Rory Sebastian
Pronouns:
Research Mentor(s): Matthias Wolf
Research Mentor School/College/Department: Pediatrics / Medicine
Program:
Authors: Rory Sebastian, Ingrid Apel, Sang Wan An, Huiyuan Wu, Matthias Wolf
Session: Session 5: 2:40 pm – 3:30 pm
Poster: 76
Abstract
Autosomal Dominant Tubulointerstitial Kidney Disease (ADTKD), is a rare kidney disease which is characterized by renal failure and gout presenting between 20 to 40 years of age. ADTKD is caused by heterozygous mutations in five different genes. One of the most commonly affected genes encodes for a protein called Uromodulin, which is the most abundant urinary protein in humans. Mutations result in a misfolded Uromodulin protein, which is less secreted and accumulates inside the cell. As the misfolded Uromodulin protein builds up over time, it results in cellular apoptosis and kidney failure, requiring dialysis or a kidney transplant. Our research focuses on the identification of new therapeutic options for ADTKD caused by Uromodulin mutations. After transfecting cell lines with luciferase-tagged Uromodulin, we conducted a cell-based assay designed to mimic the disease phenotype. This cell-based assay was used to screen 8,000 compounds from a small molecule library. We screened for compounds which enhance luciferase activity in the cell culture medium, which would indicate that the compound enhanced secretion of the luciferase-tagged mutant Uromodulin. Among the 8,000 small molecules, we identified four compounds which increased the secretion of mutant Uromodulin from 10-15% to 80-100% of the wild-type Uromodulin. This project aims to confirm the activity of these four candidates. To counter-screen the best candidates, we employ additional assays with the goal to ensure cell viability and a reduction in cellular apoptosis. To exclude toxicity, we use the Cell-Titer-Glo assay, and to investigate apoptosis we include the Caspase 3/7 assay. To detect Uromodulin secretion independently of the luciferase-tag, we will also use a Uromodulin ELISA. The overarching goal of our research is to find a small molecule which can help patients with ADTKD to secrete the mutant Uromodulin starting early in life so that the mutant Uromodulin does not build up in the kidney cells and cause kidney failure.
