PDGF-D和JAK/STAT信号通路在IgA肾病发病中的作用以及雷帕霉素对IgA肾病的影响
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摘要
目的:IgA肾病(IgA nephropathy, IgAN)是目前世界上最常见的慢性进行性的原发性肾小球疾病。其主要病理特征是IgA免疫复合物在系膜区沉积,导致系膜细胞的增殖和细胞外基质的沉积,引起一系列临床及病理生理改变,是导致终末期肾病(ESRD)的主要原因之一。随着肾脏活组织穿刺技术的逐渐推广,儿童IgA肾病的诊断率也在逐年提高,而有关IgA肾病的发病机制则是目前众学者研究的热点。Janus kinase(JAK)/ signal transducer and activators of transcription(STAT)介导的信号途径是一条重要的信号通道,可以发挥信号转导和基因转录活化子蛋白的双重作用,介导多种细胞因子和生长因子的细胞内信号转导过程并活化相应靶基因,从而产生生物学效应。其中STAT3参与了许多细胞因子及生长因子介导的细胞增殖及分化过程;细胞因子抑制因子(suppressor of cytokine Signaling, SOCS)目前被认为是经典的JAK/STAT信号通路的抑制剂,其中SOCS1和SOCS3是JAK/STAT信号途径中主要的抑制因子。血小板源性生长因子(platelet-derived growth factor, PDGF)在体内外都是最重要的促有丝分裂原,其家族中包括A、B、C、D四个成员,PDGF-A、B在各种肾脏疾病中的作用以及与JAK/STAT信号转导的关系已被大量的实验所证实。而PDGF-D是2001年发现的PDGF家族的新成员,关于PDGF-D和JAK/STAT在IgA肾病发病中的作用机制研究目前鲜见报道。雷帕霉素(rapamycin)是一种新型免疫抑制剂,具有抗自身免疫性疾病和抗细胞增殖的作用,临床上尚未用于IgA肾病的治疗,且其作用机制未完全阐明。本研究以IgA肾病患儿、IgA动物模型以及PDGF-D培养的系膜细胞为对象,采用多种实验技术,从整体、细胞和分子等不同水平检测IgA肾病肾小球细胞JAK/STAT信号途径的变化以及PDGF-D在IgA肾病发病过程中的动态变化,同时还观察了雷帕霉素对IgA肾病的作用及影响。
方法:
1. IgA肾病儿童PDGF-D和JAK/STAT信号蛋白的检测
标本均来自2004年1月~2007年10月在我科住院并行肾活检确诊的52例IgA肾病患儿,其中男29例,女23例;年龄5~16岁,平均(10.7±3.1)岁。实验分四组:对照组16例;轻度增生组17例(HassⅠ、Ⅱ型);局灶增生组18例(HassⅢ型);增生硬化组17例(HassⅣ、Ⅴ型)。正常对照组儿童32例,其中16例来自我院儿保门诊健康查体的儿童,无各种原发及继发性肾脏疾患,16例肾组织标本均取自肾脏疾病术后病理检查正常的患儿。ELISA法检测IgA肾病患儿血、尿PDGF-D和PDGF-B水平,免疫组织化学染色法检测PDGF-D、PDGF-B、PDGFR-β、STAT3、p-STAT3、p-JAK2、SOCS1和SOCS3蛋白表达。
2.大鼠IgA肾病模型的制备和PDGF-D及JAK/STAT信号蛋白的检测
Wistar雄性大鼠随机分为正常对照组和IgA肾病组,IgA肾病模型制备:隔日牛血清白蛋白400mg·kg-1·d-1灌胃,持续6周,皮下注射蓖麻油0.5ml+四氯化碳0.1ml,每周一次,持续9周,脂多糖分别于第6、8周以0.05mg尾静脉注射,观察至10周末。将肾皮质组织切成3μm的冰冻切片,采用兔抗大鼠IgA和FITC标记羊抗兔IgG间接免疫荧光染色,荧光显微镜下观察肾小球系膜区IgA沉积阳性,PASM和Masson染色显示系膜细胞增生和系膜基质的沉积表明造模成功,成功率为90%。分别于成模后1、2、4、8周每组取7只大鼠,切取肾脏。取部分肾组织置于4%多聚甲醛固定用于光镜观察及免疫组化检测;部分肾皮质组织用于提取肾组织总RNA和肾组织总蛋白。免疫组织化学染色法和Western blot检测肾组织PDGF-D、PDGF-B、PDGFR-β、p-JAK2、STAT3、p-STAT3、SOCS1和SOCS3蛋白表达;半定量RT-PCR检测肾组织PDGF-D mRNA、PDGF-B mRNA、STAT3 mRNA、SOCS1 mRNA和SOCS3 mRNA表达。
3. PDGF-DD对体外培养大鼠肾小球系膜细胞JAK/STAT信号途径的影响
取人系膜细胞株,用无血清RPMI1640培养液洗一次,换无血清RPMI1640培养液孵育24h,使细胞同步化。用MTT法观察不同时间(6、12、24、48h)和不同浓度(0、5ng/ml、10ng/ml、20ng/ml、40ng/ml)PDGF-DD对细胞增殖状态的影响。同时观察JAK2激酶特异抑制剂AG490对PDGF-DD条件下细胞增殖的影响。将细胞分成3组:对照组;PDGF-DD刺激组(20ng/ml)和PDGF-DD+AG490组(20ng/ml PDGF-DD+10μmol/L AG490)。分别在24孔板和25 cm2塑料培养瓶内培养。于刺激的0、30min、60min、120min和240min收集细胞,提取细胞总蛋白、RNA。采用免疫细胞化学和Western blot检测p-JAK2、STAT3、p-STAT3、SOCS1和SOCS3等的表达;半定量RT-PCR检测细胞SOCS1 mRNA和SOCS3 mRNA的表达。
4.雷帕霉素对IgA肾病大鼠和体外培养系膜细胞和JAK/STAT信号途径的影响
IgA肾病大鼠随机分成5组:正常对照组、IgA肾病组和雷帕霉素不同剂量组(0.5 mg·kg-1·d-1、1 mg·kg-1·d-1、2 mg·kg-1·d-1)。于治疗后4周收集血、尿和肾组织。部分肾组织经4%多聚甲醛固定、石蜡包埋,用于形态学观察和免疫组化。应用免疫组化、Western blot以及RT-PCR等方法,观察雷帕霉素对IgA肾病大鼠肾组织p-JAK2、STAT3、p-STAT3、SOCS1和SOCS3蛋白及STAT3 mRNA、SOCS1 mRNA和SOCS3 mRNA表达的影响。
取人系膜细胞株种植于96孔板,采用MTT法分别检测对照组、PDGF-DD刺激组以及干预药物雷帕霉素在不同时间、不同药物浓度对系膜细胞增殖状态的影响。采用25cm2塑料培养瓶培养细胞,将细胞分成3组:对照组;PDGF-DD刺激组(20ng/ml);PDGF-DD+RPM组(20ng/ml PDGF-DD+20μg/ml RPM)。于刺激的24h收集细胞,提取细胞总蛋白和RNA。采用Western blot检测p-JAK2、STAT3、p-STAT3、SOCS1、SOCS3的表达;半定量RT-PCR检测细胞SOCS1 mRNA和SOCS3 mRNA的表达。
结果:
1. JAK/STAT信号蛋白在IgA肾病人体、大鼠肾组织及系膜细胞中的表达
人体实验结果:免疫组化显示正常对照组STAT3在肾小球系膜区和肾小管呈灶状微量表达,其中个别肾小管可见p-STAT3表达;STAT3和p-STAT3在IgA肾病各组肾小球系膜区和肾小管的表达增加,p-STAT3的活化程度随病变程度的加重而逐渐增加,各病变组与对照组相比均有显著差异。SOCS1和SOCS3在正常对照组肾小球无明显表达,在IgA肾病组呈阳性表达。与正常对照组相比,IgA肾病各组患儿随着肾小球病变程度的加重,SOCS1和SOCS3的阳性率呈逐渐增高趋势。
动物实验结果:①光镜下观察,IgA肾病组大鼠在成模后4周内肾小球均未见明显增大,到第8周时肾小球体积有轻度增大,但无统计学意义。②免疫组化显示STAT3、p-STAT3、SOCS1和SOCS3在对照组和IgA肾病组大鼠肾组织中均有阳性表达。STAT3在细胞浆和细胞核均有表达,p-STAT3主要在细胞核表达,SOCS1和SOCS3主要在细胞浆表达。主要分布在肾小球系膜细胞、肾小管上皮细胞和间质细胞。与同期对照组相比,IgA肾病大鼠肾组织中STAT3、p-STAT3、SOCS1和SOCS3均从第1周表达开始增高,第2周时达到高峰,第4周至第8周时表达有所下降,但明显高于相应对照组。③Western blot显示,p-JAK2自IgA肾病第1周起表达开始增高,随时间延长而逐渐增高;肾组织STAT3、p-STAT3和SOCS1蛋白水平自IgA肾病成模后第1周起表达开始增加,在第2周时表达水平最高,第4周至第8周时表达水平开始有所下降,但仍明显高于对照组;SOCS3蛋白水平自IgA肾病成模后第2周起表达开始增加,在第4周时表达水平最高,第8周时表达水平开始有所下降,但仍明显高于对照组。④半定量RT-PCR结果显示,STAT3 mRNA、SOCS1 mRNA和SOCS3 mRNA在对照组肾小球有少量基础表达,不同时间表达无差异。IgA肾病组自成模后第1周起STAT3 mRNA和SOCS1 mRNA表达开始增加,随着时间延长,其表达也逐渐增加,于第2周表达最高,第4周到第8周出现回落,但仍高于对照组。SOCS3 mRNA从第2周起表达开始增加,于第4周达到高峰,第8周开始出现回落。
细胞培养结果:①免疫细胞化学显示,对照组STAT3信号蛋白在系膜细胞细胞核和细胞浆均有表达。p-STAT3在细胞核呈弱表达。SOCS1和SOCS3在细胞浆呈弱表达。STAT3和p-STAT3在PDGF-DD作用30min后表达增强,60min达到高峰,240min后表达减弱。SOCS1和SOCS3在PDGF-DD刺激30min后表达增强,120min达到高峰,240min后表达减弱。应用AG490后STAT3、p-STAT3、SOCS1和SOCS3的表达减弱。②Western blot显示,与对照组相比,PDGF-DD刺激后30min STAT3、p-STAT3和SOCS1表达开始增强,60min达到高峰,240min后表达减弱。SOCS3在PDGF-DD刺激后60min表达明显增强,120min达到高峰,240min后表达减弱。p-JAK2在PDGF-DD刺激后表达逐渐增强。AG490处理组上述蛋白表达明显下调(P<0.05或P<0.01)。③半定量RT-PCR结果显示,SOCS1 mRNA在PDGF-DD刺激后30min后表达明显上调,60min达到高峰,120min和240min后表达减弱。SOCS3 mRNA在PDGF-DD刺激60min后表达明显增强,120min达到高峰,240min后表达减弱。AG490组系膜细胞SOCS1 mRNA和SOCS3 mRNA的表达明显降低,与PDGF-DD组相比有统计学意义。
2. PDGF-D、PDGF-B和PDGFR-β在IgA肾病人体及大鼠肾组织中的表达
人体实验结果:①IgA肾病患儿血液及尿液中PDGF-D和PDGF-B水平明显高于正常对照组,且随病理分级二者的水平逐渐升高,有统计学意义(P<0.01)。②免疫组化结果显示,正常肾组织中PDGF-D和PDGFR-β只在血管内皮细胞中有微量表达;PDGF-B在正常肾组织中未见阳性表达。在IgA肾病患儿肾组织中PDGF-D、PDGF-B和PDGFR-β的表达呈阳性。轻度增生性IgA肾病患儿肾组织PDGF-D的表达呈阳性,主要分布在肾小球系膜细胞、肾小管上皮细胞和血管内皮细胞;局灶增生和增生硬化性IgA肾病患儿肾组织PDGF-D除上述部位表达增强外,还在肾间质有阳性表达。PDGF-B在轻度增生性IgA肾病患儿肾组织中呈弱阳性表达,在局灶增生和增生硬化性IgA肾病患儿肾组织呈强阳性,且分布部位与PDGF-D相同。③相关分析结果显示, IgA肾病患儿血、尿PDGF-D水平与24h尿蛋白定量均呈正相关(r=0.546, P<0.05; r=0.76, P<0.01),与血白蛋白水平呈负相关(r=-0.649, P<0.01; r=-0.528, P<0.05);血、尿PDGF-B与24h尿蛋白定量均呈正相关(r=0.634, P<0.01; r=0.577, P<0.05),与血白蛋白水平呈负相关(r=-0. 613, P<0.01; r=-0.531, P<0.05);二者与血尿、血BUN和Cr均无相关关系。
动物实验结果:①免疫组化结果显示,PDGF-D在正常对照组大鼠肾组织中无阳性表达,PDGF-B在正常对照组大鼠的系膜细胞和间质细胞中有微量表达。与同期对照组相比,PDGF-D和PDGF-B在IgA肾病大鼠肾组织中的表达从第1周开始表达增高,到第8周达到高峰,二者在IgA肾病大鼠肾组织中的表达部位相同(P均<0.05)。②Western印迹结果显示,IgA肾病大鼠从第1周起,肾组织PDGF-B蛋白表达开始增加,并且随时间延长表达更加明显。从第2周起PDGF-D、PDGFR-β蛋白表达开始增加,到第8周时表达水平最高。③半定量RT-PCR结果显示,PDGF-D mRNA和PDGF-B mRNA在对照组肾组织中呈微量表达,IgA肾病组从第1周起PDGF-B mRNA表达就开始增加,随着时间延长,其表达也逐渐增加,于第2周表达最高,第4至第8周出现回落,但仍高于对照组;从第2周起PDGF-D mRNA表达开始增加,随着时间延长,其表达也逐渐增加,于第4周表达最高,第8周出现回落,但仍高于对照组。
3.雷帕霉素对IgA肾病大鼠和体外培养系膜细胞JAK/STAT信号途径的影响
动物实验结果:①免疫细胞化学结果显示,STAT3、p-STAT3、SOCS1和SOCS3在对照组大鼠肾组织中有微弱表达,而在模型组的相对含量显著增加,与对照组相比有显著性差异(P<0.05或P<0.01),主要在肾小球系膜细胞和肾小管上皮细胞表达,间质表达呈弱阳性;雷帕霉素各剂量治疗组随药物剂量的增加,大鼠肾组织中上述蛋白表达逐渐降低。②Western印迹结果显示,模型组大鼠肾组织中p-JAK2、STAT3、p-STAT3、SOCS1和SOCS3的表达水平明显高于对照组(P<0.05或P<0.01);雷帕霉素干预组随药物剂量的增加,上述蛋白表达逐渐降低。③半定量RT-PCR结果显示,STAT3 mRNA、SOCS1 mRNA和SOCS3 mRNA在对照组大鼠肾组织中有少量表达,模型组大鼠肾组织上述蛋白表达明显上调;雷帕霉素干预组随药物剂量的增加,mRNA表达显著下调。
细胞培养结果:①与对照组相比,PDGF-DD能够促进系膜细胞增殖,并随时间延长更加明显; 4、20和100μg/ml雷帕霉素对PDGF-DD培养系膜细胞增殖都有抑制作用,并且随药物浓度的增加抑制作用越明显,20μg/ml雷帕霉素作用12h和24h后,可抑制PDGF-DD对系膜细胞的促增殖作用。②Western印迹结果显示,与对照组相比,PDGF-DD刺激组系膜细胞p-JAK2、STAT3、p-STAT3、SOCS1和SOCS3的表达上调;雷帕霉素干预后上述蛋白表达明显降低(P<0.05或P<0.01)。③半定量RT-PCR结果显示, SOCS1 mRNA和SOCS3 mRNA在对照组有少量表达。在PDGF-DD刺激组二者的表达明显上调,和对照组相比差异有统计学意义。雷帕霉素干预后系膜细胞SOCS1 mRNA和SOCS3 mRNA的表达明显降低,与PDGF-DD组相比有统计学意义。
结论:①IgA肾病患者和IgA肾病模型动物肾组织细胞信号蛋白p-JAK2、STAT3、p-STAT3、SOCS1、SOCS3和PDGF-D表达均明显升高,STAT3、SOCS1、SOCS3和PDGF-D mRNA亦明显上调,表明PDGF-D和JAK/STAT信号转导途径可能参与了IgA肾病的发病过程。②PDGF-DD能够活化体外培养的人肾小球系膜细胞JAK/STAT信号途径,使信号蛋白JAK2和STAT3的磷酸化增强,SOCS1和SOCS3蛋白及mRNA的表达增加。JAK2特异性抑制剂AG490能够降低PDGF-DD刺激引起的系膜细胞p-JAK2、STAT3、p-STAT3、SOCS1和SOCS3蛋白及mRNA的表达,提示PDGF-D可能通过激活JAK/STAT信号途径参与了IgA肾病的发病过程。③雷帕霉素治疗能够明显抑制IgA肾病大鼠肾组织JAK/STAT信号蛋白的表达,使大鼠的血、尿生化指标明显缓解;雷帕霉素能够抑制PDGF-DD刺激引起的系膜细胞JAK/STAT信号途径的活化,提示其肾脏保护作用可能部分是通过影响JAK/STAT信号途径活化实现的。雷帕霉素有望成为临床治疗IgA肾病患者的有效药物。④PDGF-D中和抗体及PDGF-D受体拮抗剂可能成为临床治疗IgA肾病的新趋势。JAK/STAT信号转导抑制剂可能成为临床治疗IgA肾病患者的新途径。
Objectives: IgA nephropathy (IgAN), a chronic and prosessive glomerular disease, is one of the major reasons of glomerulosclerosis and end-stage renal disease (ESRD), which induces a series of clinical and pathophysiological changes. IgAN is histologically characterized by overaccumulation of mensangial cell and extracellular matrix. With the development of renal tissue biopsy, diognosis ratio of children IgAN is increasing gradually. Janus kinase/signal transducer and activators of transcription (JAK/STAT) is an important signaling pathway, which may play roles as signal transducers and activators in transcription and was confirmed to mediate the signaling of numerous cytokines and growth factors and to be implicated in the regulation of a wide range of cellular processes. STAT3 mediated a series of cellular processes such as proliferation, differentiation and apoptosis. Suppressor of cytokine signaling (SOCS) proteins are classic inhibitors of JAK/STAT signaling pathway, and SOCS1 and SOCS3 are the two main inhibitors. Platelet-derived growth factor (PDGF) family is a major mitogen in vivo and in vitro, which has four members, A, B, C and D. A large number of experiments had verified the roles of PDGF-A and PDGF-B in various of renal diseases by inducing the activation of JAK/STAT. PDGF-D is a new member which was discovered in 2001. Few studies about PDGF-D and the JAK/STAT signaling pathway in IgAN were reported. Rapamycin is a new immunosuppressive agent which playes important roles in antiautoimmune and antiproliferation. Rapamycin is not yet used in clinical therapy and the precise mechanisms for rapamycin's effects were not fully understood. In the present study, we examined the expression of PDGF-D and JAK/STAT signaling pathway, investigated the dynamic change of PDGF-D, and observed the effects of rapamycin in IgA patients using Wistar rats model of experimental IgAN and human glomerular mesangial cells cultured in PDGF-D medium, as well as IgAN child patients.
Methods:
1. Detection of PDGF-D and JAK/STAT in IgAN children
Fifty-two children diagnosed as primary IgAN by renal biopsy at our hospital from January 2004 to October 2007 were involved in this study. There were 29 boys and 23 girls among them, whose ages were from 5 to 16 (10.7±3.1) -years old. These patients were divided into four groups according to their pathological changes: control group (n=16); mild proliferation group (MP, n=17); focal proliferation group (FP, n=18) and proliferaiorr sclerosis group (PS, n=17). Children in control group were all healthy and without any primary and secondary renal diseases. ELISA was used to detect the level of PDGF-D and PDGF-B in blood and urine. Immunohistochemistry was used to detect protein expressions of PDGF-D, PDGF-B, PDGFR-β, STAT3, p-STAT3, p-JAK2, SOCS1 and SOCS3 in renal tissue.
2. Induction of IgAN and detection of PDGF-D and JAK/STAT in renal tussue of IgAN rats
Male Wistar rats were randomly divided into two groups: control group and IgAN group. The rats of IgAN group had been administered with bovine serum albumin by gavage at a dose of 400mg·kg-1 every other day for 6 weeks and injected 0.4ml castor oil plus 0.1ml CCl4 once a week for 9 weeks. Lipoplysaccharides were injected at weeks 6 and 8. These rats were observrd until 10th week. Renal tissue was cut into 3μm frozen secion. IgA accumulation in glomerular mensangial areas was examined under the microscope after stained with flurorescein-conjugated affinipure goat anti-rabbit IgG and rabbit anti-rat IgA by immunofluorescence. Proliferation of mensangial cell and overaccumulation of extracellular matrix were observed by PASM and Masson stains. These results indicated that the successful ratio of the IgAN model was 90%. Seven rats from each group were respectively sacrificed at weeks 1, 2, 4 and 8 after the models were established. Partial renal tissue was fixed in 4% formaldehydum and embeded with paraffin. Partial renal cortices were used to abstract total RNA and protein. The protein levels of PDGF-D, PDGF-B, PDGFR-β, p-STAT3, STAT3, p-JAK2, SOCS1 and SOCS3 were evaluated by immunohistochemistry and Western blot; The mRNA levels of PDGF-D, PDGF-B, STAT3, SOCS1 and SOCS3 were measured by reverse transcription and polymerase chain reaction (RT-PCR).
3. Cell culture and examination of JAK/STAT signaling pathway
Human glomerular mesangial cells were incubated in serum-free RPMI 1640 for 24 hours to synchronize the cell growth, and randomly divided into three groups: control group; PDGF-D group (20ng/ml PDGF-D) and PDGF-D+AG490 group (20ng/ml PDGF-D +10μmol/L AG490). MTT assay was used to measure the proliferation of mesangial cells at 6, 12, 24 and 48 hours, meanwhile the effect of AG490 on proliferation of mesangial cells in PDGF-D medium was investigate. Mesangial cells were harvested to abstract total RNA and protein at 0, 30min, 60min, 120min and 240min after incubation. The expressions of STAT3, p-STAT3, p-JAK2, SOCS1 and SOCS3 proteins were examined by immunohistochemistry and Western blot. The levels of SOCS1 mRNA and SOCS3 mRNA were examined by RT-PCR.
4. Effects of rapamycin on expression of JAK/STAT signaling pathway in human glomerular mesangial cells and renal tissues of rats with IgAN
Male Wistar rats were divided into five groups: control group, IgAN group and rapamycin treated group at three different doses (0.5mg·kg-1·d-1, 1mg·kg-1·d-1 and 2mg·kg-1·d-1). After the IgAN model was affirmed to be successful, the rats of rapamycin treated group were administered daily with rapamycin by gavage for 4 weeks. Blood, urine and kidney samples were collected at week 4. Partial renal tissure was fixed in 4% formaldehydum and embeded with paraffin. Partial renal cortices were used to abstract total RNA and protein. Immunohistochemistry and Western blot were used to investigate the expression of STAT3, p-STAT3, p-JAK2, SOCS1 and SOCS3 proteins. RT-PCR was used to detect the expression of STAT3, SOCS1 and SOCS3 mRNAs in the renal tissue of IgAN rats.
Human glomerular mesangial cells were cultured in 96-pore plate. The cells were washed once with serum-free RPMI 1640 and further incubated for 24 hours to synchronize the cell growth. MTT assay was used to investigate the effect of rapamycin on proliferation of mesangial cell in PDGF-D medium at different time and different drug concentration. Human glomerular mesangial cells were cultured in 25cm2 plastic culture flask and divided into three groups: control group; PDGF-D group (20ng/ml PDGF-D); and PDGF-D+RPM group (20ng/ml PDGF-D +20μg/ml RPM). Total RNA and protein were abstacted 24 hours after incubation. The levels of STAT3, p-STAT3, p-STAT3, SOCS1 and SOCS3 proteins were measured by Western blot. SOCS1 mRNA and SOCS3 mRNA was measured by RT-PCR.
Results:
1. Expression of JAK/STAT signaling proteins in renal tissue of IgAN children, renal tissue of IgAN rats and human glomerular mensangial cell Immunohistochemical positive signals of STAT3 and p-STAT3 were increased in glomerular mensangial area and renal tubular epithelial area of the kidneys of patients with IgAN, and a correlation was found in the severity of glomerular injury and expression of these four proteins.
Rats with IgAN:①No obvious morphological change was seen in glomeruli within 4 weeks and glomeruli was enlarged slightly at week 8, which hadn’t statistical significance.②From immunohistochemical staining, the expression of p-STAT3, STAT3, SOCS1 and SOCS3 was positive in glomerular mensangial cells, renal tubular epithelial cells and interstitial cells of both control group and IgAN group, and the expression of p-STAT3, STAT3, SOCS1 and SOCS3 was increased in IgAN group.③Western blot analysis indicated that the expression of p-JAK2 was increased at week 1, and progressively increased with duration of IgAN. The expression levels of STAT3, p-STAT3 and SOCS1 were increased at week 1 in IgAN group, peaked at week 2, and slightly decreased at week 4 and week 8. SOCS3 was increased at week 2 in IgAN group, peaked at week 4, and slightly decreased at week 8.④RT-PCR analysis indicated that the mRNA levels of STAT3 and SOCS1 were increased at week 1 in IgAN group, peaked at week 2, and slightly decreased at week 4 and week 8. SOCS3 mRNA were increased at week 2 in IgAN group, peaked at week 4, and slightly decreased at week 8.
In vitro:①STAT3 proteins were expressed in nuclei and cytoplasm of mesangial cells, p-STAT3 was expressed in nuclei, SOCS1 and SOCS3 were expressed in cytoplasm. Compared with those in control group, the expression levels of STAT3 and p-STAT3 were increased at 30min, peaked at 60min, and decreased at 120min and 240min after stimulation, whereas the levels of SOCS1 and SOCS3 were increased at 30min and 60min, peaked at 120min, and decreased at 240min. The expression levels of these four proteins in AG490 group were significantly lower than those of PDGF-D group.②Western blot analysis indicated that the expression levels of STAT3, p-STAT3 and SOCS1 were increased at 30min, peaked at 60min, and decreased at 120min and 240min after stimulation, whereas the levels of SOCS3 were increased at 30min and 60min, peaked at 120min, and decreased at 240min. The levels of p-JAK2 proteins were gradually increased. The levels of these five proteins were decreased in AG490 group comparing with those in PDGF-D group (P<0.05 or P<0.01).③RT-PCR showed that the mRNA levels of SOCS1 were increased at 30min, peaked at 60min, and decreased at 120min and 240min after stimulation, whereas the mRNA levels of SOCS3 were increased at 30min and 60min, peaked at 120min, and decreased at 240min. The increased expressions were inhibited by the treatment with AG490.
2. The expression of PDGF-D, PDGF-B and PDGFR-βin the kidneys of patients and animal models with IgAN
Clinical experiments:①The levels of PDGF-D and PDGF-B were progressively increased in blood and urine of IgAN children with the severity of glomerular injury.②Immunocytochemical staining showed that the expressions of PDGF-D, PDGF-B and PDGFR-βwere colocalized and increased in renal tissues of IgAN children.③Relevant analyze showed that the levels of PDGF-D had positive correlation with 24h urine protein excretion rate in blood and urine of IgAN children (r=0.546, P<0.05; r=0.76, P<0.01). There was negative correlation between the levels of PDGF-D and serum albumin (r=-0.649, P<0.01; r=-0.528, P<0.05); the levels of PDGF-B had positive correlation with 24h urine protein excretion rate in blood and urine of IgAN children (r=0.634, P<0.01; r=0.577, P<0.05). There was negative correlation between the levels of PDGF-B and serum albumin (r=-0.613, P<0.01; r=-0.531, P<0.05).
Animal experiments: From the results of immunocytochemical staining, the expressions of PDGF-D and PDGF-B were increased at week 1 in IgAN group, and progressively increased with duration of IgAN.②Western blot analysis indicated that the expression of PDGF-B was increased at week 1 in IgAN group, and progressively increased with duration of IgAN. The expressions of PDGF-D and PDGFR-βwas increased at week 2 in IgAN group, and progressively increased with duration of IgAN.③RT-PCR showed the mRNA levels of PDGF-B were increased at week 1 in IgAN group, peaked at week 2, and slightly decreased at week 4 and week 8. PDGF-D mRNA were increased at week 2 in IgAN group, peaked at week 4, and slightly decreased at week 8.
3. Effects of rapamycin on expression of JAK/STAT signaling pathway in the kidney of IgAN rats and human glomerular mesangial cells
In vivo:①Immunocytochemical staining showed that the expression levels of p-STAT3, STAT3, SOCS1 and SOCS3 proteins were increased significantly in IgAN group, and the increased expressions were down-regulated in rapamycin treatment group.②Western blot showed the expression levels of p-JAK2, STAT3, p-STAT3, SOCS1 and SOCS3 were increased in IgAN group.③The mRNA levels of STAT3, SOCS1 and SOCS3 were up-regulated in IgAN group, and down-regulated by rapamycin treatment.
In vitro:①Compared with control group, PDGF-D could stimulate the proliferation of mesangial cells, rapamycin at a concentration of 4, 20 and 100μg/ml significantly inhibited the increased proliferation of mesangial cells in PDGF-D . In rapamycin treated group, the suppressing effect became apparent at a dose of 20μg/ml after 12h and 24h.②Western blot analysis indicated that levels of p-JAK2, STAT3, p-STAT3, SOCS1 and SOCS3 proteins were increased in PDGF-D group, and decreased in rapamycin treated group (P<0.05 or P<0.01).③RT-PCR analysis indicated that the expression of SOCS1 mRNA and SOCS3 mRNA was higher in PDGF-D group than in control group, while rapamycin down-regulated the expression of SOCS1 mRNA and SOCS3 mRNA in mesangial cells cultured in PDGF-D medium.
Conclusions:
From above results we can draw the following conclusions:①In IgAN, the expression levels of p-JAK2, STAT3, p-STAT3, SOCS1, SOCS3 and PDGF-D were up-regulated in renal tissue of the patients with IgAN. PDGF-D and JAK/STAT signaling pathway may be of pathogenic significance in IgAN.②PDGF-D could activate JAK/STAT signaling pathway in human glomerular mesangial cells. PDGF-D enhanced phosphorylation of JAK2/STAT3 and increased the expression of SOCS1 and SOCS3. AG490, specific inhibitor of JAK2, could inhibit activation of JAK/STAT signaling pathway in glomerular mesangial cells under PDGF-D stimulation, which suggested PDGF-D may play an important role in the pathogenesis of IgAN through JAK/STAT signaling pathway.③Rapamycin could inhibit phosphorylation of JAK2 and STAT3 in IgAN, which may be responsible for the renal protective effects of rapamycin in rats with IgAN. Rapamycin may be a promising new drug in the treatment of IgAN.④PDGF-D may have therapeutic effects on IgAN. JAK/STAT inhibitors may be new target of treatment of IgAN.
方法:
1. IgA肾病儿童PDGF-D和JAK/STAT信号蛋白的检测
标本均来自2004年1月~2007年10月在我科住院并行肾活检确诊的52例IgA肾病患儿,其中男29例,女23例;年龄5~16岁,平均(10.7±3.1)岁。实验分四组:对照组16例;轻度增生组17例(HassⅠ、Ⅱ型);局灶增生组18例(HassⅢ型);增生硬化组17例(HassⅣ、Ⅴ型)。正常对照组儿童32例,其中16例来自我院儿保门诊健康查体的儿童,无各种原发及继发性肾脏疾患,16例肾组织标本均取自肾脏疾病术后病理检查正常的患儿。ELISA法检测IgA肾病患儿血、尿PDGF-D和PDGF-B水平,免疫组织化学染色法检测PDGF-D、PDGF-B、PDGFR-β、STAT3、p-STAT3、p-JAK2、SOCS1和SOCS3蛋白表达。
2.大鼠IgA肾病模型的制备和PDGF-D及JAK/STAT信号蛋白的检测
Wistar雄性大鼠随机分为正常对照组和IgA肾病组,IgA肾病模型制备:隔日牛血清白蛋白400mg·kg-1·d-1灌胃,持续6周,皮下注射蓖麻油0.5ml+四氯化碳0.1ml,每周一次,持续9周,脂多糖分别于第6、8周以0.05mg尾静脉注射,观察至10周末。将肾皮质组织切成3μm的冰冻切片,采用兔抗大鼠IgA和FITC标记羊抗兔IgG间接免疫荧光染色,荧光显微镜下观察肾小球系膜区IgA沉积阳性,PASM和Masson染色显示系膜细胞增生和系膜基质的沉积表明造模成功,成功率为90%。分别于成模后1、2、4、8周每组取7只大鼠,切取肾脏。取部分肾组织置于4%多聚甲醛固定用于光镜观察及免疫组化检测;部分肾皮质组织用于提取肾组织总RNA和肾组织总蛋白。免疫组织化学染色法和Western blot检测肾组织PDGF-D、PDGF-B、PDGFR-β、p-JAK2、STAT3、p-STAT3、SOCS1和SOCS3蛋白表达;半定量RT-PCR检测肾组织PDGF-D mRNA、PDGF-B mRNA、STAT3 mRNA、SOCS1 mRNA和SOCS3 mRNA表达。
3. PDGF-DD对体外培养大鼠肾小球系膜细胞JAK/STAT信号途径的影响
取人系膜细胞株,用无血清RPMI1640培养液洗一次,换无血清RPMI1640培养液孵育24h,使细胞同步化。用MTT法观察不同时间(6、12、24、48h)和不同浓度(0、5ng/ml、10ng/ml、20ng/ml、40ng/ml)PDGF-DD对细胞增殖状态的影响。同时观察JAK2激酶特异抑制剂AG490对PDGF-DD条件下细胞增殖的影响。将细胞分成3组:对照组;PDGF-DD刺激组(20ng/ml)和PDGF-DD+AG490组(20ng/ml PDGF-DD+10μmol/L AG490)。分别在24孔板和25 cm2塑料培养瓶内培养。于刺激的0、30min、60min、120min和240min收集细胞,提取细胞总蛋白、RNA。采用免疫细胞化学和Western blot检测p-JAK2、STAT3、p-STAT3、SOCS1和SOCS3等的表达;半定量RT-PCR检测细胞SOCS1 mRNA和SOCS3 mRNA的表达。
4.雷帕霉素对IgA肾病大鼠和体外培养系膜细胞和JAK/STAT信号途径的影响
IgA肾病大鼠随机分成5组:正常对照组、IgA肾病组和雷帕霉素不同剂量组(0.5 mg·kg-1·d-1、1 mg·kg-1·d-1、2 mg·kg-1·d-1)。于治疗后4周收集血、尿和肾组织。部分肾组织经4%多聚甲醛固定、石蜡包埋,用于形态学观察和免疫组化。应用免疫组化、Western blot以及RT-PCR等方法,观察雷帕霉素对IgA肾病大鼠肾组织p-JAK2、STAT3、p-STAT3、SOCS1和SOCS3蛋白及STAT3 mRNA、SOCS1 mRNA和SOCS3 mRNA表达的影响。
取人系膜细胞株种植于96孔板,采用MTT法分别检测对照组、PDGF-DD刺激组以及干预药物雷帕霉素在不同时间、不同药物浓度对系膜细胞增殖状态的影响。采用25cm2塑料培养瓶培养细胞,将细胞分成3组:对照组;PDGF-DD刺激组(20ng/ml);PDGF-DD+RPM组(20ng/ml PDGF-DD+20μg/ml RPM)。于刺激的24h收集细胞,提取细胞总蛋白和RNA。采用Western blot检测p-JAK2、STAT3、p-STAT3、SOCS1、SOCS3的表达;半定量RT-PCR检测细胞SOCS1 mRNA和SOCS3 mRNA的表达。
结果:
1. JAK/STAT信号蛋白在IgA肾病人体、大鼠肾组织及系膜细胞中的表达
人体实验结果:免疫组化显示正常对照组STAT3在肾小球系膜区和肾小管呈灶状微量表达,其中个别肾小管可见p-STAT3表达;STAT3和p-STAT3在IgA肾病各组肾小球系膜区和肾小管的表达增加,p-STAT3的活化程度随病变程度的加重而逐渐增加,各病变组与对照组相比均有显著差异。SOCS1和SOCS3在正常对照组肾小球无明显表达,在IgA肾病组呈阳性表达。与正常对照组相比,IgA肾病各组患儿随着肾小球病变程度的加重,SOCS1和SOCS3的阳性率呈逐渐增高趋势。
动物实验结果:①光镜下观察,IgA肾病组大鼠在成模后4周内肾小球均未见明显增大,到第8周时肾小球体积有轻度增大,但无统计学意义。②免疫组化显示STAT3、p-STAT3、SOCS1和SOCS3在对照组和IgA肾病组大鼠肾组织中均有阳性表达。STAT3在细胞浆和细胞核均有表达,p-STAT3主要在细胞核表达,SOCS1和SOCS3主要在细胞浆表达。主要分布在肾小球系膜细胞、肾小管上皮细胞和间质细胞。与同期对照组相比,IgA肾病大鼠肾组织中STAT3、p-STAT3、SOCS1和SOCS3均从第1周表达开始增高,第2周时达到高峰,第4周至第8周时表达有所下降,但明显高于相应对照组。③Western blot显示,p-JAK2自IgA肾病第1周起表达开始增高,随时间延长而逐渐增高;肾组织STAT3、p-STAT3和SOCS1蛋白水平自IgA肾病成模后第1周起表达开始增加,在第2周时表达水平最高,第4周至第8周时表达水平开始有所下降,但仍明显高于对照组;SOCS3蛋白水平自IgA肾病成模后第2周起表达开始增加,在第4周时表达水平最高,第8周时表达水平开始有所下降,但仍明显高于对照组。④半定量RT-PCR结果显示,STAT3 mRNA、SOCS1 mRNA和SOCS3 mRNA在对照组肾小球有少量基础表达,不同时间表达无差异。IgA肾病组自成模后第1周起STAT3 mRNA和SOCS1 mRNA表达开始增加,随着时间延长,其表达也逐渐增加,于第2周表达最高,第4周到第8周出现回落,但仍高于对照组。SOCS3 mRNA从第2周起表达开始增加,于第4周达到高峰,第8周开始出现回落。
细胞培养结果:①免疫细胞化学显示,对照组STAT3信号蛋白在系膜细胞细胞核和细胞浆均有表达。p-STAT3在细胞核呈弱表达。SOCS1和SOCS3在细胞浆呈弱表达。STAT3和p-STAT3在PDGF-DD作用30min后表达增强,60min达到高峰,240min后表达减弱。SOCS1和SOCS3在PDGF-DD刺激30min后表达增强,120min达到高峰,240min后表达减弱。应用AG490后STAT3、p-STAT3、SOCS1和SOCS3的表达减弱。②Western blot显示,与对照组相比,PDGF-DD刺激后30min STAT3、p-STAT3和SOCS1表达开始增强,60min达到高峰,240min后表达减弱。SOCS3在PDGF-DD刺激后60min表达明显增强,120min达到高峰,240min后表达减弱。p-JAK2在PDGF-DD刺激后表达逐渐增强。AG490处理组上述蛋白表达明显下调(P<0.05或P<0.01)。③半定量RT-PCR结果显示,SOCS1 mRNA在PDGF-DD刺激后30min后表达明显上调,60min达到高峰,120min和240min后表达减弱。SOCS3 mRNA在PDGF-DD刺激60min后表达明显增强,120min达到高峰,240min后表达减弱。AG490组系膜细胞SOCS1 mRNA和SOCS3 mRNA的表达明显降低,与PDGF-DD组相比有统计学意义。
2. PDGF-D、PDGF-B和PDGFR-β在IgA肾病人体及大鼠肾组织中的表达
人体实验结果:①IgA肾病患儿血液及尿液中PDGF-D和PDGF-B水平明显高于正常对照组,且随病理分级二者的水平逐渐升高,有统计学意义(P<0.01)。②免疫组化结果显示,正常肾组织中PDGF-D和PDGFR-β只在血管内皮细胞中有微量表达;PDGF-B在正常肾组织中未见阳性表达。在IgA肾病患儿肾组织中PDGF-D、PDGF-B和PDGFR-β的表达呈阳性。轻度增生性IgA肾病患儿肾组织PDGF-D的表达呈阳性,主要分布在肾小球系膜细胞、肾小管上皮细胞和血管内皮细胞;局灶增生和增生硬化性IgA肾病患儿肾组织PDGF-D除上述部位表达增强外,还在肾间质有阳性表达。PDGF-B在轻度增生性IgA肾病患儿肾组织中呈弱阳性表达,在局灶增生和增生硬化性IgA肾病患儿肾组织呈强阳性,且分布部位与PDGF-D相同。③相关分析结果显示, IgA肾病患儿血、尿PDGF-D水平与24h尿蛋白定量均呈正相关(r=0.546, P<0.05; r=0.76, P<0.01),与血白蛋白水平呈负相关(r=-0.649, P<0.01; r=-0.528, P<0.05);血、尿PDGF-B与24h尿蛋白定量均呈正相关(r=0.634, P<0.01; r=0.577, P<0.05),与血白蛋白水平呈负相关(r=-0. 613, P<0.01; r=-0.531, P<0.05);二者与血尿、血BUN和Cr均无相关关系。
动物实验结果:①免疫组化结果显示,PDGF-D在正常对照组大鼠肾组织中无阳性表达,PDGF-B在正常对照组大鼠的系膜细胞和间质细胞中有微量表达。与同期对照组相比,PDGF-D和PDGF-B在IgA肾病大鼠肾组织中的表达从第1周开始表达增高,到第8周达到高峰,二者在IgA肾病大鼠肾组织中的表达部位相同(P均<0.05)。②Western印迹结果显示,IgA肾病大鼠从第1周起,肾组织PDGF-B蛋白表达开始增加,并且随时间延长表达更加明显。从第2周起PDGF-D、PDGFR-β蛋白表达开始增加,到第8周时表达水平最高。③半定量RT-PCR结果显示,PDGF-D mRNA和PDGF-B mRNA在对照组肾组织中呈微量表达,IgA肾病组从第1周起PDGF-B mRNA表达就开始增加,随着时间延长,其表达也逐渐增加,于第2周表达最高,第4至第8周出现回落,但仍高于对照组;从第2周起PDGF-D mRNA表达开始增加,随着时间延长,其表达也逐渐增加,于第4周表达最高,第8周出现回落,但仍高于对照组。
3.雷帕霉素对IgA肾病大鼠和体外培养系膜细胞JAK/STAT信号途径的影响
动物实验结果:①免疫细胞化学结果显示,STAT3、p-STAT3、SOCS1和SOCS3在对照组大鼠肾组织中有微弱表达,而在模型组的相对含量显著增加,与对照组相比有显著性差异(P<0.05或P<0.01),主要在肾小球系膜细胞和肾小管上皮细胞表达,间质表达呈弱阳性;雷帕霉素各剂量治疗组随药物剂量的增加,大鼠肾组织中上述蛋白表达逐渐降低。②Western印迹结果显示,模型组大鼠肾组织中p-JAK2、STAT3、p-STAT3、SOCS1和SOCS3的表达水平明显高于对照组(P<0.05或P<0.01);雷帕霉素干预组随药物剂量的增加,上述蛋白表达逐渐降低。③半定量RT-PCR结果显示,STAT3 mRNA、SOCS1 mRNA和SOCS3 mRNA在对照组大鼠肾组织中有少量表达,模型组大鼠肾组织上述蛋白表达明显上调;雷帕霉素干预组随药物剂量的增加,mRNA表达显著下调。
细胞培养结果:①与对照组相比,PDGF-DD能够促进系膜细胞增殖,并随时间延长更加明显; 4、20和100μg/ml雷帕霉素对PDGF-DD培养系膜细胞增殖都有抑制作用,并且随药物浓度的增加抑制作用越明显,20μg/ml雷帕霉素作用12h和24h后,可抑制PDGF-DD对系膜细胞的促增殖作用。②Western印迹结果显示,与对照组相比,PDGF-DD刺激组系膜细胞p-JAK2、STAT3、p-STAT3、SOCS1和SOCS3的表达上调;雷帕霉素干预后上述蛋白表达明显降低(P<0.05或P<0.01)。③半定量RT-PCR结果显示, SOCS1 mRNA和SOCS3 mRNA在对照组有少量表达。在PDGF-DD刺激组二者的表达明显上调,和对照组相比差异有统计学意义。雷帕霉素干预后系膜细胞SOCS1 mRNA和SOCS3 mRNA的表达明显降低,与PDGF-DD组相比有统计学意义。
结论:①IgA肾病患者和IgA肾病模型动物肾组织细胞信号蛋白p-JAK2、STAT3、p-STAT3、SOCS1、SOCS3和PDGF-D表达均明显升高,STAT3、SOCS1、SOCS3和PDGF-D mRNA亦明显上调,表明PDGF-D和JAK/STAT信号转导途径可能参与了IgA肾病的发病过程。②PDGF-DD能够活化体外培养的人肾小球系膜细胞JAK/STAT信号途径,使信号蛋白JAK2和STAT3的磷酸化增强,SOCS1和SOCS3蛋白及mRNA的表达增加。JAK2特异性抑制剂AG490能够降低PDGF-DD刺激引起的系膜细胞p-JAK2、STAT3、p-STAT3、SOCS1和SOCS3蛋白及mRNA的表达,提示PDGF-D可能通过激活JAK/STAT信号途径参与了IgA肾病的发病过程。③雷帕霉素治疗能够明显抑制IgA肾病大鼠肾组织JAK/STAT信号蛋白的表达,使大鼠的血、尿生化指标明显缓解;雷帕霉素能够抑制PDGF-DD刺激引起的系膜细胞JAK/STAT信号途径的活化,提示其肾脏保护作用可能部分是通过影响JAK/STAT信号途径活化实现的。雷帕霉素有望成为临床治疗IgA肾病患者的有效药物。④PDGF-D中和抗体及PDGF-D受体拮抗剂可能成为临床治疗IgA肾病的新趋势。JAK/STAT信号转导抑制剂可能成为临床治疗IgA肾病患者的新途径。
Objectives: IgA nephropathy (IgAN), a chronic and prosessive glomerular disease, is one of the major reasons of glomerulosclerosis and end-stage renal disease (ESRD), which induces a series of clinical and pathophysiological changes. IgAN is histologically characterized by overaccumulation of mensangial cell and extracellular matrix. With the development of renal tissue biopsy, diognosis ratio of children IgAN is increasing gradually. Janus kinase/signal transducer and activators of transcription (JAK/STAT) is an important signaling pathway, which may play roles as signal transducers and activators in transcription and was confirmed to mediate the signaling of numerous cytokines and growth factors and to be implicated in the regulation of a wide range of cellular processes. STAT3 mediated a series of cellular processes such as proliferation, differentiation and apoptosis. Suppressor of cytokine signaling (SOCS) proteins are classic inhibitors of JAK/STAT signaling pathway, and SOCS1 and SOCS3 are the two main inhibitors. Platelet-derived growth factor (PDGF) family is a major mitogen in vivo and in vitro, which has four members, A, B, C and D. A large number of experiments had verified the roles of PDGF-A and PDGF-B in various of renal diseases by inducing the activation of JAK/STAT. PDGF-D is a new member which was discovered in 2001. Few studies about PDGF-D and the JAK/STAT signaling pathway in IgAN were reported. Rapamycin is a new immunosuppressive agent which playes important roles in antiautoimmune and antiproliferation. Rapamycin is not yet used in clinical therapy and the precise mechanisms for rapamycin's effects were not fully understood. In the present study, we examined the expression of PDGF-D and JAK/STAT signaling pathway, investigated the dynamic change of PDGF-D, and observed the effects of rapamycin in IgA patients using Wistar rats model of experimental IgAN and human glomerular mesangial cells cultured in PDGF-D medium, as well as IgAN child patients.
Methods:
1. Detection of PDGF-D and JAK/STAT in IgAN children
Fifty-two children diagnosed as primary IgAN by renal biopsy at our hospital from January 2004 to October 2007 were involved in this study. There were 29 boys and 23 girls among them, whose ages were from 5 to 16 (10.7±3.1) -years old. These patients were divided into four groups according to their pathological changes: control group (n=16); mild proliferation group (MP, n=17); focal proliferation group (FP, n=18) and proliferaiorr sclerosis group (PS, n=17). Children in control group were all healthy and without any primary and secondary renal diseases. ELISA was used to detect the level of PDGF-D and PDGF-B in blood and urine. Immunohistochemistry was used to detect protein expressions of PDGF-D, PDGF-B, PDGFR-β, STAT3, p-STAT3, p-JAK2, SOCS1 and SOCS3 in renal tissue.
2. Induction of IgAN and detection of PDGF-D and JAK/STAT in renal tussue of IgAN rats
Male Wistar rats were randomly divided into two groups: control group and IgAN group. The rats of IgAN group had been administered with bovine serum albumin by gavage at a dose of 400mg·kg-1 every other day for 6 weeks and injected 0.4ml castor oil plus 0.1ml CCl4 once a week for 9 weeks. Lipoplysaccharides were injected at weeks 6 and 8. These rats were observrd until 10th week. Renal tissue was cut into 3μm frozen secion. IgA accumulation in glomerular mensangial areas was examined under the microscope after stained with flurorescein-conjugated affinipure goat anti-rabbit IgG and rabbit anti-rat IgA by immunofluorescence. Proliferation of mensangial cell and overaccumulation of extracellular matrix were observed by PASM and Masson stains. These results indicated that the successful ratio of the IgAN model was 90%. Seven rats from each group were respectively sacrificed at weeks 1, 2, 4 and 8 after the models were established. Partial renal tissue was fixed in 4% formaldehydum and embeded with paraffin. Partial renal cortices were used to abstract total RNA and protein. The protein levels of PDGF-D, PDGF-B, PDGFR-β, p-STAT3, STAT3, p-JAK2, SOCS1 and SOCS3 were evaluated by immunohistochemistry and Western blot; The mRNA levels of PDGF-D, PDGF-B, STAT3, SOCS1 and SOCS3 were measured by reverse transcription and polymerase chain reaction (RT-PCR).
3. Cell culture and examination of JAK/STAT signaling pathway
Human glomerular mesangial cells were incubated in serum-free RPMI 1640 for 24 hours to synchronize the cell growth, and randomly divided into three groups: control group; PDGF-D group (20ng/ml PDGF-D) and PDGF-D+AG490 group (20ng/ml PDGF-D +10μmol/L AG490). MTT assay was used to measure the proliferation of mesangial cells at 6, 12, 24 and 48 hours, meanwhile the effect of AG490 on proliferation of mesangial cells in PDGF-D medium was investigate. Mesangial cells were harvested to abstract total RNA and protein at 0, 30min, 60min, 120min and 240min after incubation. The expressions of STAT3, p-STAT3, p-JAK2, SOCS1 and SOCS3 proteins were examined by immunohistochemistry and Western blot. The levels of SOCS1 mRNA and SOCS3 mRNA were examined by RT-PCR.
4. Effects of rapamycin on expression of JAK/STAT signaling pathway in human glomerular mesangial cells and renal tissues of rats with IgAN
Male Wistar rats were divided into five groups: control group, IgAN group and rapamycin treated group at three different doses (0.5mg·kg-1·d-1, 1mg·kg-1·d-1 and 2mg·kg-1·d-1). After the IgAN model was affirmed to be successful, the rats of rapamycin treated group were administered daily with rapamycin by gavage for 4 weeks. Blood, urine and kidney samples were collected at week 4. Partial renal tissure was fixed in 4% formaldehydum and embeded with paraffin. Partial renal cortices were used to abstract total RNA and protein. Immunohistochemistry and Western blot were used to investigate the expression of STAT3, p-STAT3, p-JAK2, SOCS1 and SOCS3 proteins. RT-PCR was used to detect the expression of STAT3, SOCS1 and SOCS3 mRNAs in the renal tissue of IgAN rats.
Human glomerular mesangial cells were cultured in 96-pore plate. The cells were washed once with serum-free RPMI 1640 and further incubated for 24 hours to synchronize the cell growth. MTT assay was used to investigate the effect of rapamycin on proliferation of mesangial cell in PDGF-D medium at different time and different drug concentration. Human glomerular mesangial cells were cultured in 25cm2 plastic culture flask and divided into three groups: control group; PDGF-D group (20ng/ml PDGF-D); and PDGF-D+RPM group (20ng/ml PDGF-D +20μg/ml RPM). Total RNA and protein were abstacted 24 hours after incubation. The levels of STAT3, p-STAT3, p-STAT3, SOCS1 and SOCS3 proteins were measured by Western blot. SOCS1 mRNA and SOCS3 mRNA was measured by RT-PCR.
Results:
1. Expression of JAK/STAT signaling proteins in renal tissue of IgAN children, renal tissue of IgAN rats and human glomerular mensangial cell Immunohistochemical positive signals of STAT3 and p-STAT3 were increased in glomerular mensangial area and renal tubular epithelial area of the kidneys of patients with IgAN, and a correlation was found in the severity of glomerular injury and expression of these four proteins.
Rats with IgAN:①No obvious morphological change was seen in glomeruli within 4 weeks and glomeruli was enlarged slightly at week 8, which hadn’t statistical significance.②From immunohistochemical staining, the expression of p-STAT3, STAT3, SOCS1 and SOCS3 was positive in glomerular mensangial cells, renal tubular epithelial cells and interstitial cells of both control group and IgAN group, and the expression of p-STAT3, STAT3, SOCS1 and SOCS3 was increased in IgAN group.③Western blot analysis indicated that the expression of p-JAK2 was increased at week 1, and progressively increased with duration of IgAN. The expression levels of STAT3, p-STAT3 and SOCS1 were increased at week 1 in IgAN group, peaked at week 2, and slightly decreased at week 4 and week 8. SOCS3 was increased at week 2 in IgAN group, peaked at week 4, and slightly decreased at week 8.④RT-PCR analysis indicated that the mRNA levels of STAT3 and SOCS1 were increased at week 1 in IgAN group, peaked at week 2, and slightly decreased at week 4 and week 8. SOCS3 mRNA were increased at week 2 in IgAN group, peaked at week 4, and slightly decreased at week 8.
In vitro:①STAT3 proteins were expressed in nuclei and cytoplasm of mesangial cells, p-STAT3 was expressed in nuclei, SOCS1 and SOCS3 were expressed in cytoplasm. Compared with those in control group, the expression levels of STAT3 and p-STAT3 were increased at 30min, peaked at 60min, and decreased at 120min and 240min after stimulation, whereas the levels of SOCS1 and SOCS3 were increased at 30min and 60min, peaked at 120min, and decreased at 240min. The expression levels of these four proteins in AG490 group were significantly lower than those of PDGF-D group.②Western blot analysis indicated that the expression levels of STAT3, p-STAT3 and SOCS1 were increased at 30min, peaked at 60min, and decreased at 120min and 240min after stimulation, whereas the levels of SOCS3 were increased at 30min and 60min, peaked at 120min, and decreased at 240min. The levels of p-JAK2 proteins were gradually increased. The levels of these five proteins were decreased in AG490 group comparing with those in PDGF-D group (P<0.05 or P<0.01).③RT-PCR showed that the mRNA levels of SOCS1 were increased at 30min, peaked at 60min, and decreased at 120min and 240min after stimulation, whereas the mRNA levels of SOCS3 were increased at 30min and 60min, peaked at 120min, and decreased at 240min. The increased expressions were inhibited by the treatment with AG490.
2. The expression of PDGF-D, PDGF-B and PDGFR-βin the kidneys of patients and animal models with IgAN
Clinical experiments:①The levels of PDGF-D and PDGF-B were progressively increased in blood and urine of IgAN children with the severity of glomerular injury.②Immunocytochemical staining showed that the expressions of PDGF-D, PDGF-B and PDGFR-βwere colocalized and increased in renal tissues of IgAN children.③Relevant analyze showed that the levels of PDGF-D had positive correlation with 24h urine protein excretion rate in blood and urine of IgAN children (r=0.546, P<0.05; r=0.76, P<0.01). There was negative correlation between the levels of PDGF-D and serum albumin (r=-0.649, P<0.01; r=-0.528, P<0.05); the levels of PDGF-B had positive correlation with 24h urine protein excretion rate in blood and urine of IgAN children (r=0.634, P<0.01; r=0.577, P<0.05). There was negative correlation between the levels of PDGF-B and serum albumin (r=-0.613, P<0.01; r=-0.531, P<0.05).
Animal experiments: From the results of immunocytochemical staining, the expressions of PDGF-D and PDGF-B were increased at week 1 in IgAN group, and progressively increased with duration of IgAN.②Western blot analysis indicated that the expression of PDGF-B was increased at week 1 in IgAN group, and progressively increased with duration of IgAN. The expressions of PDGF-D and PDGFR-βwas increased at week 2 in IgAN group, and progressively increased with duration of IgAN.③RT-PCR showed the mRNA levels of PDGF-B were increased at week 1 in IgAN group, peaked at week 2, and slightly decreased at week 4 and week 8. PDGF-D mRNA were increased at week 2 in IgAN group, peaked at week 4, and slightly decreased at week 8.
3. Effects of rapamycin on expression of JAK/STAT signaling pathway in the kidney of IgAN rats and human glomerular mesangial cells
In vivo:①Immunocytochemical staining showed that the expression levels of p-STAT3, STAT3, SOCS1 and SOCS3 proteins were increased significantly in IgAN group, and the increased expressions were down-regulated in rapamycin treatment group.②Western blot showed the expression levels of p-JAK2, STAT3, p-STAT3, SOCS1 and SOCS3 were increased in IgAN group.③The mRNA levels of STAT3, SOCS1 and SOCS3 were up-regulated in IgAN group, and down-regulated by rapamycin treatment.
In vitro:①Compared with control group, PDGF-D could stimulate the proliferation of mesangial cells, rapamycin at a concentration of 4, 20 and 100μg/ml significantly inhibited the increased proliferation of mesangial cells in PDGF-D . In rapamycin treated group, the suppressing effect became apparent at a dose of 20μg/ml after 12h and 24h.②Western blot analysis indicated that levels of p-JAK2, STAT3, p-STAT3, SOCS1 and SOCS3 proteins were increased in PDGF-D group, and decreased in rapamycin treated group (P<0.05 or P<0.01).③RT-PCR analysis indicated that the expression of SOCS1 mRNA and SOCS3 mRNA was higher in PDGF-D group than in control group, while rapamycin down-regulated the expression of SOCS1 mRNA and SOCS3 mRNA in mesangial cells cultured in PDGF-D medium.
Conclusions:
From above results we can draw the following conclusions:①In IgAN, the expression levels of p-JAK2, STAT3, p-STAT3, SOCS1, SOCS3 and PDGF-D were up-regulated in renal tissue of the patients with IgAN. PDGF-D and JAK/STAT signaling pathway may be of pathogenic significance in IgAN.②PDGF-D could activate JAK/STAT signaling pathway in human glomerular mesangial cells. PDGF-D enhanced phosphorylation of JAK2/STAT3 and increased the expression of SOCS1 and SOCS3. AG490, specific inhibitor of JAK2, could inhibit activation of JAK/STAT signaling pathway in glomerular mesangial cells under PDGF-D stimulation, which suggested PDGF-D may play an important role in the pathogenesis of IgAN through JAK/STAT signaling pathway.③Rapamycin could inhibit phosphorylation of JAK2 and STAT3 in IgAN, which may be responsible for the renal protective effects of rapamycin in rats with IgAN. Rapamycin may be a promising new drug in the treatment of IgAN.④PDGF-D may have therapeutic effects on IgAN. JAK/STAT inhibitors may be new target of treatment of IgAN.
引文
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