In the present study, we have demonstrated that linagliptin has protective effects against endothelial cell barrier dysfunction induced by HG conditions. Furthermore, as HRGECs were exposed to high concentrations of glucose in vitro, DPP-4 activity and permeability of endothelial cells increased. Moreover, linagliptin has protective effects on HG-treated HRGECs by a mechanism associated with inhibiting the RhoA/ROCK pathway through activating AMPK and modulating cytoskeleton dynamics of F-actin in hyperglycemia conditions. We further confirmed that inhibiting activation of RhoA/ROCK and phosphorylation of MLC by linagliptin leads to a decline in F-actin stress fiber formation and endothelial cell hyperpermeability, which is relevant to the activation of AMPK. This signaling pathway likely is a valuable target for the management of GFB dysfunction induced by HG.
Hyperglycemia-related glomerulopathy is characterized by developing proteinuria following initial glomerular damage and hyperfiltration[1, 14]. The dysfunction of endothelial cells in the renal microvessels is considered to contribute to the pathogenesis of albuminuria in diabetic nephropathy[5, 14]. The mechanism includes the damage of the endothelial barrier owing to chronic diabetes and inflammation[7, 9]. DPP-4 plays a critical role in nephropathy in diabetes because of its enzymatic activity in the kidney. Recently, it has been recognized that hyperglycemia can enhance DPP-4 activity in renal endothelial cells in a dose-dependent manner. Consistent with these findings, we observed that DPP-4 activity was observed increasing and then reduced by linagliptin in HRGECs exposed to HG in our study. Several previous experiments demonstrated that vildagliptin significantly attenuated proteinuria in a diabetic model of the rat by improving the dysfunction of glomerular filtration, which is associated with increased DPP-4 activity in the early stage[17, 22, 29–31]. Linagliptin can decrease the glomerular permeability through remodeling the filtration barrier and maintaining the integrity of the endothelial function, leading to the reduction in proteinuria[23, 32, 33]. Similar effects were reported in the diabetes clinical studies, which was found that linagliptin reductions the risk of albuminuria in patients with T2DM beyond their anti-hyperglycemic impact[34–36]. Consistent with these results, our data show that linagliptin has remarkably protective effects on the cellular barrier of endothelial cells that are challenged by HG. The action of linagliptin may be assessed by its ability to maintain the endothelial barrier’s integrity through TEER and prevent the leakage of albumin in an HG status. In conclusion, linagliptin has beneficial effects on barrier dysfunction of HRGECs by inhibiting DPP-4 activity, likely associated with a marked reduction in albuminuria under HG conditions.
Additionally, our study showed that linagliptin protects against F-actin cytoskeleton remodeling, which improves the hyperglycemia-mediated endothelial barrier dysfunction. MLC phosphorylation-dependent generation of F-actin stress fiber plays a significant role in increased endothelial permeability induced by HG. Several studies reported that in renal and on-renal endothelial cells, HG causes increases in transendothelial migration of albumin and THP-1 cells, which are associated with the phosphorylation of MLC and the activation of RhoA. DPP-4 inhibitor effectively prevented HG-induced apoptosis via the activation of AMPKα in endothelial cells[37, 38]. In the present study, we noted that linagliptin protected the endothelial barrier function by preventing MLC activation, accompanied by decreased Pa and the value of TEER in HRGECs exposed to HG. The inhibition RhoA/ROCK pathway by Y-27632 diminished the linagliptin-mediated endothelial barrier protective effect. Consistent with the previous reports, our data showed that linagliptin has a beneficial impact on the barrier function of HRGECs by inhibiting disruption of F-actin cytoskeleton rearrangement and RhoA/ROCK pathway activation.
Furthermore, the mechanism underlying the action of linagliptin in modulating RhoA activation requires further investigation. AMPK plays an essential in the regulation of metabolic homeostasis[26, 39]. Thus, it is considered a crucial target to prevent microvascular endothelium damage in diabetes. Recently, some studies have demonstrated that DPP-4 inhibitor prevents endothelial cell dysfunction in diabetic conditions by activating the AMPK pathway[40, 41]. Omarigliptin, (a DPP-4 inhibitor), improved the inflammation induced by HG in glomerular endothelial cells by inhibiting the AMPK-mediated activation of the NLRP3 inflammasome. Our findings emphasized that activation of AMPK by linagliptin inhibited HG-induced RhoA activation, resulting in a further inhibition increased phosphorylation of MLC and hyperpermeability in cultured HRGECs. And then, inhibition of AMPK with compound C abolished this effect. Consistent with our finding, Maria S currently showed that the activation of AMPKα and downregulation of Rho-family small GTPase was necessary for the protective effect of metformin on HG-dependent motivation of the cytoskeletal contraction in the podocytes. Thus, the results from our study suggest that RhoA/ROCK function regulated by AMPK activation is crucial for linagliptin-mediated protective effect on glomerular endothelial hyperpermeability induced by HG.