G ZHENG1, D HARRIS1, V LEE1, T CHEN1, ST ALEXANDER3
1University Of Sydney, 2Shanxi Medical University, , 3Children’s Hospital at Westmead
Aim: To examine the role of β-catenin/Foxo in kidney injury healing.
Background: TGF-β is known to promote healing, but also drives a maladaptive fibrotic response that leads to fibrosis and organ failure. We propose that targeting TGF-β signaling pathway by using inhibitors (ICG-001) of β-catenin/TCF will promote β-catenin/Foxo which results in physiological healing with epithelial rather than mesenchymal cells.
Methods: Scratch assay using murine proximal tubule-like epithelial C1.1 cells was used to assess healing in vitro. CRISPR/Cas9 was used to knockout Foxo1 or TCF1. In vivo kidney injury healing was evaluated in murine unilateral ischemia reperfusion injury (UIR). β-catenin/Foxo and β-catenin/TCF interactions were examined by proximity ligation assay (PLA). Epithelial (E-cadherin) or mesenchymal (α-SMA) healing was examined by immunofluorescence staining and measured as percentage area of positive staining (%).
Results: TGF-β1 and ICG-001 treatment in C1.1 cells and UIR mouse caused increased β-catenin/Foxo interaction as demonstrated by PLA, and sooner closure of wound in wild type but not Foxo1 knockout C1.1 cells in scratch assay (98.3± 5.1% versus 70.0±6.8%, P<0.01 ). The combined treatment of rhTGF-β with ICG-001 inhibited rhTGF-β-induced α-SMA expression and showed dominant E-cadherin expression; α-SMA ,4.9±1.2% versus 40.4±2.6%, P<0.01 in vitro, and 17.6±1.4% versus 42.1±2.8%, P<0.01 in vivo. E-cadherin, 29.1±3.6% versus 2.4±2.6%, P<0.05 in vitro, and 21.5±4.7% versus 10.5±1.8%, P<0.05 in vivo. TGF-β1 combined with ICG-001 treatment significantly reduced the kidney fibrosis in UIR model, compared with UIR control, TGF-β1 or ICG-001 alone treatment groups (41.6±10.1% vs 66.3±16.1%, 73.3±11.2%, 54.4±16.5%, P<0.05).
Conclusions: β-catenin/Foxo may serve as a therapeutic target to prevent pathological fibrotic healing and fibrosis in the treatment of kidney diseases.
Dr Guoping Zheng is a associate professor of Sydney University. He is leading an NHMRC-funded research group focusing on experimental research in molecular and cellular signaling mechanism of kidney injury repair and fibrosis.