PTK7通过激活TGF-β1/Smad通路促进肾小管上皮细胞纤维化

PTK7 promotes fibrosis in renal tubular epithelial cells by activating the TGF-β1/Smad pathway

摘要:

为了探讨蛋白酪氨酸激酶样分子7 (protein tyrosine kinase-like 7, PTK7)在肾小管上皮细胞纤维化进程中的作用及其分子机制，评估其作为肾纤维化干预靶点的潜在价值，本研究采用免疫组化、蛋白免疫印迹、实时荧光定量聚合酶链式反应(quantitative real-time polymerase chain reaction, qRT-PCR)检测肾纤维化小鼠模型中PTK7表达变化；结合公共单细胞转录组数据库，分析PTK7在肾脏不同细胞类型中的表达分布。利用CRISPR/Cas9技术构建PTK7稳定敲除的小鼠肾小管上皮细胞系(transformed C3H mouse kidney-1, TCMK-1)，经转化生长因子β1 (transforming growth factor-beta 1, TGF-β1)刺激后，观察细胞形态变化，检测上皮-间充质转化(epithelial-mesenchymal transition, EMT)相关蛋白(E-cadherin、vimentin)及纤维化相关蛋白(α-SMA、fibronectin、collagen I)的表达水平，通过细胞计数试剂盒-8 (cell counting kit-8, CCK-8)和划痕实验评估细胞增殖活力与迁移能力，并进一步分析Smad家族成员2 (Smad family member 2, Smad2)/Smad家族成员3 (Smad family member 3, Smad3)磷酸化水平以探索其潜在机制。研究结果发现，PTK7在小鼠纤维化肾组织中表达显著上调，且在肾小管上皮细胞中表达量相对较高；PTK7敲除能够显著抑制TGF-β1诱导的EMT进程和纤维化相关蛋白表达(P0.001)，并显著抑制TGF-β1诱导的肾小管上皮细胞迁移能力；在机制层面，PTK7缺失可有效阻断TGF-β1触发的Smad2/Smad3磷酸化激活。PTK7通过激活TGF-β1/Smad通路促进肾小管细胞纤维化进程，敲除其编码基因可在细胞水平有效缓解纤维化相关表型，研究结果为进一步探索PTK7作为抗肾纤维化潜在干预靶点提供了实验依据。

Abstract:

This study aimed to investigate the role of protein tyrosine kinase-like 7 (PTK7) in the progression of fibrosis of renal tubular epithelial cells and to elucidate its underlying molecular mechanisms, thereby evaluating the potential of PTK7 as a therapeutic target for renal fibrosis. Immunohistochemistry, Western blotting, and quantitative real-time polymerase chain reaction (qRT-PCR) were performed to examine PTK7 expression in a mouse model of renal fibrosis. Public single-cell transcriptomic datasets were employed to analyze the expression distribution of PTK7 among different renal cell types. A stable PTK7-knockout transformed C3H mouse kidney-1 (TCMK-1) cell line was generated via clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9), and following transforming growth factor-beta 1 (TGF-β1) stimulation, cellular morphological changes were observed. The expression levels of epithelial-mesenchymal transition (EMT)-related proteins (E-cadherin and vimentin) and fibrosis-related proteins (α-SMA, fibronectin, and collagen I) were assessed. Cell proliferative activity and migratory capacity were evaluated by cell counting kit-8 (CCK-8) and wound healing assays, respectively, and the phosphorylation levels of Smad family member 2 (Smad2)/Smad family member 3 (Smad3) were analyzed to explore the potential mechanisms. PTK7 expression was significantly upregulated in the kidney tissue of the mouse model and was highly expressed in renal tubular epithelial cells. PTK7 knockout suppressed TGF-β1-induced EMT progression and the expression of fibrosis-related proteins (P0.001), and it attenuated the migratory ability of renal tubular epithelial cells under TGF-β1 stimulation. From a mechanism perspective, PTK7 deficiency effectively inhibited TGF-β1-triggered activation of Smad2/Smad3 phosphorylation. PTK7 promotes the fibrosis of renal tubular epithelial cells through activation of the TGF-β1/Smad signaling pathway, and PTK7 knockout can effectively alleviate fibrosis-related phenotypes at the cellular level. This study provides experimental evidence for further exploration of PTK7 as a potential therapeutic target against renal fibrosis.