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
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ABSTRACT |
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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(ab
)], 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
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INTRODUCTION |
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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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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.
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METHODS |
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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 -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).
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RESULTS |
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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(ab
) 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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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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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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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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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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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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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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DISCUSSION |
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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 -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(ab
), 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).
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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* 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.
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