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First published online December 26, 2008
Journal of Experimental Biology 212, 249-256 (2009)
Published by The Company of Biologists 2009
doi: 10.1242/jeb.019703
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Localization and regulation of a facilitative urea transporter in the kidney of the red-eared slider turtle (Trachemys scripta elegans)

Minoru Uchiyama*, Ryosuke Kikuchi, Norifumi Konno, Tatsuya Wakasugi and Kouhei Matsuda

Department of Biological Science, Graduate School of Science and Engineering, University of Toyama, 3190 Gofuku, Toyama, 930-8555, Japan


Figure 1
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  		Fig. 1. (A) Primary structure of the urea transporter (UT) isolated from the kidney of the red-eared slider turtle, Trachemys scripta elegans. The deduced amino acid sequence is aligned with those of rat UT-A2 (U09957) and rat UT-B2 (U81518) using the Clustal algorithm. Asterisks denote identical amino acid residues to turtle UT. The horizontal bars indicate the predicted transmembrane regions. The box indicates putative N-glycosylation sites (NIT). The ALE domain, which is considered to be a signature sequence for the UT-B, is underlined. (B) Kyte–Doolittle hydropathy profile of the deduced Turtle UT amino acid sequence predicts the presence of transmembrane regions (1–10). (C) Phylogenetic tree showing the relationship between vertebrate UTs. The tree was constructed by the neighbor-joining method using ClustalW based on UT sequences. Numbers at branch points are derived from bootstrap analysis (1000 repetitions). UTs of bacteria, Desulfovibrio and Ochrobactrum, were regarded as outgroups. Scale bar represents a phylogenetic distance of 0.1 amino acid substitutions per site. The position of turtle UT is boxed. Each sequence appears in the protein database with the following accession nos: Desulfovibrio UT,YP-010379; Ochrobactrum UT, YP-001370983; fugu (pufferfish) UT-C, NP001033079; eel UT-C, BAD66672; human UT-B, AAH50539; mouse UT-B, AAI00571; rat UT-B, EDL84685; Triakis UT, BAC75980; stingray UT, AAQ23382; eel eUT, BAC53976; toadfish UT, AAD53976; tilapia UT, AAG49891; fugu UT, NP001027896; toad UT, BAF16706; frog UT, CAA73322; turtle UT, AB308450; rat UT-A2, AAA84392; whale UT-A2, BAF46914; and human UT-A2, CAA65657.

 

Figure 2
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  		Fig. 2. Tissue-specific expression of turtle UT mRNA determined using RT-PCR. PCR was performed using specific primers for turtle UT and turtle glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Turtle UT mRNA was expressed in kidney but was not detected in the other tissues examined. A smaller band that was shown by sequencing to be non-specific was found in the stomach. In this experiment the expression level of turtle UT was normalized by RT-PCR amplification of the same cDNA templates using the GAPDH primer.

 

Figure 3
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  		Fig. 3. Analysis of urea uptake in Xenopus oocytes injected with turtle UT cRNA or water. Summary of [14C]urea accumulated over a 10 min period by water-injected control oocytes or oocytes expressing turtle UT. Oocytes injected with turtle UT cRNA showed significant increases in urea uptake compared with the water-injected control oocytes; phloretin significantly inhibited turtle UT-mediated urea influx. Open bar represents urea uptake in the water-injected oocyte group after incubation in Barth's medium for 10 min. Filled bars show uptake after incubation in the solution with or without 0.5 mmol l–1 phloretin for 10 min. Values are presented as means ± s.e.m. of six oocytes in each group. **P<0.01 versus control or UT with phloretin treatment (ANOVA followed by Bonferroni's test).

 

Figure 4
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  		Fig. 4. Confocal microscopic and western blot analyses of Xenopus oocytes injected with turtle UT cRNA. (A) Immunofluorescence images for turtle UT protein in turtle UT cRNA-injected oocytes using affinity-purified turtle UT antibody. Immunoreactive proteins were detected in the plasma membrane (arrowheads) of turtle cRNA-injected oocytes (a). The protein was not detected in the plasma membrane (arrowheads) of water-injected oocytes (b). Scale bars, 500 µm. (B) Western blot analysis of the membrane fractions prepared from the turtle UT-injected Xenopus oocytes, and those from the desiccated turtle kidney, heart and liver using the anti-turtle UT antibody. An immunoreactive band was detected at an approximately 58 kDa in extract of the UT-injected oocytes (lane 1) and no band was observed in the water-injected oocytes (lane 2). The 58 kDa band was detected in extract of the kidney (lane 3). No band was observed in either heart (lane 4) or liver (lane 5).

 

Figure 5
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  		Fig. 5. Immunohistochemical localization of the UT (stained brown) in the turtle kidney using the affinity-purified UT antibody. Using the anti-turtle UT and anti-Na+,K+-ATPase antibodies in adjacent mirror sections, immunoreactivity for UT and Na+,K+-ATPase was detected on the apical cell membrane of epithelia along the late distal tubule (A) and the distal nephron (B), respectively. In contrast, in adjacent mirror sections immunolabeling with the turtle UT and the H+-ATPase antibodies was observed in the apical membrane of some epithelial cells in the late distal tubules (C) and in the intercalated cells along the late distal tubules (D), respectively. In C and D, filled and open arrowheads show the UT and H+-ATPase immunopositive tubules, and H+-ATPase immunopositive cells along the distal tubules, respectively. In adjacent mirror sections, no UT immunoreactivity was detected in the collecting duct (E) where H+-ATPase was expressed (F). Abbreviations: G, glomerulus; ED, early distal tubule; LD, late distal tubule; P, proximal tubule. Scale bars for A–F, 50 µm.

 

Figure 6
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  		Fig. 6. Expression of turtle UT mRNA relative to GAPDH mRNA in the kidney of control turtles and experimental turtles acclimated to dry conditions for 7 days. The signal for each band was measured by densitometry and is presented as a ratio (turtle UT/GAPDH). The UT mRNA expression was significantly increased more than 2-fold following dry acclimation. Values are means ± s.e.m., N=6. **Significant difference from control (Student's t-test, P<0.01).

 

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