Role of homocysteine in renal tubular epithelial-mesenchymal transition: N-homocysteinylation of β-catenin impairs FUNDC1-dependent autophagy and mitochondrial quality control.

Given the nephrotoxicity of hyperhomocysteinemia and the key role of mitophagy in the homeostasis of kidney, this study investigated the β-catenin/FUNDC1/mitochondrial quality control axis to elucidate the mechanisms underlying homocysteine-induced renal tubular fibrosis. A mouse model of hyperhomocysteinemia was established via continuous supplementation of homocysteine. Western blot quantified key proteins associated with autophagy (FUNDC1, LC3, p62), fibrosis (fibronectin, α-SMA, Snail1), β-catenin signaling, and kidney injury (KIM-1). Immunostaining assessed renal spatial distribution of β-catenin, FUNDC1, α-SMA, and fibronectin, while transmission electron microscopy visualized mitochondrial ultrastructural changes. ChIP and dual-luciferase reporter assays verified the transcriptional target. Meanwhile, IP combined with mass spectrometry (MS), molecular modeling, and site-directed mutagenesis identified the modification site. β-catenin signaling inhibition by homocysteine resulted in diminished FUNDC1 expression within renal tubular epithelial cells, simultaneously suppressing autophagy and facilitating renal tubular fibrosis. β-catenin overexpression enhanced FUNDC1 expression, thereby rescuing homocysteine-suppressed autophagy and effectively mitigating renal tubular epithelial-mesenchymal transition (EMT). FUNDC1 overexpression mitigated homocysteine-induced cellular stress, further enhanced autophagy and mitochondrial quality control, and alleviated renal tubular EMT. Further, dual-luciferase reporter assays and ChIP confirmed FUNDC1 as a β-catenin transcriptional target. IP combined with MS, molecular modeling, and site-directed mutagenesis experiments revealed that homocysteine induced N-homocysteinylation of β-catenin at Lys49, which remodeled β-catenin's local structure to promote Ser45 phosphorylation and subsequent β-catenin degradation. Conclusively, homocysteine induces N-homocysteinylation of β-catenin at Lys49 to promote its degradation, thereby inhibiting β-catenin-mediated transcriptional activation of FUNDC1, disrupting autophagy and mitochondrial quality control, and ultimately inducing renal tubular EMT.