Renal Regeneration After Cisplatin Induced Acute Kidney Injury in Zebrafish and Expression Map of Tight Junction Components in Development

The kidneys perform vital homeostatic functions including osmoregulation, nutrient reabsorption, and the excretion of concentrated nitrogenous waste. They are able to perform these tasks through the coordinated activities of nephrons, which are their basic functioning unit. Each nephron contains a proximally located glomerulus that filters the blood as well as a segmented epithelial tubule that drains into a central collecting duct system. During the course of their everyday function the nephrons encounter toxins that in high concentrations can lead to an episode of acute kidney injury (AKI), characterized by a rapid loss of renal function. AKI can progress to end stage renal disease, a condition in which the kidneys are no longer functional and dialysis or transplantation are required to sustain life. Zebrafish, unlike mammals, are known to undergo robust renal regeneration after gentamicin inducted AKI. To date, the study of nephron formation and regeneration in zebrafish has several limitations, including a relatively small cohort of reliable markers for distinct cell types within the nephron tubule. In this thesis, the first goal was to identify and characterize a suite of useful epithelial cell markers. My project specifically focused on the collection and characterization of tight junction genes that were expressed during nephrogenesis. A detailed spatiotemporal expression study was completed that revealed several aspects of conservation between zebrafish and mammals, including but not limited to, the gradient of tight junction transcript expression across the proximo-distal length of the nephron, such that distal segments were found to express the greatest number of factors. The second goal of this thesis was to further interrogate the ability of the zebrafish kidney to regenerate following injury in the embryonic and adult setting. To date, the bulk of studies assessing how zebrafish undergo renal repair after injury have employed the antibiotic gentamicin to induce damage and therefore, the extent to which the kidney can respond to different types of injury remains unknown. For this project, I both developed and further refined aspects of two parallel methodologies for the delivery of small molecules to the zebrafish: microinjection in the embryo and intraperitoneal injection in the adult. Using the latter, I established for the first time that the zebrafish kidney can regenerate after catastrophic nephron damage from the chemotherapeutic, cisplatin. Hallmarks of this process include intratubular repair as well as de novo nephrogenesis, which can be visualized with histological staining. I observed regeneration of epithelial tubules in recovering fish via in situ hybridization and through histological analysis with Periodic Acid-Schiff staining, thereby establishing the time course of cellular events that will enable future studies to assay cell proliferation and death during the regeneration process