Activation of mTOR mediates hyperglycemia-induced renal glomerular endothelial hyperpermeability via the RhoA/ROCK/pMLC signaling pathway.

objectiveHyperglycemia 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. MATERIALS AND

methodsHRGECs 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.

resultsHG 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.

conclusionsOur 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.