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Kidney Week

Abstract: FR-PO956

Dysregulated 4E-BP1 Pathway in Polycystic Kidney Disease

Session Information

Category: Genetic Diseases of the Kidney

  • 1001 Genetic Diseases of the Kidney: Cystic

Authors

  • Holditch, Sara, UC Denver Anschutz Medical Campus, Aurora, Colorado, United States
  • Atwood, Daniel, University of Colorado Anschutz, Aurora, Colorado, United States
  • Brown, Carolyn Nicole, UC Denver Anschutz Medical Campus, Aurora, Colorado, United States
  • Edelstein, Charles L., UC Denver Anschutz Medical Campus, Aurora, Colorado, United States
Background

Unchecked proliferation of cystic epithelial cells is a major contributor to cyst growth in PKD. The 4E-BP1 pathway is a crucial checkpoint in translation and cellular proliferation, regulated by multiple stimulatory factors like PKCα, ERK, and AKT. The aim of this study was to 1) determine whether the 4E-BP1 pathway is dysregulated in human PKD1-/- cells, 2) examine the effects of an AKT and mTOR insensitive 4E-BP1 (F113A) on protein translation, proliferation, and 3) assess the feasibility of in vivo gene therapy of F113A 4E-BP1 adeno-associated virus (AAV9) in neonatal and adult mice.

Methods

Immunoblot, proliferation, and luciferase assays were performed on human epithelial cells from normal renal cortical tubular epithelium (PKD1+/+) and ADPKD cyst-lining epithelium (PKD1-/-) transfected with pCAG-TdTomato or pCAG-F113A. AAV9 F113A, and AAV9-TdTomato vectors were prepared and administered from D3 and to D17, or D120 to D180.

Results

There was increased phospho (p4E-BP1) species and increased pPKCa(3.6±0.2 vs 0.6±0.0DU***), pERK(3.6±0.2 vs 0.5±0.2DU***), and pAKT(0.6±0.1 vs 0.2±0.1DU*), in PKD1-/- vs. PKD1+/+ cells respectively. In vitro, F113A expression reduced p4E-BP1 S65 (1.4±0.2 vs 0.02±0.01DU***), reduced cyclin D1 (0.65±0.04 vs 0.51±0.10DU*) and increased autophagosome marker, LC3-II, (0.02±0.00 vs 0.31±0.13DU*) expression in PKD1-/- cells. Stimulation with insulin resulted in maintained p4E-BP1 suppression with F113A in PKD1-/- (2.1±0.3 vs 0.2±0.1DU***). F113A also reduced cap-dependent protein translation (by 37%**), and 72hr proliferation (250±4 vs 180±5 480/528nm O.D***). In neonatal and adult mice, administration of AAV9 resulted in detectable F113A RNA in the heart, kidney, and liver, and reduced p4E-BP1 S65 expression in the heart (2.0±1.3 vs 0.6±0.4DU*). *p<0.05, **p<0.01, *** p<0.001 vs to control.

Conclusion

In PKD, a setting of cystic tubular epithelial hyperproliferation, the 4E-BP1 pathway is dysregulated with increased phosphoregulation of 4E-BP1 by multiple overactive kinases such as; PKCa, ERK, AKT. In vitro, F113A expression in PKD1-/- cells, results in hypophosphorylated 4E-BP1 species, reduced cap dependent protein translation, reduced proliferation, and increased autophagosomes. F113A gene therapy to counter the dysregulated 4E-BP1 pathway in in vivo models of PKD is feasible to inhibit a pathway seemingly integral to the pathobiology of PKD.

Funding

  • Other U.S. Government Support