罗格列酮联合雷帕霉素抑制多囊肾囊肿衬里上皮细胞的增殖及机制研究
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摘要
常染色体显性多囊肾病(ADPKD)是人类最常见的单基因遗传性肾病,发病率在1/400-1／1000,我国约有150万患者,占终末期肾衰竭病因5%-10%。ADPKD主要特征表现为双侧肾脏出现许多个进行性增大的液性囊泡,最终破坏肾脏的结构和功能。50%患者在60岁时会进展至终末期肾衰竭。ADPKD除累及肾脏外,还引起肝、胰囊肿、心瓣膜病、结肠憩室和颅内动脉瘤等肾外病变,是一种严重危害人类健康的系统性疾病。
多年来,肾脏病学者一直致力于寻找治疗多囊肾病的有效方法,基因治疗目前存在许多障碍有待克服,通过药物治疗延缓肾衰竭进展是目前的主要研究方向。多囊肾病治疗需要早期、长期用药,因此药物的安全性成为研究者关注的焦点。由于ADPKD的分子发病及调控机制非常复杂,很多问题尚未阐明,因此针对不同发病环节研发不同药物,联合用于治疗不仅可增强疗效,而且减少不良反应,将具有潜在的的应用前景。
噻唑烷二酮类PPARy激动剂,如吡格列酮和罗格列酮等,主要用于糖尿病治疗。近年来研究发现,噻唑烷二酮类PPARy激动剂可抑制多种肿瘤细胞的增殖。2001年Muto等首先观察到吡格列酮可延长多囊蛋白1基因敲除小鼠(pkdl-/-)的生存期(通常在胎儿期死亡)；我科先前动物实验发现,罗格列酮可抑制Han:SPRD大鼠囊肿的进展,延长大鼠生存期,但其机制尚未完全阐明。
mTOR信号通路异常激活被认为是多囊肾病发生的重要机制,新生血管的形成也可促进多囊肾病的进展；因此我们探讨罗格列酮对mTOR信号通路的作用,研究其对新生血管形成中起重要作用的血管内皮生长因子(VEGF)的作用；同时观察罗格列酮与mTOR抑制剂雷帕霉素联合应用对延缓ADPKD进展的作用,为治疗多囊肾病寻找新的途径。
为此,我们首先研究了罗格列酮对多囊肾囊肿衬里上皮细胞增殖、细胞周期及细胞凋亡的作用。多囊肾囊肿衬里上皮细胞系(WT9-12)由美国哈佛大学周晶教授馈赠。培养的WT9-12细胞给予不同浓度的罗格列酮培养24、48、72h,采用MTT法检测结果显示：罗格列酮明显抑制WT9-12细胞的增殖,呈时间、剂量依赖性。预72h时,50%抑制率浓度为100μM。采用流式细胞技术检测细胞周期及凋亡,结果显示罗格列酮明显抑制WT9-12细胞周期,使细胞停滞于G0／G1期,G0／G1期细胞增加30%,与对照组比较有差异有显著性意义(P<0.05),其对细胞凋亡的影响不大,罗格列酮浓度高达200μM时,仅使细胞凋亡增加6%。多囊肾病的发展过程伴有胰岛素生长因子1(IGF-1)表达的增加,我们的研究发现囊肿衬里上皮细胞对IGF-1的刺激反应较其它永生化肾小管上皮细胞(RCTEC)更为明显。IGF-150ng/ml可诱导囊肿衬里上皮细胞增殖增加20%,而对RCTEC无明显影响。罗格列酮可完全抑制IGF-1诱导的WT9-12细胞增殖,该作用在罗格列酮12.5μM时即表现明显的抑制作用。说明罗格列酮在有IGF-1作用下对囊肿衬里上皮细胞的增殖抑制作用更为明显。
mTOR信号通路异常激活在多囊肾病发病及发展中起重要作用,为多种发病机制的共同环节,抑制mTOR信号通路可明显抑制多囊肾病的进展,p70S6K及4E-Bp1是mTOR两个主要下游底物。用不同浓度的罗格列酮作用WT9-12细胞24h,收集细胞,提取总蛋白,采用Western blot方法检测p-mTOR/mTOR及其下游p-p70S6K/p70S6K、p-4E-Bp1/4E-Bp1。结果显示罗格列酮下调p70S6K磷酸化水平,呈时间及剂量依赖性,而对mTOR及其另一个下游底物4E-Bp1的磷酸化水平无明显影响。mTOR特异性抑制剂雷帕霉素联合罗格列酮抑制p70S6K的磷酸化作用强于单用罗格列酮,不仅表明罗格列酮是不经由mTOR抑制p70S6K的磷酸化,且雷帕霉素联合罗格列酮可对mTOR信号通路下游p70S6K的磷酸化抑制有叠加作用,也为mTOR抑制剂雷帕霉素联合罗格列酮治疗ADPKD提供理论基础。
罗格列酮的作用有PPARy依赖和非依赖两种途径。为研究罗格列酮对多囊肾囊肿衬里上皮细胞mTOR/p70S6K信号通路的影响是否通过PPARy依赖途径,在给予PPARy特异性抑制剂GW9662预孵后再给予罗格列酮发现：GW9662仅部分抵消了罗格列酮对细胞增殖的抑制作用。同样,在给予GW9662抑制剂后再给予罗格列酮可抑制罗格列酮对p-p70S6K的下调效应,进一步采用siRNA干扰技术,将PPARy siRNA瞬时转染WT9-12细胞后,采用Western blot检测发现,罗格列酮仍部分抑制p70S6K的磷酸化,这些结果表明罗格列酮对细胞增殖抑制以及对mTOR/p70S6K信号通路的抑制作用可能部分通过PPARy依赖性和非依赖性两条途径实现的。
新生血管的形成是肿瘤进展及转移的重要机制,也是多囊肾病进展的主要因素。VEGF在新生血管形成中起着重要作用。低(缺)氧可诱导低氧诱导因子-1α(HIF1-α)高表达,继而促进VEGF表达。我们对多囊肾病模型Han:SPRD大鼠的观察发现,在4周龄大鼠肾脏,VEGF表达已增加,此时局部缺氧不明显,HIF1-α的表达明显上调。但是,TNF-α表达在早期大鼠肾脏出现增加,表明VEGF的表达上调早于HIF1-α,且与局部的炎症同步。细胞实验进一步证实,TNF-a不仅能诱导多囊肾囊肿衬里上皮细胞表达VEGF,其他炎症因子如LPS也同样可诱导VEGF的表达；说明诱导VEGF的表达并不是TNF-α所特有,是炎症的共有特征。细胞实验还证实TNF-a并不能诱导HIF1-α的表达,也提示TNF-α对VEGF的作用并不通过HIF1-α。因此,早期抑制炎症反应也可减缓多囊肾病的进展。罗格列酮可下调囊肿衬里上皮细胞VEGF的表达,呈剂量依赖性,而且罗格列酮对TNFα诱导的VEGF高表达抑制更明显,可使其下降到应用TNFa前的水平。罗格列酮长期用于糖尿病的治疗,副作用小,因此适用于多囊肾病的早期治疗及长期治疗。
基于以上结果,我们联合应用罗格列酮和雷帕霉素抑制多囊肾病囊肿衬里上皮细胞的增殖及VEGF的表达,从而为多囊肾病的治疗提供新的方法。我们给予WT9-12细胞不同浓度的罗格列酮、雷帕霉素、罗格列酮+雷帕霉素,作用72h,采用MTT法检测细胞增殖,计算联合用药指数(R值)、流式细胞技术检测细胞周期及凋亡,采用realtime-PCR技术检测VEGFmRNA的表达。结果显示：罗格列酮和雷帕霉素均能抑制囊肿衬里上皮细胞增殖,抑制细胞周期的进程,并且二者联合应用有叠加作用,罗格列酮单独应用对细胞凋亡的作用不明显,但与雷帕霉素联合应用可明显促进凋亡,具有协同效应。二者具有协同效应的机制可能与罗格列酮抑制mTOR下游p70S6K的磷酸化,雷帕霉素抑制mTOR活性,双重阻滞促进了细胞凋亡。我们采取序贯用药的方式研究发现：先用雷帕霉素再序贯应用罗格列酮可增强抑制细胞增殖的作用,具有明显的协同效应(R>1)。雷帕霉素和罗格列酮可分别抑制多囊肾囊肿衬里上皮细胞VEGF的表达(VEGF表达下调30%和18%),但两药联合作用细胞时,二者的作用可明显加强(VEGF下降54%)。一方面可能与罗格列酮及雷帕霉素对mTOR信号通路的共同作用有关,另一方面,罗格列酮可抑制炎症,抑制TNFα的表达,而我们的研究发现VEGF的表达与TNFα有关,因此罗格列酮也可能通过下调TNFα抑制了VEGF的表达,从而通过多途径,不同的信号通路共同作用,抑制多囊肾病的进展。
总之,本研究表明噻唑烷二酮类PPARy激动剂罗格列酮可通过直接抑制mTOR的下游p70S6K的磷酸化而抑制囊肿衬里上皮细胞的增殖、抑制细胞周期进程及促进细胞凋亡,此作用为PPARy依赖性的。多囊肾囊肿衬里上皮细胞较RCTEC对IGF-1刺激更为敏感,而罗格列酮可完全阻断IGF-1所诱导的囊肿衬里上皮细胞的增殖。罗格列酮可下调囊肿衬里上皮细胞VEGF的表达,从而抑制多囊肾病的血管形成,此作用在有TNFα刺激下更明显,可完全抑制TNFα诱导的VEGF上调。罗格列酮与雷帕霉素联合应用可明显抑制细胞增殖和细胞周期、促进细胞凋亡及VEGF表达,二者联合应用较单药有明显的叠加效应,而序贯用药方式,即先用罗格列酮再序贯应用雷帕霉素,对细胞的增殖有明显的协同效应。
目前多囊肾病的治疗仍是个难题,我们致力于研究联合用药及不同联合用药方式对多囊肾病的作用,期望通过多种药物的联合应用,减少药物的副作用,增强疗效,将在多囊肾病的治疗中有更好的应用前景。本实验的不足之处在于缺乏动物实验结果的支持,但这项工作已经在进行中。
Autosomal dominant polycystic kidney disease (ADPKD) is one of the most common human hereditary kidney diseases with a prevalence of 1/400-1/1000, affecting more than 1.5 million people and accounting for about 5% of end-stage renal disease patients who require renal replacement therapy in China. The disease is characterized by progressive formation of multiple renal cysts affecting all segments of renal tubules. About 50% ADPKD patients eventually develop renal insufficiency in the fifth or sixth decades of life. Renal involvement is often accompanied by extra-renal manifestations, including hepatic and pancreatic cysts, cardiac valvular defects, colonic diverticulosis and intracranial aneurysm. So it is a fatal systematic disease. Great efforts have been attempted for years to find a cure for PKD. Although gene therapy seems to be a candidate, many problems need to be resolved before it could be used clinically. Seeking new drugs remains to be the focus of research at present. Dai Bing etc al from our lab reported that PPARy agonist pioglitazone prolonged survival of spontaneous mutational strain Han:SPRD rats and reduced renal cystogenesis, but the mechanism of TZDs on PKD is unknown. A report suggested mTOR pathway as a converging mechanism leading to renal cyst formation. Knowing that TZDs decrease phosphorylation and activity of p70S6 kinase (a downstream target of mTOR pathway), we hypothesized that TZDs might suppress cyst growth via inhibiting mTOR pathway. Multidrug therapy is usually required for optimal treatment of cancer. It is very likely that the treatment of ADPKD requires the same approach. Thus, we also examined the effect of rosiglitazone plus rapamycin on cystic cell growth. Rosiglitazone is an anti-glycemic agent and used clinically for patients with type 2 diabetes. We showed here for the first time that rosiglitazone decreased the proliferation of cystic epithelial cells. Addition of rosiglitazone to WT9-12 cell, an immortalized cystic epithelial cell line, inhibited cell growth in a dose-and time-dependent manner with an IC50 of 100μM. It was found in our study that 50 and 100μM rosiglitazone did not induce apoptosis in WT9-12 cells, but 200μM rosiglitazone induced an 8.18% increase in apoptotic cell death. As IC50 of growth inhibition by rosiglitazone occurred at a concentration of 100μM, the suppressive effect of rosiglitazone on cystic cell growth seemed to be largely mediated by its inhibition of cell proliferation. This was supported by the result of cell cycle analysis in which we found an increase in the number of cells in G0/G1 phase and a decrease in the number of cells in S phase after 50-100μM rosiglitazone treatment. mTOR pathway plays a critical role in regulating protein synthesis, cell growth and proliferation. The activation of mTOR results in increased p70S6K activity and translation machinery. mTOR pathway is shown to be activated and plays a role in the progression of ADPKD in both patients and mice. We therefore determined whether rosiglitazone would also affect mTOR/p70S6K in ADPKD cells. The result showed that rosiglitazone induced a dose-dependent decrease in p70S6K phosphorylation. The inhibition occurred 1 h after the treatment and was most obvious after 24 h. However, mTOR phosphorylation and 4E-BP1 phosphorylation, another downstream target of mTOR, remained unchanged after rosiglitazone treatment, indicating that the effect of rosiglitazone on p70S6K in ADPKD cells was not likely to be mediated by mTOR. The mechanisms responsible for the effects of rosiglitazone seem to involve both PPARγ-dependent and PPARγ-independent signals. Our study showed that the activation of PPARγmay play an important role in rosiglitazone-induced inhibition of p70S6K phosphorylation in WT9-12 cells, since blocking of PPARy activity by GW9662 or by knocking-down PPARγexpression largely prevented the effect of rosiglitazone.
There is angiogenesis in ADPKD. Vascular endothelial growth factor (VEGF) is also known as a potent agent in angiogenesis. VEGF is strongly induced in hypoxic conditions via hypoxia inducible factor (HIF) regulated elements of the VEGF gene. We observed the expression of VEGF, HIFαand TNFαin urine and kidney tissue of Han.SPRD in different stages (4,8,12,16 and 24 weeks) by real-time PCR. The result showed that the expression of VEGF and TNFαmRNA was upregulated in Han:SPRD in the early stage of 4 weeks, but the expression of HIF1αwas normal. The expression of HIF1αbegan increasing from 8 weeks, indicating that VEGF upregulation might be related to TNFa in the early stage. To examine whether TNFαcontributed to the regulation of VEGF, WT9-12 cells were treated with TNFα, and the expression was detected by real-time PCR. TNFα(20ng/ml) led to a 15-fold increase of VEGF mRNA, but TNFαdid not induce increase of HIF1α. To determine whether other cytokines could also upregulate VEGF expression, WT9-12 cells were treated by LPS (20ng/ml), IGF-1(20ng/ml) and TGFβ(20ng/ml). The result showed that VEGF was increased by LPS, but not by IGF-1 and TGFβ, indicating that inflammatory cytokines, but not mitogens, could induce VEGF expression.
In addition, we investigated whether rosiglitazone could inhibit angiogenesis in ADPKD. WT9-12 cells were treated with different concentrations of rosiglitazone, and the expression of VEGF was detected by real-time PCR. The result showed that rosiglitazone downregulated the level of VEGF, and 50μM rosiglitazone decreased VEGF level by 30%. However, rosiglitazone was able to block TNFa-induced VEGF expression in WT9-12 cells completely. Rosiglitazone has been used to treat diabetes for many years with few reported adverse effects. We supposed that rosiglitazone would also be suitable for ADPKD therapy, especially in the early stages of the disease.
Since both rosiglitazone and rapamycin are clinical drugs that may have potential for ADPKD therapy, we tested the effects of combined use of the two drugs on cystic cell growth.50ng/ml rapamycin plus 50μM rosiglitazone significantly increased the inhibitory effect on cell growth as compared with either of the two drugs alone (p<0.05). R Value was 1.01, indicating an additive effect. This additive effect was still present when the dosage of rosiglitazone was increased to 100 and 200μM, R value being 1.14 and 1.08 respectively. Knowing that sequence-specific synergism is optimal cancer chemotherapy, we supposed that the same strategy may also be suitable for ADPKD treatment. We therefore further assessed the effect of sequential treatment with rapamycin and rosiglitazone on cystic cell proliferation. Interestingly, combination use in the sequence of rapamycin plus rosiglitazone, the R value was greater than 1 (mean 1.62), indicating that the interaction was synergistic. However, no such a synergistic effect was observed when rapamycin was used after discontinuation of rosiglitazone. Further, we investigated the effect of concomitant use of the two drugs on VEGF. Combination of 50ng/ml rapamycin and 50μM rosiglitazone decreased the expression of VEGF by 54%, as compared with either of the two drugs alone (rosiglitazone 18% and rapamycin 30%).
In conclusion, rosiglitazone (a thiazolidinedione derivative) suppressed cystic cell growth. This effect was mediated partially via an mTOR-independent inhibition of p70S6K phosphorylation and PPARγ-independent manner. Rosiglitzone also inhibited angiogenesis by downregulating VEGF expression. Combination of rosiglitazone and rapamycin increased the efficiency of cell growth and angiogenesis inhibition, as compared with either of the two drugs alone. As rosiglitazone is an anti-diabetic drug clinically used for long-term treatment, it may have a potential for ADPKD therapy. Combination therapy may be a potential and a trend for ADPKD therapy.
多年来,肾脏病学者一直致力于寻找治疗多囊肾病的有效方法,基因治疗目前存在许多障碍有待克服,通过药物治疗延缓肾衰竭进展是目前的主要研究方向。多囊肾病治疗需要早期、长期用药,因此药物的安全性成为研究者关注的焦点。由于ADPKD的分子发病及调控机制非常复杂,很多问题尚未阐明,因此针对不同发病环节研发不同药物,联合用于治疗不仅可增强疗效,而且减少不良反应,将具有潜在的的应用前景。
噻唑烷二酮类PPARy激动剂,如吡格列酮和罗格列酮等,主要用于糖尿病治疗。近年来研究发现,噻唑烷二酮类PPARy激动剂可抑制多种肿瘤细胞的增殖。2001年Muto等首先观察到吡格列酮可延长多囊蛋白1基因敲除小鼠(pkdl-/-)的生存期(通常在胎儿期死亡)；我科先前动物实验发现,罗格列酮可抑制Han:SPRD大鼠囊肿的进展,延长大鼠生存期,但其机制尚未完全阐明。
mTOR信号通路异常激活被认为是多囊肾病发生的重要机制,新生血管的形成也可促进多囊肾病的进展；因此我们探讨罗格列酮对mTOR信号通路的作用,研究其对新生血管形成中起重要作用的血管内皮生长因子(VEGF)的作用；同时观察罗格列酮与mTOR抑制剂雷帕霉素联合应用对延缓ADPKD进展的作用,为治疗多囊肾病寻找新的途径。
为此,我们首先研究了罗格列酮对多囊肾囊肿衬里上皮细胞增殖、细胞周期及细胞凋亡的作用。多囊肾囊肿衬里上皮细胞系(WT9-12)由美国哈佛大学周晶教授馈赠。培养的WT9-12细胞给予不同浓度的罗格列酮培养24、48、72h,采用MTT法检测结果显示：罗格列酮明显抑制WT9-12细胞的增殖,呈时间、剂量依赖性。预72h时,50%抑制率浓度为100μM。采用流式细胞技术检测细胞周期及凋亡,结果显示罗格列酮明显抑制WT9-12细胞周期,使细胞停滞于G0／G1期,G0／G1期细胞增加30%,与对照组比较有差异有显著性意义(P<0.05),其对细胞凋亡的影响不大,罗格列酮浓度高达200μM时,仅使细胞凋亡增加6%。多囊肾病的发展过程伴有胰岛素生长因子1(IGF-1)表达的增加,我们的研究发现囊肿衬里上皮细胞对IGF-1的刺激反应较其它永生化肾小管上皮细胞(RCTEC)更为明显。IGF-150ng/ml可诱导囊肿衬里上皮细胞增殖增加20%,而对RCTEC无明显影响。罗格列酮可完全抑制IGF-1诱导的WT9-12细胞增殖,该作用在罗格列酮12.5μM时即表现明显的抑制作用。说明罗格列酮在有IGF-1作用下对囊肿衬里上皮细胞的增殖抑制作用更为明显。
mTOR信号通路异常激活在多囊肾病发病及发展中起重要作用,为多种发病机制的共同环节,抑制mTOR信号通路可明显抑制多囊肾病的进展,p70S6K及4E-Bp1是mTOR两个主要下游底物。用不同浓度的罗格列酮作用WT9-12细胞24h,收集细胞,提取总蛋白,采用Western blot方法检测p-mTOR/mTOR及其下游p-p70S6K/p70S6K、p-4E-Bp1/4E-Bp1。结果显示罗格列酮下调p70S6K磷酸化水平,呈时间及剂量依赖性,而对mTOR及其另一个下游底物4E-Bp1的磷酸化水平无明显影响。mTOR特异性抑制剂雷帕霉素联合罗格列酮抑制p70S6K的磷酸化作用强于单用罗格列酮,不仅表明罗格列酮是不经由mTOR抑制p70S6K的磷酸化,且雷帕霉素联合罗格列酮可对mTOR信号通路下游p70S6K的磷酸化抑制有叠加作用,也为mTOR抑制剂雷帕霉素联合罗格列酮治疗ADPKD提供理论基础。
罗格列酮的作用有PPARy依赖和非依赖两种途径。为研究罗格列酮对多囊肾囊肿衬里上皮细胞mTOR/p70S6K信号通路的影响是否通过PPARy依赖途径,在给予PPARy特异性抑制剂GW9662预孵后再给予罗格列酮发现：GW9662仅部分抵消了罗格列酮对细胞增殖的抑制作用。同样,在给予GW9662抑制剂后再给予罗格列酮可抑制罗格列酮对p-p70S6K的下调效应,进一步采用siRNA干扰技术,将PPARy siRNA瞬时转染WT9-12细胞后,采用Western blot检测发现,罗格列酮仍部分抑制p70S6K的磷酸化,这些结果表明罗格列酮对细胞增殖抑制以及对mTOR/p70S6K信号通路的抑制作用可能部分通过PPARy依赖性和非依赖性两条途径实现的。
新生血管的形成是肿瘤进展及转移的重要机制,也是多囊肾病进展的主要因素。VEGF在新生血管形成中起着重要作用。低(缺)氧可诱导低氧诱导因子-1α(HIF1-α)高表达,继而促进VEGF表达。我们对多囊肾病模型Han:SPRD大鼠的观察发现,在4周龄大鼠肾脏,VEGF表达已增加,此时局部缺氧不明显,HIF1-α的表达明显上调。但是,TNF-α表达在早期大鼠肾脏出现增加,表明VEGF的表达上调早于HIF1-α,且与局部的炎症同步。细胞实验进一步证实,TNF-a不仅能诱导多囊肾囊肿衬里上皮细胞表达VEGF,其他炎症因子如LPS也同样可诱导VEGF的表达；说明诱导VEGF的表达并不是TNF-α所特有,是炎症的共有特征。细胞实验还证实TNF-a并不能诱导HIF1-α的表达,也提示TNF-α对VEGF的作用并不通过HIF1-α。因此,早期抑制炎症反应也可减缓多囊肾病的进展。罗格列酮可下调囊肿衬里上皮细胞VEGF的表达,呈剂量依赖性,而且罗格列酮对TNFα诱导的VEGF高表达抑制更明显,可使其下降到应用TNFa前的水平。罗格列酮长期用于糖尿病的治疗,副作用小,因此适用于多囊肾病的早期治疗及长期治疗。
基于以上结果,我们联合应用罗格列酮和雷帕霉素抑制多囊肾病囊肿衬里上皮细胞的增殖及VEGF的表达,从而为多囊肾病的治疗提供新的方法。我们给予WT9-12细胞不同浓度的罗格列酮、雷帕霉素、罗格列酮+雷帕霉素,作用72h,采用MTT法检测细胞增殖,计算联合用药指数(R值)、流式细胞技术检测细胞周期及凋亡,采用realtime-PCR技术检测VEGFmRNA的表达。结果显示：罗格列酮和雷帕霉素均能抑制囊肿衬里上皮细胞增殖,抑制细胞周期的进程,并且二者联合应用有叠加作用,罗格列酮单独应用对细胞凋亡的作用不明显,但与雷帕霉素联合应用可明显促进凋亡,具有协同效应。二者具有协同效应的机制可能与罗格列酮抑制mTOR下游p70S6K的磷酸化,雷帕霉素抑制mTOR活性,双重阻滞促进了细胞凋亡。我们采取序贯用药的方式研究发现：先用雷帕霉素再序贯应用罗格列酮可增强抑制细胞增殖的作用,具有明显的协同效应(R>1)。雷帕霉素和罗格列酮可分别抑制多囊肾囊肿衬里上皮细胞VEGF的表达(VEGF表达下调30%和18%),但两药联合作用细胞时,二者的作用可明显加强(VEGF下降54%)。一方面可能与罗格列酮及雷帕霉素对mTOR信号通路的共同作用有关,另一方面,罗格列酮可抑制炎症,抑制TNFα的表达,而我们的研究发现VEGF的表达与TNFα有关,因此罗格列酮也可能通过下调TNFα抑制了VEGF的表达,从而通过多途径,不同的信号通路共同作用,抑制多囊肾病的进展。
总之,本研究表明噻唑烷二酮类PPARy激动剂罗格列酮可通过直接抑制mTOR的下游p70S6K的磷酸化而抑制囊肿衬里上皮细胞的增殖、抑制细胞周期进程及促进细胞凋亡,此作用为PPARy依赖性的。多囊肾囊肿衬里上皮细胞较RCTEC对IGF-1刺激更为敏感,而罗格列酮可完全阻断IGF-1所诱导的囊肿衬里上皮细胞的增殖。罗格列酮可下调囊肿衬里上皮细胞VEGF的表达,从而抑制多囊肾病的血管形成,此作用在有TNFα刺激下更明显,可完全抑制TNFα诱导的VEGF上调。罗格列酮与雷帕霉素联合应用可明显抑制细胞增殖和细胞周期、促进细胞凋亡及VEGF表达,二者联合应用较单药有明显的叠加效应,而序贯用药方式,即先用罗格列酮再序贯应用雷帕霉素,对细胞的增殖有明显的协同效应。
目前多囊肾病的治疗仍是个难题,我们致力于研究联合用药及不同联合用药方式对多囊肾病的作用,期望通过多种药物的联合应用,减少药物的副作用,增强疗效,将在多囊肾病的治疗中有更好的应用前景。本实验的不足之处在于缺乏动物实验结果的支持,但这项工作已经在进行中。
Autosomal dominant polycystic kidney disease (ADPKD) is one of the most common human hereditary kidney diseases with a prevalence of 1/400-1/1000, affecting more than 1.5 million people and accounting for about 5% of end-stage renal disease patients who require renal replacement therapy in China. The disease is characterized by progressive formation of multiple renal cysts affecting all segments of renal tubules. About 50% ADPKD patients eventually develop renal insufficiency in the fifth or sixth decades of life. Renal involvement is often accompanied by extra-renal manifestations, including hepatic and pancreatic cysts, cardiac valvular defects, colonic diverticulosis and intracranial aneurysm. So it is a fatal systematic disease. Great efforts have been attempted for years to find a cure for PKD. Although gene therapy seems to be a candidate, many problems need to be resolved before it could be used clinically. Seeking new drugs remains to be the focus of research at present. Dai Bing etc al from our lab reported that PPARy agonist pioglitazone prolonged survival of spontaneous mutational strain Han:SPRD rats and reduced renal cystogenesis, but the mechanism of TZDs on PKD is unknown. A report suggested mTOR pathway as a converging mechanism leading to renal cyst formation. Knowing that TZDs decrease phosphorylation and activity of p70S6 kinase (a downstream target of mTOR pathway), we hypothesized that TZDs might suppress cyst growth via inhibiting mTOR pathway. Multidrug therapy is usually required for optimal treatment of cancer. It is very likely that the treatment of ADPKD requires the same approach. Thus, we also examined the effect of rosiglitazone plus rapamycin on cystic cell growth. Rosiglitazone is an anti-glycemic agent and used clinically for patients with type 2 diabetes. We showed here for the first time that rosiglitazone decreased the proliferation of cystic epithelial cells. Addition of rosiglitazone to WT9-12 cell, an immortalized cystic epithelial cell line, inhibited cell growth in a dose-and time-dependent manner with an IC50 of 100μM. It was found in our study that 50 and 100μM rosiglitazone did not induce apoptosis in WT9-12 cells, but 200μM rosiglitazone induced an 8.18% increase in apoptotic cell death. As IC50 of growth inhibition by rosiglitazone occurred at a concentration of 100μM, the suppressive effect of rosiglitazone on cystic cell growth seemed to be largely mediated by its inhibition of cell proliferation. This was supported by the result of cell cycle analysis in which we found an increase in the number of cells in G0/G1 phase and a decrease in the number of cells in S phase after 50-100μM rosiglitazone treatment. mTOR pathway plays a critical role in regulating protein synthesis, cell growth and proliferation. The activation of mTOR results in increased p70S6K activity and translation machinery. mTOR pathway is shown to be activated and plays a role in the progression of ADPKD in both patients and mice. We therefore determined whether rosiglitazone would also affect mTOR/p70S6K in ADPKD cells. The result showed that rosiglitazone induced a dose-dependent decrease in p70S6K phosphorylation. The inhibition occurred 1 h after the treatment and was most obvious after 24 h. However, mTOR phosphorylation and 4E-BP1 phosphorylation, another downstream target of mTOR, remained unchanged after rosiglitazone treatment, indicating that the effect of rosiglitazone on p70S6K in ADPKD cells was not likely to be mediated by mTOR. The mechanisms responsible for the effects of rosiglitazone seem to involve both PPARγ-dependent and PPARγ-independent signals. Our study showed that the activation of PPARγmay play an important role in rosiglitazone-induced inhibition of p70S6K phosphorylation in WT9-12 cells, since blocking of PPARy activity by GW9662 or by knocking-down PPARγexpression largely prevented the effect of rosiglitazone.
There is angiogenesis in ADPKD. Vascular endothelial growth factor (VEGF) is also known as a potent agent in angiogenesis. VEGF is strongly induced in hypoxic conditions via hypoxia inducible factor (HIF) regulated elements of the VEGF gene. We observed the expression of VEGF, HIFαand TNFαin urine and kidney tissue of Han.SPRD in different stages (4,8,12,16 and 24 weeks) by real-time PCR. The result showed that the expression of VEGF and TNFαmRNA was upregulated in Han:SPRD in the early stage of 4 weeks, but the expression of HIF1αwas normal. The expression of HIF1αbegan increasing from 8 weeks, indicating that VEGF upregulation might be related to TNFa in the early stage. To examine whether TNFαcontributed to the regulation of VEGF, WT9-12 cells were treated with TNFα, and the expression was detected by real-time PCR. TNFα(20ng/ml) led to a 15-fold increase of VEGF mRNA, but TNFαdid not induce increase of HIF1α. To determine whether other cytokines could also upregulate VEGF expression, WT9-12 cells were treated by LPS (20ng/ml), IGF-1(20ng/ml) and TGFβ(20ng/ml). The result showed that VEGF was increased by LPS, but not by IGF-1 and TGFβ, indicating that inflammatory cytokines, but not mitogens, could induce VEGF expression.
In addition, we investigated whether rosiglitazone could inhibit angiogenesis in ADPKD. WT9-12 cells were treated with different concentrations of rosiglitazone, and the expression of VEGF was detected by real-time PCR. The result showed that rosiglitazone downregulated the level of VEGF, and 50μM rosiglitazone decreased VEGF level by 30%. However, rosiglitazone was able to block TNFa-induced VEGF expression in WT9-12 cells completely. Rosiglitazone has been used to treat diabetes for many years with few reported adverse effects. We supposed that rosiglitazone would also be suitable for ADPKD therapy, especially in the early stages of the disease.
Since both rosiglitazone and rapamycin are clinical drugs that may have potential for ADPKD therapy, we tested the effects of combined use of the two drugs on cystic cell growth.50ng/ml rapamycin plus 50μM rosiglitazone significantly increased the inhibitory effect on cell growth as compared with either of the two drugs alone (p<0.05). R Value was 1.01, indicating an additive effect. This additive effect was still present when the dosage of rosiglitazone was increased to 100 and 200μM, R value being 1.14 and 1.08 respectively. Knowing that sequence-specific synergism is optimal cancer chemotherapy, we supposed that the same strategy may also be suitable for ADPKD treatment. We therefore further assessed the effect of sequential treatment with rapamycin and rosiglitazone on cystic cell proliferation. Interestingly, combination use in the sequence of rapamycin plus rosiglitazone, the R value was greater than 1 (mean 1.62), indicating that the interaction was synergistic. However, no such a synergistic effect was observed when rapamycin was used after discontinuation of rosiglitazone. Further, we investigated the effect of concomitant use of the two drugs on VEGF. Combination of 50ng/ml rapamycin and 50μM rosiglitazone decreased the expression of VEGF by 54%, as compared with either of the two drugs alone (rosiglitazone 18% and rapamycin 30%).
In conclusion, rosiglitazone (a thiazolidinedione derivative) suppressed cystic cell growth. This effect was mediated partially via an mTOR-independent inhibition of p70S6K phosphorylation and PPARγ-independent manner. Rosiglitzone also inhibited angiogenesis by downregulating VEGF expression. Combination of rosiglitazone and rapamycin increased the efficiency of cell growth and angiogenesis inhibition, as compared with either of the two drugs alone. As rosiglitazone is an anti-diabetic drug clinically used for long-term treatment, it may have a potential for ADPKD therapy. Combination therapy may be a potential and a trend for ADPKD therapy.
引文
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