Effect of low environmental salinity on plasma composition and renal function of the Atlantic stingray, a euryhaline elasmobranch.

Marine elasmobranchs maintain internal osmolality higher than their external environment, resulting in an osmotic gradient for branchial water uptake. This gradient is markedly increased in low-salinity habitats. The subsequent increase in water uptake presents a challenge to volume homeostasis. The Atlantic stingray is a marine elasmobranch that inhabits a remarkable range of environmental salinities. We hypothesized that the ability of these stingrays to regulate fluid volume in low-salinity environments is due primarily to a renal glomerular and tubular functional reserve. We tested this hypothesis by measuring renal excretory function after a rapid and sustained 50% reduction in the osmolality of the external medium. Atlantic stingrays were maintained in harbor water [control salinity (CS) approximately 850 mosmol/kgH(2)O] for 1 wk. Rays were then either transferred to diluted harbor water [low salinity (LS) approximately 440 mosmol/kgH(2)O] or maintained in CS for a further 24 h. Renal excretory function was markedly higher in the rays subjected to low salinity. Glomerular filtration rate was threefold higher and urine flow rate ninefold higher in the LS group. The clearance of solute-free water was greater, and solute-free water comprised a significantly larger proportion of the urine output for the stingrays transferred to dilute harbor water. We conclude that 1) the kidneys of Atlantic stingrays have a remarkable glomerular and tubular functional reserve, and 2) the marked increase in renal function attenuates the increase in fluid volume when these fish move into low-salinity habitats.

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

The cloning of cDNAs encoding facilitated urea transporters (UTs) from the kidneys of the elasmobranchs indicates that in these fish renal urea reabsorption occurs, at least in part, by passive processes. The previously described elasmobranch urea transporter clones from shark (shUT) and stingray (strUT-1) differ from each other primarily because of the COOH-terminus of the predicted strUT-1 translation product being extended by 51-amino acid residues compared with shUT. Previously, we noted multiple UT transcripts were present in stingray kidney. We hypothesized that a COOH terminally abbreviated UT isoform, homologous to shUT, would also be present in stingray kidney. Therefore, we used 5'/3' rapid amplification of cDNA ends to identify a 3'UTR-variant (strUT-1a) of the cDNA that encodes (strUT-1), as well as three, 3'UTR-variant cDNAs (strUT-2a,b,c) that encode a second phloretin-sensitive, urea transporter (strUT-2). The 5'UTR and the first 1,132 nucleotides of the predicted coding region of the strUT-2 cDNAs are identical to the strUT-1 cDNAs. The remainder of the coding region contains only five novel nucleotides. The strUT-2 cDNAs putatively encode a 379-amino acid protein, the first 377 amino acids identical to strUT-1 plus 2 additional amino acids. We conclude that 1) a second UT isoform is expressed in the Atlantic stingray and that this isoform is similar in size to the UT previously cloned from the kidney of the dogfish shark, and 2) at least five transcripts encoding the 2 stingray UTs are derived from a single gene product through alternative splicing and polyadenylation.

A thromboxane A(2) system in the Atlantic stingray, Dasyatis sabina.

Thromboxane B(2)(TXB(2)) is the stable metabolite of thromboxane A(2)(TXA(2)) and thromboxane B(2)-like immunoreactivity (iTXB(2)) has been identified in the plasma of the Atlantic stingray, Dasyatis sabina (0.57+/-0.03 ng/ml). Plasma levels of iTXB(2) increase if the blood is allowed to clot (3.0+/-0.27 ng/ml). When clotting occurs in the presence of indomethacin, this increase is partially inhibited (1.5+/-0.17 ng/ml), indicating the presence of a cyclooxygenase activity. Radioligand binding analysis using the TXA(2) analog [125I]BOP in isolated kidney membranes revealed a receptor of K(d)=2.88+/-0.51 nM and B(max)=25.6+/-5.9 fmol/mg protein. [125I]BOP binding was displaced by the TXA(2) receptor (TP receptor) agonists U46619 (IC(50)=106.4+/-15.7 nM) and U44069 (IC(50)=88.7+/-13.0 nM), and the antagonist SQ29548 (IC(50)=51.0+/-12.9 nM). Binding was also displaced stereoselectively by the antagonists (-)L657925 (IC(50)=18.9+/-3.8 nM) and (+)L657926 (IC(50)=2025+/-280 nM). Tissue bath studies revealed that U46619, a stable TXA(2) mimetic, elicited concentration-dependent contractions in the ventral aorta which were inhibited in a concentration-dependent manner by the TP receptor antagonist SQ29548. Using a human TP receptor riboprobe, Northern blotting of mRNA isolated from the stingray kidney identified transcripts of 2.8 and 6kb. The 2.8kb transcript is similar to a 2.8kb transcript found in human cells or tissues, but the 6kb transcript may be unique. These data indicate the presence of a TXB(2)-like substance in the blood, a TP receptor in the kidney, TXA(2) biological activity in the ventral aorta, and expression of a TP receptor-like gene.

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

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.