In this study, we confirmed that HG induced an increase in HRGEC permeability that was highly dependent on RhoA/ROCK activation and MLC phosphorylation. First, we found that HG activated the RhoA/ROCK pathway, which resulted in increased MLC phosphorylation and actin stress fiber formation, leading to endothelial hyperpermeability. Second, we observed that RhoA/ROCK pathway regulated HG-induced GFB dysfunction via mTOR activation. This pathway may be a useful therapeutic target for treating HG-induced GFB dysfunction.
GFB dysfunction is the leading cause of glomerular injury and albuminuria[2, 4]. The GFB is a composite, multilayered structure : glomerular endothelium, glomerular basement membrane, and podocytes[6, 13]. Previous studies have shown that in GFB dysfunction, glomerular endothelial cells play a key role independent of podocytes , and it is well established that the toxic effect of HG on the endothelial barrier eventually causes GFB[4, 9, 24]. MLC phosphorylation and F-actin stress fiber formation are crucial factors of endothelial hyperpermeability[14, 16, 23]. It has previously been reported that in human umbilical vein endothelial cells (HUVECs), increased transendothelial migration of albumin and THP1 cells occurs in response to HG, mediated by MLC phosphorylation and RhoA activation. However, the underlying mechanisms remain largely unexplored.
We observed that HG exposure increased the permeability of endothelial monolayers, which is affected by MLC phosphorylation, actin stress fiber formation, and a contractile endothelial cell phenotype. The increase in permeability was highly dependent on mTOR activation and MLC phosphorylation. Inhibition of MLC phosphorylation by Y-27632 or of mTOR by rapamycin resulted in reductions in the rearrangement of the F-actin cytoskeleton and in HG-induced endothelial permeability. Our findings emphasized the significance of mTOR activation and confirmed the role of endothelial MLC-dependent F-actin rearrangement in establishing renal endothelium hyperpermeability.
Additionally, we observed upregulated RhoA expression and activity in HG-treated HRGECs. The RhoA/ROCK pathway has been shown to be involved in renal microvascular complications caused by HG conditions, and it has been observed that the activation of RhoA/ROCK signaling in podocytes and/or endothelial cells by HG conditions is required for the development of hyperpermeability in glomerular cells[10, 11, 17]. Consistent with these prior findings, we observed increased permeability and RhoA activity in HG-treated HRGECs. Furthermore, inhibition of RhoA/ROCK by Y-27632 attenuated MLC phosphorylation and hyperpermeability in HG-treated HRGECs. This indicates that HG-induced glomerular endothelial hyperpermeability may be mediated by the RhoA/ROCK pathway, in alignment with previous reports of RhoA/ROCK signaling in endothelial cells.
mTOR signaling has been shown to be physiologically and pathologically critical in diabetic kidney disease. There is a growing body of evidence indicating that mTOR inhibitors are the preferred treatment for diabetic nephropathy, which is primarily associated with reduced proteinuria and albuminuria[18, 26]. Conversely in non-diabetic disease, mTOR inhibition can produce de novo proteinuria in transplant patients, which is associated with the effects of increased glomerular protein leakage induced by podocyte injury. Therefore, the role of the mTOR signaling pathway in the formation of diabetic nephropathy may be cell specific, and mTOR in different cells may play different roles in the various stages of diabetic nephropathy[19, 20, 26].
Our current results demonstrated that mTOR inhibition by rapamycin suppressed HG-induced endothelial hyperpermeability in HRGECs. A recently report demonstrated that IGF-1–stimulated F-actin reorganization and cell motility occurs via the upregulation of RhoA protein expression and activity through the mTOR signaling pathway in tumor cells. Similarly, our data demonstrated that RhoA expression and activity was upregulated in HG-treated cells and that this effect was partially diminished by rapamycin-mediated suppression of the mTOR pathway.
Previous studies have shown that mTOR upregulates RhoA activity in HG-treated HUVECs. Here, for the first time, we demonstrated that mTOR controlled the activity and expression of RhoA in HG-treated HRGECs. This result is supported by the finding that rapamycin-mediated inhibition of mTOR inhibited the expression and activity of RhoA induced by HG conditions[19, 22]. This is consistent with previous findings suggesting that RhoA/ROCK activation and subsequent F-actin cytoskeleton contraction result in increased membrane permeability following HG exposure[11, 17, 27].