小G蛋白Rho/Rock信号转导通路在大鼠肾间质纤维化中的作用机制探讨
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摘要
肾间质纤维化(Renal interstitial fibrosis，RIF)是多种肾脏疾病慢性进展的共同病理改变，是各种原因引起的肾间质炎型浸润、肾脏固有细胞损伤、活化与表型改变，最终导致细胞外基质过度沉积与肾小管间质正常结构毁损。RIF是一个由多种分子和多种靶细胞相互作用的复杂过程，而其中何种因素在RIF的发生与发展阶段起关键作用，尚无明确的结论。因此，研究RIF形成过程中复杂的信号转导通路及其相互作用，对于阐明其发病机制、寻找延缓RIF进展的有效分子靶标具有重要意义。
近年来，Rho／Rock信号转导通路在多种器官组织慢性炎性纤维化中的作用受到了日益广泛的关注。有研究表明，在RIF动物模型中选择性阻断该信号通路可以改善肾小管间质损伤的进展和预后，然而其确切的作用机制尚不清楚。
为了证实Rho／Rock信号转导通路在RIF形成中的意义，并对其可能的作用机制进行初步探讨，我们进行了下面体内和体外两部分研究。
第一部分：Rho／Rock信号转导通路在实验性肾间质纤维化大鼠模型中的意义
目的：观察小G蛋白Rho／Rock信号转导通路关键信号分子Rock-I在肾间质纤维化大鼠不同阶段肾组织中的表达规律、激活状态，及其与肾间质纤维化进展程度的相关性。并对其在肾间质纤维化形成中的重要性和可能的作用机制进行初步探讨。
方法：构建单侧输尿管结扎(unilateral ureteral obstruction，UUO)大鼠模型，分别于纤维化进展不同阶段处死大鼠，留取标本供形态学和分子生物学检查。采用RT-PCR、Western blotting法从基因和蛋白水平检测Rock-I表达水平；采用Western blotting检测Rock-I底物——肌球蛋白磷酸酶结合亚单位第697位苏氨酸(MBS Thr-697)的磷
南京医科大学硕士学位论文
酸化水平，作为Rho/Rock信号转导通路活化指标。将上述指标与肾
间质纤维化指数进行相关分析，了解该信号通路的活性与肾间质纤维
化进展之间的关系。
结果:(1)UUO模型组肾组织第3天即可见皮质区小管间质少量炎
性细胞浸润，于第7天达高峰并伴有集合管扩张和间质水肿，部分近
髓肾小球球囊周围和间质出现轻度纤维化;随着模型进展于第14天
出现近曲小管扩张、上皮萎缩和广泛间质纤维化;至模型晚期(第21
天)’肾组织基本为纤维样结构替代，多数肾小球发生球性硬化。(2)
Rock一ImRNA在肾间质纤维化早期(第3天)即明显上调(F二47.73，
尸<0.01)，于第7天达高峰后呈下降趋势，至uuo模型后期(第21
天)降至基础表达水平。其表达较肌成纤维细胞标志基因。一平滑肌肌
动蛋白(a一smooth musele aetin，a一SMA)提前达到高峰，二者呈显
著正相关(r=0.61，p<0.01)。(3)Western blotting结果显示UUO模
型大鼠肾组织中Rock一I蛋白表达水平明显高于对照组(F=43 .39，
尸<0.01)，并随肾间质纤维化相对面积的扩大而持续增加，二者呈显
著正相关(:=0.82，P<0.01)。(4)UUO模型组大鼠肾组织MBS Thr一697
磷酸化水平较对照组显著增高(F二33.32，尸<0 .01)，并且与肾间质纤
维化指数呈显著正相关(:二0.63，尸<0.05)。
结论:(1)’肾间质纤维化形成过程中存在Rock一I的异常表达与激活。
(2) Rho瓜ock信号转导通路参与UUO大鼠肾间质纤维化的形成过
程，并且可能通过调节肌成纤维细胞的增生来发挥作用。
第二部分:Rho/Rock信号转导通路在大鼠肾脏成纤维细胞表型改变
中的作用
目的:观察Rho/R ock信号通路选择性阻断剂丫27632对正常大鼠肾
脏成纤维细胞株(NRK)a一SMA表达的影响。探讨该信号通路在肌
成纤维细胞表型形成与维持中的作用。
方法:正常大鼠肾脏成纤维细胞株(NRK)能够较为均一地表达高
水平的肌成纤维细胞标志基因—a一SMA。本实验以Rho/R ock信号
南京医科大学硕士学位论文
转导通路选择性阻断剂丫27632干预NRK细胞，采用免疫荧光法观
察细胞内F一aetin聚合状态、Western blotting法观察MBs磷酸化水平，
将这两项作为该信号通路的活性指标。同时观察丫27632对a一SMA
表达的影响，并分析a一SMA表达水平与Rho/Rock信号通路的活性状
态之间的相关性。
结果:(1)30协MY-27632干预NRK细胞，30分钟后即开始细胞形
态的显著改变与细胞内肌动蛋白纤维的解聚，12~24小时后微丝结构
重现，与胞浆MBS磷酸化水平的改变趋势一致(30分钟、6，卜时、
12，J、时、24，J、时分另，」为下降至43.6%、39.8%、58.2%、79.9%)。
(2)a一SMA蛋白表达在30分钟时较对照组即开始下降(t=一11.1，
p<0 .01)，12小时降至对照组的50%以下。(3)a一SMA表达水平与
MBs磷酸化状态呈显著正相关(r=0.708，p<0.01)。
结论:Rock选择性阻断剂丫27632能够阻断NRK细胞Rho/Rock信
号通路的活性，从而抑制a一SMA的表达。Rho/R ock可能是调控肌成
纤维细胞标志基因一一a一SMA表达和肌成纤维细胞表型形成的关键
信号通路，并且可能通过这一机制在RIF的形成过程中扮演重要角
色。
Renal interstitial fibrosis ( RIF) , a common finding in patients with progressive chronic renal disease of all types, arises because of a complex interplay between factors in the tubular lumen, tubular epithelial cells, peritubular capillaries, resident and infiltrating interstitial cells and extracellular matrix. Several factors: macrophages, growth factors, hypoxia, cytokines are involved in the pathogenesis of interstitial fibrosis. All these changes result in excessive matrix deposition that leads to tissue destruction and impairment of renal function . It is evident that the process of renal fibrosis is a complicated one with several cellular and molecular mediators interacting in concert, however, the mechanisms of initiation and maintenance of renal fibrosis remain obscure. Studies concerning the complex signal transduction and their interplay may contribute to the illustration of the mechanism of RIF and the prevention of RIF progression.
Great inportance has been attached to the contribution of Rho/Rock signaling to chronic inflammatory fibrosis of several organs, such as lung, heart and liver. Evidence accumulated that this signaling had protective effects on renal function, however, the mechanisms underlying are to be elucidated.
During the progression of RIF, fibroblast and tubular epithelial cell undergo a phenotipic change into myofibroblast, the major source of ECM. Recently, it was reported that Rho/Rock signaling may play a key role in the transformation and maintainence of myofibroblast. So it was suggested the fibrogenic effect of Rho/Rock signaling may result from the induction of myofibroblast transformation.
The aim of our invo and in vitro experiments was to illustrate the significance of Rho/Rock signaling in the pathogenesis of RIF, and to
clarify the mechanism underlying.
Part I: the contribution of Rho/Rock signaling in the development of Renal interstitial in UUO rat
Objective To observe the expression law of Rock-I , a key effector of Rho, and its functional activation in renal tissue from UUO kidneys, and to elucidate the role of Rho/Rock signaling in the progression of renal interstitial fibrosis.
Methods Expression of Rock-I mRNA and protein were measured by RT-PCR and Western blotting, respectively. The phosphorylation of MBS
(binding subunit of myosin phosphatase)--a substrate of Rock-I was
detected by Western blotting, as the mark of functional activation of the kinase. And the relationship between the activation of Rho/Rock signaling and the development of RIF was clarified as well.
Results ( 1) The expression of Rockl mRNA was elevated before the onset of RIF(the 3rd day after the experiment) (F=47.73, P<0.01). The expression peak of Rock-I mRNA was prior to that of ct-SMA, and there was a positive correlation between them ( r= 0.61, P<0.01) . (2) The expression of Rock-I protein was significantly enhanced in the UUO group than in the Vehicle control (F=43.39, .P<0.01) , and its increase accompanied the the fibrostic index ( r=0.82, P<0.01 ) . ( 3 ) The phosphorylation of MBS was significantly upregulated in the UUO group ( F=33.32, P<0.01) , and it was positive correlated with the fibrostic index (r=0.63, P<0.05) .
Conclusions (1 ) We found the enhanced expression and functional activation of Rock-I in renal tissue from UUO rats . Rho/Rock signaling may play an important role in the progression of RIF. ( 2) Rock-I may be an useful marker and a potential target for therapeutic intervention with a view to slowing, preventing, and in some senses even reversing the
progression of renal diseases.
Part II: the contribution of Rho/Rock signaling in the transformation and maintainence of myofibroblast
Objective To evaluate whether the activation of Rho/Rock signaling
modulates the expression of a characteristic marker of myofibroblast--
a-SMA in a fibroblastic clone of normal rat kidney cells (NRK cell lines).
Methods The rat kidney fibroblast cell line NRK was treated with a selective inhibitor of Rock, Y-27632. Immunofluorescence was applied to display the polyme
近年来，Rho／Rock信号转导通路在多种器官组织慢性炎性纤维化中的作用受到了日益广泛的关注。有研究表明，在RIF动物模型中选择性阻断该信号通路可以改善肾小管间质损伤的进展和预后，然而其确切的作用机制尚不清楚。
为了证实Rho／Rock信号转导通路在RIF形成中的意义，并对其可能的作用机制进行初步探讨，我们进行了下面体内和体外两部分研究。
第一部分：Rho／Rock信号转导通路在实验性肾间质纤维化大鼠模型中的意义
目的：观察小G蛋白Rho／Rock信号转导通路关键信号分子Rock-I在肾间质纤维化大鼠不同阶段肾组织中的表达规律、激活状态，及其与肾间质纤维化进展程度的相关性。并对其在肾间质纤维化形成中的重要性和可能的作用机制进行初步探讨。
方法：构建单侧输尿管结扎(unilateral ureteral obstruction，UUO)大鼠模型，分别于纤维化进展不同阶段处死大鼠，留取标本供形态学和分子生物学检查。采用RT-PCR、Western blotting法从基因和蛋白水平检测Rock-I表达水平；采用Western blotting检测Rock-I底物——肌球蛋白磷酸酶结合亚单位第697位苏氨酸(MBS Thr-697)的磷
南京医科大学硕士学位论文
酸化水平，作为Rho/Rock信号转导通路活化指标。将上述指标与肾
间质纤维化指数进行相关分析，了解该信号通路的活性与肾间质纤维
化进展之间的关系。
结果:(1)UUO模型组肾组织第3天即可见皮质区小管间质少量炎
性细胞浸润，于第7天达高峰并伴有集合管扩张和间质水肿，部分近
髓肾小球球囊周围和间质出现轻度纤维化;随着模型进展于第14天
出现近曲小管扩张、上皮萎缩和广泛间质纤维化;至模型晚期(第21
天)’肾组织基本为纤维样结构替代，多数肾小球发生球性硬化。(2)
Rock一ImRNA在肾间质纤维化早期(第3天)即明显上调(F二47.73，
尸<0.01)，于第7天达高峰后呈下降趋势，至uuo模型后期(第21
天)降至基础表达水平。其表达较肌成纤维细胞标志基因。一平滑肌肌
动蛋白(a一smooth musele aetin，a一SMA)提前达到高峰，二者呈显
著正相关(r=0.61，p<0.01)。(3)Western blotting结果显示UUO模
型大鼠肾组织中Rock一I蛋白表达水平明显高于对照组(F=43 .39，
尸<0.01)，并随肾间质纤维化相对面积的扩大而持续增加，二者呈显
著正相关(:=0.82，P<0.01)。(4)UUO模型组大鼠肾组织MBS Thr一697
磷酸化水平较对照组显著增高(F二33.32，尸<0 .01)，并且与肾间质纤
维化指数呈显著正相关(:二0.63，尸<0.05)。
结论:(1)’肾间质纤维化形成过程中存在Rock一I的异常表达与激活。
(2) Rho瓜ock信号转导通路参与UUO大鼠肾间质纤维化的形成过
程，并且可能通过调节肌成纤维细胞的增生来发挥作用。
第二部分:Rho/Rock信号转导通路在大鼠肾脏成纤维细胞表型改变
中的作用
目的:观察Rho/R ock信号通路选择性阻断剂丫27632对正常大鼠肾
脏成纤维细胞株(NRK)a一SMA表达的影响。探讨该信号通路在肌
成纤维细胞表型形成与维持中的作用。
方法:正常大鼠肾脏成纤维细胞株(NRK)能够较为均一地表达高
水平的肌成纤维细胞标志基因—a一SMA。本实验以Rho/R ock信号
南京医科大学硕士学位论文
转导通路选择性阻断剂丫27632干预NRK细胞，采用免疫荧光法观
察细胞内F一aetin聚合状态、Western blotting法观察MBs磷酸化水平，
将这两项作为该信号通路的活性指标。同时观察丫27632对a一SMA
表达的影响，并分析a一SMA表达水平与Rho/Rock信号通路的活性状
态之间的相关性。
结果:(1)30协MY-27632干预NRK细胞，30分钟后即开始细胞形
态的显著改变与细胞内肌动蛋白纤维的解聚，12~24小时后微丝结构
重现，与胞浆MBS磷酸化水平的改变趋势一致(30分钟、6，卜时、
12，J、时、24，J、时分另，」为下降至43.6%、39.8%、58.2%、79.9%)。
(2)a一SMA蛋白表达在30分钟时较对照组即开始下降(t=一11.1，
p<0 .01)，12小时降至对照组的50%以下。(3)a一SMA表达水平与
MBs磷酸化状态呈显著正相关(r=0.708，p<0.01)。
结论:Rock选择性阻断剂丫27632能够阻断NRK细胞Rho/Rock信
号通路的活性，从而抑制a一SMA的表达。Rho/R ock可能是调控肌成
纤维细胞标志基因一一a一SMA表达和肌成纤维细胞表型形成的关键
信号通路，并且可能通过这一机制在RIF的形成过程中扮演重要角
色。
Renal interstitial fibrosis ( RIF) , a common finding in patients with progressive chronic renal disease of all types, arises because of a complex interplay between factors in the tubular lumen, tubular epithelial cells, peritubular capillaries, resident and infiltrating interstitial cells and extracellular matrix. Several factors: macrophages, growth factors, hypoxia, cytokines are involved in the pathogenesis of interstitial fibrosis. All these changes result in excessive matrix deposition that leads to tissue destruction and impairment of renal function . It is evident that the process of renal fibrosis is a complicated one with several cellular and molecular mediators interacting in concert, however, the mechanisms of initiation and maintenance of renal fibrosis remain obscure. Studies concerning the complex signal transduction and their interplay may contribute to the illustration of the mechanism of RIF and the prevention of RIF progression.
Great inportance has been attached to the contribution of Rho/Rock signaling to chronic inflammatory fibrosis of several organs, such as lung, heart and liver. Evidence accumulated that this signaling had protective effects on renal function, however, the mechanisms underlying are to be elucidated.
During the progression of RIF, fibroblast and tubular epithelial cell undergo a phenotipic change into myofibroblast, the major source of ECM. Recently, it was reported that Rho/Rock signaling may play a key role in the transformation and maintainence of myofibroblast. So it was suggested the fibrogenic effect of Rho/Rock signaling may result from the induction of myofibroblast transformation.
The aim of our invo and in vitro experiments was to illustrate the significance of Rho/Rock signaling in the pathogenesis of RIF, and to
clarify the mechanism underlying.
Part I: the contribution of Rho/Rock signaling in the development of Renal interstitial in UUO rat
Objective To observe the expression law of Rock-I , a key effector of Rho, and its functional activation in renal tissue from UUO kidneys, and to elucidate the role of Rho/Rock signaling in the progression of renal interstitial fibrosis.
Methods Expression of Rock-I mRNA and protein were measured by RT-PCR and Western blotting, respectively. The phosphorylation of MBS
(binding subunit of myosin phosphatase)--a substrate of Rock-I was
detected by Western blotting, as the mark of functional activation of the kinase. And the relationship between the activation of Rho/Rock signaling and the development of RIF was clarified as well.
Results ( 1) The expression of Rockl mRNA was elevated before the onset of RIF(the 3rd day after the experiment) (F=47.73, P<0.01). The expression peak of Rock-I mRNA was prior to that of ct-SMA, and there was a positive correlation between them ( r= 0.61, P<0.01) . (2) The expression of Rock-I protein was significantly enhanced in the UUO group than in the Vehicle control (F=43.39, .P<0.01) , and its increase accompanied the the fibrostic index ( r=0.82, P<0.01 ) . ( 3 ) The phosphorylation of MBS was significantly upregulated in the UUO group ( F=33.32, P<0.01) , and it was positive correlated with the fibrostic index (r=0.63, P<0.05) .
Conclusions (1 ) We found the enhanced expression and functional activation of Rock-I in renal tissue from UUO rats . Rho/Rock signaling may play an important role in the progression of RIF. ( 2) Rock-I may be an useful marker and a potential target for therapeutic intervention with a view to slowing, preventing, and in some senses even reversing the
progression of renal diseases.
Part II: the contribution of Rho/Rock signaling in the transformation and maintainence of myofibroblast
Objective To evaluate whether the activation of Rho/Rock signaling
modulates the expression of a characteristic marker of myofibroblast--
a-SMA in a fibroblastic clone of normal rat kidney cells (NRK cell lines).
Methods The rat kidney fibroblast cell line NRK was treated with a selective inhibitor of Rock, Y-27632. Immunofluorescence was applied to display the polyme
引文
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29. Mack CP, Somlyo AV, Hautmann M, e tal. Smooth muscle differentiation marker gene expression is regulated by RhoA-mediated actin polymerization[J]. J Biol Chem. 2001, 276(1):341-347
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22. Tada S, Iwamoto H,Nakamuta M, e tal. A selective ROCK inhibitor, Y27632, prevents dimethylnitrosamine-induced hepatic fibrosis in rats[J]. J Hepatol, 2001, 34(4):529-536.
23. Iwamoto H, Nakamuta M, Tada S, e tal. A p160ROCK-specific inhibitor, Y-27632, attenuates rat hepatic stellate cell growth[J]. J Hepatol, 2000, 32(5):762-770.
24. Satoh S, Yamaguchi T, Hitomi A, e tal. Fasudil attenuates interstitial fibrosis in rat kidneys with unilateral ureteral obstruction [J]. Eur J Pharmacol, 2002,455(2-3):169-174
25．林琼真．肌成纤维细胞与肾间质纤维化[R]．河北医科大学学报，2001，22(05)：304-307
26. Schurch W, Seemayer TA, Gabbiani G. The myofibroblast: a quarter century after its discovery[R]. Am J Surg Pathol, 1998, 22(2):141-147
27. el Nahas AM, Muchaneta-Kubara EC, Zhang G, e tal. Phenotypic modulation of renal cells during experimental and clinical renal scarring[R]. Kidney Int Suppl, 1996, 54: S23-27
28. Masszi A, Di Ciano C, Sirokmany G, e tal. Central role for Rho in TGF-betal-induced alpha-smooth muscle actin expression during epithelial-mesenchymal transition[J]. Am J Physiol Renal Physiol. 2003, 284(5):F911-924
29. Mack CP, Somlyo AV, Hautmann M, e tal. Smooth muscle differentiation marker gene expression is regulated by RhoA-mediated actin polymerization[J]. J Biol Chem. 2001, 276(1):341-347
30. Muller GA, Schettler V, Muller CA, e tal. Prevention of progression of renal fibrosis: how far are we? [R] Kidney Int Suppl, 1996, 54:S75-82
31. Ishizaki T, Uehata M, Tamechika I, e tal. Pharmacological properties of Y-27632, a specific inhibitor of rho-associated kinases[J]. Mol Pharmacol, 2000, 57(5):976-983
32. Klahr S and Morrissey J. Obstructive nephropathy and renal fibrosis[J]. Am J Physiol Renal Physiol, 2002,283(5):F861-875.
33. Klahr S. New insight into the consequences and mechanisms of renal impairment in obstructive nephrology[J]. Am T kidney Dis, 1991,18:689-699.
34. Katafuchi R, Kiyoshi Y, OH Y, e tal. Glomerular score as a prognosticator in IgA nephropathy: its usefulness and limitation[J]. Clin Nephr. 1998, 49(1): 1-8.
35. Kawano Y, Fukata Y, Oshiro N, e tal. Phosphorylation of myosin-binding subunit (MBS) of myosin phosphatase by Rho-kinase in vivo[J]. J Cell Biol, 1999, 147(5): 1023-1038.
36. Evans RA, Tian YC, Steadman R, e tal. TGF-betal-mediated fibroblast-myofibroblast terminal differentiation-the role of Smad proteins[J]. Exp Cell Res, 2003, 282(2):90-100.
37. Lewis MP and Norman JT. Differential response of activated versus
non-activated renal fibroblasts to tubular epithelial cells: a model of initiation and progression of fibrosis? [J] Exp Nephrol, 1998, 6(2):132-143.
38. Schoecklmann HO, Rupprecht HD, Zauner I, e tal. TGFbetal-induced cell cycle arrest in renal mesangial cells involves inhibition of cyclin E-cdk2 activation and retinoblastoma protein phosphorylation[J] Kidney Int, 1997, 51: 1228-1236.
39. Kaartinen V. Haataja L. Nagy A, e tal. TGFbeta3-induced activation of RhoA/Rho-kinase pathway is necessary but not sufficient for epithelio-mesenchymal transdifferentiation: implications for palatogenesis[J]. Int J Mol Med, 2002, 9(6): 563-570
40. Sobue K, Hayashi K, Nishida W. Expressional regulation of smooth muscle cell-specific genes in association with phenotypic modulation [R]. Mol Cell Biochem. 1999, 190(1-2):105-118.
41. Settleman J. A nuclear MAL-function links Rho to SRF [R]. Mol Cell, 2003, 11(5): 1121-1123.
42. Patel K. Harding P. Haney LB, e tal. Regulation of the mesangial cell myofibroblast phenotype by actin polymerization[J]. J Cell Physiol, 2003, 195(3): 435-445
43. Schelling JR, Sinha S, Konieczkowski M, e tal. Myofibroblast differentiation: plasma membrane microdomains and cell phenotype[R]. Exp Nephrol, 2002, 10(5-6):313-319.