To determine whether calcium polyvalent cation-sensing receptors (CaRs) are salinity sensors in fish, we used a homology-based cloning strategy to isolate a 4.1-kb cDNA encoding a 1,027-aa dogfish shark (Squalus acanthias) kidney CaR. Expression studies in human embryonic kidney cells reveal that shark kidney senses combinations of Ca 2؉ , Mg 2؉ , and Na ؉ ions at concentrations present in seawater and kidney tubules. Shark kidney is expressed in multiple shark osmoregulatory organs, including specific tubules of the kidney, rectal gland, stomach, intestine, olfactory lamellae, gill, and brain. Reverse transcriptase-PCR amplification using specific primers in two teleost fish, winter flounder (Pleuronectes americanus) and Atlantic salmon (Salmo salar), reveals a similar pattern of CaR tissue expression. Exposure of the lumen of winter flounder urinary bladder to the CaR agonists, Gd 3؉ and neomycin, reversibly inhibit volume transport, which is important for euryhaline teleost survival in seawater. Within 24 -72 hr after transfer of freshwater-adapted Atlantic salmon to seawater, there are increases in their plasma Ca 2؉ , Mg 2؉ , and Na ؉ that likely serve as a signal for internal CaRs, i.e., brain, to sense alterations in salinity in the surrounding water. We conclude that CaRs act as salinity sensors in both teleost and elasmobranch fish. Their tissue expression patterns in fish provide insights into CaR functions in terrestrial animals including humans.

The mammalian kidney responds to partial nephrectomy with glomerular and tubular hypertrophy, but without renal regeneration. In contrast, renal regeneration in lower vertebrates is known to occur. Understanding the underlying mechanisms of renal regeneration is highly important; however, a serviceable animal model has not been developed. A neonephrogenic zone has been identified in the European lesser spotted dogfish, Scyliorhinus caniculus (Hentschel H. Am J Anat 190: 309 -333, 1991), as well as in the spiny dogfish Squalus acanthias and the little skate, Leucoraja erinacea. The zone features the production of new nephrons complete with a countercurrent system. To analyze this nephrogenic region of elasmobranch fish further, a renal reduction model was established. The neonephrogenic zone in the adult kidney of the little skate resembles the embryonic metanephric kidney and contains stem cell-like mesenchymal cells, tips of the branching collecting duct system, and outgrowth of the arterial system. Four stages of nephron development were analyzed by serial sections and defined: stage I, aggregated mesenchymal cells; stage II, S-shaped body-like structure with high-pris-

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The zebrafish pronephric kidney provides a simplified model of nephron development and epithelial cell differentiation which is amenable to genetic analysis. The pronephros consists of two nephrons with fused glomeruli and paired pronephric tubules and ducts. Nephron formation occurs after the differentiation of the pronephric duct with both the glomeruli and tubules being derived from a nephron primordium. Fluorescent dextran injection experiments demonstrate that vascularization of the zebrafish pronephros and the onset of glomerular filtration occurs between 40 and 48 hpf. We isolated fifteen recessive mutations that affect development of the pronephros. All have visible cysts in place of the pronephric tubule at 2–2.5 days of development. Mutants were grouped in three classes: (1) a group of twelve mutants with defects in body axis curvature and manifesting the most rapid and severe cyst formation involving the glomerulus, tubule and duct, (2) the fleer mutation with distended glomerular capillary loops and cystic tubules, and (3) the mutation pao pao tang with a normal glomerulus and cysts limited to the pronephric tubules. double bubble was analyzed as a representative of mutations that perturb the entire length of the pronephros and body axis curvature. Cyst formation begins in the glomerulus at 40 hpf at the time when glomerular filtration is established suggesting a defect associated with the onset of pronephric function. Basolateral membrane protein targeting in the pronephric duct epithelial cells is also severely affected, suggesting a failure in terminal epithelial cell differentiation and alterations in electrolyte transport. These studies reveal the similarity of normal pronephric development to kidney organogenesis in all vertebrates and allow for a genetic dissection of genes needed to establish the earliest renal function.

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Light and electron microscopy of the excretory kidney of adolescent dogfish, Scyliorhinus caniculus (L.), revealed immature and mature nephrons as well as four developmental stages of nephrons. At stage I the nephron was characterized by a condensed mass of mesenchymal cells in the center of several concentric layers of connective tissue. At stage II of the nephron, the S-shaped body was an elongate cyst with a high prismatic epithelium that was connected by a developing collecting tubule with the collecting duct system. At stage III, the developing nephrons already possess the essential features of the mature nephron but lack complete differentiation. Developing renal corpuscles had one afferent arteriole and two efferent vessels. Developing tubules ran four times between the lateral bundle zone and the mesial tissue zone before they joined the collecting duct system. A continuous sheath of flat cells, encompassing the collecting duct system, extended around the developing lateral bundle. A rudimentary central vessel ran from the developing lateral bundle to the venous sinusoid capillaries between the mesial convolutions. Developmental stage IV was similar to the mature nephron, however, renal corpuscles and tubular segments were smaller than those of mature nephrons. Conclusive evidence for morphological homology of elasmobranch nephron segments and collecting tubule-collecting duct system with those of other vertebrates is provided. The origin and nature of the central vessel and the bundle sheath is clarified. These specific structures of marine elasmobranch kidney supposedly are of great functional relevance for the renal countercurrent system that in turn is essential for ion- and osmo-regulation.

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The cloning of a renal Na-Pi contransport in system from winter flounder (P eudopleuronectes americanus) has recently been reported. We used this information to answer the questions 1) what is the distribution of the transport protein along the nephron? and 2) how are renal and intestinal transporters related? The distribution of the flounder NaPi-II protein was tested using two antisera raised against partial sequences (amino acids 1-14 and 388-441) of the transporter. Antibody-specific fluorescence was detected at the basolateral membrane of epithelial cells in the proximal tubular segment PII. Two clones corresponding to the renal Na-Pi cotransporter were isolated from a flounder intestinal cDNA library. Their functional properties were determined using Xenopus laevis oocytes. The apparent affinities for Pi [Michaelis constant (K(m)) = 0.063 mM] and Na (K(m) = 45.3 mM), as well as the pH dependency (increasing transport activity with increasing pH), showed the same characteristics in both intestinal and the renal systems. Sequence analysis revealed that the two intestinal clones were 100% homologous to the renal cDNA, Flounder NaPi-II-specific immunofluorescence was observed predominantly at the apical membrane on intestinal cross sections. We report the cloning and expression of the first intestinal Na-Pi cotransport system. This transporter belongs to the small group of proteins that exhibit the same function in the apical and the basolateral membranes of different cells.