Front Med (Lausanne). 2025 Dec 1;12:1547410. doi: 10.3389/fmed.2025.1547410. eCollection 2025.
ABSTRACT
BACKGROUND: Diabetic nephropathy (DN) is characterized by tubular injury and tubulointerstitial fibrosis, leading to progressive renal dysfunction. While dysregulation of autophagy has been linked to DN pathogenesis, the underlying regulatory mechanisms remain incompletely understood. This study aimed to test the hypothesis that transcription factor 3 (TCF3) serves as a critical upstream regulator of autophagy dysfunction in DN by suppressing Netrin-1 expression, thereby promoting epithelial-mesenchymal transition (EMT) through activation of the PI3K/Akt/mTOR pathway.
METHODS: We established a DN rat model using high-fat diet followed by low-dose streptozotocin injection (25 mg/kg). Thirty-five male Sprague-Dawley rats were divided into five groups (n = 6-7/group, with specific numbers clearly defined for each experimental condition): control, DN, DN + vector, DN + TCF3-shRNA lentivirus, and DN + TCF3-shRNA + 3-methyladenine (3-MA). All key experiments were performed with at least three independent biological replicates. In vitro, HK-2 cells were categorized into four groups: normal glucose (NG, 5.5 mmol/L), high glucose (HG, 30 mmol/L), HG with negative control siRNA (HG + si-NC), and HG with TCF3-targeting siRNA (HG + TCF3-siRNA). Western blotting was utilized to determine the expression levels of autophagy-related proteins, EMT-associated proteins, and PI3K/Akt/mTOR signaling pathway-related proteins. Quantitative real-time polymerase chain reaction (qRT-PCR) was employed to assess the mRNA expression levels of TCF3 and Netrin-1. Additionally, a dual-luciferase reporter gene assay was performed to investigate the interaction between TCF3 and Netrin-1. Statistical analyses were performed using one-way ANOVA followed by Tukey’s post-hoc test, with p < 0.05 considered statistically significant.
RESULTS: We first confirmed that TCF3 expression was significantly elevated in both DN rat kidneys (2.8-fold increase at protein level, p < 0.001) and high glucose-treated HK-2 cells (2.5-fold at protein level, p < 0.001) compared to controls. Both the DN rat model and HG-stimulated HK-2 cells exhibited enhanced EMT markers, with significantly increased α-SMA and vimentin expression (p < 0.001), and decreased E-cadherin levels (p < 0.001). TCF3 knockdown significantly attenuated these EMT changes and increased autophagy markers, as evidenced by decreased P62 levels (p < 0.01) and increased LC3-II/I ratio (p < 0.001) and Beclin-1 expression (p < 0.01). The dual luciferase assay confirmed direct binding of TCF3 to the Netrin-1 promoter, with a 57% ± 4.3% reduction (p < 0.001) in luciferase activity. Mechanistically, TCF3 silencing mitigated HG-induced fibrosis and promoted autophagy by increasing Netrin-1 expression and suppressing the PI3K/Akt/mTOR signaling pathway.
CONCLUSION: Our findings demonstrate that TCF3 functions as a critical negative regulator of autophagy in DN, establishing a novel TCF3-Netrin-1-autophagy regulatory axis. This study provides new mechanistic insights distinct from previous work by demonstrating the direct transcriptional repression of Netrin-1 by TCF3 in renal pathophysiology. The limitation of our study includes the lack of human DN tissue validation and TCF3-specific pharmacological inhibitors. These findings suggest TCF3 as a potential therapeutic target for preventing renal fibrosis in DN through restoration of autophagy function.
PMID:41404576 | PMC:PMC12702734 | DOI:10.3389/fmed.2025.1547410
