UT-A3: localization and characterization of an additional urea
transporter isoform in the IMCD
James M.
Terris1,2,
Mark
A.
Knepper1, and
James B.
Wade3
1 Laboratory of Kidney and Electrolyte Metabolism, National
Heart, Lung, and Blood Institute, National Institutes of Health,
Bethesda; 2 Department of Physiology, Uniformed Services
University of the Health Sciences, Bethesda; and 3 Department of
Physiology, University of Maryland School of Medicine, Baltimore,
Maryland 21201
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ABSTRACT |
UT-A3 has recently been
identified as a splicing variant transcript of the UT-A gene present in
the kidney. To study the cellular and subcellular localization of
UT-A3, we raised a new polyclonal antibody to its COOH-terminal end.
Immunoblots identified bands at 44 and 67 kDa predominately in the
inner medulla and showed that the antibody does not recognize UT-A1.
Deglycosylation with PNGase decreased the molecular mass of both forms
to 40 kDa. UT-A3 is most abundant in the inner third of the inner
medulla and is present in membrane fractions. Cell fractionation
studies showed that UT-A3 is only detectable in inner medullary
collecting duct (IMCD) cells. These observations were confirmed with
immunolocalization studies demonstrating an exclusive labeling of IMCD
cells. Double-labeling studies with anti-Na-K-ATPase demonstrated UT-A3
in intracellular membranes and in the apical region but were
incompatible with a basolateral site for UT-A3. Although the function
of this isoform in the inner medulla is unknown, the large abundance
suggests that it may be important in the renal handling of urea.
urinary concentrating mechanism; inner medullary collecting duct
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INTRODUCTION |
THE PHYSIOLOGICAL
BASIS of renal urea transport has been greatly clarified by the
identification of distinct epithelial urea transporters via molecular
cloning (21, 22, 29). Initially, two transporters were
identified that were transcribed from the same gene from two distinct
promoters. This gene has been referred to as "UT-A" to distinguish
it from the gene coding for a urea transporter expressed in
erythrocytes and endothelial cells, termed "UT-B"
(18). The larger of the UT-A isoforms, termed "UT-A1," (originally called UT1) has an open reading frame of 929 amino acids
(21) and is expressed solely in the inner medullary
collecting duct (IMCD) (19). A second isoform, termed
"UT-A2" [originally called "UT2" (22, 29)]
corresponds to the COOH-terminal 397 amino acids of UT-A1. RT-PCR
studies in microdissected renal tubules (19) found UT-A2
mRNA in the descending thin limbs of short loops of Henle in outer
medulla and in descending limbs of long loops of Henle in inner
medulla. UT-A2 expression appears to be absent in descending limbs of
long loops in outer medulla. Northern blotting studies have revealed
that the abundance of the UT-A2 transcript is increased in rats in
response to water restriction (22) and by arginine
vasopressin (AVP) infusion (15, 20). UT-A1 mRNA abundance,
however, does not appear to be consistently increased by AVP infusion
(15) and may even be decreased slightly (20).
Immunolocalization studies of UT-A isoforms showed that
peptide-directed antibodies raised to the COOH terminal of UT-A1 (whose COOH-terminal sequence is identical to that of UT-A2) labeled both the
IMCD and the thin descending limbs of Henle's loop (13). Immunoblots run with membrane fractions from the inner medulla revealed
bands of 97 and 117 kDa (13, 25). The two sizes are the
result of different amounts of glycosylation of the same core protein
(1). Chronic elevation of AVP in rats does not appear to
change the abundance of the 97-kDa form and decreased the abundance of
the 117-kDa form (25). A recent study used surface
biotinylation to carefully examine the possibility that UT-A1 might
traffic to the apical surface of IMCD cells in response to AVP
(6). Although trafficking of aquaporin-2 (AQP2) could be
clearly shown, the distribution of UT-A1 was unaltered under conditions
known to strongly stimulate IMCD urea permeability. On the basis of these data, the prevailing view in the field is that UT-A1 responds to
AVP by mechanisms different from AQP2. Recently, the UT-A gene has been
shown to produce at least two other transcripts with the cloning of
cDNAs for UT-A3 and UT-A4 (8). The renal tubular localization and functional roles of these transcripts remain undefined. The present work describes a new antibody raised to the COOH
terminal of UT-A3 that does not appear to recognize UT-A1 or other
known urea transporters. This work localizes UT-A3 to the terminal
portion of the IMCD.
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MATERIALS AND METHODS |
Experimental animals.
All experiments were conducted in accord with animal protocols for rats
approved by the Animal Care and Use Committee of the National Heart,
Lung, and Blood Institute. Pathogen-free Sprague-Dawley (Taconic Farms,
Germantown, NY; National Cancer Institute, Frederick Cancer Research
Facility, Frederick, MD) male rats, weighing 192-225 g, were used
in these studies. Body weights of all rats were carefully matched in
each protocol, and animals were fed an ad libitum intake of a standard
rat chow containing 18% protein and 130 meq Na+/kg (NIH-31
autoclavable rodent diet, Ziegler Brothers, Gardner, PA). The rats were
kept in filter-top microisolator cage with autoclaved feed and bedding,
to maintain a pathogen-free state.
Antibodies.
To obtain the anti-UT-A3, the polyclonal antibody used in these
studies, a synthetic peptide was designed for specificity, antigenicity
and absence of posttranslational modifications using computer analysis.
They were produced by standard solid-phase, peptide-synthesis
techniques, purified by high-performance liquid chromatography, and
conjugated to maleimide-activated keyhole limpet hemocyanin via
covalent linkage to the NH2-terminal cysteine. For each
antibody, two rabbits were immunized by using a combination of
Freund's complete and incomplete adjuvants. The antisera obtained were
affinity purified by using a column on which 2 mg of the immunizing
peptide were immobilized via covalent linkage to agarose beads
(SulfoLink Kit; Pierce, Rockford, IL). The following published and
characterized antibodies were used for these studies: rabbit L194 and
L403 [UT-A1, UT-A2, and UT-A4 (characterized in Refs. 13
and 25; COOH-terminal 19 amino acids 911-929; sequence: H2N-QEKNRRASMITKYQAYDVS-COOH)]; rabbit L448 [UT-A1
(characterized in Ref. 25; middle loop, amino acids
500-522; sequence:
NH2-KVFGKSEHQERQTKEPLPYLYRK-COOH)]; rabbit Q2
[UT-A3 (amino acids 447-460; sequence:
NH2-TAKRSDEQKPPNGD-COOH)]; and rabbit L446 [UT-A1,
UT-A3, UT-A4 (characterized in Refs. 1 and
27; amino acids 56-78; sequence:
NH2-EEKDLRSSDEDSHIVKIEKPNER-COOH)].
Other antibodies used were to aquaporin-1 (AQP1; L266, characterized in
Ref. 24), AQP2 (L751, sequence:
NH2-VELHSPQSLPRGSKA-COOH) and to Na-K-ATPase (Upstate
Biotechnology, Lake Placid, NY).
Tissue preparation and immunoblotting.
The rats were euthanized by rapid decapitation. Kidney homogenates were
prepared according to Terris et al. (25). Briefly, the
kidneys were removed and one kidney was dissected into inner medulla,
outer medulla, and cortex. The tissues were homogenized with an Omni
1000 fitted with a microsawtooth generator in ice-cold isolation
solution (adjusted to pH 7.6 with NaOH) containing 250 mM sucrose/10 mM
triethanolamine, 1 µg/ml leupeptin, and 0.1 mg/ml phenylmethylsulfonylfluoride. Some samples were centrifuged at 200,000 g for 1 h (Beckman L8-M ultracentrifuge with a
type-80TI rotor) to produce a plasma membrane- and intracellular
vesicle-free supernatant. The total protein concentration of all
samples was determined with a Pierce BCA Protein Assay reagent kit
(Pierce) and adjusted to ~1 µg/µl with isolation solution.
Laemmli buffer (5×; 7.5% SDS, 30% glycerol, 1 M Tris, pH 6.8, bromophenol blue) with 30 mg/ml dithiothreitol (DTT) was added to the
samples in a ratio of 1:4 and heated to 60°C for 15 min.
Electrophoresis was performed on Bio-Rad polyacrylamide minigels, and
the proteins were transferred electrophoretically to nitrocellulose
membranes. After blocking with 5% nonfat dry milk for 30 min, the
nitrocellulose membranes were probed with affinity-purified antibody,
described above, for 24 h at 4°C, washed, and exposed to
secondary antibody (donkey anti-rabbit IgG conjugated with horseradish
peroxidase, Pierce no. 31458). Sites of antibody-antigen reaction were
visualized by using luminol-based enhanced chemiluminescence (LumiGLO,
Kirkegaard and Perry Laboratories, Gaithersburg, MD, or SuperSignal,
Pierce). The blots were quantitated by densitometry (model PDSI-P90,
Molecular Dynamics, Sunnyvale, CA).
Fixation of tissue and immunocytochemistry.
Kidneys of ketamine-xylazine-anesthetized rats were fixed with 2%
paraformaldehyde in PBS by retrograde perfusion through the abdominal
aorta, and antibodies were immunolocalized on frozen sections as
previously described (27). Sections were incubated overnight at 4°C with primary antibodies diluted to 10 µg/ml. Secondary antibodies were species-specific donkey anti-rabbit and
donkey anti-mouse antibodies (Jackson Immunoresearch Labs, West Grove,
PA) coupled to Alexa 488 and Alexa 568, respectively (Molecular Probes,
Eugene, OR).
Preparation of IMCD suspensions.
Suspensions were prepared as described by Chou et al. (2).
The rats were injected with 0.5 ml furosemide (10 mg/ml), and 20-30 min later were rapidly decapitated. Both kidneys were
perfused with 15 ml of ice-cold dissection fluid to wash out blood and perfused with 20 ml of prewarmed digestion solution containing 2 mg/ml
collagenase B (Boehringer Mannheim, Indianapolis IN), 540 U/ml
hyaluronidase (Worthington Biochemical, Freehold, NJ), and 0.5 mg/ml
bovine serum albumin dissolved in the basic bicarbonate-buffered tubule
suspension solution containing (in mM) 118 NaCl, 25 NaHCO3, 4 Na2HPO4, 2 CaCl2, 1.2 MgSO4, 5 glucose, and 5 sodium acetate (300 mosmol).
After removal of both kidneys, the inner medullas were dissected and
finely minced with a razor blade into 1-mm cubes. The minced tissue was
transferred into 12 × 75-mm glass tubes, suspended in the same
digestion solution, and incubated at 37°C with 95% air-5%
CO2 superfusion. After a 30-min initial incubation period, DNase I (Boehringer Mannheim) was added to a final concentration of
0.001%, and the incubation continued for another 20 min. The suspensions were aspirated with a large-bore Pasteur pipette every 15 min to break up large tissue fragments. After the incubation, the
suspensions were transiently (10 s) centrifuged at 50 g, and the pellet was resuspended in tubule suspension fluid containing 0.001% DNase I. The supernatants were discarded. This procedure was
repeated two more times, and the supernatants were combined. The
resulting inner medullary pellet suspensions consisted almost entirely
of collecting duct fragments with only very few thin limb fragments, as
confirmed by viewing the suspensions under a dissection microscope. The
final pellet was resuspended with 1.5-2 ml of tubule suspension
fluid to yield a protein concentration of ~10 µg/50 µl of suspension.
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RESULTS |
Figure 1 shows a schematic
representation of UT-A1 and UT-A3 proteins illustrating epitope
locations recognized by the antibodies employed in this study. H1-H4
represent hydrophobic regions likely to contain membrane-spanning
domains. The L403/L194 antibodies, previously characterized (13,
25), label an epitope at the COOH terminus of UT-A1 not found in
UT-A3. For the studies reported here we also utilized antibodies that
recognize epitopes in the intracellular loop of UT-A1 (L448) and the
NH2 terminus of UT-A1 and UT-A3 (27). The
UT-A3 isoform is homologous to the first 460 amino acids of the
NH2-terminal end of UT-A1, except that the COOH-terminal
amino acid of UT-A3 is aspartic acid whereas the corresponding amino
acid in UT-A1 is glycine. The anti-UT-A3 antibody (designated "Q2")
was raised to amino acids 447-460 of the COOH terminus of UT-A3.

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Fig. 1.
UT-A1 (top) and UT-A3 (bottom)
polyclonal antibody recognition sites. Rabbit antiserum L446 recognizes
amino acids (a.a.) 56-78 of the NH2 terminus of UT-A1
and UT-A3; rabbit antisera L403 and L194 recognize amino acids
911-929 of the COOH terminus of UT-A1; L448, amino acids
500-522 of UT-A1 H1-H4 represent postulated membrane-spanning
domains. Antiserum Q2 was raised against the COOH-terminal amino acids
of UT-A3, which differ from the corresponding sequence in UT-A1 in that
the COOH-terminal amino acid of UT-A3 is aspartic acid whereas the
corresponding amino acid in UT-A1 is glycine.
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Characterization of UT-A3-specific antibody.
Figure 2 is a Western blot probed with
the Q2 antibody and shows regional localization of bands at 44 and 67 kDa, consistent with the predicted molecular weight of UT-A3 plus
glycosylation. For this blot, tissue from the inner medulla, outer
medulla, and cortex was homogenized and centrifuged at 17,000 g to obtain membrane pellets. The inner medulla was cut into
thirds, IM3 representing the inner third (papillary tip), IM2 the
middle third, and IM1 the initial third (base). The greatest abundance
of both bands was in the inner third of the inner medulla. A light band
at 67 kDa was also found in the outer medulla, consistent with the
detection of mRNA for UT-A3 there (8). Neither of these
bands was detected in the cortex. Interestingly, despite the homology
of all but one amino acid with UT-A1, no significant amount of UT-A1
protein (97 and 117 kDa) was detected in any region by this antibody. Other studies (not shown) suggest that the lighter, higher molecular weight proteins labeled in the IM3 and IM2 regions are dimers of UT-A3
proteins.

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Fig. 2.
Western blot of 17,000-g samples probed with
the Q2 antibody showing the renal distribution of UT-A3 as 44- and
67-kDa proteins. IM, inner medulla; IM3, inner third of inner medulla
(papillary tip); IM2, middle third of inner medulla; IM1, initial third
of inner medulla (base); OM, outer medulla; CTX, cortex. The greatest
abundance of both proteins occurs in the inner third of the inner
medulla, decreasing in abundance toward the base of the inner medulla.
A weak band at 67 kDa appears in the outer medulla.
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To further characterize the 44- and 67-kDa proteins, fresh aliquots of
the samples used for Fig. 2 were probed with the L446 antibody,
specific for a site near the NH2 terminus of UT-A1, UT-A3,
and UT-A4. This blot was compared with one probed with antibody L448,
specific for an epitope found only in UT-A1. Figure 3 shows the results. As expected, L448
labeled proteins in the inner medulla in a pattern and with molecular
masses (97 and 117 kDa) consistent with UT-A1. In addition to these
UT-A1 proteins, L446 also identified bands at 44 and 67 kDa, the same
size as those recognized by the Q2 antibody. Detection of 44- and
67-kDa bands by both Q2 and L446 supports the view that the two
antibodies are detecting the same proteins, i.e., UT-A3. Note also that
antibody L446 recognizes bands in the cortex that may represent other
short UT isoforms.

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Fig. 3.
Western blot of 17,000-g samples probed with
the L446 (left) and L448 (right) antibodies.
UT-A1 and UT-A3 proteins are detected by the UT-A1
NH2-terminal L446 antibody and UT-A1 by the UT-A1
middle-loop antibody L448.
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To evaluate the specificity of the Q2 antibody for UT-A3 protein,
preadsorption controls were carried out. Blots were prepared utilizing
the 17,000-g fraction from the inner third of the inner medulla (Fig. 4,
A-C). The blot in A was carried
out with Q2 antibody previously incubated overnight with the immunizing
peptide, whereas the blot in B was done with the Q2 antibody
incubated alone. All bands were ablated after preincubation with the
specific peptide. Note that there are no UT-A1 protein bands at 97 and
117 kDa in B. The blot in B was then stripped of
IgG and reprobed with L403 to verify the position of the UT-A1 bands in
the blot (C).

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Fig. 4.
Western blot of 17,000-g samples of IM3.
Lane B was probed with the Q2 antibody. Proteins
at 44 and 67 kDa were recognized as in Figs. 2 and 3. In addition
lane A is an identical blot probed with antibody incubated
with the immunizing peptide. Both bands shown in lane B are
ablated, demonstrating that the antibody is specific for UT-A3.
Lane B was then stripped of IgG and reprobed with the UT-A1
COOH-terminal antibody L403 (lane C). UT-A1 protein is shown
at 97 and 117 kDa. It is significant to note that the 97- and 117-kDa
protein bands are absent in the blot probed with Q2. As expected, UT-A3
protein was not detected by L403.
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Figure 5 shows the immunoblots using
17,000- and 200,000-g pellets and the 200,000-g
supernatant from the three regions of the inner medulla. As previously
reported for UT-A1, the greatest abundance of UT-A3 was also in the
inner third of the inner medulla, decreasing in abundance in the middle
third and base in both the membrane-enriched and intracellular
vesicle-enriched fractions. In these heavily loaded gels,
high-molecular-mass bands are also detected that appear to be multimers
of the UT-A3 protein. There was no detectable protein in the
200,000-g supernatant.

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Fig. 5.
Western blot of the IM distribution of UT-A3. Consistent
with Figs. 2 and 3, the greatest abundance of UT-A3 protein in the
17,000-g fraction was found in IM3 (papilla) relative to IM1
(base). Additionally, a similar distribution of UT-A3 protein was also
observed in the 200,000-g pellet. There was no detectable
protein in the 200,000-g supernatant.
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Glycosylation of UT-A3.
To test whether the 44- and 67-kDa proteins are glycosylated,
17,000- and 200,000-g aliquots from the inner third
(IM3) of the inner medulla were incubated at 37°C for 1 h with
vehicle (
) or PNGase (+) (Fig. 6).
Another aliquot (c) was incubated at 4°C to control for possible
nonspecific effects of the 37°C incubation. Figure 6 shows that,
after incubation with the enzyme, there is a single band at ~40 kDa,
suggesting that the two bands represent different glycosylation states
of a single protein. It is significant to note that the 97- and 117-kDa
protein bands of UT-A1 are again absent in these blots probed with Q2.
The absence of these bands further supports the view that the Q2
antibody does not significantly recognize UT-A1 protein, although, with the exception of the COOH amino acid, it is specific for an epitope also found in the middle loop of UT-A1. The lack of recognition of
UT-A1 could be explained if the epitope were inaccessible to the
antibody when the loop is intact, as it would be in UT-A1. With UT-A3,
the COOH-terminal end is freely available to the antibody.

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Fig. 6.
Deglycosylation of samples of IM3 with (+) and without
( ) PNGase revealed a single band at ~40 kDa in both the 17,000- and
200,000-g fractions, suggesting 2 different glycosylation
states of a single protein. Lane c is an equivalent amount
of protein that was incubated on ice without PNGase as a reference
sample.
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Localization of UT-A3 to IMCD.
To characterize further the localization of UT-A3 protein, IMCD
enriched and non-IMCD enriched suspensions, consisting primarily of
thin limb segments, were probed with the Q2 antibody. As can be seen in
Fig. 7, UT-A3 protein was not detected in
the non-IMCD enriched sample, whereas it was found in the whole inner
medulla and IMCD enriched fractions, strongly suggesting localization to the collecting duct. This UT-A3 blot was then sequentially stripped
of IgG and reprobed for UT-A1, AQP2, and AQP1 proteins. UT-A1 and AQP2,
marker proteins for the IMCD (4, 13), showed a
distribution similar to that of UT-A3. To verify the presence of
protein in the non-IMCD lane, the blot was probed for AQP1 (thin
descending limb marker). As expected, the greatest abundance of AQP1
was found in the non-IMCD enriched fraction.

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Fig. 7.
Western blot of inner medullary collecting duct
(IMCD)-enriched and non-IMCD-enriched suspensions. Aliquots of whole
inner medulla, IMCD-enriched, and non-IMCD-enriched samples were
probed with (left to right) anti-UT-A3
(Q2), -UT-A1 (L403), -AQP2, and -AQP1 antibodies. There was no UT-A3,
UT-A1, or AQP2 protein (a collecting duct marker) detected in the
non-IMCD-enriched samples. Additionally, UT-A3 protein was not detected
by the UT-A1-specific antibody L403, and no significant UT-A1 protein
was detected by the Q2 antibody Finally, the presence of another
protein in the non-IMCD-enriched samples was confirmed by the detection
of AQP1 that is present in thin descending limbs of Henle's loop.
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Immunolocalization of UT-A3.
Antibody Q2 was used to localize UT-A3 by immunocytochemistry. Figure
8 shows a low-magnification localization
of Q2 labeling in inner medulla (A) with respect to
Na-K-ATPase (B). No specific labeling was detectable in the
cortex or outer medulla with antibody Q2. Very weak labeling was
present in the base of the inner medulla in the IM1 zone, with
progressively increased labeling of IMCDs in the intermediate IM2 zone
and very strong labeling in the IM3 zone at the tip of the papilla.
This labeling pattern corresponds closely to the pattern of labeling
seen in immunoblots of these zones (Fig. 2).

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Fig. 8.
A: immunolocalization of Q2 at low
magnification in longitudinal sections of renal cortex (C), OM, and
regions IM1, IM2, and IM3 of the IM. Labeling is undetectable in the OM
and cortex and increases progressively from region IM1 to IM3.
B: immunolocalization of Na-K-ATPase in the same section.
Scale bar, 0.5 mm.
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The abundance of UT-A3 protein in the IMCD suggests it may be important
in renal urea handling. One possible function for another urea
transporter in the IMCD would be to function as the basolateral urea
transporter. We tested this hypothesis by immunolocalizing Q2 labeling
with respect to Na-K-ATPase as shown in Fig.
9. Q2 labeling (A) is
distributed throughout IMCD cells (presumably in small vesicles) but
fails to label the lateral cell membrane (arrows), which are strongly
labeled by anti-Na-K-ATPase (B). In Fig. 9C,
labeling by Q2 antibody (shown in red) is directly compared with
respect to anti-Na-K-ATPase (shown in green) and shows that UT-A3
labeling overlaps with anti-Na-K-ATPase (shown in yellow) very
minimally at the basolateral surface. This localization is consistent
with an insignificant expression of UT-A3 in the basolateral plasma
membrane.

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Fig. 9.
A: high-magnification
immunolocalization of Q2 in cross sections of IMCD in IM3. Strong
cellular labeling is seen, but the lateral intercellular membranes are
poorly labeled (arrows). B: immunolocalization of
Na-K-ATPase in the same section shows that the basolateral surfaces are
strongly labeled by this antibody. C: combined Q2 labeling
(red) and Na-K-ATPase labeling (green) shows little or no labeling by
Q2 of the basolateral surface. Scale bar, 10 µm.
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DISCUSSION |
Urea transport by the kidney is critically important to the
urinary concentrating process and regulation of renal water excretion (10, 11). Our understanding of renal urea transport in has been greatly advanced by the identification of distinct epithelial urea
transporters via molecular cloning (21, 22, 29). Thus far
three urea isoforms have been characterized and localized in the
kidney. Of the two UT-A isoforms, UT-A1, also called UT1 (21), produces bands at 117 and 97 kDa (13,
25) and is expressed only in the IMCD (19). A
second UT-A isoform, termed "UT-A2" (originally called "UT2")
(22, 29) corresponds to the COOH-terminal 397 amino acids
of UT-A1. UT-A2 is a 55-kDa protein expressed in the descending thin
limbs of short loops of Henle in the outer medulla and in descending
limbs of long loops of Henle in the inner medulla (19,
27). A third renal urea transporter originally called HUT11
(14) and now known as UT-B1 (18) is produced by another gene and localizes to the vasa recta of the renal medulla (26, 28). On the basis of the finding that previous urea
transporter isoforms have each localized to distinct renal sites, it
was of interest to determine whether the newly identified UT-A3 isoform (8) would be expressed in a distinct renal segment.
Development of UT-A3-specific antibodies.
Previous immunolocalizations of UT-A isoforms (13, 27)
used antibodies raised to the COOH-terminal 19 amino acids of
UT-A1 whose COOH terminus is identical to that of UT-A2 and UT-A4.
These antibodies labeled not only the IMCDs but also the thin
descending limbs of Henle's loop, consistent with the expectation that
UT-A1 and UT-A2 would both be recognized. Because the different urea transporter isoforms that have been described are alternative splice
forms of UT-A1, we believed that it would not be possible to produce
antibodies to the short isoforms that do not recognize UT-A1. This
represents a major problem for strategies to understand the function of
each isoform. Here we characterized an antibody to the COOH terminal of
UT-A3 (called "Q2") that would be expected to also recognize UT-A1
because it differs from the sequence for that isoform by only one amino
acid. However, immunoblots with this antibody show no labeling of the
97/117-kDa bands known to be UT-A1. There are two strong bands at 44 and 67 kDa in the size range expected for UT-A3 that are ablated by the
immunizing peptide. Furthermore, deglycosylation of samples with PNGase
showed that both forms are glycosylated forms of the same core protein.
The absence of the UT-A1 bands from blots with Q2 supports the view that this antibody does not significantly recognize UT-A1 protein, at
least to the extent detectable by immunoblotting. The lack of
recognition of UT-A1 could be explained if the epitope were inaccessible to the antibody when the loop is intact, as it would be in
UT-A1 or because the epitope depends on the one amino acid that differs
between UT-A1 and UT-A3. With UT-A3, the COOH-terminal end could be
freely available to the antibody.
Site of UT-A3 expression.
Our immunolocalization studies show that UT-A3 localizes to the
terminal portion of the IMCD in a pattern very similar to UT-A1. This
was established by two independent assessments. Purified suspensions of
IMCDs were prepared using established methods (2), and
these blots showed that labeling by the Q2 antibody to UT-A3 was
strongly enriched in the IMCD over non-IMCD fractions. The IMCD
fraction was rich in AQP2 and had less AQP1 than the non-IMCD fraction,
as expected. These findings suggested that UT-A3 is expressed in IMCDs.
This was also demonstrated by immunofluorescent localizations showing
that Q2 labels IMCD cells with progressively greater intensity toward
the tip of the inner medulla.
Possible functional role of UT-A3.
The large abundance of the protein in samples from the tip of the inner
medulla suggests UT-A3 may be important in renal urea handling.
Physiological measurements have developed evidence for the presence of
a phloretin-sensitive pathway for urea at the basolateral as well as
the apical surface of the IMCD (3, 23). Because one
possible function for UT-A3 in the IMCD would be to function as the
basolateral urea transporter, we tested that hypothesis by
immunolocalizing Q2 with respect to Na-K-ATPase. The absence of overlap
at the basolateral surface is inconsistent with a basolateral function
for UT-A3, making it very unlikely that UT-A3 represents the
basolateral urea transporter. This function might be accomplished by
AQP3, which has been shown to be permeable to urea as well as water
(7). Alternatively, basolateral urea flux might be carried
out by UT-A4 or another uncharacterized UT (5).
UT-A3 may have a role in mediating the well-established action of AVP
on IMCD urea permeability (11, 17). On the basis of the
localization of UT-A1 in IMCD cells, it has been presumed that UT-A1 is
involved in this action of AVP (21). If so, the regulatory
mechanism involved is very different from the action of AVP action on
water permeability because neither translocation of UT-A1
(6) nor changes in its abundance appear to occur in response to AVP stimulation (25). UT-A1 expression is,
however, regulated by other factors (9, 12, 16, 25). Our
finding that the IMCD expresses more than one urea transporter isoform raises the possibility that UT-A3 may mediate the effect of AVP on IMCD
urea permeability. Additional work will be required to determine
whether UT-A3 functions in this or some other role in the IMCD.
 |
ACKNOWLEDGEMENTS |
We thank Jie Liu for expert technical help with immunolocalizations.
 |
FOOTNOTES |
This work was supported by National Diabetes and Digestive and Kidney
Diseases Grant DK-32839 (to J. B. Wade) and by the intramural budget of the National, Heart, Lung, and Blood Institute (project no.
Z01-HL-01282-KE; to M.A. Knepper). The Confocal Microscope Facility
used for the immunolocalizations was funded by National Science
Foundation Grant BIR9318061.
Address for reprint requests and other correspondence: J. B. Wade, Dept. of Physiology, 655 W. Baltimore St., Univ. of Maryland, Baltimore, MD 21201 (E-mail: jwade{at}umaryland.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.
 |
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