The First Clinical Medical College, Lanzhou University, Lanzhou, Gansu, China.
Tianjin Normal University, Tianjin, China.
Front Endocrinol (Lausanne). 2023 Jan 26;13:1009507. doi: 10.3389/fendo.2022.1009507. eCollection 2022.
Glycation is a common post-transcriptional modification of proteins. Previous studies have shown that advanced glycation end modified transferrin (AGE-Tf) levels in diabetic rat kidney tissues were increased; however, its role in diabetic nephropathy remains unclear. In this study, differences in glycation degree and Tf sites induced by differing high glucose concentrations and the effect on total iron binding capacity (TIBC) were observed. Moreover, the effect of AGE-Tf on human renal tubular epithelial cells (HK-2) was investigated.
Tf was incubated with increasing glucose concentrations (0 mM, 5.6 mM, 11.1 mM, 33.3 mM, 100 mM, 500 mM, and 1,000 mM) for AGE-Tf. Differences in AGE-Tf glycation degree and TIBC level were analyzed colorimetric method. The AGE-Tf glycation sites were identified with LC-MS/MS. HK-2 cells were treated with AGE-Tf prepared with different glucose concentrations (33.3 mM and 500 mM) . The effects of AGE-Tf on HK-2 cell viability, proliferation, oxidative stress index, and Tf receptor expression levels were then observed.
With increasing glucose concentrations (100 mM, 500 mM, and 1,000 mM) , Tf glycation degree was significantly increased. The TIBC levels of AGE-Tf were decreased significantly with increasing glucose concentrations (33.3 mM, 100 mM, 500 mM, and 1,000 mM). Four glycated modification sites in Tf and 17 glycated modification sites were detected in AGE-Tf (500 mM) by LC-MS/MS. The structural types of AGEs were CML, G-H1, FL-1H2O, FL, and MG-H1. No significant differences were found in the survival rate of HK-2 cells among the AGE-Tf (500 mM), AGE-Tf (33.3 mM), and Tf groups (all > 0.05). The apoptosis rate of HK-2 cells in the AGE-Tf (500 mM) group was significantly higher than that in the AGE-Tf (33.3 mM) group. Additionally, both of them were significantly higher than that in the Tf group (both < 0.05). The MDA levels of HK-2 cells in the AGE-Tf (500 mM) and AGE-Tf (33.3 mM) groups were higher than that in the Tf group, but not significantly (both > 0.05). The T-AOC level of HK-2 in the AGE-Tf (500 mM) group was significantly lower than that in the AGE-Tf (33.3 mM) and Tf groups (both < 0.001). The GSH level of HK-2 cells in the AGE-Tf (500 mM) group was significantly lower than that in the Tf group ( < 0.05). The expression level of TfR in the AGE-Tf (500 mM) group was also significantly lower than that in the Tf group ( < 0.05).
The degree and sites of Tf glycation were increased secondary to high-glucose exposure; however, the binding ability of Tf to iron decreased gradually. After HK-2 was stimulated by AGE-Tf , the apoptosis of cells was increased, antioxidant capacity was decreased, and TfR expression levels were downregulated.
糖基化是蛋白质常见的转录后修饰。先前的研究表明，糖尿病大鼠肾组织中晚期糖基化终产物转铁蛋白（AGE-Tf）水平升高，但在糖尿病肾病中的作用尚不清楚。本研究观察了不同高葡萄糖浓度诱导的 Tf 糖化程度和铁结合能力（TIBC）的差异，以及 AGE-Tf 对人肾小管上皮细胞（HK-2）的影响。
将 Tf 与不同浓度的葡萄糖（0 mM、5.6 mM、11.1 mM、33.3 mM、100 mM、500 mM 和 1000 mM）孵育，制备 AGE-Tf。采用比色法分析 AGE-Tf 糖化程度和 TIBC 水平的差异。采用 LC-MS/MS 鉴定 AGE-Tf 的糖化位点。用不同浓度（33.3 mM 和 500 mM）的葡萄糖制备 AGE-Tf 处理 HK-2 细胞，观察 AGE-Tf 对 HK-2 细胞活力、增殖、氧化应激指数和 Tf 受体表达水平的影响。
随着葡萄糖浓度（100 mM、500 mM 和 1000 mM）的增加，Tf 的糖化程度显著增加。随着葡萄糖浓度的增加（33.3 mM、100 mM、500 mM 和 1000 mM），AGE-Tf 的 TIBC 水平显著降低。通过 LC-MS/MS 在 AGE-Tf（500 mM）中检测到 4 个糖化修饰位点和 17 个糖化修饰位点。AGEs 的结构类型为 CML、G-H1、FL-1H2O、FL 和 MG-H1。AGE-Tf（500 mM）、AGE-Tf（33.3 mM）和 Tf 组之间 HK-2 细胞存活率无显著差异（均>0.05）。AGE-Tf（500 mM）组 HK-2 细胞凋亡率明显高于 AGE-Tf（33.3 mM）组，差异有统计学意义（均<0.05）。与 Tf 组相比，AGE-Tf（500 mM）和 AGE-Tf（33.3 mM）组 HK-2 细胞的 MDA 水平升高，但差异无统计学意义（均>0.05）。AGE-Tf（500 mM）组 HK-2 的 T-AOC 水平明显低于 AGE-Tf（33.3 mM）和 Tf 组，差异有统计学意义（均<0.001）。AGE-Tf（500 mM）组 HK-2 细胞的 GSH 水平明显低于 Tf 组，差异有统计学意义（<0.05）。AGE-Tf（500 mM）组 TfR 的表达水平也明显低于 Tf 组，差异有统计学意义（<0.05）。
高葡萄糖暴露可导致 Tf 糖化程度和位点增加，但 Tf 与铁的结合能力逐渐降低。AGE-Tf 刺激 HK-2 后，细胞凋亡增加，抗氧化能力降低，TfR 表达下调。
