Effects of Rapamycin on the Epithelial-to-mesenchymal Transition of Human Peritoneal Mesothelial Cells

Abstract

The preservation of the peritoneal membrane is crucial for long-term survival in peritoneal dialysis. Epithelial-to-mesenchymal transition (EMT) is a process demonstrated in mesothelial cells (MC), responsible for negative peritoneal changes and directly related to PD. EMT enables neovascularization and fibrogenic capabilities in MC. Vascular endothelial growth factor (VEGF) is the mediator for neo-vascularization. Rapamycin is a potent immunosuppressor with antifibrotic action in renal allografts and has a demonstrated anti-VEGF effect. We performed this study with the hypothesis that rapamycin may regulate the EMT of MC. MC from human omentum were cultured. When mesothelial cells reached confluence, some of them were stimulated with r-TGF-ß (1 ng/mL) to induce EMT, co-administered with rapamycin (0.2, 2, 4, 20 and 40 nM). Other groups of cells received similar doses of rapamycin or r-TGF-ß, separately. Cells were analyzed at 6, 24, 48 hours and 7 days. As markers of EMT we included α-SMA, E-cadherin and snail nuclear factor by quantitative RT-PCR. EMT markers and regulators demonstrated the following changes with rapamycin: E-cadherin (a protective gene for EMT) increased 2.5-fold relative to controls under 40 nM, at 24h. Importantly, rapamycin inhibited snail expression induced by TGF-ß at 6h, whereas TGF-ß increased snail 10fold. At day 7, rapamycin showed no anti-EMT properties. An important decrease in α-SMA expression by MC after rapamycin addition was observed. In conclusion, rapamycin shows a mild protective effect on EMT, as it increases E-cadherin and decreases α-SMA expression. Consequently, rapamycin might partially regulate the epithelial-to-mesenchymal transition of mesothelial cells.

Rapamycin inhibits epithelial‐to‐mesenchymal transition of peritoneal mesothelium cells through regulation of Rho GTPases

“…We specifically analyzed the E-cadherin, a-SMA, fibronectin, and collagen I expressions, which are the representative markers implicated in EMT. A little different from Aguilera's study [24], rapamycin showed a remarkable protective effect on HG-induced EMT of RPMCs, as it increased E-cadherin expression and decreased a-SMA, fibronectin, and collagen I expressions. The acquisition of migratory capacity is another typical characteristic of cells undergoing EMT [13].…”

“…The concentration of HG and rapamycin were used according to our premier data and previous studies [9,24,35]. As shown in Fig.…”

“…Briefly, 25 000 cells/well were plated in a 96-well plate with 10% FBS media for 24 h before being exposed to rapamycin. RPMCs were then treated with different doses of rapamycin (10, 25, 50, 100 nM) for different time point (12,24,48, 72 h). After treatment, 10 lL of CCK-8 solution was added to each well and incubated for 2 h. Absorbance was measured at 450 nm.…”

“…It has been proven to have antifibrotic effects in a number of tissues, such as the kidney [18,19], liver [20], and skin [21]. In the peritoneum, recent studies have reported that rapamycin is also effective in suppressing the EMT of PMCs and preventing peritoneal fibrosis [22][23][24][25]. However, the underlying molecular mechanisms remain poorly understood.…”

Effects of Mycophenolate Mofetil and Rapamycin on Peritoneal Fibrosis in an Experimental Model of Peritoneal Dialysis

“…Previous research in this field, using animal models, has examined the efficacy of treatment with ACE (angiotensinconverting enzyme) inhibitors, angiotensin receptor blockers (ARBs), octreotide and corticosteroids on peritoneal alterations that are induced by hypertonic PD solutions (36)(37)(38). There are very limited studies, no matter experimental or clinical, that attempt to investigate and report the effects of MMF and RAP in fibrotic diseases (25,26,30,39,40). Besides, previous work in this field has so managed to produce little direct evidence that would suggest additional biocompatible dialysis solutions can preserve peritoneal function.…”