Methylglyoxal-derived nucleoside adducts drive vascular dysfunction in a RAGE-dependent manner
Diabetic kidney disease (DKD) is a leading cause of death in patients with diabetes. An early precursor to DKD is endothelial cell dysfunction (ECD), a factor that often precedes and exacerbates vascular disease progression. We previously discovered that covalent adducts formed on DNA, RNA, and proteins by reactive metabolic by-product methylglyoxal (MG) predict DKD risk in patients with type 1 diabetes up to 16 years pre-diagnosis. However, the mechanisms by which MG-adducts contribute to vascular disease onset and progression remain unclear. Here, we report that the most predominant MG-induced nucleoside adducts, N2-(1-carboxyethyl)-deoxyguanosine (CEdG) and N2-(1-carboxyethyl)-guanosine (CEG), drive endothelial dysfunction. Following CEdG or CEG exposure, primary human umbilical vein endothelial cells (HUVECs) undergo endothelial dysfunction, resulting in enhanced monocyte adhesion, increased reactive oxygen species production, endothelial permeability, impaired endothelial homeostasis, and exhibit a dysfunctional transcriptomic signature. These effects were discovered to be mediated through the receptor for advanced glycation end products (RAGE), as an inhibitor for intracellular RAGE signaling diminished these dysfunctional phenotypes. Therefore, we find that not only are MG-adducts biomarkers for DKD, this study reveals they may also have a dual role as potential drivers of vascular disease onset and progression and a new therapeutic modality. Overall design: Human unbillical vein endothelial cells (HUVEC) were treated with vehicle or either Mg-adduct with or without RAGE inhibitor Ri. Total RNA was isolated using Direct-zol RNA Miniprep Plus (Zymo). RNA se-quencing libraries were generated with Kapa RNA HyperPrep kit with RiboErase (Kapa Biosystems, KR1351) according to the manufacturer's protocol. Total RNA (250 ng) from each sample was used for sequencing library preparation. The final libraries were val-idated with the Agilent Bioanalyzer DNA High Sensitivity Kit. Sequencing was per-formed on Illumina NovaSeq6000 with S4 Reagent Kit v1.5 (Illumina, 20028313) with the sequencing length of 2x101. Real-time analysis (RTA) 3.4.4 software was used to process the image analysis.