Renal Physiology

UT-A3: localization and characterization of an additional urea transporter isoform in the IMCD

James M. Terris, Mark A. Knepper, James B. Wade


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

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.


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.


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. 13and 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 and27; 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 200,000g 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.


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.

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.

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 17,000g 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.

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.

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. Figure3 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.

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.

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,AC). 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).

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 additionlane 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.

Figure 5 shows the immunoblots using 17,000- and 200,000-g pellets and the 200,000-gsupernatant 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.

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.

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.

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.

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.

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.

Immunolocalization of UT-A3.

Antibody Q2 was used to localize UT-A3 by immunocytochemistry. Figure8 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).

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.

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. 9 C, 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.

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.


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.


We thank Jie Liu for expert technical help with immunolocalizations.


  • 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}

  • 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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