Testing Novel Podocyte Slit Diaphragm Protein Conservation in Danio rerio (Zebrafish)

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  • September 23, 2022
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  • Imseis, Zachary
    • Affiliation: College of Arts and Sciences
  • Gerlach, Gary
    • Affiliation: School of Medicine, Department of Cell Biology and Physiology
  • O'Brien, Lori
    • Affiliation: School of Medicine, Department of Cell Biology and Physiology
Abstract
  • Kidneys are vital organs responsible for filtering and removing toxins from the blood. The major filtration unit of the kidney is the nephron. The nephron consists of the glomerulus and tubules. The glomerulus consists of three components: fenestrated endothelium, the glomerular basement membrane (GBM), and podocytes. Podocytes are highly specialized epithelial cells with long protrusions called foot processes that connect to each other via specialized junctions called slit diaphragms (SD). The SD is largely responsible for the integrity of the glomerulus. Lack of the SD contributes to a loss of podocyte integrity; however, only a small number of proteins are known to localize in the SD. One protein known to localize in the SD is podocin. We utilized proximity-dependent Biotin Identification (BioID). We utilized CRISPR-Cas9-based gene editing to knock in the BioID moiety to the endogenous NPHS2 locus (Podocin). BioID allows for the biotinylation of a protein of interest (POI) by fusing a mutated promiscuous prokaryotic biotin ligase to the endogenous locus of our target gene, NPHS2 (Podocin). This biochemical assay allowed us to identify the repertoire of proteins within the podocyte foot process. We identified a novel Immunoglobulin-like domain-containing receptor 2 (Ildr2) within the podocyte SD. Recent data from sc-RNAseq databases evince Ildr2 expression in human podocytes. By utilizing zebrafish and mice, the importance of this novel immunoglobulin family protein can be tested in podocyte integrity. Our innovative method has allowed us to identify novel proteins of the SD and potential new therapeutic targets for kidney disease.
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