P RAO 1, M PANG 1,2, X QIAO 1,2, H YU 1, H WANG 1,2, M HU1, Q CAO 1, Y WANG 1, G ZHANG 3, Y WANG 3, CH P’NG 4, B NANKIVELL4, VW LEE 1, S ALEXANDER 3, G ZHENG 1, D HARRIS 1
1University of Sydney at Westmead Institute for Medical Research, Westmead, New South Wales; 2Shanxi Medical University, Taiyuan, China; 3Children’s Hospital at Westmead, Westmead, New South Wales; 4Westmead Hospital, New South Wales
Aim: To examine the role of β-catenin/Foxo in kidney fibrosis.
Background: TGF-β causes fibrosis by cross-talk with major profibrotic pathways. β-catenin is a common co-factor in different TGF-β signalling pathways. β-catenin/TCF is known to activate profibrotic genes, while β-catenin also binds to Foxo in competition with TCF. We propose that promoting β-catenin/Foxo will protect against β-catenin/TCF mediated profibrotic changes and kidney fibrosis.
Methods: Human kidney biopsies from patients with a kidney transplant and diabetes patients were assessed 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 with or without ICG-001 (5µM), an inhibitor of β-catenin/TCF. Foxo1 and TCF1 were knocked out by CRISPR/Cas9-mediated gene knockout. We evaluated kidney fibrosis in unilateral ureteric obstruction (UUO). Profibrotic changes were examined by Western blot and immunofluorescence analysis of profibrotic proteins. Duolink – Proximity Ligation Assay (PLA) and co-immunoprecipitation assays (co-IP) were used to examine β-catenin/Foxo and β-catenin/TCF interactions.
Results: PLA of human kidney biopsies showed that β-catenin/Foxo correlated negatively (r=-0.877) whilst β-catenin/TCF correlated positively (r=0.926) with kidney fibrosis (P<0.01). ICG-001 promoted β-catenin/Foxo interaction by inhibiting β-catenin/TCF binding in TGF-β1-treated C1.1 cells, as shown by co-IP and PLA. TGF-β1-induced β-catenin/TCF activity and expression of fibrotic genes (vimentin, N-cadherin, collagen I, III & IV) were reduced by ICG-001 and TCF1 knockout, while Foxo1 knockout prevented the reduction of the fibrotic gene expression. Kidney fibrosis was significantly reduced in UUO mice with TGF-β1 and ICG-001 treatment, which redirected TGF-β1 signalling from β-catenin/TCF to β-catenin/Foxo1 as shown by PLA.
Conclusions: These results indicate that β-catenin/Foxo plays a protective role against TGF-β’s profibrotic activity by inhibiting β-catenin/TCF interaction and thereby preventing kidney fibrosis.