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Most of the cartilaginous fishes live principally in seawater (SW) environments, while a limited number of species including the bull shark, Carcharhinus leucas, inhabit both SW and freshwater (FW) environments during their life cycle. Euryhaline elasmobranchs maintain high internal urea and ion levels even in FW environments, but little is known about the osmoregulatory mechanisms that enable them to maintain internal homeostasis in hypoosmotic environments. In the present study, we focused on the kidney because this is the only organ that can excrete excess water from the body in a hypoosmotic environment. We conducted a transfer experiment of bull sharks from SW to FW and performed differential gene expression analysis between the two conditions using RNA-seq. A search for genes up-regulated in the FW-acclimated bull shark kidney indicated that the expression of the Na+-Cl− cotransporter (NCC; Slc12a3) was ten times higher in the FW-acclimated fish compared to that in SW fish. In the kidney, apically-located NCC was observed in the late distal tubule and in the anterior half of collecting tubule where basolateral Na+/K+-ATPase was also expressed, implying that these segments contribute to NaCl reabsorption from the filtrate for diluting the urine. This expression pattern was not observed in the houndshark, Triakis scyllium, that had been transferred to 30% SW; this species cannot survive in FW environment. The salinity transfer experiment combined with a comprehensive gene screening approach demonstrates that NCC is a key renal protein that contributes to the remarkable euryhaline ability of the bull shark.

For adaptation to high-salinity marine environments, cartilaginous fishes (sharks, skates, rays, and chimaeras) adopt a unique urea-based osmoregulation strategy. Their kidneys reabsorb nearly all filtered urea from the primary urine, and this is an essential component of urea retention in their body fluid. Anatomical investigations have revealed the extraordinarily elaborate nephron system in the kidney of cartilaginous fishes, e.g., the four-loop configuration of each nephron, the occurrence of distinct sinus and bundle zones, and the sac-like peritubular sheath in the bundle zone, in which the nephron segments are arranged in a countercurrent fashion. These anatomical and morphological characteristics have been considered to be important for urea reabsorption; however, a mechanism for urea reabsorption is still largely unknown. This review focuses on recent progress in the identification and mapping of various pumps, channels, and transporters on the nephron segments in the kidney of cartilaginous fishes. The molecules include urea transporters, Na+/K+-ATPase, Na+-K+-Cl−cotransporters, and aquaporins, which most probably all contribute to the urea reabsorption process. Although research is still in progress, a possible model for urea reabsorption in the kidney of cartilaginous fishes is discussed based on the anatomical features of nephron segments and vascular systems and on the results of molecular mapping. The molecular anatomical approach thus provides a powerful tool for understanding the physiological processes that take place in the highly elaborate kidney of cartilaginous fishes.

SummaryMarine cartilaginous fish retain a high concentration of urea to maintain the plasma slightly hyperosmotic to the surrounding seawater. In adult fish, urea is produced by hepatic and extrahepatic ornithine urea cycles (OUCs). However, little is known about the urea retention mechanism in developing cartilaginous fish embryos. In order to address the question as to the mechanism of urea-based osmoregulation in developing embryos, the present study examined the gene expression profiles of OUC enzymes in oviparous holocephalan elephant fish (Callorhinchus milii) embryos. We found that the yolk sac membrane (YSM) makes an important contribution to the ureosmotic strategy of the early embryonic period. The expression of OUC enzyme genes was detectable in the embryonic body from at least stage 28, and increased markedly during development to hatching, which is most probably due to growth of the liver. During the early developmental period, however, the expression of OUC enzyme genes was not prominent in the embryonic body. Meanwhile, we found that the mRNA expression of OUC enzymes was detected in extraembryonic YSM; the mRNA expression of cmcpsIII in the YSM was much higher than that in the embryonic body during stages 28-31. Significant levels of enzyme activity and the existence of mitochondrial-type cmgs1 transcripts in YSM supported the mRNA findings. We also found that the cmcpsIII transcript is localized in the vascularized inner layer of the YSM. Taken together, our findings demonstrate for the first time that the YSM is involved in urea-based osmoregulation during the early to mid phase of development in oviparous cartilaginous fish.