Novel Quantification and Localization of Water and Solute Transporters in the Tissues of the Spiney Dogfish (Squalus acanthias)

• Main Author: Ojo, Tolulope B
• Format: Text
• Language: unknown
• Published: Digital Commons@Georgia Southern 2021
• Subjects: Squalus acanthias; Elasmobranch; Aquaporin; Urea transporter; Osmoregulation; AQP3; AQP3-2; UT-1 long and short; NCC; Brain UT; Spliceoform; Western blotting; Immunohistochemistry; qPCR; Tissue PCR; Kidney; Biology; Cellular and Molecular Physiology; Marine Biology; Physiology; Pacific; North Atlantic
• Online Access: https://digitalcommons.georgiasouthern.edu/etd/2306; https://digitalcommons.georgiasouthern.edu/context/etd/article/3499/viewcontent/CORRECTEDR.Novel_Quantification_and_Localization_of_Water_and_Solute_Transpo.pdf

The dogfish, Squalus acanthias is a marine cartilaginous elasmobranch found in the North Atlantic and Pacific oceans. Dogfish synthesize and excrete urea as a product of nitrogen metabolism. They also convert ammonia into urea and retain this urea, such that their plasma is isosmotic or slightly hyperosmotic to the surrounding seawater. To facilitate the regulation of body fluid and to maintain ionic concentrations and osmotic pressure, Dogfish use ion and solute transporters (e.g., NCC, UT-1) and aquaporin water channel proteins. Studies have identified some of the aquaporin genes in the elasmobranch genome, but their functions are mostly uncharacterized. Recent transcriptomic studies deposited sequence data for duplicate copies of AQP3 (AQP3-2) and its splice variant as well as ‘long’ and ‘short’ splice variants of UT-1 and a second novel UT (Brain UT) expressed in Dogfish. To investigate changes in expression of AQP3-2, UT-1, Brain-UT and NCC in the kidney as salinity changes, the fish were acclimated to 75%, 100% and 120% seawater for 7days. Cloning and sequencing experiments were used to generate primers and antibodies for further studies. Through this, the sequence of a novel gene splice variant of the Brain UT was identified in the brain. The tissue PCRs and western blots confirmed the presence of these genes. Proteins with expected molecular weights were identified for these genes except for NCC. Western blots for AQP3-2 and UT-1 had additional bands which implies the potential presence of other splice variants. The quantitative PCR (qPCR) quantification of mRNA/cDNA expression of these genes showed that only the AQP3-2 spliceoform and UT-1 long splice variant had statistically significant differences and only AQP3 expression increased with increasing salinity as expected. Imunohistochemical localization experiments showed that AQP3-2 is located in the basolateral membranes of the EDT and LDT. UT-1 was localized to most of the nephron, suggesting that urea reabsorption occurs along most of its length. ...