摘要
背景和目的:内皮素-1(ET-1)是一种小分子生物活性物质,除了具有很强的收缩血管作用外,还可以促进细胞外基质的合成,在多种器官的纤维化中发挥重要的作用。ET-1的受体有两种,分别为内皮素受体A(ETAR)、内皮素受体B(ETBR),ET-1是通过和其受体的结合而发挥作用的。本实验通过建立模拟持续性非卧床式腹膜透析(CAPD)大鼠模型,测定不同浓度腹膜透析液组大鼠腹膜ET-1、ETAR、ETBR、以及纤维连接蛋白(FN)的表达水平,分析ET-1、ETAR、ETBR与FN的相关性,探讨CAPD相关性腹膜纤维化的发生机制。
方法:S-D雄性大鼠40只(体重280g～350g),按体重分层随机分为四组,每组10只。正常对照组、1.5%腹膜透析液组、2.5%腹膜透析液组、4.25%腹膜透析液组。除正常对照组外分别给予大鼠腹腔注射不同浓度的腹透液,20ml/d,连续四周,第29天注射20ml入腹腔保留4小时后处死大鼠。免疫组化法检测壁层腹膜ET-1、ETAR、ETBR、FN的表达。
结果:
1.各腹膜透析液组ET-1、ETAR、ETBR、FN的表达水平与正常对照组相比较,均具有显著性差异(P＜0.05)。4.25%腹膜透析液组ET-1、ETAR、ETBR、FN的表达水平最高,2.5%腹膜透析液组又高于1.5%腹膜透析液组;
2.4.25%腹膜透析液组ET-1、ETAR、ETBR的表达水平和1.5%、2.5%腹膜透析液组相比,均具有显著性差异(P＜0.05),2.5%腹膜透析液组ET-1、ETAR、ETBR的表达水平和1.5%腹膜透析液组相比,均无显著性差异(P＞0.05);
3.1.5%、2.5%、4.25%腹膜透析液组FN的表达水平相比均具有显著性差异(P＜0.05)。
4.大鼠腹膜间皮细胞ET-1、ETAR、ETBR的表达和腹膜FN的表达呈正相关(r=0.960,p＜0.01;r=0.908,p＜0.01;r=0.934,p＜0.01)。
结论:
1.腹透液可以导致大鼠腹膜问皮细胞ET-1、ETAR、ETBR、以及腹膜FN表达水平的升高,且随着葡萄糖浓度的升高而升高。
2.腹膜FN的水平和腹膜间皮细胞ET-1、ETAR、ETBR的水平呈正相关,提示FN的堆积和ET-1、ETAR、ETBR的升高有关,故推断ET-1、ETAR、ETBR的升高可能是CAPD相关性腹膜纤维化的原因之一。
3.高糖引起ET-1、ETAR、ETBR的升高是高糖导致FN升高的原因之一。
Objections:Endothelin -1(ET-1),a 21-amino peptide hormone,has potent vasoconstrictor activity which also induces production of extracellular matrix proteins and involves in fibrotic processes of numerous organs.These ET-1 effects are elicited through the activation of two receptor subtypes,ETAR and ETBR.In the present study,we detected the expressions of ET-1,ETAR,ETBR and FN in peritoneum treated with different concentrations of PDF through the rat CAPD models,and then to investigate the effect of PDF on ET-1,ETAR,ETBR,FIg and analyze the relationship of ET-1,ETAR,ETBR and FN in non-infection situations to gain a better understanding of the mechanism of CAPD related peritoneal fibrosis.
Methods:Experiments were performed on 40 adult male Sprague-Dawley rats weighing between 280～350g.Rats were randomized into 4 groups randomly and received daily intraperitoneal injection of 20ml 1.5%,2.5%,4.25%PDF into the respective group for 4 weeks except the control group,on the 29th day,after a 4-hour dwell,the rats were put to death,parietal peritoneum tissue were taken to determine the ET-1,ETAR,ETBR and FN protein expression by immunohistochemistry.
Results:
1.The expression of ET-1,ETAR,ETBR and FN in experimental groups was higher than that of control group,and the differences were significant.The ET-1, ETAR,ETBR and FN protein expression in 4.25%group were the highest of the experimental groups,and the values of 2.5%group were higher than that of 1.5% group.
2.The ET-1,ETAR and ETBR protein in 4.25%group were higher than that of 1.5%group and 2.5%group,and the differences between 4.25%group with 2.5%group,4.25%group with 1.5%group were statistically significant(P＜0.05), but the differences between 2.5%group with 1.5%group had no statistic significance(P＞0.05).
3.The FN protein expression in 4.25%group were higher than that of 2.5%group, and the values of 2.5%group were higher than that of 1.5%group,and the differences of the 3 groups had statistic significance(P＜0.05).
4.There were positive correlations between expression of ET-lor ETAR or ETBR and FN(r=0.960,p＜0.01;r=0.908,p＜0.01;r=0.934,p＜0.01).
Conclusion:
1.PDF could induced the increase of ET-1,ETAR,ETBR and FN protein expression of CAPD rat peritoneum,and the increase followed by the increase of glucose concentrations of PDF.
2.Accumulation of FN was associated with the increase expression of ET-1, ETAR and ETBR,so we can conclude that the increase expression of ET-1, ETAR and ETBR may be one of the reasons for CAPD relative peritoneal fibrosis.
3.The increased expression of ET-1,ETAR and ETBR induced by glucose may be one of the reasons that glucose induce FN incresing.
引文
[1]Oreopoulos DG,Lobbedez T,Gupta S.Peritoneal dialysis:where is it now and where is it going?[J].Int J Artif Organs,2004,27(2):88-94.
[2]Honda K,Nitta K,Horxta S,et al.Morphological changes in the peritoneal vasculature of patients on capd with ultrafiltration failure[J].Nephron,1996,72(2):171-176.
[3]Coles GA,Topley N.Long-term peritoneal membrane changes[J].Adv Ren Replace Ther,2000,7(4)289-301.
[4]Shostak A,Pivnik K,Gotloib L.Daily short exposure of cultured mesothelial cells to lactated,high-glucose,low-pH peritoneal dialysis fluid induces a lowprofile regenerative steady state[J].Nephrol Dial Transplant,1996,11:608-613.
[5]Ito T,Yorioka N,Yamamoto M,et al.Effect of glucose on intercellular junctions of cultured human peritoneal mesothelial cells[J].J Am Soc Nephrol,2000,11:1969-1979.
[6]Leung JC,Chan LY,Li FF,et al.Glucose degradation products down-regulate ZO-1 expression in human peritoneal mesothelial cells:the role of VEGF[J].Nephrol Dial Transplant,2005,20(7):1336-1349.
[7]邵维斌,钱家麒,蒋季杰.高浓度葡萄糖对人HPMC c-myc和p21基因表达的影响.南通医学院学报,2002,22(2):142-144.
[8]Oh JH,Ha H,Yu MR,et al.Sequential effects of high glucose on mesangial cell transforming growth factor-beta 1 and fibronectin synthesis[J].Kidney Int,1998,54(6):1872-1878.
[9]Dobbie JW,Lloyd JK,Gall CA.Categorization of ultrastructural changes in peritoneal mesothelium,stroma and blood vessels in uremia and CAPD patients[J].Adv Petit Dial,1990,6:3-12.
[10]Ogata S,Yorioka N.Effect of prednisolone on increased expression of laminin by human peritoneal mesothelial cells cultured with high glucose[J].Adv Perit Dial,2001,17:2-4.
[11]Ha H,Cha MK,Choi HN,et al.Effects of peritoneal dialysis solutions on the secretion of growth factors and extracellular matrix proteins by human peritoneal mesothelial cells[J].Petit Dial Int,2002,22(2):171-177.
[12]Morgera S,Schlenstedt J,Hambach P,et al.Combined ETA/ETB receptor blockade of human peritoneal mesothelial cells inhibits collagen I RNA synthesis[J].Kidney Int,2003,64(6):2033-2040.
[13]孙芳,洪富源,姚建,等.内皮素-1对人腹膜间皮细胞合成细胞外基质及转化生长因子β-1的影响[J].肾脏病与透析肾移植杂志,2005,14(3):235-239.
[14]孙芳,洪富源,姚建,等.高糖对人腹膜间皮细胞局部内皮素系统表达的影响[J].中国中西医结合肾病杂志,2006,7(2):83-87.
[15]Shimizu M,Ishibashi Y,Taki F,et al.Endothelin(B)receptor blocker inhibits high glucose-induced synthesis of fibronectin in human peritoneal mesothelial cells[J].Petit Dial Int,2006,26(3):393-410.
[16]Yanagisawa M,Katihara H,Kimura S,et al.A novel potent vasoconstrictor peptide produced by vascular endothelial cell[J].Nature,1988,322(31):411-415.
[17]Block KD,Fridrick SP,Lee M E,et al.Structural organization and chromosomal assignment of the gene encoding endothelin[J].J Bio Chem,1989,264:10851-10857.
[18]Inoue A,Yanaglsawa M,Kimura S,et al.The human endothelin family:three structurally and pharmacologically distinct isopeptides predicted by three separate genes[J].Proc Natl Acad Sci USA,1989,86:2863-2867.
[19]Teller P,Noble PW.A cytokine reborn? Endothelin-1 in pulmonary inflammation and fibrosis[J].Am J Respir Cell Mol Biol,2000,23(1):7-10.
[20]Reinehr RM,Kubitz R,Peters-Regehr T,et al.Activation of rat hepatic stellate cells in culture is associated with increased sensitivity to endothelin 1[J].Hepatology,1998,28(6):1566-1577.
[21]Shi-Wen X,Denton CP,Dashwood MR,et al.Fibroblast matrix gene expression and connective tissue remodeling:role of endothelin-1[J].Invest Dermatol,2001,116(3):417-425.
[22]田雪飞,唐功耀,谌贻璞.内皮素-1及内皮素受体A拮抗剂对人肾间质成 纤维细胞的作用[J].中华医学杂志,2002,82(1):5-9.
[23]Dube J,Chakir J,Dube C,et aI.Synergistic action of endothelin(ET)-1 on the activation of bronchial fibroblast isolated from normal and asthmatic subjects[J].Int J Exp Pathol,2000,81(6):429-437.
[24]Morgera S,Kuchinke S,Budde K,et al.Volume stress-induced peritoneal endothelin-1 release in continuous ambulatory peritoneal dialysis[J].J Am Soc Nephrol,1999,10(12):2585-2590.
[25]Hargrove GM,Dufresne J,Whiteside C,et al.Diabetes mellitus increases endothelin-1 gene transcription in rat kidney[J].Kidney Int,2000,58(4):1534-1545.
[26]Picard P,Smith PJ,Monge JC,et al.Coordinated upregulation of the cardiac endothelin system in a rat model of heart failure[J].J Cardiovasc Pharmacol,1998,31 Suppl 1:S294-297.
[27]Yokomori H,Oda M,Ogi M,et al.Enhanced expression of endothelin B receptor at protein and gene levels in human cirrhotic liver[J].Am J Pathol,2001,159(4):1353-1362.
[28]Pinzani M,Milani S,De-Franco R,et al.Endothelin-1 is overexpressed in humancirrhotic liver and exerts multiple effects onactivated hepatic stellate cells[J].Gastroen-terology.1996,110:534-548.
[29]Ha H,Yu MR,Lee HB.High glucose-induced PKC activation mediates TGF-beta-1 and fibronectin synthesis by peritoneal mesothelial cells[J].Kidney Int,2001,59(2):463-470.
[30]Xu ZG,Kim KS,Park HC,et al.High glucose activates the p38 MAPK pathway in cultured human peritoneal mesothelial cells[J].Kidney Int,2003,63(3):958-968
[31]唐知还,张政,刘军,等.活性氧在高糖诱导人腹膜间皮细胞产生IL-8、MCP-1中的作用[J].中国血液净化,2006,1(12):19-22.
[32]Al-Jayyousi R,Medcalf JF,Harris KP.Role of transforming growth factor-beta in peritoneal fibrosis[J].Nephrology,2002,7:216-219.
[33]Horstmeyer A,Licht C,Scherr G,et al.Signalling and regulation of collagen I synthesis by ET-1 and TGF-betal[J].FEBS J,2005,272(24):6297-6309.
[34] Gonzalez W, Chen Z, Damon DH. Transforming growth factor-beta regulation of endothelin expression in rat vascular cell and organ cultures[J]. Cardiovasc Pharmacol,2001,37(2):219 -226.
[35] Rodriguez-Pascual F, Redondo-Horcajo M, Lamas S. Functional cooperation between Smad proteins and activator protein-1 regulates transforming growth factor-beta-mediated induction of endothelin-1 expression [J]. Circ Res, 2003,92 (12):1288-1295.
[36] Shi-Wen X, Rodriguez-Pascual F, Lamas S, et al. Constitutive ALK5-independent c-Jun N-terminal kinase activation contributes to endothelin-1 overexpression in pulmonary fibrosis: evidence of an autocrine endothelin loop operating through the endothelin A and B receptors [J]. Mol Cell Biol,2006,26 (14):5518-5527.
[37] Ksiazek K, Piwocka K, Brzezinska A, et al. Early loss of proliferative potential of human peritoneal mesothelial cells in culture: the role of p16~(INK4a)-mediated premature senescence [J]. Appl Physiol,2006,100 (3):988-995.
[38] Ksiazek K, Breborowicz A, Jorres A, et al. Oxidative stress contributes to accelerated development of the senescent phenotype in human peritoneal mesothelial cells exposed to high glucose[J]. Free Radic Biol Med, 2007,42(5):636-641.
[39] Kaneda K, Miyamoto K, Nomura S, et al. Intercellular localization of occludins and ZO-1 as a solute transport barrier of the mesothelial monolayer[J]. J Artif Organs,2006,9(4):24-250.
[40] Shi-Wen X, Chen Y, Denton CP, et al. Endothelin-1 promotes myofibroblast induction through the ETA receptor via a rac/phosphoinositide 3-kinase /Akt-dependentpathway and is essential for the enhanced contractile phenotype offibrotic fibroblasts [J]. Mol Biol Cell,2004,15(6):2707-2719.
[41] Lee HB, Yu MR, Song JS, et al. Reactive oxygen species amplify proteinkinaseC signaling in high glucose-inducedfibronectin expression by human peritoneal mesothelial cells[J]. Kidney Int,2004,65:1170-1179.
[42] Chen S, Mukherjee S, Chakraborty C, et al. High glucose-induced, endothelin-dependent fibronectin synthesis is mediated via NF-κB and AP-1[J]. Am J Physiol cell Physiol.2003,284: 263-272.
[43]Xin X, Khan ZA, Chen S, et al. Extracellular signal-regulated kinase (ERK) in glucose-induced and endothelin-mediated fibronectin synthesis[J]. Lab Invest. 2004,84(11):1451-1459.
[44] Chen S, Khan ZA, Cukiernik M. Differential activation of NF-kappa B and AP-1 in increased fibronectin synthesis in target organs of diabetic complications [J]. Am J Physiol Endocrinol Metab,2003,284 (6):E1089-1097. [45] Rodriguez-Vita J, Ruiz-Ortega M, Ruperez M, et al. Endothelin-1, via ETA receptor and independently of transforming growth factor-beta, increases the connective tissue growth factor in vascular smooth muscle cells[J]. Circ Res,2005,97(2):125-134.
[46] Nishida M, Okada Y, Akiyoshi K, et al. Role of endothelin ETB receptor in the pathogenesis of monocrotaline-induced pulmonary hypertension in rats[J]. Eur J Pharmacol, 2004,496 (1-3):159-165.
[47] Kuo HT, Shin SJ, Kuo MC, et al. Effects of specific endothelin-1 receptor antagonists on proliferation and fibronectin production of glomerular mesangial cells stimulated with Angiotensin II[J]. Kaohsiung J Med Sci,2006,22(8):371-376.
[48] Shi-wen X, Denton CP, Holmes A, et al. Endothelins: effect on matrix biosynthesis and proliferation in normal and scleroderma fibroblasts [J]. J Cardiovasc Pharmacol, 1998,31(Suppl 1):S360-363.
[49] Mulder P, Boujedaini H, Richard V, et al. Selective endothelin-A versus combined endothelin-A/endothelin-B receptor blockade in rat chronic heart failure[ J ]. Circulation,2000,102(5):491-493.
[1]Yanagisawa M,Karihara H,Kimura S,et al.A novel potent vasoconstrictor peptide produced by vascular endothelial cell[J].Nature,1988,322(31):411-415.
[2]Morgera S,Schlenstedt J,Hambach P,et al.Combined ETA/ETB receptor blockade of human peritoneal mesothelial cells inhibits collagen I RNA synthesis[J].Kidney Int,2003,64(6):2033-2040.
[3]孙芳,洪富源,姚建,等.内皮素-1对人腹膜间皮细胞合成细胞外基质及转化生长因子β-1的影响[J].肾脏病与透析肾移植杂志,2005,14(3):235-239.
[4]孙芳,洪富源,姚建,等.高糖对人腹膜间皮细胞局部内皮素系统表达的影响[J].中国中西医结合肾病杂志,2006,7(2):83-87.
[5]Shimizu M,Ishibashi Y,Taki F,et al.Endothelin(B)receptor blocker inhibits high glucose-induced synthesis of fibronectin in human peritoneal mesothelial cells[J].Perit Dial Int,2006,26(3):393-401.
[6]Morgera S,Kuchinke S,Budde K,et al.Volume stress-induced peritoneal endothelin-1 release in continuous ambulatory peritoneal dialysis[J].J Am Soc Nephrol,1999,10(12):2585-2590.
[7]Hargrove GM,Dufresne J,Whiteside C,et al.Diabetes mellitus increases endothelin-1 gene transcription in rat kidney[J].Kidney Int,2000,58(4):1534-1545.
[8]Ksiazek K,Breborowicz A,Jorres A,et al.Oxidative stress contributes to accelerated development of the senescent phenotype in human peritoneal mesothelial cells exposed to high glucose[J].Free Radic Biol Med,2007,42(5):636-641.
[9]Lee HB,Yu MR,Song JS,et al.Reactive oxygen species amplify protein kinase C signaling in high glucose-induced fibronectin expression by human peritoneal mesothelial cells[J].Kidney Int,2004,65(4):1170-1179.
[10]Ha H,Yu MR,Lee HB.High glucose-induced PKC activation mediates TGF-beta 1 and fibronectin synthesis by peritoneal mesothelial cells[J].Kidney Int,2001,59(2):463-470.
[11]唐知还,张政,刘军,等.活性氧在高糖诱导人腹膜间皮细胞产生IL-8、MCP-1中的作用[J].中国血液净化,2006,1(12):12-15.
[12]Horstmeyer A,Licht C,Scherr G..Signalling and regulation of collagen I synthesis by ET-1 and TGF-betal[J].FEBS J,2005,272(24):6297-6309.
[13]Gonzalez W,Chen Z,Damon DH.Transforming growth factor-beta regulation of endothelin expression in rat vascular cell and organ cultures[J].Cardiovasc Pharmacol,2001,37(2):219-226.
[14]Rodriguez-Pascual F,Redondo-Horcajo M,Lamas S.Functional cooperation between Smad proteins and activator protein-1 regulates transforming growth factor-beta-mediated induction of endothelin-1 expression[J].Circ Res,2003,92(12):1288-1295.
[15]Shi-Wen X,Rodriguez-Pascual F,Lamas S,et al.Constitutive ALK5-independent c-Jun N-terminal kinase activation contributes to endothelin-1 overexpression in pulmonary fibrosis:evidence of an autocrine endothelin loop operating through the endothelin A and B receptors[J].Mol Cell Biol,2006,26(14):5518-5527.
[6]Morgera S,Schlenstedt J,Hambach P,et al.Glucose-mediated transforming growth factor-betal release in human mesothelial cells is endothelin independent[J].Cardiovasc Pharmacol,2004,44(suppl):216-218.
[17]Ksiazek K,Piwocka K,Brzezinska A,et al.Early loss of proliferative potential of human peritoneal mesothelial cells in culture:the role of p16~(INK4a)-mediated premature senescence[J].Appl Physiol,2006,100(3):988-995.
[18]Shi-Wen X,Chen Y,Denton CP,et al.Endothelin-1 promotes myofibroblast induction through the ETA receptor via a rac/phosphoinositide 3-kinase/Akt-dependentpathway and is essential for the enhanced contractile phenotype offibrotic fibroblasts[J].Mol Biol Ce11,2004,15(6):2707-2719.
[19]Teder P,Noble PW.A cytokine rebom?Endothelino-1 in pulmonary inflammation and fibrosis[J].Am J Respir Cell Mol Bio,2000,23(1):7-10.
[20]Kaneda K,Miyamoto K,Nomura S,et al.Intercellular localization of occludins and ZO-1 as a solute transport barrier of the mesothelial monolayer[J].J Artif Organs,2006,9(4):241-250.
[21]Chen S,Apostolova MD,Cherian MG,et al.Interaction of endothelin-1 with vasoactive factors in mediating glucose-induced increased permeability in endothelial cells[J].Lab Invest,2000,80(8):1311-1321.
[22]Spinella F,Rosano L,Di Castro V,et al.Endothelin-1 induces vascular endothelial growth factor by increase hypoxia-inducible factor-lalpha in ovarian carcinoma cells[J].Biol Chem,2002,277(31):27850-27855.
[23]洪富源,孙芳,刘军,等.细胞内活性氧在甲基乙二醛促人腹膜间皮细胞分 泌血管内皮生长因子中的作用[J].肾脏病与透析肾移植杂志,2005,14(4):329-332.
