INTRODUCTION

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TO COMBAT the osmotic stress of living in a marine environment, sharks, skates, and rays, collectively belonging to the elasmobranchs, maintain relatively high tissue concentrations of urea (~350 mM). Consequently, a large concentration gradient for urea exists between elasmobranch plasma and seawater (2). Despite this, elasmobranchs excrete a relatively small amount of urea. In spiny dogfish (Squalus acanthias), as with other elasmobranchs, the gills represent a large surface area for loss and are the primary site of urea excretion [>95% of total urea lost from body (33)]. Although urea is freely filtered by the glomerulus, >5% of the total body urea excretion occurs via the kidney (33). In euryhaline elasmobranchs, such as the little skate Raja erinacea, the kidney is the primary site for the regulation of tissue urea levels. When transferred to dilute seawater, renal tubular urea reabsorption decreases resulting in an increase in the renal clearance of urea (8, 22). In contrast, the urea permeability of the gills remains constant. Clearly, substantial regulated reabsorption of urea occurs in elasmobranch kidney, yet the molecular mechanism of this process and how it is regulated is unknown.

Since the times of H. W. Smith (29, 30), it has been speculated that a carrier-mediated process is involved in elasmobranch renal urea reabsorption. The fact that urea is reabsorbed against a sizable urea gradient (16, 17, 25, 29) has led to the proposal that the reabsorption mechanism is active, possibly coupled to the movement of Na⁺ (10, 26); however, there is little empirical evidence supporting this notion. Ion-coupled (i.e., secondary active) urea transporters have been described in the rat inner medullary collecting duct (IMCD) (13, 15); however, the molecular identity of this transporter has not yet been determined. An alternative passive reabsorption mechanism has been proposed for marine elasmobranchs that is similar to that in the mammalian collecting duct but, again, remains to be fully tested (3, 7). In mammalian species, facilitative urea transporter proteins play a crucial role in the urinary concentration mechanism and in regulating urea excretion by the kidney (1, 28). Two groups of urea transporter (UT) proteins have been isolated that are the products of two genes, types A and B (24). In the terminal mammalian nephron, proteins belonging to the urea transporter protein family UT-A have been isolated and characterized (5, 27, 28). These transporters are facilitators of urea diffusion and are not capable of transporting urea against an opposing urea gradient. They are inhibited by millimolar concentrations of phloretin, and some are stimulated by vasopressin (27, 28). Proteins belonging to the B isoform were originally isolated from erythropoietic tissue (20) but have since been found to have a wider tissue distribution (24). They are similar in structure to the UT-A proteins, and urea transport via these proteins is inhibited by phloretin and urea analogs (18, 20). As a first step to understanding the molecular basis of how elasmobranch kidney retains urea, we have isolated and characterized a cDNA from the kidney of the spiny dogfish, S. acanthias, encoding a homologue of the mammalian UT-A2 urea transporter.

	METHODS

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cDNA clones were isolated from a nonamplified S. acanthias kidney gt10 cDNA library. Approximately 300,000 clones were screened using a gel-purified, ³²P-labeled (^T7QuickPrime) rat UT2 cDNA. Hybridization was at 35°C (50% formamide), and the final wash was in 0.1× standard sodium citrate (SSC)-0.1% SDS at 37°C. Initial screening yielded 33 clones, out of which a 2.2-kb cDNA was isolated and subcloned into the Not1 site of pBluescript. Both strands of the cDNA clone were sequenced using an ABI dye terminator ready reaction kit.

cRNA was synthesized in vitro and microinjected (30 ng) into collagenase-treated and manually defolliculated Xenopus oocytes, as previously described (28). After incubation in modified Barth's solution (9) containing 50 µg/ml gentamicin for 3 days at 18°C, oocytes were preincubated in (in mM) 200 mannitol, 2 KCl, 1 MgCl₂, 1 CaCl₂, 10 HEPES buffer, and 5 Tris, pH 7.4, for 1 h. To measure urea uptake, 2.7 µCi [¹⁴C]urea/ml and 1 mM urea were added to the preincubation solution. Unlabeled urea was freshly deionized before use by passing through an ion-exchange column [AG501-X8(D), 20-50 mesh; Bio-Rad, Hercules, CA]. After uptake, oocytes were washed with ice-cold uptake solution containing 10 mM unlabeled urea and dissolved in 10% SDS, and the radioactivity was measured by scintillation counting. Inhibition of [¹⁴C]urea uptake by 0.35 mM phloretin was measured by addition of phloretin to the preincubation solution 15 min before addition of [¹⁴C]urea, as previously described (35).

To study the distribution of urea transporter mRNA in shark tissue, poly(A⁺) RNA was isolated from S. acanthias liver, blood, kidney, gill, intestine, muscle, rectal gland, and brain by the guanidine isothyocyanate method using Trizol (GIBCO, Grand Island, NY) followed by oligo(dT)-cellulose column chromatography (Collaborative Biochemical, Bedford, MA). Poly(A⁺) RNA (3 µg/lane) was separated in a 1% agarose gel in the presence of 2.2 M formaldehyde and transferred to nylon filters. Filters were probed using a ³²P-labeled full-length random-primed ShUT cDNA probe and hybridized at 35°C (low stringency) or 42°C (high stringency), both with 50% formamide. Final washing was in 0.1× SSC-0.1% SDS at 37°C (low stringency) or 65°C (high stringency).

	RESULTS

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Low-stringency screening of a S. acanthias kidney cDNA library with the full-length rat UT-A2 cDNA probe yielded a 2,191-bp cDNA. This cDNA has a polyadenylation sequence (ATTAAA) at position 2,154 and an open reading frame (ORF) from nucleotides 103 to 1,245 and putatively encodes a 380-residue protein (ShUT) that has 66% amino acid identity with rat UT-A2 (28), 61% identity with rat UT-B (6), and 67% identity with the vasopressin-regulated urea transporter from Rana esculenta (31) (Fig. 1). There are two putative N-glycosylation sites (N-I-T/S) at N174 and N204.

View larger version (63K): [in this window] [in a new window]   Fig. 1.   Alignment of urea transporter (UT) proteins. Amino acid sequence alignment of Squalus acanthias UT (ShUT), rat UT-A2, rat UT-B2, and Rana esculenta UT (Ran-UT) using multiple alignment Clustral algorithm (12) in DNASTAR software package. Numbers correspond to ShUT residues. Conserved potential N-glycosylation site (NIT) at N204 (shaded region) is indicated.

Injection of ShUT cRNA into Xenopus oocytes induced a 10-fold increase in the uptake (90 s) of ¹⁴C-labeled urea (1 mM) compared with water-injected control oocytes. Uptake (90 s) of urea by ShUT was inhibited 60% by 0.35 mM phloretin (Fig. 2). A time course of urea uptake (data not shown) demonstrated that urea uptake by ShUT cRNA-injected oocytes reached a maximum after 30 min and did not exceed the concentration of the bathing solution. This result indicates that, like the mammalian urea transporters, ShUT is a facilitative transporter. These properties are consistent with those of previously characterized, mammalian, phloretin-sensitive, facilitative urea transporters (11).

View larger version (12K): [in this window] [in a new window]   Fig. 2.   Expression of ShUT in Xenopus oocytes. Uptake (90 s) of urea (1 mM) by ShUT cRNA-injected oocytes (30 ng/oocyte, filled bars) compared with water-injected oocytes (open bars). Each bar represents mean ± SE of 6-8 oocytes. Unpaired t-test (P < 0.01) was used to compare ShUT-injected vs. water-injected group and ShUT-injected vs. ShUT-injected + phloretin (0.35 mM) group.

High-stringency Northern analysis showed hybridization of ShUT cDNA to mRNA species at 2.2 and >10 kb in kidney and brain (Fig. 3A). These transcripts were not detected in any other tissue including gill. Low-stringency Northern analysis revealed additional signals at 3.0 kb in liver and kidney, 6.5 kb in all tissues analyzed except brain, and 8 kb in intestine (Fig. 3B).

View larger version (70K): [in this window] [in a new window]   Fig. 3.   Tissue distribution of ShUT using Northern analysis. Filters were probed using 32P-labeled full-length random-primed ShUT cDNA probe and hybridized at 42°C (high stringency) or 35°C (low stringency), both with 50% formamide. Final washes were in 0.1× standard sodium citrate-0.1% SDS at 65°C (high stringency) or 37°C (low stringency). A: high-stringency Northern analysis of S. acanthias mRNA (3 µg/lane). Strong signals at 2.2 and >10 kb are seen in kidney and brain. B: same filter probed at low stringency. Signals were detected at 3.0 kb (liver and kidney), 6.5 kb (all tissues analyzed except brain), and 8 kb (intestine).

	DISCUSSION

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Urea transport across cell membranes may involve specialized transporter proteins or be dependent on simple lipid permeation. For example, urea excretion across the gills of marine sculpin (34) or urea uptake by fish red blood cells (4, 14, 32) is not affected by urea analogs (e.g., acetamide, thiourea) or inhibitors (e.g., phloretin) and therefore is considered to be simple lipid-phase diffusion. On the other hand, specialized urea transporter proteins have been cloned and characterized in amphibians (31) and a number of mammalian species (6, 27, 28, 35). Most of our understanding of facilitated urea transporters is derived from studies of the mammalian kidney. Urea transport in the IMCD is regulated by vasopressin (23), and the cDNA encoding this urea transporter protein UT-A1 has been cloned (27) and localized to the apical membrane of the IMCD (19).

Our experiments clearly show that the kidney of S. acanthias contains at least one protein capable of urea transport and indicate the existence of other homologues. The ORF we have predicted encodes a 380-amino acid protein. There are, however, two other putative ORF initiation sites [5'-end (bp 70) and 3'-end (bp 205)] to our predicted methionine start codon. These encode proteins of 391 and 346 amino acids, respectively. Currently, we have no direct evidence indicating which initiator codon is preferred but have based our ORF initiation site prediction on the codon having the highest consensus to a typical Kozak initiation site (GCC[A/G]CCATGG).

The homology of ShUT to similar-length mammalian UT-A2 and UT-B2 urea transporter proteins is ~60% amino acid identity. The identity with UT-A2 is slightly higher than with UT-B2, and ShUT lacks residues that, to date, have been found only in UT-B2 family members, including the ALE domain (A214-E216 of UT-B2). This indicates that the protein encoded by our cDNA belongs to the UT-A2 family.

The similarity between the two known families of urea transporters suggests that they share a common ancestor. It is unlikely, however, that ShUT represents this ancestor because of its similarity to UT-A2 compared with UT-B2 family members. We suggest that ShUT constitutes a phylogenetic ancestral form of UT-A2 alone and that other urea transporter homologues may be present in elasmobranch species. This prediction is borne out by the results of low-stringency Northern analysis, which revealed ShUT homologues in several tissues, including gill and blood. We suggest that these signals represent UT-B2 mRNAs or mRNAs encoding proteins of an as yet undefined family of urea transporter proteins.

We propose that ShUT expression in the kidney plays an important role in osmoregulation; that is, urea is reabsorbed from the tubule filtrate back into the blood. Obviously, further experiments are required to test this hypothesis. The physiological role of brain ShUT expression is less clear. Couriaud et al. (6) report expression of a UT-B2 protein in the rat brain, speculating that its role may be in osmoregulation or waste product excretion. Currently, we are unable to present direct evidence in support of this hypothesis, but we suggest the following. In elasmobranch species, central nervous system urea transporters may simply allow the rapid entry of urea into brain tissue so as to dissipate any osmotic gradient between intracellular compartments and plasma containing high concentrations of urea.

ShUT homologues are likely to be present in a variety of marine elasmobranch species and tissues, inasmuch as we have preliminary evidence for a similar urea transport protein in another elasmobranch species, the little skate R. erinacea. Low-stringency Northern analysis of R. erinacea RNA using a rat UT-A2 cDNA probe revealed two distinct bands at 0.6 and 3.4 kb in kidney, gill, heart, and liver tissues (unpublished observation).

Previous to our discovery, there is circumstantial evidence supporting the existence of urea transporters in elasmobranch gill. In dogfish, relatively little urea escapes across the gills despite a large blood-to-water gradient (2, 21, 33). Results of urea analog and inhibitor studies in the dogfish isolated head (gill) preparation (21) and in the intact animal (33) suggest that a transporter may be present in the basolateral but not apical membrane. The low-stringency Northern analysis revealed that a 6.5-kb homologue belonging to the UT family is present in gill tissue. Further investigation is required to determine the role of this ShUT homologue in gill tissue.

The question remains as to whether facilitative transporter proteins are solely responsible for urea reabsorption in the elasmobranch kidney. A passive, i.e., thermodynamically dissipative, model of urea reabsorption has been proposed by Friedman and Hebert (7). This model relies on the presence of a countercurrent multiplication system and differential permeabilities of tubular segments to urea, water, and sodium, as is present in mammalian species. The presence of ShUT in the S. acanthias and countercurrent arrangement of nephrons in the bundle zone of the dogfish kidney (17) support this model and indicate that perhaps a similar process is present in S. acanthias as is in mammals. However, without knowledge of segmental urea permeabilities and transport characteristics or the distribution of ShUT and its homologues within the kidney, it is too early to rule out the involvement of active urea reabsorption.

In conclusion, we have isolated a cDNA encoding a functional urea transporter from S. acanthias belonging to the UT-A2 family of urea transporter proteins. This transporter is the first urea transporter to be isolated from a marine fish.

Perspectives