Chen Xiaolin, Chen Jianhui, Li Xianfan, Yu Zengpu
Department of Clinical Laboratory, Pingxiang People's Hospital, Pingxiang, 337000, Jiangxi, China.
Department of Clinical Laboratory, The Sixth Clinical College of Gannan Medical University, Pingxiang, Jiangxi, China.
Diabetol Metab Syndr. 2021 Oct 9;13(1):105. doi: 10.1186/s13098-021-00723-7.
Hyperglycemia is associated with albuminuria and renal glomerular endothelial dysfunction in patients with diabetic nephropathy. The mTOR and RhoA/ROCK signaling pathways are involved in glomerular filtration barrier (GFB) regulation, but their role in high glucose (HG)-induced GFB dysfunction in human renal glomerular endothelial cells (HRGECs) has not been investigated. This study aimed to investigate the mechanisms of HG-induced GFB dysfunction in vitro.
HRGECs were cultured in vitro and exposed to HG. The horseradish peroxidase-albumin leakage and transendothelial electrical resistance of the endothelial monolayer were measured after HG treatment with or without rapamycin preincubation. A fluorescence probe was used to study the distribution of F-actin reorganization. The phosphorylation levels of myosin light chain (MLC) and mTOR were measured via western blotting. RhoA activity was evaluated via GTPase activation assay. The effects of blocking mTOR or the RhoA/ROCK pathway on endothelial permeability and MLC phosphorylation under HG conditions were observed.
HG exposure induced F-actin reorganization and increased MLC phosphorylation, leading to EC barrier disruption. This effect was attenuated by treatment with rapamycin or Y-27632. Phospho-MLC (pMLC) activation in HRGECs was mediated by RhoA/ROCK signaling. mTOR and RhoA/ROCK inhibition or knockdown attenuated pMLC activation, F-actin reorganization and barrier disruption that occurred in response to HG exposure.
Our results revealed that HG stimulation upregulated RhoA expression and activity through an mTOR-dependent pathway, leading to MLC-mediated endothelial cell cytoskeleton rearrangement and glomerular endothelial barrier dysfunction.
糖尿病肾病患者的高血糖与蛋白尿和肾小球内皮功能障碍有关。mTOR和RhoA/ROCK信号通路参与肾小球滤过屏障（GFB）调节，但它们在高糖（HG）诱导的人肾小球内皮细胞（HRGECs）GFB功能障碍中的作用尚未得到研究。本研究旨在探讨体外HG诱导GFB功能障碍的机制。
体外培养HRGECs并使其暴露于HG。在用或不用雷帕霉素预孵育后，测量HG处理后内皮单层的辣根过氧化物酶-白蛋白渗漏和跨内皮电阻。使用荧光探针研究F-肌动蛋白重组的分布。通过蛋白质免疫印迹法测量肌球蛋白轻链（MLC）和mTOR的磷酸化水平。通过GTP酶激活试验评估RhoA活性。观察在HG条件下阻断mTOR或RhoA/ROCK通路对内皮通透性和MLC磷酸化的影响。
HG暴露诱导F-肌动蛋白重组并增加MLC磷酸化，导致内皮细胞屏障破坏。雷帕霉素或Y-27632处理可减弱这种作用。HRGECs中的磷酸化MLC（pMLC）激活由RhoA/ROCK信号介导。mTOR和RhoA/ROCK抑制或敲低减弱了HG暴露后发生的pMLC激活、F-肌动蛋白重组和屏障破坏。
我们的结果表明，HG刺激通过mTOR依赖性途径上调RhoA表达和活性，导致MLC介导的内皮细胞细胞骨架重排和肾小球内皮屏障功能障碍。
