Sun Yan, Deng Ming, Ke Xiao, Lei Xiangyang, Ju Hao, Liu Zhiming, Bai Xiaosu
Department of Endocrinology, Southern University of Science and Technology Hospital, Shenzhen, People's Republic of China.
Department of Cardiology, Fuwai Hospital, Chinese Academy of Medical Sciences, Shenzhen Sun Yat-Sen Cardiovascular Hospital, Shenzhen, 518057, People's Republic of China.
Diabetes Metab Syndr Obes. 2021 May 19;14:2255-2268. doi: 10.2147/DMSO.S299562. eCollection 2021.
Diabetic nephropathy (DN) is a serious health problem worldwide. Epidermal growth factor (EGF) has suggested as a potential biomarker for the progression of chronic kidney disease. In this study, we examined the effects of EGF on the high glucose (HG)-induced podocyte injury and explored the underlying molecular mechanisms.
The cell proliferation, toxicity, and cell apoptosis of podocytes were determined by CCK-8 assay, lactate dehydrogenase release assay, and flow cytometry, respectively, and protein levels in the podocytes were determined by Western blot assay. Mechanistically, DNA methylation analysis, bioinformatic analysis, methylation‑specific PCR and quantitative real-time PCR were used to analyze functional pathways in differentially methylated genes and the expression of the key methylated genes in the podocytes after different interventions.
EGF treatment significantly increased the protein expression level of LC3 and decreased the protein level of P62 in HG-stimulated podocytes, which was attenuated by autophagy inhibitor, 3-methyladenine. EGF increased the cell proliferation and the protein expression levels of nephrin and synaptopodin, but reduced cell toxicity and cell apoptosis and protein expression level of cleaved caspase-3, which was partially antagonized by 3-methyladenine. DNA methylation expression profiles revealed the differential hypermethylation sites and hypomethylation sites among podocytes treated with normal glucose, HG and HG+EGF. GO enrichment analysis showed that DNA methylation was significantly enriched in negative regulation of phosphorylation, cell-cell junction and GTPase binding. KEGG pathway analysis showed that these genes were mainly enriched in PI3K-Akt, Hippo and autophagy pathways. Further validation studies revealed that six hub genes (ITGB1, GRB2, FN1, ITGB3, FZD10 and FGFR1) may be associated with the protective effects of EGF on the HG-induced podocyte injury.
In summary, our results demonstrated that EGF exerted protective effects on HG-induced podocytes injury via enhancing cell proliferation and inhibiting cell apoptosis. Further mechanistic studies implied that EGF-mediated protective effects in HG-stimulated podocytes may be associated with modulation of autophagy and PI3K/AKT/mTOR signaling pathway.
糖尿病肾病（DN）是全球范围内严重的健康问题。表皮生长因子（EGF）已被认为是慢性肾脏病进展的潜在生物标志物。在本研究中，我们研究了EGF对高糖（HG）诱导的足细胞损伤的影响，并探讨了其潜在的分子机制。
分别通过CCK-8法、乳酸脱氢酶释放法和流式细胞术检测足细胞的增殖、毒性和细胞凋亡情况，并通过蛋白质免疫印迹法检测足细胞中的蛋白质水平。机制上，采用DNA甲基化分析、生物信息学分析、甲基化特异性PCR和定量实时PCR分析不同干预后足细胞中差异甲基化基因的功能通路及关键甲基化基因的表达。
EGF处理显著增加了HG刺激的足细胞中LC3的蛋白表达水平，降低了P62的蛋白水平，自噬抑制剂3-甲基腺嘌呤可减弱这种作用。EGF增加了细胞增殖以及nephrin和synaptopodin的蛋白表达水平，但降低了细胞毒性和细胞凋亡以及裂解的caspase-3的蛋白表达水平，3-甲基腺嘌呤可部分拮抗这种作用。DNA甲基化表达谱揭示了正常葡萄糖、HG和HG+EGF处理的足细胞之间的差异高甲基化位点和低甲基化位点。基因本体（GO）富集分析表明，DNA甲基化在磷酸化的负调控、细胞间连接和GTP酶结合中显著富集。京都基因与基因组百科全书（KEGG）通路分析表明，这些基因主要富集于PI3K-Akt、Hippo和自噬通路。进一步的验证研究表明，六个枢纽基因（ITGB1、GRB2、FN1、ITGB3、FZD10和FGFR1）可能与EGF对HG诱导的足细胞损伤的保护作用有关。
总之，我们的结果表明，EGF通过增强细胞增殖和抑制细胞凋亡对HG诱导的足细胞损伤发挥保护作用。进一步的机制研究表明，EGF在HG刺激的足细胞中介导的保护作用可能与自噬和PI3K/AKT/mTOR信号通路的调节有关。
