Leptin induces TGF-beta synthesis through functional leptin receptor expressed by human peritoneal mesothelial cell

Marked increase in leptin concentration in spent peritoneal dialysate has been reported following continuous ambulatory peritoneal dialysis treatment. The present study was designed to determine whether functional leptin receptor is expressed by human peritoneal mesothelial cells and if so, the possible implication in dialysis. Expression of leptin receptors in cultured mesothelial cells and omental tissue was examined. The effect of leptin on the production of transforming growth factor-beta (TGF-beta) by mesothelial cells in the presence or absence of high glucose was determined using in vitro culture model of human peritoneal mesothelial cells and adipocytes. The signaling mechanism involved in leptin-induced TGF-beta synthesis by mesothelial cells was studied. Both mRNA and protein of the full-length leptin receptor are constitutively expressed in mesothelial cells. The leptin receptor expression in mesothelial cells was upregulated by glucose but not leptin. In adipocytes, glucose increased the mRNA expression and synthesis of leptin. The Janus kinase-signal transducers and activation (JAK-STAT) signal transduction pathway in mesothelial cells was activated by either exogenous or adipocytes-derived leptin. Exogenous leptin induced the release of TGF-beta by mesothelial cells. The TGF-beta synthesis induced by leptin was amplified by glucose through increased leptin receptor expression. Our novel findings reveal that functional leptin receptor is present on human peritoneal mesothelial cells. The leptin-induced TGF-beta synthesis in mesothelial cells is associated with the expression of leptin receptor and the activation of the JAK-STAT signal transduction pathway.     

The TGF-beta-induced gene product, betaig-h3: its biological implications in peritoneal dialysis.

BACKGROUND: TGF-beta is involved in peritoneal changes during long-term peritoneal dialysis (PD). TGF-beta induces betaig-h3 in several cell lines, and betaig-h3 may be a marker for biologically active TGF-beta. However, no study has reported induction of betaig-h3 in human peritoneal mesothelial cells (HPMCs) or its involvement in PD-related peritoneal membrane changes. METHODS: We used cultured HPMCs to investigate the biological roles of betaig-h3 during mesothelial cell injury and repair, employing the adhesion, spreading, scratching and cell migration assays. Changes in betaig-h3 expression after high glucose exposure in vivo were also evaluated using an animal chronic PD model. RESULTS: In vitro, TGF-beta1 induced betaig-h3 in cultured HPMCs, and betaig-h3-mediated mesothelial cell adhesion occurred via alphavbeta3 integrin. betaig-h3 enhanced mesothelial cell adhesion and migration and, in part, wound healing during mesothelial cell injury. The animal study demonstrated that compared to the control group, betaig-h3 concentrations in the dialysate effluent increased in the dialysis group with alterations in peritoneal structure and function during PD, and betaig-h3 positively correlated with peritoneal solute transport. Immunohistochemical and immunoblotting results showed that betaig-h3 localizes in the mesothelium and submesothelial matrix of the parietal peritoneum, and in the vascular endothelium of omentum. betaig-h3 protein expression was higher in the dialysis group. CONCLUSION: In vitro, betaig-h3 induced by TGF-beta1 in HPMCs improved adhesion and migration of HPMCs during wound healing. In the chronic infusion model of PD, betaig-h3 played a role in the functional deterioration of the peritoneal membrane, which is associated with fibrosis.     

Involvement of TGF-beta signal for peritoneal sclerosing in continuous ambulatory peritoneal dialysis

BACKGROUND: Functional failure of the peritoneal membrane is the most serious problem in long-term continuous ambulatory peritoneal dialysis (CAPD). Transforming growth factor-beta (TGF-ss) is one of the key mediators of fibrosis in some organs, and is thought to be involved in peritoneal alterations. In this study, we examined the role of TGF-beta1/TGF-ss receptors for human peritoneal mesothelial cells (HPMCs) and fibroblasts, and their interactions in CAPD patients. METHODS: HPMCs were cultured for 48 h in a medium containing normal- dose glucose (7 mM), high-dose glucose (30 mM) and mannitol as an osmotic agent, equal to 30 mM glucose. Cell proliferation was observed using the Tetra Color One assay. The concentration of TGF-beta1 in culture supernatants was measured by enzyme-linked immunosorbent assay (ELISA). The expression of TGF-ss receptor types I and II was observed by flow cytometry. HPMCs and fibroblasts were co-cultured and assayed using transwell inserts in order to identify the effects of the high-concentration glucose solution. RESULTS: HPMC proliferation was inhibited by the high concentration of glucose but not by mannitol. The inhibition was abrogated by the neutralizing antibody for TGF-beta1. TGF-beta1 was induced by a high concentration of glucose but not by mannitol. The expression of both TGF-ss receptors was augmented in culture with the high concentration of glucose but not with mannitol. In the co-culture assay, the number of HPMCs was decreased and fibroblasts were significantly increased in culture with the high concentration of glucose. CONCLUSIONS: A high concentration of glucose induced a large amount of TGF-beta1 and enhanced the expression of TGF-ss receptors. HPMCs were sensitive to TGF-beta1 in response to a high concentration of glucose. These data suggest that TGF-beta1 from HPMCs exposed to a high concentration of glucose down-regulates the proliferation of HPMCs and accelerates peritoneal fibrosis.     

Dipyridamole inhibits TGF-beta-induced collagen gene expression in human peritoneal mesothelial cells

BACKGROUND: Peritoneal matrix accumulation is characteristic of peritoneal fibrosis (PF). Continuous ambulatory peritoneal dialysis (CAPD) patients who had persistent transforming growth factor-beta (TGF-beta) in their drained effluent had an increased risk of PF. We previously reported that TGF-beta stimulates the expression of types I and III collagen mRNA in cultured human peritoneal mesangial cells (HPMCs), which may predispose them to develop PF. Pharmacological interventions to attenuate TGF-beta-stimulated matrix accumulation in HPMC may have therapeutic potential for the treatment of PF. The SMAD family and the extracellular signal-regulated protein kinase (ERK1/2, p44/p42) pathways have been shown to participate in TGF-beta signaling. Our current study identified these signal pathways in HPMCs and investigated the molecular mechanisms involved in the inhibitory effects of dipyridamole on TGF-beta-induced collagen gene expression in HPMCs. METHODS: HPMCs were cultured from human omentum by an enzyme digestion METHOD: Expression of collagen alpha1(I) mRNA was determined by Northern blotting. The SMAD proteins and the ERK1/2 activity were determined by Western blotting. RESULTS: TGF-beta-stimulated collagen alpha1(I) mRNA expression of HPMC was inhibited by dipyridamole in a dose-dependent manner. Smad2 and ERK1/2 were activated in response to TGF-beta; however, TGF-beta had little effect on the protein expression of Smad4. The addition of PD98059, which blocked activation of ERK1/2, suppressed TGF-beta-induced collagen alpha1(I) mRNA expression in a dose-dependent manner. At a concentration that inhibited collagen gene expression (17 microg/mL), dipyridamole suppressed ERK1/2 activation by TGF-beta. In contrast, the same concentration of dipyridamole had no effect on TGF-beta-induced activation of Smad2. CONCLUSION: Dipyridamole inhibits TGF-beta-induced collagen gene expression in HPMC through modulation of the ERK pathway. Our study of dipyridamole may provide therapeutic basis for clinical applications in the prevention of PF.     

PI3K/Akt signaling regulates epithelial-mesenchymal transition of peritoneal mesothelial cells in peritoneal dialysis

Objective To investigate the role of PI3K/Akt signaling in the regulation of epithelial-mesenchymal transition (EMT) of peritoneal mesothelial cells (PMCs) in peritoneal dialysis in vitro and in vivo. Methods The level of Phosphorylated serine/threonine kinase Akt and the expression of EMT associated gene and protein, including ZO-1, Vimentin and FN, were measured in mice EMT model. In vitro study, phosphorylation level and nuclear translocation of Akt, ZO - 1 and Vimentin expression induced by TGF - β1 in human peritoneal mesothelial cells (HPMCs) were also observed. Moreover, HPMCs were pre-treated by one of PI3K/Akt inhibitor, LY294002, or transfected with dominant-negative Akt plasmid to inhibit PI3K/Akt signaling, then analyzed its effect on Zo-1 and Vimentin expression induced by TGF-β1. Results Compared with the control, thickened parietal peritoneum and remarkable decrease in mRNA and protein of the epithelial marker ZO-1, and notable increased in the expression of mesenchymal markers Vimentin and FN were observed in PD mice (all P＜0.01). Moreover, the phosphorylation of Akt also significantly increased under above condition (P＜ 0.01). In vitro study, with the stimulation of TGF-β1, the expression of Zo-1 was down-regulated, while the expression of Vimentin increased (all P＜0.01). In addition, TGF-β1 remarkably increased pAkt in HPMCs (all P＜0.01) in dose-dependent. However, LY294002 and DN-Akt dramatically inhibited the vimentin expression in HPMCs induced by TGF-β1 after inhibition of pAkt. On the other hand, the expression of ZO - 1 also was restored (P＜0.01). Conclusion PI3K/Akt signaling is involved in EMT of peritoneal mesothelial cells in peritoneal dialysis, and may be a new target for the prevention and treatment of peritoneal fibrosis during PD.     

p38信号通路对腹膜间皮细胞p27kip1和纤连蛋白的影响

目的 探讨p38有丝分裂素激活蛋白激酶(MAPK)对高糖诱导的人腹膜问皮细胞(HPMC)p27^kip1的表达和纤连蛋白(FN)分泌的影响。方法 同步化生长融合的HPMC在有或无p38 MAPK抑制剂SB203580的条件下和不同浓度的葡萄糖共同孵育。采用Bradford法测定细胞内总蛋白量。采用逆转录．聚合酶链反应(RT．PCR)方法检测p27^kip1 mRNA的表达。p27^kip1和p38 MAPK蛋白用Western印迹法测定。采用酶联免疫吸附试验(ELISA)测定细胞培养液FN水平。结果 高糖刺激HPMC时细胞内总蛋白量、p27^kip1蛋白及细胞培养液里的FN蛋白水平明显升高。抑制p38 MAPK活性可有效阻断高糖介导的腹膜间皮细胞p27^kip1的表达及FN的分泌。结论 高糖通过p38 MAPK的活化可上调HPMC p27^kip1的表达和FN的分泌。     

Effect of lactate and bicarbonate on human peritoneal mesothelial cells, fibroblasts and vascular endothelial cells, and the role of basic fibroblast growth factor.

BACKGROUND: In patients on long-term continuous ambulatory peritoneal dialysis (CAPD), peritoneal dysfunction may occur due to loss of peritoneal mesothelial cells, peritoneal fibrosis and neovascularization. Lactate, long used as a buffer in peritoneal dialysates, has been substituted by bicarbonate in recent years. However, their effects on the peritoneum of CAPD patients are unknown. This study investigated the influence of lactate and bicarbonate on peritoneal dysfunction in CAPD patients. METHODS: The mitochondrial activity of human peritoneal mesothelial cells (HPMCs) and their expression of basic fibroblast growth factor (bFGF) were studied after culture under various conditions. We also assessed the mitochondrial-activating effect of the supernatant of those cultures on human peritoneal fibroblasts (HPFBs) and human umbilical vein endothelial cells (HUVECs) and the effect of recombinant human bFGF on the mitochondrial activity of HPFBs and HUVECs. We used the WST-1 assay to determine mitochondrial activity in HPMC. RESULTS: At pH 7.4, the mitochondrial activity of HPMCs was lowest in a medium containing 40 mM (Lac), intermediate in a lactate (15 mM) plus bicarbonate (25 mM) medium (Lac/Bic), and highest in a 40 mM bicarbonate medium (Bic). In culture supernatant, the increase of bFGF was: Lac > Lac/Bic > Bic. Mitochondrial activation of HPFBs and HUVECs was stimulated by HPMC culture supernatants in the following decreasing order: Lac > Lac/Bic > Bic. The effects of these supernatants were suppressed by a bFGF-neutralizing antibody, while recombinant bFGF caused concentration-dependent mitochondrial activation in HPFBs and HUVECs. CONCLUSIONS: The role of bFGF in peritoneal fibrosis and neovascularization may be important. A bicarbonate-containing medium is better than a lactate-containing medium for preserving cell viability in HPMCs and preventing bFGF expression by these cells.     

Expression of Na＋-dependent glucose transporter 1 and 2 in peritoneum of peritoneal dialysis patients

Objective To investigate the expression of Na＋-dependent glucose transporter (SGLT) in human peritoneal mesothelial cells (HPMCs) and vascular endothelial cells in peritoneal tissues of peritoneal dialysis (PD) patients at different dialysis vintages, and to study the influence of high glucose treatment on the expression of SGLT1 and SGLT2 in primary HPMCs. Methods According to the dialysis vintage, PD patients were divided into four groups: 0 year group, ＞0-2 years group, ＞2-4 years group and＞4 years group. HE and Masson staining were used to observe the morphologic changes of peritoneal tissues in PD patients. Immunohistochemical staining was used to detect the expression of SGLT1 and SGLT2 in peritoneal HPMCs and vascular endothelial cells. The primary HPMCs were extracted from the peritoneal dialysis fluid, and treated with high-glucose or high-mannitol for 0 h, 12 h, 24 h, 48 h, 72 h and 96 h. Western blotting was used to investigate the SGLT1 and SGLT 2 expression in HPMCs. The cell viability was detected by using cell counting kit (CCK-8). Results HE and Masson staining showed that the peritoneum of PD patients in 0 year group was smooth and continuous, with a flat layer of HPMCs. The number of HPMCs in＞0-2 years group decreased compared with that in 0 year group. The HPMCs size increased in＞2-4 years group, but the number decreased. The peritoneum of PD patients in＞4 years group was significantly thickened and fibrotic, and HPMCs almost disappeared. Immunohistochemical staining showed that the expression of SGLT1 and SGLT2 in HPMCs gradually decreased with the increase of dialysis vintage (P＜0.05). The wall of peritoneal blood vessel became thicken, but the expression of SGLT1 and SGLT2 was not statistically different among four groups (P＞0.05). SGLT1 in primary HPMCs could be up-regulated (0 h, 12 h and 24 h), and then down-regulated (24 h, 48 h, 72 h, 96 h) with the treatment of 60 mmol/L glucose (P=0.029); but there was no significant difference of SGLT2. Conclusion High glucose and the increase of dialysis vintage can reduce the number and the viability of HPMCs, and decrease the expression of SGLT1 and SGLT2, but there was no significant influence on SGLT1 and SGLT2 in peritoneal vascular endothelial cells.     

Heme oxygenase-1 attenuates epithelial-to-mesenchymal transition of human peritoneal mesothelial cells

Background

Epithelial-to-mesenchymal transition (EMT) of peritoneal mesothelial cells has been regarded as an early mechanism of peritoneal fibrosis. A substantial and rapidly growing literature indicates that HO-1 provides the provenance for pathways that can interrupt virtually all major mechanisms of tissue injury. The effects of HO-1 expression on EMT, which plays a critical role in the development of peritoneal membrane (PM) fibrosis, are unknown and its roles in peritoneal fibrosis has not been studied, yet.

Methods

A piece of human omentum obtained from consenting patients undergoing elective abdominal surgery was used for study. We treated the human peritoneal mesothelial cells (HPMCs) with high glucose solution and HO-1 inducer (hemin, 10 μmol/L). To further investigate the pure effect of HO-1 on EMT of mesothelium, gene transfer of recombinant Adenovirus-harboring human HO-1 (Adv-HO-1 gene) to HPMCs was done.

Results

Exposure of HPMCs to HG solution resulted in an increase of the expression of mesenchymal markers such as α-smooth muscle actin (α-SMA) and was associated with a decrease in the expression of epithelial markers, E-cadherin. HO-1 protein expression was decreased in the same situation. Treatment of HPMCs with HO-1 inducer, hemin showed a dosage-dependent amelioration of HG induced changes in markers of EMT with increase of expression of HO-1. Human HO-1 gene transfection resulted in a significant increase in HO-1 expression and ameliorated HG-induced changes in expression of E-cadherin and α-SMA.

Conclusion

Taken together, our results suggest that HO-1 has a critical role in the modulation of peritoneal fibrosis, and, more important, the suppression of EMT. This study is the first to show the beneficial effect of HO-1 on reversing EMT in MC.     

Hepatocyte growth factor prevents peritoneal fibrosis in an animal model of encapsulating peritoneal sclerosis

BACKGROUND: Ultrafiltration failure associated with peritoneal fibrosis can lead patients to discontinue continuous ambulatory peritoneal dialysis (CAPD). It has been reported that the reciprocal imbalance between transforming growth factor-beta1 (TGF-beta1) and hepatocyte growth factor (HGF) is closely involved in the progression of tissue fibrosis. We previously showed that exogenous HGF restores the growth of human peritoneal mesothelial cells suppressed by a high concentration of D-glucose or TGF-beta1. In this study, we examined whether constitutive exposure to HGF prevents peritoneal fibrosis in an animal model of encapsulating peritoneal sclerosis (EPS). METHODS: To establish the model, a daily intraperitoneal injection of 0.1% chlorhexidine gluconate was given to male Wister rats for 35 days. Rat peritoneal mesothelial cells (RPMCs) transfected with full-length human HGF cDNA in an expression vector (pUCSRalpha/HGF) were injected into the peritoneal cavity of the rats. Thereafter, pathological changes to the peritoneal membrane were observed, and the effect on peritoneal ultrafiltration volume was examined. RESULTS: In the model, microscopic examination revealed a progressive thickening of the submesothelial layer, and an increase in the number of capillary vessels. Peritoneal ultrafiltration volume was decreased. Interestingly, the pathological changes to the peritoneal membrane were reversed by the intraperitoneal injection of pUCSRalpha/HGF-transfected RPMCs. Furthermore, peritoneal ultrafiltration volume was increased. CONCLUSIONS: The constitutive production of HGF by UCSRalpha/HGF-transfected RPMCs can improve peritoneal fibrosis resulting in an increase in peritoneal ultrafiltration volume. This approach may have clinical application.     

Myofibroblast transdifferentiation of mesothelial cells is mediated by RAGE and contributes to peritoneal fibrosis in uraemia.

BACKGROUND: Uraemia is associated with fibrosis of the peritoneal membrane, even prior to the start of peritoneal dialysis. Increased carbonyl stress and the resultant formation of advanced glycation end-products (AGEs) are potentially involved. The interaction of AGEs with their cell surface receptor for AGE (RAGE) induces sustained cellular activation, including the production of the fibrogenic growth factor-beta (TGF-beta). TGF-beta is pivotal in the process of epithelial-to-mesenchymal transition with the acquisition of myofibroblast characteristics. We investigated whether antagonism of RAGE prevents uraemia-induced peritoneal fibrosis. In addition, we examined whether myofibroblast transdifferentiation of mesothelial cells contributes to peritoneal fibrosis in uraemia. METHODS: Uraemia was induced in rats by subtotal nephrectomy. Uraemic and age-matched sham-operated rats were treated for 6 weeks with neutralizing monoclonal anti-RAGE antibodies or placebo. Expression of AGE, RAGE, cytokeratin and alpha-smooth muscle actin was evaluated using immunohistochemistry. TGF-beta expression was examined with immunostaining and western blotting, and Snail expression with western blotting. Fibrosis was quantified with a picro-sirius red staining and measurement of the hydroxyproline content of the tissue. RESULTS: Uraemia resulted in the accumulation of AGE, up-regulation of RAGE and TGF-beta and the development of interstitial fibrosis and vascular sclerosis in the peritoneal membrane. Prominent myofibroblast transdifferentiation of mesothelial cells was identified by colocalization of cytokeratin and alpha-smooth muscle actin in submesothelial and interstitial fibrotic tissue. The antagonism of RAGE prevented the up-regulation of TGF-beta, epithelial-to-mesenchymal transition of mesothelial cells and fibrosis in uraemia. CONCLUSION: The ligand engagement of RAGE and the subsequent up-regulation of TGF-beta induces peritoneal fibrosis in chronic uraemia. The process may be mediated by the conversion of mesothelial cells into myofibroblasts.     

Antibiotics induce apoptosis of human peritoneal mesothelial cells

SUMMARY:   The peritoneal mesothelial cell is a critical component of the peritoneal membrane. The intraperitoneal use of several antibiotics to treat bacterial peritonitis is current clinical practice. Our previous study showed that cephalothin (CPL) and cefotaxime (CFT) have cytotoxic effects on human peritoneal mesothelial cells (HPMC), however, the exact mechanism of cytotoxicity has not been elucidated. In the present study, flow cytometry, TdT-mediated dUTP nick-end labelling (TUNEL) staining and electron microscopy were used to detect the apoptosis of HPMCs. Immunofluorescent staining was used to evaluate the cytochrome c distribution pattern. Western blotting was used to assess apoptotic signalling proteins. We found that CPL (0.5 mg/mL) and CFT (1 mg/mL) induced apoptosis of HPMCs, whereas cefazolin (0.5 mg/mL) and ceftriaxone (0.5 mg/mL) failed to induce apoptosis of HPMCs. While the DNA content of CFT- or CPL-treated cells was reduced, as determined by flow cytometry, cefazolin and ceftriaxone had no such effect. The CFT- or CPL-treated cells displayed the features of apoptosis both under the electron microscope and by using TUNEL staining. However, cefazolin and ceftriaxone produced the same result as the medium controls. Furthermore, CFT and CPL increased the expression of Bax and p53, and caused the translocation of cytochrome c from the mitochondria to the cytoplasm. The HPMC treated by CFT but not by CPL induced the cleavage of procaspase-3 to form active caspase-3. In conclusion, cefotaxime and cephalothin induce apoptosis of HPMCs in vitro . Signal transduction may be through the mitochondrial pathway.     

线粒体呼吸链在高糖腹膜透析液诱导人腹膜间皮细胞层高通透性中的作用

目的 研究线粒体呼吸链在高糖腹膜透析液（PDS）诱导人腹膜间皮细胞（HPMC）通透性升高中的作用,探讨高糖PDS导致腹膜高通透状态的分子机制。 方法 体外培养HPMC,用不同浓度葡萄糖PDS加或不加抗氧化剂谷胱甘肽,培养24 h。采用跨细胞电阻（TER）技术测定HPMC通透性的变化;免疫荧光及Western印迹观察claudin-1的表达;线粒体活性氧（ROS）荧光探针检测细胞线粒体ROS的生成;线粒体呼吸链酶复合物活性检测试剂盒检测复合物Ⅰ~Ⅳ的活性变化。 结果 各组细胞的TER值均随时间的延长而下降,4.25% PDS组TER值24 h内从（34.7±1.5） Ω&#8226;cm2下降至（4.3±1.4） Ω&#8226;cm2;高糖PDS使细胞间连接蛋白claudin-1的表达显著下调,葡萄糖浓度越高作用越明显;同时高糖PDS导致线粒体呼吸链酶复合物Ⅲ活性较对照组下降89.2%（P < 0.01）,进而使线粒体ROS产生增加。谷胱甘肽可逆转上述指标的变化。 结论 高浓度葡萄糖PDS通过抑制线粒体呼吸链酶复合物Ⅲ的活性,引起HPMC的氧化损伤,进而导致腹膜间皮细胞层的高通透性增高。