The versatile role of Foxp2 in chronic kidney disease

Abstract

Chronic kidney disease (CKD) affects over 10% of the world’s population with limited effective treatment. Underlying its mechanisms to unveil novel therapeutic targets is of high clinical significance. In CKD, injured tubular epithelial cells (TECs) often undergo incomplete repair and enter a proinflammatory phenotype by overexpressing multiple cytokines to orchestrate the development of kidney fibrosis. Forkhead box protein P2 (Foxp2) is a transcription factor closely associated with organ development and tumorigenesis, but its putative role in CKD remains unexplored. This work aims to demonstrate the role of Foxp2 in CKD and explore the anti-fibrotic mechanism of Foxp2 as a potential treatment. In vivo, I observed upregulation of Foxp2 protein in renal tubules in kidney biopsies of patients with CKD and in kidneys of mice underwent unilateral ureteral obstruction (UUO) and unilateral ischemia reperfusion injury (UIRI). To investigate the role of renal tubular Foxp2 in CKD, tubule-specific Foxp2 knockout (Foxp2 KO) were generated. In the UUO model, tubular Foxp2 deletion reduced interstitial fibrosis and epithelial-mesenchymal transition (EMT) with reduction of fibronectin (Fn), collagens, plasminogen activator inhibitor-1 (PAI-1), vimentin and α-smooth muscle actin (α-SMA) expression in the injured kidney versus sham animals. Suppression of ERK1/2-mediated inflammatory cytokines with reduction of neutrophil and macrophage infiltration was demonstrated in Foxp2 KO mice at day 7 post-UUO surgery. Apoptosis and cell cycle arrest of TECs were decreased in Foxp2 KO mice with downregulated expressions of p53, Bax, cyclin-dependent kinase inhibitor 1 (p21), cyclin-dependent kinase inhibitor 1B (p27) and phosphorylated Histone 3 (pH3). In the UIRI-induced CKD model that aims to explore the role of tubular Foxp2 in acute kidney injury (AKI)-to-CKD transition, kidney function was preserved and tubular injury reduced in Foxp2 KO mice at day 7 after UIRI surgery versus sham animals. Inhibition of interstitial fibrosis and EMT was demonstrated whereas activation of ERK1/2 and NF-κB-mediated inflammation was suppressed with reduced macrophage infiltration in Foxp2 KO mice at day 7 post UIRI. Renal apoptosis and cell cycle arrest were prevented in tubular Foxp2 KO mice after UIRI. In vitro, expression of Foxp2 was induced by TGF-β1 through Smad3-dependent pathway in mouse tubular epithelial cells (TECs). The relationship between Foxp2 and TGF-β/Smad3 signaling pathway was further elucidated by demonstration of heterozygous dimerization of Foxp2/Smad3 through co-immunoprecipitation (Co-IP) assay. Gene silencing of Foxp2 in TECs led to a reduction of fibrotic and EMT marker expression under a profibrotic milieu with TGF-1. To further explore the renoprotective mechanisms of Foxp2 knockdown in driving fibrosis, chromatin immunoprecipitation (ChIP) followed by dual luciferase reporter assay were performed to show that Foxp2 mediated collagen 1 (Col-1) and E-cadherin (Ecad) gene transcription by direct binding to the promoter region, thus regulating fibrotic responses and EMT in TECs. Collectively, these findings suggest that upregulation of Foxp2 in renal tubules affects EMT and cell cycle regulation in tubular repair. However, dysregulation of Foxp2 expression subsequently leading to interstitial fibrosis and development of CKD. Targeting Foxp2 in TECs may provide a promising strategy for kidney fibrosis and progressive CKD.