| Objectives: To investigate the effect of dialysate interleukin-6(IL-6)on the alteration of peritoneal solute transport rate(PSTR)and development of peritonitis in continuous ambulatory peritoneal dialysis(CAPD)patients and to explore the mechanisms and signaling pathways of IL-6 involved in peritoneal structural and functional changes.Methods: 1.Stable CAPD patients were enrolled in this prospective study.IL-6,Angpt-1,Angpt-2,s Tie-2 and VEGF concentrations in the overnight effluent was determined and expressed as the appearance rate(AR).The study tried to examine the association between IL-6 AR and change in MTACcr as well as the risk of peritonitis;2.Human peritoneal mesothelial cells(HPMCs)were separated from the drained dialysate fluid of incident PD patients.When HPMCs were incubated in high glucose and mannitol,protein secretion of IL-6 in the supernatant was determined by enzyme-linked immunosorbent assay(ELISA).HPMCs were stimulated by IL-6/s IL-6R and protein expression of VEGF and Angpt-2 were evaluated by Western blotting.HUVECs were also stimulated by IL-6/s IL-6R and protein expression of Angpt-1 as well as VE-cadherin,Occludin,Claudin-5 in HUVECs were examined by Western blotting.A co-culture system of HPMCs and HUVECs was built using Transwell closet.The co-culture system was stimulated by IL-6/s IL-6R to detect the protein secreting of Angpt-1,Angpt-2 and VEGF in the supernatant and the protein expression of VE-cadherin,occludin,claudin-5 in HUVECs.The matrigel was used to evaluated the ability of vessel formation.Both HPMCs and HUVECs were incubated with IL-6/s IL-6R or STAT3 specific inhibitor S3I-201 or sgp130.Phosphorylated STAT3 were examined by Western blotting.The HPMCs were incubated with100ng/m L IL-6/s IL-6R or STAT3 specific inhibitor S3I-201 or sgp130 or STAT3 si RNA or transforming growth factor-β1(TGF-β1)specific inhibitor SB431542,then the expression of EMT related protein was examined.The HPMCs were incubated with IL-6/s IL-6R for different time and phosphprylated Smad3 expression were detected.3.Intraperitoneal injection of mice with 4.25% glucose based PDF(2ml/one/day)and sgp130(250 ng/one/day)for 28 days,parietal peritoneum morphology changes were detected and protein expression of α-SMA,collagen I and phosphorylation STAT3 and the expression of CD31 and F4/80 in the parietal peritoneum were detected.After intraperitoneal injection of 4.25% glucose based PDF to mice for 28 days,mice were gave another intraperitoneal injection of sgp130(250ng/one/day)for 14 days.Then the expression of EMT related protein was examined.Results: A total of 326 patients were analyzed in the study.,MTACcr was significantly increased during the 3-year follow-up(P < 0.05).IL-6 AR was significantly positively correlated with 1-year △MTACcr(R = 0.164,P = 0.008)and2-year △MTACcr(R = 0.173,P = 0.014),but not significantly correlated with 3-year△MTACcr(P > 0.05).There was a significant positive correlation between IL-6 AR and Angpt-1AR(R = 0.274,P < 0.001),Angpt-2 AR(R = 0.282,P < 0.001),s Tie2 AR(R = 0.247,P < 0.001)and VEGF AR(R = 0.358,P < 0.001).Multivariate Cox regression showed that high dialysate IL-6AR(hazard ratio [HR] 1.247 [1.052–1.478];p = 0.011)and high serum C-reactive protein(HR 1.072 [1.005–1.144];p = 0.036)were independent risk factors for inferior peritonitis-free survival.1.5%,2.5% and4.25% glucose can up-regulate the IL-6 concentration in the supernatant of HPMCs(208.58 ± 37.84 vs 307.83 ± 65.86,388.88 ± 107.02,660.07 ± 135.2pg /m L,all P <0.05).IL-6/s IL-6R significantly reduced the expression of Angpt-1(0.56 ± 0.07 vs 1.0± 0.41,P < 0.05)and induced the expression of VEGF(3.09 ± 1.82 vs 1.0 ± 0,P <0.05)in HPMCs.IL-6/s IL-6R significantly increased the expression of Angpt-2(3.71± 0.89 vs 1.0 ± 0.26,P < 0.05)and unpregulated the expression of Claudin-5(0.82 ±0.05 vs 1.08 ± 0.24,P < 0.05),VE-cadherin(0.54 ± 0.36 vs 0.91 ± 0.49,P < 0.05)and Occludin(0.63 ± 0.04 vs 1.0 ± 0.17,P < 0.05)in HUVECs.In the co-culture system,Angpt-1 levels in the supernatant were significantly down-regulated by IL-6/s IL-6R(0.40 ± 0.17 vs 1.74 ± 0.6 pg/ m L,P < 0.05)and Angpt-2(99.52 ± 15.47 vs 59.23 ±17.2 pg/ m L)and VEGF(2250.9 ± 154.51 vs 1364.2± 102.4 pg/ml,P < 0.05)levels were significantly up-regulated.In the co-culture system,the expression of claudin-5(0.55 ± 0.27 vs 1.0 ± 0.4,P < 0.05),VE-cadherin protein(0.51 ± 0.12 vs 1.0 ± 0.32,P< 0.05)and Occludin protein(0.63 ± 0.29 vs 1.0 ± 0.13,P < 0.05)could be significantly down-regulated incubated with IL-6/s IL-6R.IL-6/s IL-6R could significantly increase the permeability of FITC-Detran40 and the endothelial tube formation of HUVECs(P < 0.05).IL-6/s IL-6R significantly increased the level of phosphorylated STAT3 in HPMCs(8.59 ± 3.37 vs 1.0 ± 0.45,P < 0.05)and S3I-201 significantly inhibited the phosphorylation of STAT3 in HPMCs induced IL-6/s IL6 R complex(2.18 ± 0.74 vs 3.73 ± 1.37,P < 0.05).IL-6/s IL-6R significantly increased the level of phosphorylated STAT3 in HUVECs(7.92 ± 2.23 vs 1.16 ± 1.57,P < 0.05).sgp130 and S3I-201 significantly inhibited the phosphorylation of STAT3 in HUVECs induced by the IL-6/s IL-6R complex.IL-6/s IL-6R stimulation could transform HPMCs from typical "paving stone" appearance to "spindle" appearance.IL-6/s IL-6R significantly down-regulated the expression of E-cadherin(0.16 ± 0.18 vs 1.0 ± 0.63,P < 0.05)and significantly up-regulated the expression of α-SMA(2.3 ± 0.31 vs 1.04± 0.38,P < 0.05),Fibronectin(3.44 ± 0.5 vs 1.07 ± 0.12,P < 0.05)and Collagen I(3.32 ± 0.48 vs.1.0±0.88,P < 0.05).sgp130 and S3I-201 and STAT3 si RNA can significantly inhibit the effect of IL-6/s IL-6R on EMT related protein expression of HPMCs.IL-6/s IL-6R significantly up-regulated the level of phosphorylated Smad3 in HPMCs and the peak time was 1 hour.SB431542 can significantly inhibit the effect of IL-6/s IL-6R on expression of EMT related protein in HPMCs(P all < 0.05).3.4.25% dialysate significantly promoted the peritoneal fibrosis in mice,while sgp130 intervention significantly reduced the peritoneal fibrosis.4.25% high glucose based dialysate can significantly increase the expression of Collagen I(23.73 ± 5.9 vs.1.0 ±0.7,P < 0.05)and α-SMA(16.33 ± 2.92 vs.1.0 ± 0.5,P < 0.05)and phosphorylated STAT3(2.98 ± 1.07 vs 1.0 ± 0.41,P < 0.05)in the parietal peritonium,while sgp130 intervention can significantly inhibit the expression of these protein.Treatment of sgp130 can significantly reduced the expression of Collagen I and α-SMA protein and phosphorylated STAT3(P all < 0.05).4.25% high glucose based dialysate significantly increased the positive expression of CD31 and F4/80 in the peritoneum,while sgp130 intervention significantly inhibited the expression of CD31 and F4/80 in the peritoneum and significantly increased the vascular permeability of Evans blue dye.Conclusions: Intraperitoneal IL-6 level was significantly correlated with the long-term change of peritoneal solute transport function,which may depend on its critical role in regulating the expression of angiogenic cytokines in the local cavity.Dialysate IL-6 level,might be also a potential predictor of peritonitis in patients undergoing PD.2.The IL-6 trans-signaling pathway can promote the secretion of angiogenic cytokines in HPMCs and HUVECs and is directly involved in the mesenchymal transformation of peritoneal mesothelial cells so as to participate in the process of angiogenesis and fibrosis in peritonium.3.Interleukin-6 signaling signaling pathway plays a role in peritoneal fibrosis,angiogenesis and inflammation of mice induced by high glucose based dialysate. |
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