Poster #040, Children's Hospital Los Anglese
Disabling SIRT3 Promotes Metabolic Dysfunction in the Proximal Tubular Cells
Mentors: Hasmik Soloyan (MD), PI: Sargis Sedrakyan (PhD)
Alport Syndrome is a hereditary kidney disease caused predominantly by COL4A5 mutations in the glomerulus that lead to progressive renal failure. Even if Alport Syndrome is considered a disease of the glomerulus, our studies indicate that damage to the tubules is also important in this disease’s progression. Indeed, our preliminary data shows downregulation of SIRT3 and PGC-1a expression in the proximal tubular cells: this alteration leads to metabolic abnormalities in these cells, impaired mitochondrial activity, and land accumulation. These findings suggest that disruption of the SIRT3-PGC-1a signaling axis may contribute to proximal tubule injury and disease progression. To test our hypothesis, we performed loss-of-function studies in human proximal tubular cells. We seeded 600,000 cells and SIRT3 was selectively inhibited using siRNA following the standard protocol in our laboratory. After silencing, we performed immunocytochemistry and immunoblotting on our experimental groups to evaluate the expression of SIRT3 and PGC-1a in the silenced cells compared to normal cells. We efficiently knocked down SIRT3. By light microscopy, we determined that silencing impaired tubular cell morphology and induced tubular injury. In addition, we collected cell protein, evaluated protein concentration, and performed immunoblotting. At the moment of submitting this abstract, the blots are developing, and we are also performing immunocytochemistry for PGC-1a. These studies will define a model mechanism linking SIRT3 and PGC-1a to proximal tubular injury in Alport. In addition, we are also evaluating the role of SIRT3 in tubular metabolic function since its expression regulates mitochondrial activity in these cells.