Development of an RNA-FISH Procedure for Detection of mRNA Targets in Diabetic Nephropathy
Schulte, Emma   (University of Washington)
Mentor: Akilesh, Shreeram
Diabetic nephropathy (DN) is a common complication of diabetes and is associated with significant morbidity and mortality among patients. As the most common cause of end-stage renal disease (ESRD) in the United States, DN remains a significant burden to national public health. While the glomerulus, a key structure in the kidney filter, is the principal target of diabetic kidney injury, there are currently no approved treatments that reverse the glomerular injury seen in DN. Our lab has used modern genetic approaches to identify novel glomerular target genes for DN and other kidney diseases (PMID: 30760496). As a next step, precise localization of target gene expression in key cell types within the tissue context will be essential to understand their mechanistic role in DN. In situ detection of messenger RNA (mRNA) molecules using hybridization of fluorescently labeled probes (RNA-FISH) is the most powerful and versatile technique to accomplish this, but is technically challenging. In order to establish this technique within our laboratory, we tested cells grown in vitro, and mouse and human kidney tissues that were preserved using different strategies. We initially targeted detection of polyadenylated messenger RNA (mRNA) using a fluorescently labeled oligo-dT50 probe as a test for RNA integrity and imaged our preparations on both widefield and confocal microscopes. While the initial experiments appear to have been unsuccessful, we will next focus on optimizing tissue preservation, target selection and imaging to bring this powerful methodology to bear on the study of novel glomerular targets in DN.