Molecular and functional characterization of a urea transporter from the kidney of the Atlantic stingray

Abstract

In general, marine elasmobranch fishes (sharks, skates, and rays) maintain body fluid osmolality above seawater, principally by retaining large amounts of urea. Maintenance of the high urea concentration is due in large part to efficient renal urea reabsorption. Regulation of renal urea reabsorption also appears to play a role in maintenance of fluid homeostasis of elasmobranchs that move between habitats of different salinities. We identified and cloned a novel 2.7-kb cDNA from the kidney of the euryhaline Atlantic stingray Dasyatis sabina (GenBank accession no. AF443781). This cDNA putatively encoded a 431-amino acid protein (strUT-1) that had a high degree of sequence identity (71%) to the shark kidney facilitated urea transporter (UT). However, the predicted COOH-terminal region of strUT-1 appears to contain an additional sequence that is unique among cloned renal UTs. Injection of strUT-1 cRNA into Xenopus oocytes induced a 33-fold increase in [(14)C]urea uptake that was inhibited by phloretin. Four mRNA bands were detected in kidney by Northern blot: a transcript at 2.8 kb corresponding to the expected size of strUT-1 mRNA and bands at 3.8, 4.5, and 5.5 kb. Identification of a facilitated UT in the kidney of the Atlantic stingray provides further support for the proposal that passive mechanisms contribute to urea reabsorption by elasmobranch kidney.

Cloning and functional characterization of a second urea transporter from the kidney of the Atlantic stingray,Dasyatis sabina

“…In contrast, transcripts encoding strUT-2 were detected in the extrarenal tissues by RT-PCR. These findings are in agreement with our previous findings (18). This difference in the ability to detect message for strUT is most probably due to the difference in sensitivity between these two techniques of mRNA detection.…”

“…The current findings support our previous proposal that a single gene encodes the urea transporters that have to this date been cloned from the kidneys of elasmobranchs (18). Furthermore, a comparison of the cDNAs and predicted protein sequences support the proposal that the two stingray urea transporters and the spiny dogfish urea transporter (36) originate from a common translational start site.…”

“…In a previous study, we used primers to the coding region of strUT-1 mRNA to detect its presence in erythrocytes and liver by RT-PCR (18). However, the findings of the present study indicate that the primers used in the previous study cannot distinguish between the two strUT-1 and the three strUT-2 transcripts.…”

Cloning and functional characterization of a second urea transporter from the kidney of the Atlantic stingray,Dasyatis sabina

“…In contrast, transcripts encoding strUT-2 were detected in the extrarenal tissues by RT-PCR. These findings are in agreement with our previous findings (18). This difference in the ability to detect message for strUT is most probably due to the difference in sensitivity between these two techniques of mRNA detection.…”

“…The current findings support our previous proposal that a single gene encodes the urea transporters that have to this date been cloned from the kidneys of elasmobranchs (18). Furthermore, a comparison of the cDNAs and predicted protein sequences support the proposal that the two stingray urea transporters and the spiny dogfish urea transporter (36) originate from a common translational start site.…”

“…In a previous study, we used primers to the coding region of strUT-1 mRNA to detect its presence in erythrocytes and liver by RT-PCR (18). However, the findings of the present study indicate that the primers used in the previous study cannot distinguish between the two strUT-1 and the three strUT-2 transcripts.…”

Multi-tissue RNA-seq and transcriptome characterisation of the spiny dogfish shark (Squalus acanthias) provides a molecular tool for biological research and reveals new genes involved in osmoregulation

“…The resulting transcripts code for proteins that are orthologues to the elephant shark urea transporter efUT1 as well as to two different aquaporin genes (Aqp3-like) proteins. In the case of the urea transporter, two isoforms of ut1 were identified in the kidney assembly: the first one encodes an identical protein sequence to the previously described urea transporter in spiny dogfish shUT [ 53 ] while the second one codes for a longer protein and possesses an extended carboxyl terminal part similar to the one identified in the Atlantic stingray Dasyatis sabina [ 54 , 55 ], s ee Fig 3 . In addition, three more urea transporter transcripts were found in the spiny dogfish brain transcriptome belonging to the same gene assembled by Trinity.…”

Hepatic urea biosynthesis in the euryhaline elasmobranchCarcharhinus leucas

“…However, as highlighted recently, acclimation to reduced salinity also leads to an increase in absolute renal urea reabsorption in elasmobranchs (Janech et al, 2003). This suggests that urea clearance by the kidney of elasmobranchs is governed primarily by glomerular filtration rate (GFR) and that the nephrons have the capacity to increase reabsorption significantly when presented with a 2-4-fold increase in GFR.…”

Cloning and functional characterization of a second urea transporter from the kidney of the Atlantic stingray,Dasyatis sabina

“…In contrast, transcripts encoding strUT-2 were detected in the extrarenal tissues by RT-PCR. These findings are in agreement with our previous findings (18). This difference in the ability to detect message for strUT is most probably due to the difference in sensitivity between these two techniques of mRNA detection.…”

“…The current findings support our previous proposal that a single gene encodes the urea transporters that have to this date been cloned from the kidneys of elasmobranchs (18). Furthermore, a comparison of the cDNAs and predicted protein sequences support the proposal that the two stingray urea transporters and the spiny dogfish urea transporter (36) originate from a common translational start site.…”

“…In a previous study, we used primers to the coding region of strUT-1 mRNA to detect its presence in erythrocytes and liver by RT-PCR (18). However, the findings of the present study indicate that the primers used in the previous study cannot distinguish between the two strUT-1 and the three strUT-2 transcripts.…”

Multi-tissue RNA-seq and transcriptome characterisation of the spiny dogfish shark (Squalus acanthias) provides a molecular tool for biological research and reveals new genes involved in osmoregulation

“…The resulting transcripts code for proteins that are orthologues to the elephant shark urea transporter efUT1 as well as to two different aquaporin genes (Aqp3-like) proteins. In the case of the urea transporter, two isoforms of ut1 were identified in the kidney assembly: the first one encodes an identical protein sequence to the previously described urea transporter in spiny dogfish shUT [ 53 ] while the second one codes for a longer protein and possesses an extended carboxyl terminal part similar to the one identified in the Atlantic stingray Dasyatis sabina [ 54 , 55 ], s ee Fig 3 . In addition, three more urea transporter transcripts were found in the spiny dogfish brain transcriptome belonging to the same gene assembled by Trinity.…”

Hepatic urea biosynthesis in the euryhaline elasmobranchCarcharhinus leucas

“…However, as highlighted recently, acclimation to reduced salinity also leads to an increase in absolute renal urea reabsorption in elasmobranchs (Janech et al, 2003). This suggests that urea clearance by the kidney of elasmobranchs is governed primarily by glomerular filtration rate (GFR) and that the nephrons have the capacity to increase reabsorption significantly when presented with a 2-4-fold increase in GFR.…”

Cloning and functional characterization of a second urea transporter from the kidney of the Atlantic stingray,Dasyatis sabina

“…In contrast, transcripts encoding strUT-2 were detected in the extrarenal tissues by RT-PCR. These findings are in agreement with our previous findings (18). This difference in the ability to detect message for strUT is most probably due to the difference in sensitivity between these two techniques of mRNA detection.…”

“…The current findings support our previous proposal that a single gene encodes the urea transporters that have to this date been cloned from the kidneys of elasmobranchs (18). Furthermore, a comparison of the cDNAs and predicted protein sequences support the proposal that the two stingray urea transporters and the spiny dogfish urea transporter (36) originate from a common translational start site.…”

“…In a previous study, we used primers to the coding region of strUT-1 mRNA to detect its presence in erythrocytes and liver by RT-PCR (18). However, the findings of the present study indicate that the primers used in the previous study cannot distinguish between the two strUT-1 and the three strUT-2 transcripts.…”

Multi-tissue RNA-seq and transcriptome characterisation of the spiny dogfish shark (Squalus acanthias) provides a molecular tool for biological research and reveals new genes involved in osmoregulation

“…The resulting transcripts code for proteins that are orthologues to the elephant shark urea transporter efUT1 as well as to two different aquaporin genes (Aqp3-like) proteins. In the case of the urea transporter, two isoforms of ut1 were identified in the kidney assembly: the first one encodes an identical protein sequence to the previously described urea transporter in spiny dogfish shUT [ 53 ] while the second one codes for a longer protein and possesses an extended carboxyl terminal part similar to the one identified in the Atlantic stingray Dasyatis sabina [ 54 , 55 ], s ee Fig 3 . In addition, three more urea transporter transcripts were found in the spiny dogfish brain transcriptome belonging to the same gene assembled by Trinity.…”

Hepatic urea biosynthesis in the euryhaline elasmobranchCarcharhinus leucas

“…However, as highlighted recently, acclimation to reduced salinity also leads to an increase in absolute renal urea reabsorption in elasmobranchs (Janech et al, 2003). This suggests that urea clearance by the kidney of elasmobranchs is governed primarily by glomerular filtration rate (GFR) and that the nephrons have the capacity to increase reabsorption significantly when presented with a 2-4-fold increase in GFR.…”