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Core D: Model Organisms
The Model Organisms Core employs two genetically-tractable systems: the yeast S. cerevisiae and the zebrafish D. rerio. These experimental systems are used to dissect fundamental aspects of kidney development and protein structure and function. Experiments associated with these model systems will be complemented by the use of small molecule modulators that have emerged from Core-associated activities over the past four years. During this time, more than 40 investigators have benefited from using the services provided by this Core, and our tools and expertise have been acknowledged on 32 papers. Hypotheses arising from the unique attributes of the yeast and zebrafish models and from the use of chemical modulators will continue to be tested in higher organisms via the other Cores. In turn, experiments using yeast and zebrafish provide rapid assessments of predictions that emerge from more complex systems. The objectives of the core are:

• To use the yeast Saccharomyces cerevisiae as a “tool” to understand basic aspects of renal disease. The yeast Saccharomyces cerevisiae has long-served as a model eukaryote. Genetic dissections of yeast physiology led to significant advances in our understanding of several human diseases. This is due to the fact that most yeast genes have human homologs, and nearly every cellular pathway is conserved between yeast and man. Emerging genomic tools were then co-opted to examine the molecular basis and to aid in the treatment of human diseases. More recently, yeast have served as a tool to study protein conformational diseases. Many conformational diseases affect renal cell function, and as we summarized in a recent review the yeast model has helped us understand renal function and the etiology of kidney disease. During the past funding period, we have developed and utilized yeast expression systems for proteins linked to Liddle’s Syndrome, Gittleman’s Syndrome, Pseudohypoaldosteronism Type I, Nephrogenic Diabetes Insipidus (NDI), and distal Renal Tubular Acidosis (dRTA), and for renal drug targets including NCC (thiazide) and ENaC (amiloride). These efforts are complementary to concurrent studies on misfolded proteins linked to cystic fibrosis (CFTR), and heart (apoB) and liver disease (1 antrypsin Z, AT-Z); therefore, the methods developed in our other studies have catalyzed discoveries on the quality control of proteins that impact renal cell function, and vice-versa. We will continue to employ our yeast models for kidney disease and have lined-up new collaborations. Our efforts will further delineate how proteins linked to specific diseases result in pathology, and we will continue identify conserved factors that can be targeted therapeutically.

• To develop novel tools to dissect a variety of kidney processes and provide a novel approach to study zebrafish larval and adult physiology. To fully comprehend physiological and pathophysiological processes we must understand the integrated interactions between molecules, pathways, cells, tissues, and organs. Ex vivo systems only partially capture this complexity. Therefore, the study of biological pathways, networks, and processes across multiple scales from individual molecules to specific cell types to integrated organ physiology are most informative in an animal. Of the many models, the zebrafish is ideal because of its compatibility with multi-well plate formats used in large-scale, phenotype-based chemical screens. Gene knockouts have also helped model diseases but their application is limited when it comes to processes that are temporally controlled or where gene products compensate for the knockout: deletion (or overexpression) of a gene may also lead to altered physiological functions, possibly resulting in indirect effects. A powerful, alternative approach is to use small molecule probes. These are ideal when kinetics must be tightly controlled, such as signal transduction cascades. Furthermore, if a process is perturbed with small molecules it suggests the target is “druggable”, providing a rationale for therapeutic intervention. The combination of small molecule chemical probes and zebrafish reporter methodology presents a unique interdisciplinary method that has already proven vital in the identification of PTBA, a compound that expands the embryonic renal progenitor cell (RPC) population. In the parent P30, we designed and implemented a quantitative screen that can be applied to acquire and archive images from any imaging platform. We will continue to use this technology with new lines, those that are currently in development, and those requested by Core users.

• To use small molecules to offset the consequences of protein conformational disorders linked to renal cells, kidney injury, and viral infection. A significant effort of this Core over the past ~4 years has been to identify and characterize small molecules that improve renal cell health and development. We worked with new collaborators to isolate compounds that remedy kidney disease in model systems. We improved recovery after kidney injury in zebrafish and mouse models and rescued a protein linked to dRTA. Compounds that thwart the replication of a virus that results in the rejection of up to 30% of kidney transplant patients have also been isolated. These efforts will be expanded and compounds with improved properties will be identified to begin testing preclinical candidates.