College of Pharmacy, Xinjiang Medical University Key Laboratory of Active Components of Xinjiang Natural Medicine and Drug Release Technology, Urumqi, China.
State Key Laboratory of Pathogenesis, Prevention and Treatment of High Incidence Diseases in Central Asia, Urumqi, China.
Front Endocrinol (Lausanne). 2022 Jul 29;13:910907. doi: 10.3389/fendo.2022.910907. eCollection 2022.
Diabetic kidney disease (DKD), one of the main complications of diabetes mellitus (DM), has become a frequent cause of end-stage renal disease. A clinically convenient, non-invasive approach for monitoring the development of DKD would benefit the overall life quality of patients with DM and contribute to lower medical burdens through promoting preventive interventions.
We utilized 5hmC-Seal to profile genome-wide 5-hydroxymethylcytosines in plasma cell-free DNA (cfDNA). Candidate genes were identified by intersecting the differentially hydroxymethylated genes and differentially expressed genes from the GSE30528 and GSE30529. Then, a protein interaction network was constructed for the candidate genes, and the hub genes were identified by the MCODE and cytoHubba algorithm. The correlation analysis between the hydroxymethylation level of the hub genes and estimated glomerular filtration rate (eGFR) was carried out. Finally, we demonstrated differences in expression levels of the protein was verified by constructing a mouse model of DKD. In addition, we constructed a network of interactions between drugs and hub genes using the Comparative Toxicogenomics Database.
This study found that there were significant differences in the overall distribution of 5hmC in plasma of patients with DKD, and an alteration of hydroxymethylation levels in genomic regions involved in inflammatory pathways which participate in the immune response. The final 5 hub genes, including (CTNNB1, MYD88, CD28, VCAM1, CD44) were confirmed. Further analysis indicated that this 5-gene signature showed a good capacity to distinguish between DKD and DM, and was found that protein levels were increased in renal tissue of DKD mice. Correlation analysis indicated that the hydroxymethylation level of 5 hub genes were nagatively correlated with eGFR. Toxicogenomics analysis showed that a variety of drugs for the treatment of DKD can reduce the expression levels of 4 hub genes (CD44, MYD88, VCAM1, CTNNB1).
The 5hmC-Seal assay was successfully applied to the plasma cfDNA samples from a cohort of DM patients with or without DKD. Altered 5hmC signatures indicate that 5hmC-Seal has the potential to be a non-invasive epigenetic tool for monitoring the development of DKD and it provides new insight for the future molecularly targeted anti-inflammation therapeutic strategies of DKD.
糖尿病肾病（DKD）是糖尿病（DM）的主要并发症之一，已成为终末期肾病的常见病因。一种临床方便、非侵入性的方法来监测 DKD 的发展将有益于 DM 患者的整体生活质量，并通过促进预防性干预措施来降低医疗负担。
我们利用 5hmC-Seal 技术在血浆无细胞 DNA（cfDNA）中对全基因组 5-羟甲基胞嘧啶进行分析。通过将差异羟甲基化基因和 GSE30528 和 GSE30529 中的差异表达基因进行交集，确定候选基因。然后，构建候选基因的蛋白质相互作用网络，并通过 MCODE 和 cytoHubba 算法识别枢纽基因。对枢纽基因的羟甲基化水平与估计肾小球滤过率（eGFR）进行相关性分析。最后，我们通过构建 DKD 小鼠模型验证了枢纽基因的蛋白表达水平的差异。此外，我们使用比较毒理学基因组数据库构建了药物与枢纽基因相互作用的网络。
本研究发现，DKD 患者血浆中整体 5hmC 的分布存在显著差异，参与免疫反应的炎症途径中基因组区域的羟甲基化水平发生改变。最终确定了 5 个枢纽基因，包括（CTNNB1、MYD88、CD28、VCAM1、CD44）。进一步分析表明，该 5 基因标志物具有良好的区分 DKD 和 DM 的能力，并且发现 DKD 小鼠肾脏组织中蛋白水平升高。相关性分析表明，5 个枢纽基因的羟甲基化水平与 eGFR 呈负相关。毒理学分析表明，多种治疗 DKD 的药物可降低 4 个枢纽基因（CD44、MYD88、VCAM1、CTNNB1）的表达水平。
5hmC-Seal 检测法成功应用于 DM 患者伴或不伴 DKD 的血浆 cfDNA 样本。改变的 5hmC 特征表明，5hmC-Seal 有可能成为监测 DKD 发展的非侵入性表观遗传工具，并为未来针对 DKD 的分子靶向抗炎治疗策略提供新的见解。
