Department of Pathophysiology and Therapeutics of Diabetic Vascular Complications, Kurume University School of Medicine, Kurume 830-0011, Japan.
Department of Chemistry, Kurume University School of Medicine, Kurume 830-0011, Japan.
Int J Mol Sci. 2020 Apr 9;21(7):2604. doi: 10.3390/ijms21072604.
Glyceraldehyde-derived advanced glycation end products (glycer-AGEs) contribute to proximal tubulopathy in diabetes. However, what glycer-AGE structure could evoke tubular cell damage remains unknown. We first examined if deleterious effects of glycer-AGEs on reactive oxygen species (ROS) generation in proximal tubular cells were blocked by DNA-aptamer that could bind to glyceraldehyde-derived pyridinium (GLAP) (GLAP-aptamer), and then investigated whether and how GLAP caused proximal tubular cell injury. GLAP-aptamer and AGE-aptamer raised against glycer-AGEs were prepared using a systemic evolution of ligands by exponential enrichment. The binding affinity of GLAP-aptamer to glycer-AGEs was measured with a bio-layer interferometry. ROS generation was evaluated using fluorescent probes. Gene expression was analyzed by reverse transcription-polymerase chain reaction (RT-PCR). GLAP-aptamer bound to glycer-AGEs with a dissociation constant of 7.7 × 10 M. GLAP-aptamer, glycer-AGE-aptamer, or antibodies directed against receptor for glycer-AGEs (RAGE) completely prevented glycer-AGE- or GLAP-induced increase in ROS generation, MCP-1, PAI-1, or RAGE gene expression in tubular cells. Our present results suggest that GLAP is one of the structurally distinct glycer-AGEs, which may mediate oxidative stress and inflammatory reactions in glycer-AGE-exposed tubular cells. Blockade of the interaction of GLAP-RAGE by GLAP-aptamer may be a therapeutic target for proximal tubulopathy in diabetic nephropathy.
甘油醛衍生的晚期糖基化终产物（甘油-AGEs）导致糖尿病近端肾小管病变。然而，甘油-AGE 的结构如何引起肾小管细胞损伤尚不清楚。我们首先研究了针对甘油醛衍生的吡啶（GLAP）的 DNA 适体（GLAP-适体）是否可以阻断甘油-AGE 对近端肾小管细胞中活性氧（ROS）生成的有害影响，然后研究了 GLAP 是否以及如何引起近端肾小管细胞损伤。使用指数富集的配体系统进化制备了针对甘油-AGE 的 GLAP-适体和 AGE-适体。使用生物层干涉测量法测量 GLAP-适体与甘油-AGEs 的结合亲和力。使用荧光探针评估 ROS 的生成。通过逆转录-聚合酶链反应（RT-PCR）分析基因表达。GLAP-适体与甘油-AGE 的解离常数为 7.7×10 M。GLAP-适体、甘油-AGE-适体或针对甘油-AGE 受体（RAGE）的抗体完全阻止了甘油-AGE 或 GLAP 诱导的 ROS 生成、MCP-1、PAI-1 或 RAGE 基因表达的增加在肾小管细胞中。我们目前的结果表明，GLAP 是结构上不同的甘油-AGE 之一，它可能介导暴露于甘油-AGE 的肾小管细胞中的氧化应激和炎症反应。GLAP-aptamer 阻断 GLAP-RAGE 的相互作用可能是糖尿病肾病中近端肾小管病变的治疗靶点。
