P RAO1, M PANG1,2, X QIAO1,2, H YU1, T CHEN1, H WANG1,2, M HU1, Q CAO1, Y WANG1, G ZHANG3, Y WANG3, C P’NG4, B Nankivell4, V Lee1, S Alexander3, G Zheng1, D Harris1
1University of Sydney at Westmead Institute for Medical Research, Westmead, Australia, 2Shanxi Medical University, , China, 3Children’s Hospital at Westmead, Westmead, Australia, 4Westmead Hospital, Westmead, Australia
Aim: To examine the role of β-catenin/Foxo in kidney fibrosis.
Background: The conflicting effects of TGF-β (profibrotic versus anti-inflammatory) have caused a major challenge in the treatment of kidney fibrosis. β-catenin/TCF is central to all TGF-β’s profibrotic signaling pathways and activates profibrotic genes. β-catenin also binds to Foxo in competition with TCF and promotes cell survival under oxidative stress. We propose that promoting β-catenin/Foxo will protect against β-catenin/TCF mediated profibrotic changes and kidney fibrosis.
Methods: Kidney biopsies of patients with chronic kidney disease (CKD) or a kidney transplant were assessed by Proximity Ligation Assay (PLA) for β-catenin/Foxo and β-catenin/TCF interactions in relation to kidney fibrosis. Mouse tubular epithelial C1.1 cells were treated with TGF-β1 (3ng/ml) with or without ICG-001 (5µM), which inhibits β-catenin/TCF. Foxo1 and TCF1 were knocked out by CRISPR/Cas9-mediated gene knockout. We also evaluated kidney fibrosis in murine unilateral ureteric obstruction (UUO). β-catenin/Foxo and β-catenin/TCF interactions were examined by co-immunoprecipitation assays (co-IP) and PLA. Profibrotic gene expressions were examined by western blot and immunofluorescence.
Results: PLA of CKD and kidney transplant patient biopsies showed that β-catenin/Foxo correlated negatively (r=-0.7405, P<0.001) and β-catenin/TCF positively (r=0.8061, P<0.001) with kidney fibrosis. Combined treatment with TGF-β1 and ICG-001 protected against TGF-β1-induced profibrotic gene expression while the protection was not seen in Foxo1 KO C1.1 cells. PLA and co-IP showed direct evidence for the promotion of β-catenin/Foxo with TGF-β1 and ICG-001 in C1.1 cells. UUO mice treated with TGF-β1 and ICG-001 had significantly reduced kidney fibrosis, via promotion of β-catenin/Foxo interaction as shown by PLA.
Conclusions: These results indicate that β-catenin/Foxo plays a protective role against TGF-β’s profibrotic activity and thereby prevents kidney fibrosis.
Padmashree Rao is the student of the University of Sydney at Westmead Institute for Medical Research. Her research has been focused on kidney fibrosis. TGF-beta, a cytokine causing kidney fibrosis, also exhibits anti-inflammatory and wound healing effects. Her project aims to prove that targeting beta-catenin/Foxo will prevent beta-catenin/TCF-mediated fibrosis and may be used as a novel target for the treatment of kidney fibrosis.