heart, liver, lung, and testis. When kidney medulla or liver proteins are analyzed with the use of a native gel, only a single protein band is detected. UT-B protein is detected in cultured bovine endothelial cells. We conclude that UT-B protein is expressed in more rat tissues than previously reported, as well as in erythrocytes.

kidney; brain; liver; testis; lung; heart; colon; Kidd antigen; endothelial cells


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE COMPLEMENTARY DNA of the facilitated urea transporter in erythrocytes has been cloned from human (hUT-B1 or hUT11) and rat (rUT-B1 or UT3, rUT-B2 or rUT11) erythropoietic cell lines (3, 19, 28, 32). The human UT-B gene is located near the same locus (chromosome 18q12.1-q21.1) as the human UT-A gene and the human Kidd (or Jk) blood group (a minor blood group) antigen locus (13, 20, 21). The two reported rat cDNA sequences (rUT-B1 and rUT-B2) differ by only a few nucleotides at their 3' end (Fig. 1) (3, 32). The derived amino acid sequences of rUT-B1 and rUT-B2 share 80% (32) and 77% identity (3), respectively, with hUT-B1 (19). rUT-B1 and/or rUT-B2 mRNA is expressed in rat bone marrow, spleen, kidney medulla, testis, lung, thymus, and brain (2, 8, 25, 32). The methods used in these studies did not distinguish between the rUT-B1 and rUT-B2 isoforms.


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Fig. 1.   Diagram of peptide sequence used to prepare polyclonal antibody to the human urea transporter protein (hUT-B1) and comparison to the homologous sequences in rat (rUT-B1 and rUT-B2). aa, Amino acids.

At the protein level, hUT-B1 is expressed in human Jk(a+b+) erythrocytes (33). UT-B1 and/or UT-B2 proteins are also expressed in human and rat vasa recta endothelial cells (8, 33). Hu and colleagues (8) raised an antibody to the NH2 terminus of hUT-B1 that did not detect rUT-B protein in rat brain even though UT-B1 or UT-B2 mRNA was present. This antibody also detected rUT-B in testis (8). Xu and colleagues (33) raised antibodies against both the NH2 terminus and COOH terminus of UT-B1 but did not examine extrarenal expression. Therefore, the goal of our study was to prepare a new polyclonal antibody to rUT-B to determine whether rUT-B proteins are expressed in rat kidney and other rat tissues.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Preparation of a polyclonal antibody to the human erythrocyte urea transporter. Polyclonal antibodies to hUT-B1 were commercially prepared (Lampire Biologicals, Pipersville, PA). In brief, rabbits were immunized against an HPLC-purified synthetic peptide corresponding to the COOH- terminal 19 amino acids (CEENRIFYLQAKKRMVESPL) of hUT-B1 (20); an additional cysteine residue was added to the NH2 terminus to facilitate conjugation to keyhole limpet hemocyanin (KLH). The KLH-conjugated peptide was dissolved in PBS and then mixed with Freund's complete adjuvant, and two rabbits (nos. 4984 and 4985) were injected. The rabbits were reinjected at 1, 2, 4, 8, and 12 wk with the KLH-linked peptide dissolved in Freund's incomplete adjuvant. Antibody from rabbit no. 4984 was affinity purified against the immunizing polypeptide (Research Genetics, Huntsville, AL) and used in all studies.

Animal models. Pathogen-free male Sprague-Dawley rats (National Cancer Institute, Frederick, MD) weighing 60-70 g were kept in filter-top cages with autoclaved bedding and received free access to food (NIH-31; Ziegler Brothers, Gardner, PA) and water.

Tissue preparation for Western blot analysis. Rats were killed, and their erythrocytes, kidneys, liver, lungs, brain, testis, and colon were removed. Kidneys were dissected into four regions: 1) cortex; 2) outer medulla; 3) base of the inner medulla; and 4) tip of the inner medulla, as previously described (1, 18, 27). The tissues were homogenized in isolation buffer [10 mM triethanolamine, 250 mM sucrose, 1 µg/ml leupeptin, and 0.1 mg/ml phenylmethylsulfonyl fluoride (PMSF), pH 7.6, with 0.025-0.1 g of tissue per milliliter of isolation buffer] (11, 18). For SDS-polyacrylamide gel electrophoresis (PAGE), concentrated SDS was added to the homogenized samples to achieve a final concentration of 1%, after which samples were sheared with a 28-gauge needle attached to an insulin syringe and centrifuged for 15 min at 14,000 g. For native gels, homogenates did not receive SDS but were otherwise treated the same. Protein was determined in the supernatant fractions (DC protein assay kit; Bio-Rad, Hercules, CA).

Human erythrocytes were collected in heparinized tubes and packed by centrifugation. The plasma and buffy coat were removed by aspiration, and the remaining packed erythrocytes were washed twice with isotonic 110 mM MgCl2 solution and then solubilized with 2% SDS containing 0.1 mg/ml PMSF. The preparation of the rat erythrocytes was identical to that of the human erythrocytes, except that rat erythrocytes were passed over 0.25 in. of cotton in a Pasteur pipette to remove white cells and platelets not removed with the buffy coat. Protein concentrations were determined with bicinchoninic acid (BCA protein assay kit; Pierce, Rockford, IL), and 2× Laemmli sample buffer was added to a final concentration of 1 µg protein/µl.

Human kidney tissue was obtained from excess tissue samples stored in the Department of Pathology at Emory University Hospitals. Chimpanzee kidney was obtained from excess tissue samples stored at Yerkes Primate Center. Mouse kidney tissue was obtained from normal mice that were killed for other studies. No human, primate, or mouse tissue was obtained solely for its use in this study.

Western blot/protein analysis. Tissue or erythrocyte proteins (10 µg/lane), boiled for 1 min in Laemmli sample buffer, were separated on 10% SDS-polyacrylamide gels and then transferred to a polyvinylidene difluoride (PVDF) membrane. For native gels, proteins were mixed with sample buffer containing 25 mM Tris, pH 7.5, 50% glycerol, and 0.05% bromphenol blue without either SDS or beta -mercaptoethanol, separated on 6 or 7.5% polyacrylamide gels, and then transferred to a PVDF membrane. Membranes were probed with our UT-B antibody diluted 1:3,000 in Tris-buffered saline (TBS) with 0.5% Tween 20 overnight at 4°C and then washed three times in TBS/Tween. Blots were then incubated with horseradish peroxidase-linked goat anti-rabbit IgG diluted 1:5,000 (Amersham, Arlington Heights, IL) for 2 h at room temperature and washed twice with TBS/Tween, and bound antibody was visualized by enhanced chemiluminescence (ECL; Amersham) (11, 18).

Antibody competition studies. Primary UT-B antibody was preincubated with the immunizing peptide (0.1 µg/ml) for 30 min, and then the preadsorbed antibody was used to probe Western blots and compared with control samples for which nonpreadsorbed antibody was used as the probe (12, 18).

Deglycosylation studies. N-deglycosylation by peptide N-glycosidase F (PNGase F; New England Biolabs, Beverly, MA) was performed as described by Maley and colleagues (16).

Immunofluorescence. Tissue was embedded in OCT (Baxter, Deerfield, IL) and frozen in 2-methylbutane. Bovine aortic endothelial cells, cultured as previously described (10), were grown on glass coverslips and then fixed with 3.7% formaldehyde and permeabilized with 0.2% Triton X-100. Both cells and cryostat tissue sections (5 µm) were stained with our anti-UT-B antibody at 1:100 dilution, followed by staining with FITC-conjugated goat anti-rabbit IgG (Sigma). Negative controls omitted anti-UT-B and were stained only with the secondary antibody.

Immunohistochemistry. Sections (5 µm) of Formalin-fixed, paraffin-embedded human renal tissue were stained with our anti-UT-B antibody at 1:400 dilution. Control slides were stained for the human endothelial antigen factor VIII, using an anti-factor VIII mouse monoclonal antibody (Bio Tek Solutions, Santa Barbara, CA), and for keratin, using a mouse anti-keratin monoclonal antibody (Bio Tek Solutions), at 1:2 dilution. Positive antigen-antibody interaction was detected with an avidin-biotinylated enzyme complex kit (Signet Laboratories, Dedham, MA) by employing the capillary gap staining technique with the automated TechMate 1000 immunostaining system (Ventana Medical, Tucson, AZ) according to the manufacturer's instructions.

Ultrastructural localization of UT-B protein. Rat kidneys were fixed by retrograde aortic perfusion with periodate-lysine-2% paraformaldehyde (17) and cut into 50- to 70-mm transverse vibratome sections. To probe for UT-B, we incubated sections overnight at 4°C with our anti-UT-B diluted 1:800 in 1% ovalbumin in PBS, followed by incubation for 2 h in peroxidase-conjugated goat anti-rabbit IgG (Fab fragment; Jackson ImmunoResearch, West Grove, PA) diluted 1:50 in 1% ovalbumin in PBS. Tissue samples were postfixed in 2% osmium tetroxide, processed, and embedded in Taab 812 epoxy resin (Marivac, Nova Scotia, Canada) as previously described (9). Ultrathin sections were counterstained with lead citrate and examined with a Zeiss 10A transmission electron microscope.

RNA isolation and purification. Rat tissues were harvested, immediately frozen in liquid nitrogen, and then directly homogenized in TRIzol reagent (Life Technologies, Rockville, MD) according to the manufacturer's protocol. The RNA was treated with DNase (1 U/200 µg RNA), and then proteinase K (0.75 U/200 µg RNA), extracted three times with phenol-chloroform (5:1, pH 4.5), precipitated with ethanol-ammonium acetate (2.5:0.5, vol/wt), and dissolved in water. The absorbance ratio at 260 and 280 nm ranged from ~1.7 to 2.1.

Reverse transcription and PCR. Each rat tissue RNA (2 µg) was reverse transcribed (Omniscript; Qiagen, Valencia, CA) to cDNA and then amplified by PCR (Advantage 2 DNA polymerase; Clontech, Palo Alto, CA) for 35 cycles (30 s at 95°C, 2 min at 68°C). Because the intron-exon boundaries of the rat UT-B gene are not known, we designed rat UT-B primers that would correspond to regions within exons 9 (forward primer) and 11 (reverse primer) of the human UT-B gene (13) using Primer Designer v4.10 software (Science and Educational Software, Durham, NC). The forward primer includes nucleotides 910 to 931 (5'-GTCATCGCAGGTCTCAGTCTTG-3'); the reverse primer includes nucleotides 1208 to 1229 (5'-CGGTTCTCCTCGGAGTAGGTAA-3'); and the PCR product size is 320 bp. We calculated that the same-sized PCR product would result from either of the published rat UT-B clones (3, 32). PCR reaction mixtures were separated by electrophoresis on ethidium bromide-stained 1% agarose gels in TAE (Tris-acetate-EDTA) buffer, and the bands were excised under ultraviolet light and purified using the QIAquick gel extraction kit (Qiagen). Samples were fluorescently labeled by PCR (ABI Prism Dye Terminator Cycle Sequencing Ready Reaction Kit; Applied Biosystems, Foster City, CA), purified with P-30 polyacrylamide columns (Micro Bio-spin; Bio-Rad, Hercules, CA), and then sequenced using an ABI Prism 310 Genetic Analyzer (Applied Biosystems).


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

UT-B protein in erythrocytes. Western analysis performed with our affinity-purified UT-B antisera showed a broad band between 45 and 65 kDa in human erythrocytes and between 37 and 51 kDa in rat erythrocytes; detection of the bands in human erythrocytes was eliminated by preadsorbing the antibody with the immunizing peptide (Fig. 2A). Pretreating human or rat erythrocytes with PNGase F converted the broad band to a tight band at 32 kDa (Fig. 2B). In human erythrocytes, the UT-B protein is the Kidd (or Jk) blood group antigen (21). We detected the 45- to 65-kDa protein band in Jk(a+b+) erythrocytes. However, Jk(a-b-) erythrocytes showed only a single faint band at 55 kDa, suggesting a markedly reduced amount of UT-B protein (Fig. 2C).


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Fig. 2.   UT-B protein in human or rat erythrocytes. A: a representative Western blot showing a broad band between 45 and 65 kDa in human erythrocytes and between 37 and 51 kDa in rat erythrocytes. Bands in human erythrocytes were not detected when probed with antibody that was preadsorbed with the immunizing peptide (Human preads). B: deglycosylation of UT-B proteins from human or rat erythrocytes. Human or rat erythrocytes were washed and solubilized and then either treated (+) or not (-) with peptide N-glycosidase F (PNGase F). Pretreating human or rat erythrocytes with PNGase F converted the broad band to a tight band at 32 kDa. C: UT-B in erythrocytes from a normal individual [Jk(a+/b+)] and one lacking the human Kidd (Jk) blood group antigen [Jk(a-/b-)]. The 45- to 65-kDa protein band is present in Jk(a+b+) erythrocytes. However, Jk(a-b-) erythrocytes only show a single faint band at 55 kDa, suggesting a markedly reduced amount of UT-B protein.

UT-B protein in kidney. In rat kidney outer and inner medulla, a broad band between 41 and 54 kDa was detected (Fig. 3A); this band was not detected in cortex. In addition, a larger band at 98 kDa was detected in all kidney regions. Each of these bands was specifically recognized by the anti-UT-B antibody, since they are not recognized by antibody that had been preadsorbed by the immunizing peptide (Fig. 3B). The same size bands were also detected in mouse and chimpanzee inner medulla (Fig. 3C). Immunofluorescence of human or rat kidney confirmed (8, 33) the presence of UT-B protein in the endothelial cells of the medullary portions of the vasa recta (Fig. 4). In human kidney, the vasa recta, identified by the presence of red blood cells in their lumen and by positive staining for factor VIII, an endothelial cell marker (Fig. 4I), showed staining for UT-B (Fig. 4H). However, it was not possible to distinguish apical vs. basolateral plasma membrane localization in the endothelial cells. UT-B staining was not detected in inner medullary collecting ducts, which are identified by positive staining for keratin, an epithelial cell marker (Fig. 4J). Electron microscopy of several kidney sections showed UT-B staining in nonfenestrated endothelial cells (Fig. 5), characteristic of descending vasa recta (7). However, the preembedding immunoperoxidase method used in this study did not have the resolution necessary to determine whether UT-B was staining the apical plasma membrane, the basolateral plasma membrane, or both, because of the very thin, flat morphology of the endothelial cells. No UT-B staining was detected in fenestrated capillaries (Fig. 5).


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Fig. 3.   UT-B protein in kidney. Rat kidney was divided into cortex (Ctx), outer medulla (OM), and base and tip of inner medulla (IM). A: in outer and inner medulla, but not in cortex, a broad band between 41 and 54 kDa was detected. A sharp band at 98 kDa was detected in all kidney regions. B: both the 98-kDa and the 41- to 54-kDa bands in rat inner and outer medulla were ablated by preadsorption of the UT-B antibody with immunizing peptide. C: the same-sized bands (41-54 kDa and 98 kDa) were detected in inner medulla harvested from mouse and chimpanzee.



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Fig. 4.   Immunohistological detection of UT-B protein in human and rat kidney. Immunofluorescence studies of human kidney with the anti-UT-B antibody are negative in the cortex (A, original magnification ×100; scattered yellow granules are due to nonspecific autofluorescence), but positive stain is noted in the wall of the vasa recta in outer (C, original magnification ×100) and inner medulla (E, original magnification ×200). Immunofluorescence studies in rat kidney also show negative staining in the cortex (B, original magnification ×100) but positive staining of the vasa recta lining in outer (D, original magnification ×100) and inner medulla (F, original magnification ×100). Positive immunohistochemical staining of the endothelial lining of vasa recta of human renal inner medulla with the anti-UT-B antibody is indicated by arrowheads (G, original magnification ×100; and H, original magnification ×400). The UT-B-positive endothelium of medullary vasa recta is factor VIII positive (I, arrowhead; original magnification ×400) but keratin negative (J, arrowheads; original magnification ×400).



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Fig. 5.   A section of the inner stripe of the outer medulla of rat kidney showing a thin limb (TL), fenestrated capillary (FC), and nonfenestrated capillary (NFC). Only the nonfenestrated capillary is labeled (black staining) by the UT-B antibody (original magnification, ×9,500).

UT-B protein in nonrenal tissues. In rat brain, bands at 44, 53, 70, and 98 kDa were detected (Fig. 6, left). In colon, only a single band at 98 kDa was detected. In heart, bands at 44, 70, and 98 kDa were detected. In liver, bands at 48, 53, and 98 kDa were detected. In lung, bands at 44 and 98 kDa were detected. In testis, bands at 48 and 98 kDa were detected. Detection of the bands in rat tissues was reduced or eliminated by preadsorbing the antibody with the immunizing peptide (Fig. 6, right).


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Fig. 6.   Extrarenal expression of UT-B proteins in rat. In brain, colon, heart, liver, lung, and testis, several bands are detected (left). Each of these bands was specifically recognized by the UT-B antibody, since they were not detected when blots were probed with antibody that was preadsorbed with 0.1 µg/ml of the immunizing peptide (right).

UT-B mRNA in rat tissues. Figure 7A shows a diagram of the PCR primers in relation to the rat UT-B cDNA. RT-PCR yielded a single 320-bp product for each kidney region (Fig. 7B) and in heart, liver, brain, lung, colon, and testis (Fig. 7C). Control experiments (without reverse transcription) did not yield bands in any tissue (data not shown). Sequencing of both strands of the PCR product from kidney cortex, outer medulla, and inner medullary base and from heart, liver, brain, and colon confirmed that the PCR product was UT-B (data not shown).


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Fig. 7.   A: diagram of rat UT-B cDNA. The coding region of UT-B is shown in white and the untranslated regions in black. PCR primers are depicted as forward (sense) and reverse (antisense) single-headed arrows. Numbers below each primer correspond to the nucleotide position. The expected 320-bp PCR product is shown in gray. B: a single 320-bp RT-PCR product was detected in all rat kidney regions. C: a single 320-bp RT-PCR product was detected in heart, liver, brain, lung, colon, kidney inner medullary base, and testes. No PCR product was obtained when reverse transcriptase was omitted (data not shown).

Deglycosylation of UT-B protein in tissue. Treatment with PNGase F converted the broad band in kidney inner or outer medulla to a 32-kDa band but had no effect on the 98-kDa band (Fig. 8). PNGase F also had no effect on the 98-kDa band in kidney cortex, liver, or brain or on any of the smaller bands detected in these tissues (Fig. 8).


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Fig. 8.   Deglycosylation of UT-B proteins in rat. Tissue samples, control (-) or treated (+) with PNGase F, were analyzed by Western blot. In both renal inner and outer medulla, the 41- to 54-kDa UT-B protein was deglycosylated to a 32-kDa band. There was no apparent change in the size of the 98-kDa protein. No evidence of deglycosylation products is shown in renal cortex, liver, or brain.

Native gel analysis of UT-B protein in tissue. Protein samples from kidney outer medulla or liver were analyzed and compared using standard SDS-PAGE (i.e., reducing and denaturing); nondenaturing, nonreducing (i.e., native) PAGE; and denaturing, nonreducing PAGE. Only a single protein band was detected on the native gel (Fig. 9), suggesting a single protein complex containing the polypeptide forms detected by SDS-PAGE. In contrast, both the smaller and larger molecular weight bands were detected by denaturing, nonreducing PAGE (Fig. 9, right lanes), suggesting that the higher molecular weight band is a either a single polypeptide or a protein complex of the smaller bands that associate through noncovalent interactions, resistant to SDS, or through covalent bonds other than disulfide linkages.


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Fig. 9.   Separation of native vs. denatured proteins. Left: comparison of proteins from rat outer medulla that were denatured and reduced (+SDS, +beta ME) and separated on a 10% gel (left lane); a nondenaturing, nonreduced sample (native) electrophoresed on 6% or 7.5% gels (middle lanes); and a denatured, nonreduced (+SDS, -beta ME) sample on a 10% gel (right lane). beta ME, beta -mercaptoethanol. Right: a similar analysis of protein from liver. Molecular mass standards are only applicable to +SDS lanes. No UT-B proteins were identified on a control native gel electrophoresed with a reversed polarity (data not shown). In either tissue, the multiple protein bands observed in the denatured (SDS) samples appear to be derived from a single native protein species.

UT-B protein in aortic endothelial cells. Bands at 31, 44, and 98 kDa were detected in cultured bovine aortic endothelial cell lines (Fig. 10, left); detection of these bands was eliminated by preadsorbing the antibody with the immunizing peptide (data not shown). Immunofluorescence confirmed the presence of UT-B protein in cultured bovine aortic endothelial cell lines (Fig. 10, top right). As a control, cells were stained with the secondary antibody only. The secondary antibody stained the nuclei but did not stain the cell edges (Fig. 10, bottom right), suggesting that the cell edge staining is specific for UT-B while the nuclear staining is nonspecific.


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Fig. 10.   UT-B protein in cultured bovine aortic endothelial cells. Left: Western blot showing bands at 31, 44, and 98 kDa. Top right: the light areas at the periphery of the cells indicate the presence of UT-B protein by immunofluorescence (original magnification, ×1,000). Bottom right: cells show background fluorescence from FITC-conjugated goat anti-rabbit IgG in the absence of primary antibody treatment.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The major result of this study is that we have shown, for the first time, that UT-B protein is expressed in rat tissues other than kidney or erythrocytes. We obtained this result using a new polyclonal antibody to a human UT-B peptide sequence that detects UT-B in human and rat erythrocytes. Using RT-PCR and sequencing of the PCR product, we have shown that UT-B mRNA is present in every kidney region or extrarenal tissue that expresses UT-B protein. We have found that there is some variability in the protein band sizes detected in different tissues. We have no explanation for this variability in band size, and future studies are needed to address this issue.

As reported by others, UT-B protein is present in vasa recta within the renal medulla (8, 33). However, we have extended these studies by showing that UT-B protein expression is limited to nonfenestrated endothelial cells, i.e., descending vasa recta (7). UT-B protein is also expressed in chimpanzee and mouse kidney inner medulla and in cultured bovine aortic endothelial cell lines. The latter finding suggests that UT-B may be widely expressed in endothelial cells in the body.

We also have shown that UT-B is a glycoprotein in human and rat erythrocytes and in kidney medulla. Deglycosylation with PNGase F reduced the size of human or rat erythrocyte UT-B protein to 32 kDa, consistent with the size of the nonglycosylated, in vitro-translated hUT-B1 (8). Unexpectedly, we detected a 98-kDa band in kidney and all extrarenal tissues that expressed UT-B; this band is not present in erythrocytes. The 98-kDa band was eliminated when the antibody was preadsorbed with the immunizing peptide, suggesting that antibody recognition of this band is specific. Previous studies of UT-B protein expression (8, 33) did not evaluate proteins larger than 60 kDa. Thus we cannot assess whether the antibodies used in those studies (8, 33) also revealed this 98-kDa band. When kidney medulla or liver proteins were analyzed using native (nondenaturing, nonreducing) conditions, only a single protein band was detected, suggesting that UT-B protein exists as a complex. The complex does not depend solely on disulfide linkages because it remained intact in the presence of beta -mercaptoethanol. Although the 98-kDa complex should contain the lower molecular weight UT-B glycoprotein, the protein size was unaffected by treatment with PNGase F. Thus additional studies, such as peptide mapping or raising a second antibody to another site on UT-B, are needed to determine whether the 98-kDa band is really UT-B or a complex containing UT-B. In the latter case, additional studies would be needed to clarify the nature of this protein complex and whether it consists exclusively of UT-B.

The human UT-B gene is located near the same locus (chromosome 18q12.1-q21.1) as the human UT-A gene and the human Kidd blood group antigen locus (13, 20, 21). Erythrocytes from individuals lacking the Kidd antigen [Jk(a-b-), or Jk null] lack facilitated urea transport (6, 26). In this study, we found that UT-B was significantly diminished, but detectable, in Jk(a-b-) human erythrocytes. Using their antibody to human UT-B, Xu and colleagues (33) did not detect any UT-B in Jk(a-b-) human erythrocytes. We cannot explain this difference, but one possibility is a difference in the expression or conformation of UT-B protein between different individuals with the Jk(a-b-) phenotype (29).

Role in the urine-concentrating mechanism. We previously showed that individuals lacking the Kidd antigen are unable to concentrate their urine above 800 mosmol/kgH2O (26) and suggested that facilitated urea transport in erythrocytes is necessary to preserve the efficiency of countercurrent exchange as proposed by Macey (14). Phloretin-inhibitable urea transport is present in erythrocytes and in perfused rat descending vasa recta (22-24). Therefore, the expression of UT-B protein in erythrocytes and vasa recta (present study; Refs. 8 and 33) suggest that urea transport occurs via UT-B protein in erythrocytes and descending vasa recta. Mathematical modeling of microcirculatory exchange between ascending and descending vasa recta predicts that urea transporters (UT-B) are necessary to counterbalance the effect of aquaporin-1 water channels in the descending vasa recta; i.e., in the absence of UT-B, the efficiency of small solute trapping within the renal medulla is decreased, decreasing the efficiency of countercurrent exchange and urine-concentrating ability (4, 5). Thus the production of maximally concentrated urine may require UT-B protein expression in erythrocytes (15, 26) and in descending vasa recta (4, 5).


    ACKNOWLEDGEMENTS

We thank Dr. William E. Mitch (Renal Division, Department of Medicine, Emory University) for critical reading of this manuscript. We thank Patricia Rouillard, Bronislava Stockman, and Sharon Langley for technical assistance in the performance of these studies.


    FOOTNOTES

* R. T. Timmer and J. D. Klein contributed equally to this work.

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants PO1-DK-50268, RO1-DK-41707, and RO1-DK-53917.

Portions of this work have been published in abstract form (30, 31) and presented at the 31st Annual Meeting of the American Society of Nephrology, October 25-28, 1998, Philadelphia, PA, and Experimental Biology '99, April 17-21, 1999, Washington, DC.

Address for reprint requests and other correspondence: J. M. Sands, Emory Univ. School of Medicine, Renal Division, WMRB Rm. 338, 1639 Pierce Drive NE, Atlanta, GA 30322 (E-mail: jsands{at}emory.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 22 December 2000; accepted in final form 10 May 2001.


    REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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