In the setting of diabetes mellitus, mitochondrial dysfunction and oxidative stress are important pathogenic mechanisms causing end organ damage, including diabetic kidney disease (DKD), but mechanistic understanding at a cellular level remains obscure. In mouse models of DKD, glomerular endothelial cell (GEC) dysfunction precedes albuminuria and contributes to neighboring podocyte dysfunction, implicating GECs in breakdown of the glomerular filtration barrier. In the following studies we wished to explore the cellular mechanisms by which GECs become dysfunctional in the diabetic milieu, and the impact to neighboring podocytes. Mouse GECs were exposed to high glucose media (HG) or 2.5% v/v serum from diabetic mice or serum from non-diabetic controls, and evaluated for mitochondrial function (oxygen consumption), structure (electron microscopy), morphology (mitotracker), mitochondrial superoxide (mitoSOX), as well as accumulation of oxidized products (DNA lesion frequency (8-oxoG, endo-G), double strand breaks (γ-H2AX), endothelial function (NOS activity), autophagy (LC3) and apoptotic cell death (Annexin/PI; caspase 3). Supernatant transfer experiments from GECs to podocytes were performed to establish the effects on podocyte survival and transwell experiments were performed to determine the effects in co-culture. Diabetic serum specifically causes mitochondrial dysfunction and mitochondrial superoxide release in GECs. There is a rapid oxidation of mitochondrial DNA and loss of mitochondrial biogenesis without cell death. Many of these effects are blocked by mitoTEMPO a selective mitochondrial anti-oxidant. Secreted factors from dysfunctional GECs were sufficient to cause podocyte apoptosis in supernatant transfer experiments, or in co-culture but this did not occur when GECs had been previously treated with mitoTEMPO. Dissecting the impact of the diabetic environment on individual cell-types from the kidney glomerulus indicates that GECs become dysfunctional and pathological to neighboring podocytes by increased levels of mitochondrial superoxide in GEC. These studies indicate that GEC-signaling to podocytes contributes to the loss of the glomerular filtration barrier in DKD.

中文翻译：

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糖尿病微环境导致肾小球内皮细胞线粒体氧化应激和足细胞病理串扰。

在糖尿病的背景下，线粒体功能障碍和氧化应激是导致终末器官损伤的重要致病机制，包括糖尿病肾病 (DKD)，但细胞水平的机制理解仍然模糊不清。在 DKD 小鼠模型中，肾小球内皮细胞 (GEC) 功能障碍先于蛋白尿，并导致邻近的足细胞功能障碍，这表明 GEC 与肾小球滤过屏障的破坏有关。在以下研究中，我们希望探索 GEC 在糖尿病环境中功能失调的细胞机制，以及对邻近足细胞的影响。小鼠 GEC 暴露于高葡萄糖培养基 (HG) 或 2.5% v/v 糖尿病小鼠血清或非糖尿病对照血清，并评估线粒体功能（耗氧量）、结构（电子显微镜）、在上清液转移实验或共培养中，来自功能失调的 GEC 的分泌因子足以引起足细胞凋亡，但当 GEC 先前已用 mitoTEMPO 处理时，这不会发生。剖析糖尿病环境对肾小球中单个细胞类型的影响表明，GEC 通过 GEC 中线粒体超氧化物水平的增加，对邻近足细胞变得功能障碍和病理性。这些研究表明，GEC 向足细胞的信号传导导致 DKD 肾小球滤过屏障的丧失。剖析糖尿病环境对肾小球中单个细胞类型的影响表明，GEC 通过 GEC 中线粒体超氧化物水平的增加，对邻近足细胞变得功能障碍和病理性。这些研究表明，GEC 向足细胞的信号传导导致 DKD 肾小球滤过屏障的丧失。剖析糖尿病环境对肾小球中单个细胞类型的影响表明，GEC 通过 GEC 中线粒体超氧化物水平的增加，对邻近足细胞变得功能障碍和病理性。这些研究表明，GEC 向足细胞的信号传导导致 DKD 肾小球滤过屏障的丧失。