TCF3 downregulation alleviates renal fibrosis via PI3K/Akt/mTOR pathway inhibition and autophagy restoration in diabetic nephropathy

Chen-xiao Liu^1*Yan-qiu Jiang²Ai-jie Huang²Yun Zhao²Xing-na Hu²Rong Xiang²Min Feng²Ying Xie^1*

• ¹Department of Neurology, Second Affiliated Hospital of Soochow University, Suzhou, China
• ²Department of Endocrinology, The Affiliated Suzhou Hospital of Nanjing Medical University, Suzhou, Liaoning Province, China

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.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). To address the mechanistic link between Netrin-1 and PI3K/Akt/mTOR pathway, we performed co-immunoprecipitation and pathway inhibitor experiments. Western blotting (n=3 independent experiments), qRT-PCR (n=3 biological replicates with technical triplicates), and dual luciferase reporter assays (n=3 independent experiments) were used to analyze protein and mRNA expression. Statistical analyses were performed using one-way ANOVA followed by Tukey's post-hoc test, with p<0.05 considered statistically significant.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 (58±7.3%, p<0.01) and increased LC3-II/I ratio (2.4-fold, p<0.001) and Beclin-1 expression (1.8-fold, 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. Netrin-1 knockdown experiments confirmed that Netrin-1 mediates TCF3's effects on PI3K/Akt/mTOR signaling, as Netrin-1 siRNA reversed the inhibitory effects of TCF3