血管紧张素II(Ang II)在慢性肾脏病中起重要的致病作用，尽管体外研究证实TGF-β/Smad3起正调控，Smad7起负调控作用，但Smad3在Ang II 诱导的肾脏损害中的作用仍不清楚。因此，本论文在Smad3基因敲除的小鼠中通过Ang II诱导的高血压肾损伤模型研究TGF-β/Smad3通路的作用及机制。如第三章所述，敲除Smad3的小鼠不发生Ang II诱导的高血压肾损伤如尿白蛋白，血肌酐升高，肾脏炎症（如IL-1, TNFα上调，F4/80+ 巨噬细胞浸润）及肾脏纤维化（包括α-SMA+肌成纤维细胞聚集，和胶原基质沉积）。敲除Smad3对高血压肾病起保护作用是因为抑制了肾脏TGF-β1表达及Smurf2 依赖的Smad7泛素化降解，从而抑制TGF-β/Smad3介导的肾脏纤维化和NF-B介导的炎症。 / 越来越多的证据显示Ang II产生和降解的平衡在高血压肾病的发展中起重要作用。在这篇论文中，我们假设ACE2的降解可能会引起Ang II代谢通路的失衡，从而加重其介导的高血压肾病。这一假设在第四章得到验证，在单侧输尿管梗阻小鼠模型敲除ACE2加重肾内Ang II介导的肾脏纤维化和炎症。这一变化与肾内高水平的Ang II和降低的血管紧张素1-7，上调的血管紧张素受体1，及激活的TGF-β/Smad3 和 NF-κB 信号通路有关。另外，升高的Smurf2介导的Smad7泛素化降解加重了敲除ACE2 基因后Ang II介导的肾脏纤维化和炎症。 / 因为Smad7 是TGF-β/Smad和NF-κB通路的负调控因子，因此论文进一步提出假设过表达Smad7能够阻止Ang II介导的肾脏纤维化炎症。如第五章所述，ACE2基因敲除的小鼠肾内升高的Smurf2介导了肾脏Smad7 的泛素化降解， 加重了Ang II 介导的肾脏损伤如白蛋白尿，血肌酐的升高，及肾脏纤维化和炎症，这与激活的Ang II/TGF-β/Smad3/NF-κB信号有关。相反，过表达Smad7能够阻断TGF-β/Smad3 介导的肾脏纤维化和 NF-κB介导的肾脏炎症以缓解ACE2敲除小鼠中Ang II诱导的肾脏损伤。 / 总之，Smad3在Ang II诱导的高血压肾脏病中起关键作用，Smad7具有肾脏保护作用。 ACE2敲除引起Ang II产生和降解的失衡从而增加肾内Ang II的产生，加重TGF-β/Smad3介导的肾脏纤维化和NF-κB介导的肾脏炎症，而这可以被Smad7缓解。 本论文得出结论针对TGF-β/Smad3 和NF-κB通路，通过过表达Smad7可能为高血压肾脏病和慢性肾脏病提供新的治疗策略。 / Angiotensin II (Ang II) plays a pathogenic role in chronic kidney disease (CKD). Although in vitro studies find that Ang II mediates renal fibrosis via the Smad3-dependent mechanism, the functional role of Smad3 in Ang II-mediated kidney disease remains unclear. Therefore, this thesis examined the pathogenesis role and mechanisms of TGF-β/Smad3 in Ang II-mediated hypertensive nephropathy in Smad3 Knockout (KO) mice. As described in Chapter III, Smad3 deficiency protected against Ang II-induced hypertensive nephropathy as demonstrated by lowering levels of albuminuria, serum creatinine, renal inflammation such as up-regulation of pro-inflammatory cytokines (IL-1β, TNFα) and infiltration of CD3+ T cells and F4/80+ macrophages, and renal fibrosis including α-SMA+ myofibroblast accumulation and collagen matrix deposition (all p<0.01). Inhibition of hypertensive nephropathy in Smad3 KO mice was associated with reduction of renal TGF-β1 expression and Smurf2-associated ubiquitin degradation of renal Smad7, thereby blocking TGF-β/Smad3-mediated renal fibrosis and NF-κB-driven renal inflammation. / Increasing evidence shows that the balance between the generation and degradation of Ang II is also important in the development of hypertensive nephropathy. In this thesis, we also tested a hypothesis that enhanced degradation of ACE2 may result in the imbalance between the Ang II generation and degradation pathways, therefore enhancing Ang II-mediated hypertensive nephropathy and CKD. This hypothesis was examined in a mouse model of unilateral ureteral obstructive nephropathy (UUO) induced in ACE2 KO mice. As described in Chapter IV, loss of ACE2 increased intrarenal Ang II-mediated renal fibrosis and inflammation in the UUO kidney. These changes were associated with higher levels of intrarenal Ang II, reduced Ang 1-7, up-regulated AT1R, and activation of TGF-β/Smad3 and NF-κB signalling. In addition, enhanced Smurf2-associated ubiquitin degradation of Smad7 was another mechanism by which loss of ACE2 promoted Ang II-mediated renal fibrosis and inflammation. / Because Smad7 is a negative regulator for TGF-β/Smad and NF-κB signalling, this thesis also examined a hypothesis that overexpression of renal Smad7 may be able to prevent Ang II-induced, TGF-β/Smad3-mediated renal fibrosis and NF-κB-driven renal inflammation in ACE2 KO mice. As described in Chapter V, mice null for ACE2 resulted in degradation of renal Smad7 via the Smurf2 -- dependent mechanism (all p<0.01). Enhanced Ang II-mediated renal injury in ACE2 KO mice such as albuminuria, serum creatinine, and renal fibrosis and inflammation was associated with enhanced activation of Ang II/TGF-β/Smad3/NF-κB signalling. In contrast, overexpression of Smad7 was able to rescue AngII-induced progressive renal injury in ACE2 KO mice by blocking TGF-β/Smad3 and NF-κB-dependent renal fibrosis and inflammation. In conclusion, Smad3 plays an essential role in Ang II-induced hypertensive nephropathy, while Smad7 is reno-protective. Loss of ACE2 results in the imbalance between the Ang II generation and degradation pathways and thus enhances intrarenal Ang II-induced, TGF-β/Smad3-mediated renal fibrosis and NF-κB-driven renal inflammation, which can be rescued by Smad7. Results from this thesis indicate that targeting TGF-β/Smad3 and NF-κB pathways by overexpressing Smad7 may represent a novel therapy for hypertensive nephropathy and CKD. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Liu, Zhen. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 189-209). / Abstracts also in Chinese. / ABSTRACT --- p.i / DECLARATION --- p.v / ACKNOWLEDGEMENTS --- p.vi / LIST OF PUBLICATION --- p.viii / TABLE OF CONTENTS --- p.ix / LIST OF ABBREVIATIONS --- p.xiv / LIST OF FIGURES AND TABLES --- p.xvii / CHAPTER I --- p.1 / INTRODUCTION --- p.1 / Chapter 1.1 --- RAS (Renin-Angiotensin system) --- p.2 / Chapter 1.1.1 --- Circulating RAS --- p.2 / Chapter 1.1.2 --- Tissue RAS --- p.5 / Chapter 1.1.2.1 --- Angiotensinogen --- p.6 / Chapter 1.1.2.2 --- Renin Receptors --- p.7 / Chapter 1.1.2.3 --- ACE and ACE2 --- p.9 / Chapter 1.1.2.4 --- Angiontensin II and Its Receptors --- p.10 / Chapter 1.1.2.5 --- AT2 Receptors --- p.11 / Chapter 1.1.2.6 --- Chymase-Alternative Pathways of Ang II Generation --- p.13 / Chapter 1.1.2.7 --- Ang (1-7) Receptor (MAS) --- p.13 / Chapter 1.2 --- Ang II and Renal Injury --- p.15 / Chapter 1.2.1 --- Pressure Dependent Renal Injury Induced by Ang II --- p.15 / Chapter 1.2.2 --- Ang II induces production of cytokines and growth factors --- p.16 / Chapter 1.2.3 --- Ang II and Renal Fibrosis --- p.17 / Chapter 1.2.4 --- Signalling Mechanisms Involved in Ang II-Induced Renal Fibrosis --- p.18 / Chapter 1.2.5 --- Ang II in Renal Inflammation --- p.22 / Chapter 1.3 --- TGF-β/Smad Signalling Pathway in Renal Disease --- p.24 / Chapter 1.3.1 --- Mechanisms of TGF-β/Smad Activation --- p.24 / Chapter 1.3.1.1 --- Cross-talk Between Smads and Other Signalling Pathways in Renal Fibrosis --- p.26 / Chapter 1.3.1.2 --- Activation of R-Smads (Smad2 and Smad3) --- p.28 / Chapter 1.3.2 --- Inhibitory Role of Smad7 in Renal Fibrosis and Inflammation --- p.30 / Chapter CHAPTER II --- p.32 / MATERIALS AND METHODS --- p.32 / Chapter 2.1 --- MATERIALS --- p.33 / Chapter 2.1.1 --- Regents and Equipments --- p.33 / Chapter 2.1.1.1 --- Regents and Equipments for Cell Culture --- p.33 / Chapter 2.1.1.2 --- General Reagents and Equipments for Real-time PCR --- p.34 / Chapter 2.1.1.3 --- General Reagents and Equipments for Masson Trichrome Staining --- p.34 / Chapter 2.1.1.4 --- General Reagents and Equipments for Immunohistochemistry --- p.35 / Chapter 2.1.1.5 --- General Reagents and Equipments for Western Blot --- p.35 / Chapter 2.1.1.6 --- General Reagents and Equipments for ELISA --- p.37 / Chapter 2.1.1.7 --- Measurement of Blood Pressure in Mice --- p.37 / Chapter 2.1.1.8 --- Reagents and Equipment for Genotyping --- p.37 / Chapter 2.1.2 --- Buffers --- p.38 / Chapter 2.1.2.1 --- Immunohistochemistry Buffers --- p.38 / Chapter 2.1.2.2 --- Buffers for Western Blotting --- p.40 / Chapter 2.1.2.3 --- ELISA Buffers --- p.44 / Chapter 2.1.2.4 --- Primer Sequences --- p.46 / Chapter 2.1.2.5 --- Primary Antibodies --- p.47 / Chapter 2.1.2.6 --- Secondary Antibodies --- p.48 / Chapter 2.2 --- METHODS --- p.49 / Chapter 2.2.1 --- Animal --- p.49 / Chapter 2.2.1.1 --- Genotypes of Gene KO Mice --- p.49 / Chapter 2.2.1.2 --- Animal Model of Unilateral Ureteral Obstruction (UUO) --- p.50 / Chapter 2.2.1.3 --- Animal Model of Angiotensin II (Ang II)-Induced Hypertensive Nephropathy --- p.50 / Chapter 2.2.1.4 --- Measurement of Ang II and Ang 1-7 --- p.51 / Chapter 2.2.2 --- Cell Culture --- p.51 / Chapter 2.2.3 --- Microalbuminuria and Renal Function --- p.51 / Chapter 2.2.3.1 --- Urine Collection --- p.51 / Chapter 2.2.3.2 --- Plasma Collection --- p.52 / Chapter 2.2.3.3 --- Microalbuminuria --- p.52 / Chapter 2.2.3.4 --- Creatinine Measurement --- p.52 / Chapter 2.2.4 --- Real-time PCR --- p.53 / Chapter 2.2.4.1 --- Total RNA Extraction --- p.53 / Chapter 2.2.4.2 --- Reverse Transcription --- p.53 / Chapter 2.2.4.3 --- Real-time PCR --- p.54 / Chapter 2.2.4.4 --- Analysis of Real-time PCR --- p.54 / Chapter 2.2.5 --- Western Blot --- p.55 / Chapter 2.2.5.1 --- Protein Preparation --- p.55 / Chapter 2.2.5.2 --- Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) --- p.56 / Chapter 2.2.5.3 --- Protein Transfer (Wet Transfer) --- p.56 / Chapter 2.2.5.4 --- Incubation of Antibodies --- p.56 / Chapter 2.2.5.5 --- Scanning and Analysis --- p.57 / Chapter 2.2.5.6 --- Stripping --- p.57 / Chapter 2.2.6 --- Histochemistry --- p.57 / Chapter 2.2.6.1 --- Tissue Fixation --- p.57 / Chapter 2.2.6.2 --- Tissue Embedding and Sectioning --- p.58 / Chapter 2.2.6.3 --- Preparation of Paraffin Tissue Sections for PAS Staining --- p.58 / Chapter 2.2.6.4 --- PAS Staining --- p.58 / Chapter 2.2.7 --- Immunohistochemistry --- p.59 / Chapter 2.2.7.1 --- Tissue Embedding and Sectioning --- p.59 / Chapter 2.2.7.2 --- Antigen-Antibody Reaction and Immunostaining --- p.59 / Chapter 2.2.7.3 --- Semi-quantification of Immunohistochemistry --- p.60 / Chapter 2.2.8 --- Statistical Analysis --- p.60 / Chapter CHAPTER III --- p.62 / ROLE OF SMAD3 IN ANGIOTENSIN II-INDUCED RENAL FIBROSIS AND INFLAMMATION --- p.62 / Chapter 3.1 --- INTRODUCTION --- p.63 / Chapter 3.2 --- MATERIALS AND METHODS --- p.64 / Chapter 3.2.1 --- Generation of Smad3 KO Mice --- p.64 / Chapter 3.2.2 --- Mouse Model of Ang II-Induced Hypertension --- p.64 / Chapter 3.2.3 --- Histology and Immunohistochemistry --- p.65 / Chapter 3.2.4 --- Renal Function and Proteinuria --- p.65 / Chapter 3.2.5 --- Western Blot Analysis --- p.65 / Chapter 3.2.6 --- Real-time RT-PCR --- p.65 / Chapter 3.2.7 --- In Vitro Study of Mesangial Cells from Smad3 WT and KO Mice --- p.66 / Chapter 3.2.8 --- Statistical Analysis --- p.66 / Chapter 3.3 --- RESULTS --- p.66 / Chapter 3.3.1 --- Smad3 KO Mice Prevents Ang II-induced Renal Injury Independent of Blood Pressure --- p.66 / Chapter 3.3.2 --- Smad3 KO Mice Are Resistant to Renal Fibrosis in a Mouse Model of Ang II -Induced Hypertension --- p.70 / Chapter 3.3.3 --- Smad3 KO Mice Are Resistant to Renal Inflammation in a Mouse Model of Ang II-Induced Hypertension --- p.76 / Chapter 3.3.4 --- Smad3 Deficiency Inhibits Ang II-induced Renal Fibrosis and Inflammation In Vitro --- p.82 / Chapter 3.3.5 --- Smad3 Mediates Ang II-Induced Renal Fibrosis by the Positive Feedback Mechanism of TGF-β/Smad Signalling --- p.87 / Chapter 3.3.6 --- Enhancing NF-κB Signalling via the Smurf2-associated Ubiquitin Degradation of Smad7 In Vivo and In Vitro --- p.92 / Chapter 3.4 --- DISCUSSION --- p.101 / Chapter 3.5 --- CONCLUSION --- p.106 / Chapter CHAPTER IV --- p.107 / LOSS OF ANGIOTENSIN-CONVERTING ENZYME 2 ENHANCES TGF-β/SMAD-MEDIATED RENAL FIBROSIS AND NF-κB-DRIVEN RENAL INFLAMMATION IN A MOUSE MODEL OF OBSTRUCTIVE NEPHROPATHY --- p.107 / Chapter 4.1 --- INTRODUCTION --- p.108 / Chapter 4.2 --- MATERIALS AND METHODS --- p.109 / Chapter 4.2.1 --- Generation of ACE2 KO Mice --- p.109 / Chapter 4.2.2 --- Mouse Model of Unilateral Ureteral Obstruction (UUO) --- p.109 / Chapter 4.2.3 --- Histology and Immunohistochemistry --- p.110 / Chapter 4.2.4 --- Western Blot Analysis --- p.110 / Chapter 4.2.5 --- Real-time RT-PCR --- p.110 / Chapter 4.2.6 --- Measurement of Ang II and Ang 1-7 --- p.110 / Chapter 4.2.7 --- Statistical Analysis --- p.111 / Chapter 4.3 --- RESULTS --- p.111 / Chapter 4.3.1 --- ACE2 KO Mice Accelerate Renal Fibrosis and Inflammation Independent of Blood Pressure in the UUO Nephropathy --- p.111 / Chapter 4.3.2 --- Loss of ACE2 Enhances Ang II, Activation of TGF-β/Smad and NF-κB Signalling Pathways --- p.128 / Chapter 4.3.3 --- Loss of Renal Smad7 Is an Underlying Mechanism Accounted for the Progression of TGF-β/Smad-mediated Renal Fibrosis and NF-κB-Driven Renal Inflammation in the UUO Nephropathy in ACE2 KO Mice --- p.140 / Chapter 4.4 --- DISCUSSION --- p.143 / Chapter 4.5 --- CONCLUSION --- p.147 / CHAPTER V --- p.148 / PROTECTIVE ROLE OF SMAD7 IN HYPERTENSIVE NEPHROPATHY IN ACE2 DEFICIENT MICE --- p.148 / Chapter 5.1 --- INTRODUCTION --- p.149 / Chapter 5.2 --- MATERIALS AND METHODS --- p.151 / Chapter 5.2.1 --- Generation of ACE2 KO Mice --- p.151 / Chapter 5.2.2 --- Mouse Model of Ang II-Induced Hypertension --- p.151 / Chapter 5.2.3 --- Smad7 Gene Therapy --- p.151 / Chapter 5.2.4 --- Histology and Immunohistochemistry --- p.152 / Chapter 5.2.5 --- Western Blot Analysis --- p.153 / Chapter 5.2.6 --- Real-time RT-PCR --- p.153 / Chapter 5.2.7 --- Measurement of Ang II and Ang 1-7 --- p.153 / Chapter 5.2.8 --- Statistical Analysis --- p.153 / Chapter 5.3 --- RESULTS --- p.154 / Chapter 5.3.1 --- Deletion of ACE2 Accelerates Ang II-Induced Renal Injury --- p.154 / Chapter 5.3.2 --- Renal Fibrosis and Inflammation are Enhanced in ACE2 KO Mice with Ang II-Induced Renal Injury --- p.156 / Chapter 5.3.3 --- Enhanced Activation of TGF-β/Smad3 and NF-κB Signalling Pathways are Key Mechanism by Which Deletion of ACE2 Promotes Ang II-Induced Renal Injury --- p.163 / Chapter 5.3.4 --- Loss of Renal Smad7 Mediated by Smurf2-ubiquintin Degradation Pathway Contributes to Ang II-Induced Hypertensive Nephropathy in ACE2 KO Mice --- p.166 / Chapter 5.3.5 --- Overexpression of Smad7 is able to Rescue Ang II-induced Renal Injury in ACE2 KO Mice by Blocking Both TGF-β/Smad3 and NF-κB-dependent Renal Fibrosis and Inflammation --- p.168 / Chapter 5.4 --- DISCUSSION --- p.180 / Chapter 5.5 --- CONCLUSION --- p.182 / Chapter CHAPTER VI --- p.183 / SUMMARY AND DISCUSSION --- p.183 / Chapter 6.1 --- Smad3 Plays a Key Role in Ang II-Induced Hypertensive Nephropathy --- p.185 / Chapter 6.2 --- The Intrarenal Ang II Plays a Key Role in the Progress of Ang II-Mediated Renal Injury --- p.185 / Chapter 6.3 --- A Novel Finding of Ang II-Smad3-TGF-β-Smad3 amplification loop in Ang II-mediated Renal Fibrosis --- p.186 / Chapter 6.4 --- Smurf2-associated Ubiquitin-Proteasome Degradation of Smad7 Contributes to the Progression of Ang II-mediated Renal Injury in ACE2 KO Mice --- p.187 / Chapter 6.5 --- Smad7 Protects against Ang II-Mediated Hypertensive Kidney Disease by Negatively Regulating TGF-β/Samd and NF-κB Signalling --- p.187 / REFERENCE --- p.189

目的： / TGF-β/Smad信号通路在慢性肾脏纤维化疾病中有着重要的作用。大量研究证实Smad3在TGF-β/Smad信号介导的肾脏纤维化过程中发挥着关键的作用，但TGF-β/Smad3这一关键信号通路的分子机制尚不明确。该论文研究假设TGF-β通过Smad3介导的microRNA-21（miR-21）导致肾脏纤维化；特异性的针对miR-21将有助于提供有效的、创新性的方法治疗慢性肾脏纤维化疾病。 / 方法： / 该论文研究利用大鼠肾小管上皮细胞株（TEC）及系膜细胞株(MC)，探讨TGF-β1诱导miR-21表达增高的机制；通过过表达及抑制miR-21在上述细胞株的表达，研究miR-21在TGF-β1的刺激及高糖环境下，对肾脏纤维化的影响。进一步通过采用超声微泡介导基因转入技术，将miR-21 shRNA质粒特异性的诱导入梗阻性肾病小鼠模型（UUO）及糖尿病肾病db/db小鼠模型的肾脏中，体内研究抑制miR-21对纤维化的治疗作用。通过荧光素酶报告分析，检测miR-21的靶基因。 / 结果： / 通过微阵列（microarray）及实时荧光定量PCR（realtime PCR）技术，检测miR-21在TGF-β1及高糖的刺激下的表达水平，结果发现其表达在TEC及MC均明显升高。进一步通过体内体外实验，在TGF-β1及高糖的刺激下，高表达的miR-21和TGF-β/Smad3信号通路的激活有关。体外对miR-21的功能进行研究，结果表明在TGF-β1及高糖的刺激下过表达miR-21促进TEC及MC纤维化的发生，而抑制miR-21的表达有效的降低TEC及MC的纤维化损伤。体内利用梗阻性肾病小鼠模型，通过采用超声微泡介导基因转入技术，将miR-21 shRNA质粒分别于模型前后特异性的诱导入小鼠肾脏，结果发现抑制miR-21的表达能有效地阻止肾脏纤维化的进展，减轻梗阻肾纤维化的程度；利用2型糖尿病肾病db/db小鼠模型，发现抑制miR-21的表达能减轻糖尿病肾病小鼠肾脏的纤维化及炎症程度，并改善糖尿病肾病小鼠的肾脏功能。采用荧光素酶报告分析，结果发现Smad7是miR-21的直接靶基因，miR-21通过直接抑制Smad7的表达从而影响肾脏纤维化和炎症。该论文的研究结果提示miR-21在慢性肾脏纤维化疾病中的治疗作用和前景。 / 结论： / miR-21作为TGF-β/Smad3信号通路的下游因子，在肾脏纤维化的发生发展中起着重要作用。特异性针对miR-21为肾脏纤维化疾病的治疗提供了创新性的有效方法。 / Objectives: / TGF-β/Smad signaling plays a critical role in renal fibrosis in chronic kidney disease (CKD). It is well known that Smad3 is a key mediator of downstream TGF-β/Smad signaling in renal fibrosis, however, the exact mode of TGF-β/Smad3 in renal fibrosis remains unclear. In this thesis, we tested a novel hypothesis that TGF-β may act by regulating the Smad3-dependent microRNA-21（miR-21） to mediate renal fibrosis and that specific targeting miR-21 may represent an effective and novel therapy for chronic kidney disease. / Methods: / The regulatory mechanism of TGF-β1-induced miR-21 expression via the Smad3-dependent pathway was studied in a rat NRK52E tubular epithelial cell (TEC) line and mesangial cell (MC) line. The functional role of miR-21 in renal fibrosis was investigated by overexpressing or down-regulating of miR-21 both in TGF-β1 and high glucose (HG) conditions in TEC and MC. The therapeutic potential role of miR-21 in kidney diseases were examined in unilateral ureteral obstructive (UUO) mouse model and in db/db mice by applying an ultrasound-microbubble-mediated anti-miR-21 gene transfer technique. The target gene of miR-21 was identified by luciferase reporter assays. / Results: / By microarray and realtime PCR, upregulation of miR-21 was observed in tubular epithelial cells (TECs) and mesangial cells (MCs) in response to TGF-β1 and high glucose (HG). Both in vitro and in vivo studies demonstrated that the upregulation of miR-21 expression during renal fibrosis and diabetic conditions was dependent on the activation of TGF-β/Smad3 signaling. The findings that overexpression of miR-21 promoted but knockdown of miR-21 suppressed TGF-β1-induced renal fibrosis and HG-induced diabetic kidney injury demonstrated the functional importance for miR-21 in fibrosis and inflammation in vitro. More importantly, ultrasound-microbubble-mediated gene transfer of a miR-21 knockdown plasmid into the mouse kidney before and after established unilateral ureteral obstructive (UUO) nephropathy was able to prevent and halt the progression of renal fibrosis. Furthermore, we also found that blockade of miR-21 was capable of attenuating diabetic kidney injury including progressive renal fibrosis and inflammation, as well as renal functional injury in a mouse model of type 2 diabetes in db/db mice. The functional role of miR-21 on renal fibrosis and inflammation was through Smad7, which was identified as a direct target gene of miR-21. All these results revealed a therapeutic potential for targeting miR-21 in chronic kidney disease. / Conclusions: / In conclusion, miR-21 is a downstream mediator of TGF-β/Smad3 signaling and plays a critical role in the development of renal fibrosis. Targeting miR-21 may represent a novel and effective therapy to combat renal fibrosis in chronic kidney disease. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Zhong, Xiang. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 206-221). / Abstract also in Chinese. / ABSTRACT --- p.ii / TABLE OF CONTENTS --- p.vi / DECLARATION --- p.xiv / ACKNOWLEDGEMENTS --- p.xv / LISTS OF ABBREVIATION --- p.xvii / LISTS OF FIGURES AND TABLES --- p.xx / PUBLICATIONS --- p.xxvi / Chapter CHAPTER I --- INTRODUCTION --- p.1 / Chapter 1.1 --- MicroRNA --- p.1 / Chapter 1.1.1 --- Biogenesis and Function of MicroRNA --- p.2 / Chapter 1.1.2 --- Recognition of MicroRNA Target --- p.5 / Chapter 1.2 --- MicroRNA-21 --- p.6 / Chapter 1.2.1 --- The Role of miR-21 In Fibrosis-related Disease --- p.7 / Chapter 1.2.2 --- The Role of miR-21 In Inflammatory Disease --- p.10 / Chapter 1.2.3 --- The Regulation of miR-21 --- p.12 / Chapter 1.3 --- TGF-β/SMADS SIGNALING IN RENAL FIBROSIS --- p.15 / Chapter 1.3.1 --- TGF-β/Smads Signaling --- p.15 / Chapter 1.3.2 --- The Diverse Role of TGF-β/Smads Signaling In Renal Fibrosis and Inflammation --- p.19 / Chapter 1.3.2.1 --- The Diverse Role of TGF-β1 In Renal Fibrosis and Inflammation --- p.19 / Chapter 1.3.2.2 --- The Diverse Role of Smad2 and Smad3 In Renal Fibrosis --- p.20 / Chapter 1.3.2.3 --- The Inhibitory Role of Smad7 In Renal Fibrosis and Inflammation --- p.22 / Chapter 1.4 --- THE POTENTIAL ROLE OF MIR-21 IN RENAL FIBROSIS --- p.24 / Chapter CHAPTER II --- MATERIALS AND METHODS --- p.26 / Chapter 2.1 --- MATERIALS --- p.26 / Chapter 2.1.1 --- Reagents --- p.26 / Chapter 2.1.1.1 --- Reagents for Cloning --- p.26 / Chapter 2.1.1.2 --- Reagents for Cell Culture --- p.27 / Chapter 2.1.1.3 --- Reagents for Realtime RT-PCR --- p.27 / Chapter (1) --- For miR-21 Assay --- p.27 / Chapter (2) --- For Fibrotic and Inflammatory Index Assay --- p.28 / Chapter 2.1.1.4 --- Reagents for Western Blot --- p.28 / Chapter 2.1.1.5 --- Reagents for In Situ Hybridization (ISH) --- p.29 / Chapter 2.1.1.6 --- Reagents for Immunochemistry Staining --- p.30 / Chapter 2.1.1.7 --- Reagents for Luciferase Activity Assay --- p.30 / Chapter 2.1.1.8 --- Reagents for CHIP Assay --- p.31 / Chapter 2.1.1.9 --- Reagents for Urine Albumin Excretion Measurement --- p.31 / Chapter 2.1.2 --- Buffers --- p.31 / Chapter 2.1.2.1 --- Buffers for Western Blot --- p.31 / Chapter (1) --- RIPA Lysis Buffer --- p.31 / Chapter (2) --- 4× SDS Loading Sample Buffer --- p.32 / Chapter (3) --- 10% Ammonia Persulfate (10% APS) --- p.33 / Chapter (4) --- 1.5 M Tris Buffer Mix (For 15% Resolving Gel) --- p.33 / Chapter (5) --- 1.5 M Tris Buffer Mix (For 12% Resolving Gel) --- p.33 / Chapter (6) --- 1.5 M Tris Buffer Mix (For 10% Resolving Gel) --- p.33 / Chapter (7) --- 0.5 M Tris Buffer Mix (For 4% Stacking Gel) --- p.34 / Chapter (8) --- 15% Resolving Gel --- p.34 / Chapter (9) --- 12% Resolving Gel --- p.34 / Chapter (10) --- 10% Resolving Gel --- p.35 / Chapter (11) --- 4% Stacking Gel --- p.35 / Chapter (12) --- Tris Buffered Saline (TBS) --- p.35 / Chapter (13) --- TBS-Tween 20 (TBS-T) --- p.36 / Chapter (14) --- SDS-PAGE Electrophoresis Running Buffer --- p.36 / Chapter (15) --- Transfer Buffer without SDS --- p.36 / Chapter (16) --- Transfer Buffer --- p.37 / Chapter (17) --- Blocking Buffer --- p.37 / Chapter (18) --- Antibody Diluent Buffer --- p.37 / Chapter 2.1.2.2 --- Buffers for Immunochemistry Staining --- p.37 / Chapter (1) --- Methyl Carnoy's Fixative --- p.37 / Chapter (2) --- Phosphate Buffered Saline (PBS) --- p.38 / Chapter (3) --- Horseradish Peroxidase (HRP) Inactivation Solution --- p.38 / Chapter (4) --- Microwave-based Antigen-retrieval Solution --- p.38 / Chapter (5) --- Blocking Buffer --- p.39 / Chapter (7) --- Substrate Solution for Fast Blue Staining --- p.39 / Chapter (8) --- Substrate Solution for DAB Staining --- p.39 / Chapter 2.1.2.3 --- Buffers for In Situ Hybridization (ISH) --- p.40 / Chapter (1) --- Fixative Solution --- p.40 / Chapter (2) --- DEPC-treated Water --- p.40 / Chapter (3) --- DEPC-treated PBS --- p.40 / Chapter (4) --- 0.2N HCl --- p.41 / Chapter (5) --- Proteinase K Solution --- p.41 / Chapter (6) --- 5XSSC/50% Deionized Formamide --- p.41 / Chapter (7) --- 5XSSC --- p.41 / Chapter (8) --- 2XSSC --- p.41 / Chapter (9) --- 0.2XSSC --- p.42 / Chapter (10) --- Hybridization Solution --- p.42 / Chapter (11) --- Solution B1 --- p.42 / Chapter (12) --- Solution B2 --- p.42 / Chapter 2.1.3 --- Antibodies --- p.43 / Chapter 2.1.3.1 --- The Primary Antibodies --- p.43 / Chapter 2.1.3.2 --- The Second Antibodies --- p.44 / Chapter 2.1.4 --- Primers --- p.45 / Chapter 2.1.4.1 --- Primers for Realtime RT-PCR --- p.45 / Chapter 2.1.4.2 --- Primers for Luciferase Activity Assay --- p.46 / Chapter 2.1.4.3 --- Primers for CHIP Assay --- p.47 / Chapter 2.1.5 --- Equipments --- p.47 / Chapter 2.1.5.1 --- Equipments for Cloning --- p.47 / Chapter 2.1.5.2 --- Equipments for Cell Culture --- p.47 / Chapter 2.1.5.3 --- Equipments for Realtime RT-PCR --- p.48 / Chapter 2.1.5.4 --- Equipments for Immunochemistry Staining --- p.48 / Chapter 2.1.5.5 --- Equipments for Western Blot --- p.48 / Chapter 2.1.5.6 --- Equipments for Luciferase Activity Assay --- p.49 / Chapter 2.1.5.7 --- Equipments for CHIP Assay --- p.49 / Chapter 2.1.5.8 --- Equipments for Urine Albumin Excretion Measurement --- p.49 / Chapter 2.2 --- METHODS --- p.50 / Chapter 2.2.1 --- Cloning --- p.50 / Chapter 2.2.1.1 --- Cloning Doxcycline-inducible overexpression of MiR-21 and Knockdown of MiR-21 expression plasmids --- p.50 / Chapter 2.2.1.2 --- Cloning Smad7 3’UTR Luciferase Reporter Plasmids --- p.51 / Chapter 2.2.2 --- Cell Cultures --- p.52 / Chapter 2.2.2.1 --- NRK52E Cell Lines and rat Mesengial Cell Lines --- p.52 / Chapter 2.2.2.2 --- Transient Transfection with microRNAs in TECs --- p.52 / Chapter 2.2.2.3 --- Construct Doxcycline-inducible Overexpression of MiR-21 and Knockdown of MiR-21 Stable Cell Lines in NRK52E and MCs --- p.53 / Chapter 2.2.3 --- Animal Models --- p.53 / Chapter 2.2.3.1 --- Unilateral Ureteral Obstruction (UUO) Mouse Model --- p.54 / Chapter 2.2.3.2 --- Diabetes Model --- p.54 / Chapter 2.2.4 --- Ultrasound-Mediated Gene Transfer --- p.55 / Chapter 2.2.5 --- Real Time RT-PCR --- p.56 / Chapter 2.2.5.1 --- Total RNA Isolation --- p.56 / Chapter 2.2.5.2 --- Reverse Transcription --- p.56 / Chapter (1) --- RT For MiR-21 Assay --- p.57 / Chapter (2) --- RT for Fibrotic and Inflammatory Index Assay --- p.57 / Chapter 2.2.5.3 --- Realtime PCR --- p.58 / Chapter (1) --- Realtime PCR For MiR-21 Assay --- p.58 / Chapter (2) --- Realtime PCR for Fibrotic and Inflammatory Index Assay --- p.58 / Chapter 2.2.5.4 --- Analysis of Realtime RT-PCR --- p.59 / Chapter 2.2.6 --- Western Blot --- p.59 / Chapter 2.2.6.1 --- Protein Preparation --- p.59 / Chapter 2.2.6.2 --- Running in SDS-PAGE --- p.60 / Chapter 2.2.6.3 --- Transfer --- p.61 / Chapter 2.2.6.4 --- Blocking --- p.61 / Chapter 2.2.6.5 --- Incubation --- p.62 / Chapter 2.2.6.6 --- Scanning --- p.62 / Chapter 2.2.6.7 --- Stripping --- p.62 / Chapter 2.2.7 --- PAS Staining --- p.63 / Chapter 2.2.7.1 --- Tissue Handling and Fixation --- p.63 / Chapter 2.2.7.2 --- Tissue Embedding and Sectioning --- p.63 / Chapter 2.2.7.3 --- Preparation of Paraffin Tissue Sections for PAS Staining --- p.64 / Chapter 2.2.7.4 --- PAS Staining --- p.64 / Chapter 2.2.7.5 --- Quantitative Analysis of PAS Staining --- p.65 / Chapter 2.2.8 --- Immunochemistry Staining --- p.65 / Chapter 2.2.8.1 --- Tissue Handling and Fixation --- p.65 / Chapter 2.2.8.2 --- Tissue Embedding and Sectioning --- p.65 / Chapter 2.2.8.3 --- Preparation of Paraffin Tissue Sections for Immunostaining --- p.65 / Chapter 2.2.8.4 --- Immunostaining --- p.66 / Chapter (1) --- Antigen-Antibody Reaction --- p.66 / Chapter (2) --- Signal Detection --- p.67 / Chapter 2.2.8.5 --- Quantitative Analysis of Immunohistochemistry --- p.67 / Chapter 2.2.9 --- In Situ Hybridization(ISH) --- p.68 / Chapter 2.2.9.1 --- Tissue Handling and Fixation --- p.68 / Chapter 2.2.9.2 --- Tissue Embedding and Sectioning --- p.68 / Chapter 2.2.9.3 --- Deparaffinization and Dewaxing --- p.68 / Chapter 2.2.9.4 --- Digestion --- p.69 / Chapter 2.2.9.5 --- Pre-Hybridization --- p.69 / Chapter 2.2.9.6 --- Hybridization --- p.69 / Chapter 2.2.9.7 --- Washing --- p.70 / Chapter 2.2.9.8 --- Blocking --- p.70 / Chapter 2.2.9.9 --- Incubation with anti-DIG Reagent --- p.70 / Chapter 2.2.9.10 --- Equilibration --- p.71 / Chapter 2.2.9.11 --- Signaling Detection --- p.71 / Chapter 2.2.10 --- Luciferase Activity Assay --- p.71 / Chapter 2.2.11 --- CHIP Analysis --- p.72 / Chapter 2.2.12 --- Urine Albumin Excretion Measurement --- p.73 / Chapter 2.2.12.1 --- Microalbuminuria Measurement --- p.73 / Chapter 2.2.12.2 --- Creatinine Measurement --- p.74 / Chapter 2.2.13 --- Statistical Analysis --- p.74 / Chapter CHAPTER III --- THE ROLE OF MIR-21 IN TGF-BETA-INDUCED RENAL FIBROSIS IN VITRO --- p.75 / Chapter 3.1 --- INTRODUCTION --- p.75 / Chapter 3.2 --- MATERIAS AND METHODS --- p.77 / Chapter 3.2.1 --- Cell Culture --- p.77 / Chapter 3.2.2 --- Transient Transfection with microRNAs --- p.78 / Chapter 3.2.3 --- Construction of Inducible Stable Cell Lines of miR-21 Overexpression and Knockdown --- p.78 / Chapter 3.2.4 --- Realtime RT-PCR --- p.79 / Chapter 3.2.5 --- Chromatin Immunoprecipitation (ChIP) Analysis --- p.79 / Chapter 3.2.6 --- Western Blot Analysis --- p.79 / Chapter 3.2.7 --- Statistical Analysis --- p.79 / Chapter 3.3 --- RESULTS --- p.80 / Chapter 3.3.1 --- The Expression of miR-21 Is Up-regulated in TGF-β-induced Renal Fibrosis In Vitro --- p.80 / Chapter 3.3.2 --- The Up-regulation of miR-21 Is Mediated by TGF-β/Smad Signaling during Renal Fibrosis In Vitro --- p.82 / Chapter 3.3.2.1 --- The Up-regulation of miR-21 Depends On the Activation of TGF-β Signaling During Renal Fibrosis In Vitro --- p.82 / Chapter 3.3.2.2 --- The Up-regulation of miR-21 in Response to TGF-β1 Is Positively Mediated by Smad3, Negatively by Smad2 --- p.84 / Chapter 3.3.2.3 --- The Up-regulation of miR-21 in Response to TGF-β1 Is Physically Regulated by Smad3 in CHIP Assay --- p.86 / Chapter 3.3.3 --- miR-21 Plays an Important Role in TGF-β-induced Renal Fibrosis In Vitro --- p.89 / Chapter 3.3.3.1 --- The Role of miR-21 in Renal Fibrosis Is Identified by Transient Transfection with miR-21 Mimic or Anti-miR-21 --- p.89 / Chapter 3.3.3.2 --- The Role of miR-21 in Renal Fibrosis Is Identified by Applied Inducible-Stable Cell Lines which Is Overexpression of miR-21 or Knockdown of miR-21 in TECs --- p.92 / Chapter (1) --- Characterize the Inducible-Stable Cell Lines which Is Overexpression of miR-21 or Knockdown of miR-21 in TECs --- p.92 / Chapter (2) --- Overexpression of miR-21 Enhances the TGF-β-induced Renal Fibrosis In Vitro --- p.95 / Chapter (3) --- Knockdown of miR-21 Inhibits the TGF-β-induced Renal Fibrosis In Vitro --- p.99 / Chapter 3.4 --- DISCUSSION --- p.103 / Chapter 3.5 --- CONCLUSION --- p.106 / Chapter CHAPTER IV --- THE THERAPUTIC ROLE OF MIR-21 IN UNILATERAL URETERAL OBSTRUCTION (uuo)-INDUCED RENAL FIBROSIS IN VIVO --- p.107 / Chapter 4.1 --- INTRODUCTION --- p.107 / Chapter 4.2 --- MATERIAS AND METHODS --- p.109 / Chapter 4.2.1 --- Animal Model of Unilateral Ureteral Obstruction (UUO) --- p.109 / Chapter 4.2.2 --- Ultrasound-mediated Gene Transfer of Inducible miR-21 shRNA Plasmids Into the Ligated Kidneys --- p.109 / Chapter 4.2.3 --- Realtime RT-PCR --- p.110 / Chapter 4.2.4 --- Western Blot Analysis --- p.111 / Chapter 4.2.5 --- PAS Staining --- p.111 / Chapter 4.2.6 --- Immunohistochemistry Staining --- p.111 / Chapter 4.2.7 --- In Situ Hybridization --- p.111 / Chapter 4.2.8 --- Statistical Analysis --- p.112 / Chapter 4.3 --- RESULTS --- p.112 / Chapter 4.3.1 --- The Expression of miR-21 Is Up-regulated in Renal Fibrosis in UUO Mouse Model --- p.112 / Chapter 4.3.2 --- Induce miR-21 siRNA Plasmid into the Kidney by Using Ultrasound-microbubble-mediated Gene Transfer Technique --- p.114 / Chapter 4.3.2.1 --- Determine Transgene Expression --- p.114 / Chapter 4.3.2.2 --- Determine Gene Transfer Rate --- p.117 / Chapter 4.3.2.3 --- Determine Gene Transfer Safety --- p.118 / Chapter 4.3.3 --- Knockdown of miR-21 Prevents the Development of Renal Fibrosis in UUO Mice --- p.120 / Chapter 4.3.3.1 --- Delivery of miR-21 shRNA Plasmid Suppresses the Expression of miR-21 and TGF-β1 in UUO Mouse Model --- p.120 / Chapter 4.3.3.2 --- Knockdown of MiR-21 Suppresses the Deposition of Collagen I, Fibronectin and α-SMA in UUO Mouse Model --- p.122 / Chapter 4.3.3.3 --- Knockdown of MiR-21 Suppresses the mRNA Levels of Collagen I, Fibronectin and α-SMA expression in UUO Mouse Model --- p.127 / Chapter 4.3.3.4 --- Knockdown of miR-21 Suppresses the Protein Levels of Collagen I, Fibronectin and α-SMA Expression in UUO Mouse Model --- p.129 / Chapter 4.3.4 --- Knockdown of miR-21 Attenuates the Progressive of Renal Fibrosis in UUO Mice --- p.131 / Chapter 4.3.4.1 --- Delivery miR-21 shRNA Plasmid Attenuates the Expression of miR-21 and TGF-β1 in Established UUO Mouse Model --- p.131 / Chapter 4.3.4.2 --- Knockdown of MiR-21 Attenuates the Deposition of Collagen I, Fibronectin and α-SMA in Established UUO Mouse Model --- p.133 / Chapter 4.3.4.3 --- Knockdown of MiR-21 Attenuates the mRNA Levels of Collagen I, Fibronectin and α-SMA in Established UUO Mouse Model --- p.138 / Chapter 4.3.4.4 --- Knockdown of miR-21 Attenuates the Protein Levels of Collagen I, Fibronectin and α-SMA Expression in Established UUO Mouse Model --- p.140 / Chapter 4.4 --- DISCUSSION --- p.143 / Chapter 4.5 --- CONCLUSION --- p.145 / Chapter CHAPTER V --- THE ROLE OF MIR-21 IN DIABETIC KIDNEY INJURY --- p.146 / Chapter 5.1 --- INTRODUCTION --- p.146 / Chapter 5.2 --- MATERIAS AND METHODS --- p.148 / Chapter 5.2.1 --- Cell Culture --- p.148 / Chapter 5.2.2 --- Construction of Inducible Stable Cell Lines of miR-21 Overexpression and Knockdown --- p.149 / Chapter 5.2.3 --- Animal Model of db/db Mice --- p.149 / Chapter 5.2.4 --- Ultrasound-mediated Gene Transfer of Inducible miR-21 shRNA Plasmids into the Kidneys of db/db Mice --- p.150 / Chapter 5.2.5 --- Realtime RT-PCR --- p.150 / Chapter 5.2.6 --- Western Blot Analysis --- p.150 / Chapter 5.2.7 --- PAS Staining --- p.151 / Chapter 5.2.8 --- Immunohistochemistry Staining --- p.151 / Chapter 5.2.9 --- Urine Albumin Excretion Measurement --- p.151 / Chapter 5.2.10 --- Construction of Plasmids and Luciferase reporter Assay --- p.152 / Chapter 5.2.11 --- Statistical Analysis --- p.152 / Chapter 5.3 --- RESULTS --- p.153 / Chapter 5.3.1 --- The Expression of miR-21 Is Increased Under Diabetic Conditions Both In Vitro and In Vivo --- p.153 / Chapter 5.3.1.1 --- The expression of miR-21 Is Increased in High Glucose Conditions in TECs and MCs --- p.153 / Chapter 5.3.1.2 --- The Expression of miR-21 Is Increased in Diabetic Kidney Injury in db/db Mouse Model --- p.155 / Chapter 5.3.2 --- The Expression of miR-21 Depends On The Activation of TGF-β/Smad Signaling Under Diabetic Conditions --- p.156 / Chapter 5.3.3 --- The Expression of MiR-21 Affects On Renal Fibrosis Under Diabetic Conditions In Vitro --- p.158 / Chapter 5.3.3.1 --- The Role of miR-21 in Renal Fibrosis Under Diabetic Conditions Is Identified in TECs --- p.158 / Chapter (1) --- Overexpression of miR-21 Enhances Renal Fibrosis in High Glucose Condition in TECs --- p.158 / Chapter (2) --- Knockdown of miR-21 Suppresses Renal Fibrosis in High Glucose Condition in TECs --- p.160 / Chapter 5.3.3.2 --- The Role of miR-21 in Renal Fibrosis Under Diabetic Conditions Is Identified in MCs --- p.162 / Chapter (1) --- Characterize the Inducible-Stable Cell Lines Which Is Overexpression of miR-21 or Knockdown of miR-21 in MCs --- p.162 / Chapter (3) --- Knockdown of miR-21 Suppresses Renal Fibrosis in High Glucose Condition in MCs --- p.165 / Chapter 5.3.4 --- The Expression of miR-21 Affects On Renal Inflammation Under Diabetic Conditions In Vitro --- p.167 / Chapter 5.3.4.1 --- The role of miR-21 in Renal Inflammation Under Diabetic Conditions Is Identified in TECs --- p.167 / Chapter 5.3.4.2 --- The Role of miR-21 in Renal Inflammation Under Diabetic Conditions Is Identified in MCs --- p.169 / Chapter 5.3.5 --- Knockdown of miR-21 Suppresses the Renal Fibrosis and Inflammation in db/db Mice --- p.172 / Chapter 5.3.5.1 --- Delivery of miR-21 siRNA suppresses the Expression of miR-21 in db/db Mice --- p.172 / Chapter 5.3.5.2 --- Knockdown of miR-21 Improves the Microalbuminuria in db/db Mice --- p.174 / Chapter 5.3.5.3 --- Knockdown of miR-21 Suppresses the Renal Fibrosis in db/db Mice --- p.176 / Chapter 5.3.5.4 --- Knockdown of miR-21 Suppresses the Renal Inflammation in db/db Mice --- p.183 / Chapter 5.3.6 --- Identification of Smad7 Is A Directly Target of miR-21 Both In Vitro and In Vivo --- p.187 / Chapter 5.3.6.1 --- The Expression of miR-21 Negatively Regulates the Smad7 Expression Under Diabetic Conditions Both in vitro and in vivo. --- p.187 / Chapter 5.3.6.2 --- Knockdown of miR-21 Blocks the Smad7-mediated TGF-β and NF-κB Signaling Pathways. --- p.190 / Chapter 5.3.6.3 --- Smad7 Is A Directly Target of miR-21. --- p.192 / Chapter 5.4 --- DISCUSSION --- p.194 / Chapter 5.5 --- CONCLUSION --- p.197 / Chapter CHAPTER VI --- SUMMARY AND CONCLUSION --- p.198 / Chapter 6.1 --- SUMMARY AND DISCUSSION --- p.200 / Chapter 6.1.1 --- The Up-regulation of miR-21 Was Observed in TGF-β- Induced Renal Fibrosis and Under Diabetic Conditions Both In Vitro and In Vivo. --- p.200 / Chapter 6.1.2 --- The Expression of miR-21 Is Regulated by TGF-β/Smad3 Signaling. --- p.200 / Chapter 6.1.3 --- The Expression of miR-21 Plays a Critical Role in Renal Fibrosis and Inflammation. --- p.201 / Chapter 6.1.4 --- MiR-21 Directly Targets on Smad7 to Regulate Renal Fibrosis and Inflammation. --- p.202 / Chapter 6.1.5 --- The Therapeutic Effect of miR-21 on Renal Fibrosis and Inflammation Is Developed in UUO and db/db Mouse Models. --- p.203 / Chapter 6.1.6 --- The Potential Clinical Use by Targeting On miR-21 --- p.204 / Chapter 6.2 --- CONCLUSION --- p.205 / REFERENCES --- p.206

以客觀存在之腎臟損害和功能異常的臨床表現為診斷依據的腎臟疾病，業已成為全世界所共同面臨的危害人類健康的一項主要健康問題。現今，人群中有超過十分之一的人患有慢性腎臟疾病（CKD）, 該病的患病率不亞於糖尿病 (James et al., 2010)。慢性腎髒病是一種難以治癒的疾病。儘管存在有效的治療方法，但在全世界範圍內，慢性腎髒病仍舊是導致終末期腎臟疾病和死亡的首要原因。據2010年全球疾病負擔的研究報導 (Lozano et al., 2013)，在引起全球死亡總數原因列表中，慢性腎髒病由1990年的第27位（年齡標準化的每100 000人的年死亡率為15.7）躍升至2010年的第18位（每100 000人的年死亡率為16.3）。慢性腎髒病的攀升趨勢僅次於HIV和AIDS。這種狀況已經引起高度關注，應對措施的實施刻不容緩！因此，迫切需要慢性腎髒病早期診斷和治療的有效方案。 / 纖維化是慢性腎髒病這一以終末期腎髒病（ESRD）為結局的疾病腎臟損傷和進展的主要病理特徵。現已確立转化生长因子β/Smads 信号蛋白（TGF-β/Smad）在腎臟纖維化發病機制中具有主要的作用。在過去近20年對於转化生长因子β/Smads 信号蛋白在慢性腎髒病發病機制中的作用研究揭示：Smad3在腎臟纖維化中起致病作用，而Smad2和Smad7具保護作用。鑒於转化生长因子β/Smads 信号蛋白在免疫調節中也發揮關鍵作用，直接針對转化生长因子β/Smads 信号蛋白的干預方案可能引起免疫損傷的副作用。因而，針對转化生长因子β/Smads 信号蛋白特異性下游靶目標的療法是對抗慢性腎髒病更好的選擇。 / 自首個微小核糖核酸的發現至今，已歷經20年。微小核糖核酸在基因表达的转录后起著重要調節作用。現已明確，在人類疾病包括腎臟病的發生和/或進展中存在微小核糖核酸的表達或功能的失調。最近的研究明確的表明，微小核糖核酸與腎臟疾病的發病機制密切相關。另外，已有確鑿的證據顯示，一些微小核糖核酸正是转化生长因子β/Smads信号蛋白的特異性下游。並且我們發現，不僅在转化生长因子β和血管緊張素II誘導的體外纖維化反應中，而且在小鼠體內梗阻性和高血壓性腎病模型中，微小核糖核酸-433-Azin1軸不但在腎臟纖維化中起重要作用，並且和转化生长因子β/Smad3信号蛋白密切相關。據重要意義的是，藉助安全、有效的超聲微泡介導的基因轉染方法，通過逆轉失調的微小核糖核酸-433-Azin1軸，有效的抑制了小鼠腎臟疾病的進展、腎組織的損傷并減少了蛋白尿的排泄。因此，微小核糖核酸-433-Azin1軸不但可以作為慢性腎臟病早期診斷的生物學標記，也能夠成為遏制甚至逆轉慢性腎髒病的治療靶點。 / 近年來，從基礎到臨床旨在預防和治療慢性腎髒病的研究已取得碩果累累。基於以转化生长因子β/Smad3信号蛋白特異性微小核糖核酸為把目標的治療策略，預見了遏制慢性腎髒病治療的未來希望。然而， 消除慢性腎髒病這一世界性的人類健康威脅，仍需付諸長期不懈的努力。 / Kidney disease is diagnosed with objective clinic manifestation of kidney damage and renal dysfunction has been recognized as a major global health burden. Nowadays, more than one out of ten people have chronic kidney disease (CKD) and the overall prevalence is not less than that of diabetes (James et al., 2010). CKD is a kind of persistent ailment. In spite of the availability of medical treatments, CKD continues to be a leading cause of end stage of renal disease (ESRD) and death worldwide. Reported in the 2010 Global Burden of Disease study (Lozano et al., 2013), CKD was ranked from 27th in 1990 (age-standardised annual death rate of 15.7 per 100 000) and rose to 18th in 2010 (annual death rate 16.3 per 100 000) in the list of causes of total number of global deaths. The growing momentum of CKD was just second to that for HIV and AIDS. This situation has claimed our serious attention and the prompt action is imperative. Thus, effective early diagnosis biomarker and treatment of CKD are urgent needed. / Fibrosis is the key feature during renal lesion formation and progression in CKD which will be ended in ESRD eventually. It has been established that Transforming growth factor β/Combination of the Drosophila protein "Mothers against decapentaplegic" (MAD) and C. elegant protein SMA (TGF-β/Smad) signaling plays a central role in the pathogenesis of renal fibrosis. For last two decades, dissection of the critical role of TGF-β/Smad signaling in the fibrogenesis of CKD has unveiled that Smad3 plays a pathogenic but Smad2 and Smad7 play a protective role in renal fibrosis. As TGF-β/Smad signaling also plays a crucial function in immunity, targeting TGF-β/Smad signaling may cause adverse effect. Thus, targeting specific downstream of T GF-β/Smad signaling should be a better alternative to fight against CKD. / A score of years has elapsed from the first microRNA discovery. MicroRNAs are important post-transcriptional regulators of gene expression. It is now widely acknowledged that the dysregulation of microRNA expression or action underlies the onset and/or development of various human diseases including kidney disease. Recently, researches have substantial evidences that microRNAs are tightly associated with the pathogenesis of kidney disease. In addition, eye-catching tangible evidences showed that several microRNAs are specific downstream of TGF-β/Smad3 signaling. Moreover, we found that not only in TGF-β and angiotensin II induced fibrotic response in vitro, but also in mouse models of obstructive and hypertensive nephropathy, microRNA-433-Azin1 axis is vital in renal fibrosis and is closely related with TGF-β/Smad3 signaling. Last but not least, our study suggests that, using a safe, effective, ultrasound microbubble-mediated gene transfer therapeutic method can significantly halt the progression of kidney lesions and reduce renal tissue damage and the excretion of albuminuria by balancing the microRNA-433-Azin1 axis. Hence, microRNA-433-Azin1 axis may act either as biomarkers favorable early diagnosis or therapeutic targets to halt or even reverse CKD. / The bench and bedside research on preventing and managing CKD have gained fruitful results in recent decades. Therapeutic strategy against TGF-β/Smad3 specific microRNA brings us a bright future in combating against CKD. However, more endeavors are necessary to eliminate the enormous burden of CKD worldwide. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Li, Rong. / Thesis (Ph.D.) Chinese University of Hong Kong, 2014. / Includes bibliographical references (leaves 182-202). / Abstracts also in Chinese.

背景：纤维化是各种因素导致肾脏慢性损伤的最终病理过程，是决定肾功能转归的关键因素。肌纤维母细胞作为构成肾脏纤维化组织的主要细胞成分，其来源尚不清楚。本研究认为骨髓来源的巨噬细胞向肌纤维母细胞转分化（MMT）可能是肾脏纤维化中肌纤维母细胞的主要来源。我们分别在慢性肾脏病患者的肾活检组织和小鼠单侧输料管梗阻模型（UUO）中验证这一假说。 / 方法：我们用激光共聚焦技术和流式细胞染色的方法检测小鼠UUO肾脏和患者肾活检组织中的MMT细胞(F4/80⁺α-SMA⁺或CD68⁺α-SMA⁺)。为了验证骨髓来源的MMT在肾纤维化中的重要作用，UUO模型分别在以下小鼠进行：1）去除骨髓的C57BL/6J小鼠，给予或不给予绿色荧光蛋白（GFP）标记的骨髓细胞移植；2）GFP⁺骨髓的嵌合体小鼠；3）巨噬细胞敲除或不敲除的lysM-Cre/DTR小鼠；4）GFP⁺Smad3⁺/⁺ 或GFP⁺Smad3⁻/⁻骨髓的嵌合体小鼠。我们用实时定量PCR和Western blot检测小鼠肾组织collagen-I和α-SMA水平。另外，我们观察MMT细胞和PDGFR-β⁺ pericytes, CD45⁺collagen I⁺ fibrocytes的关系。最后，通过观察GFP⁺Smad3⁻/⁻骨髓嵌合体小鼠UUO模型肾纤维化程度和TGF-β1刺激下TGF-β受体II或Smad3敲除的骨髓巨噬细胞MMT的不同进一步探索TGF-β/Smad3通路对MMT的影响。 / 结果：去除骨髓后，肾脏collagen-I沉积和α-SMA⁺肌纤维母细胞生成显著受抑制，骨髓细胞移植可以恢复肾脏纤维化，免疫荧光染色显示嵌合体小鼠中多数（80-90%）肌纤维母细胞来自于骨髓巨噬细胞转分化。同时，在白喉霉素诱导的巨噬细胞敲除小鼠中，50-60%巨噬细胞被去除，伴有纤维化明显减少，并且和MMT细胞显著减少相关。进一步验证巨噬细胞通过MMT直接参与肾脏纤维化过程。患者肾活检组织亦可见不同数目MMT细胞，纤维化活跃组织中MMT细胞可占到肌纤维母细胞总数的80%。另外，我们发现无论在小鼠模型还是患者肾活检组织中，多数MMT细胞表达pericyte（PDGFR-β⁺）和fibrocyte（CD45⁺collagen-I⁺）标记物。Smad3⁻/⁻骨髓嵌合体小鼠肾纤维化程度明显低于Smad3⁺/⁺骨髓嵌合体组，TGF-β1刺激下TGF-β受体II或Smad3敲除的骨髓巨噬细胞MMT明显低于不敲除组，提示TGF-β/Smad3通路在MMT过程中起重要作用。 / 结论：骨髓来源的MMT是肾纤维化组织中肌纤维母细胞的主要来源，TGF-β/Smad3 通路在MMT 过程中起重要作用。 / Background: Fibrosis is the ultimate pathological feature and determinant process for chronic kidney disease (CKD) regardless of the underlying etiology. Myofibroblasts are a key cell type in renal fibrosis by producing excessive collagen matrix. However, the origin of myofibroblasts during renal fibrosis remains largely controversial. This thesis tested the hypothesis that bone marrow (BM)-derived macrophage myofibroblast transition (MMT) may be a key pathway leading to renal fibrosis in patients with CKD and in a mouse model of unilateral ureteral obstructive nephropathy (UUO). / Methods: Renal fibrosis was assessed by expression of fibrotic marker collagen I and α-SMA using real-time PCR and western-blot analysis. MMT was determined in both mouse and human kidneys by confocal microscopy and flow cytometry with α-SMA⁺F4/80⁺ (or CD68⁺). The critical role of BM-derived MMT in renal fibrosis was examined in a mouse model of UUO, with various conditions: 1) BM depletion followed by BM transplantation (BMT) with GFP⁺ BM cells; 2) in GFP⁺ BM chimeric mice; 3) in lysM-Cre/DTR mice with or without inducible macrophage deletion; 4) in GFP⁺Smad3⁺/⁺ or GFP⁺Smad3⁻/⁻ BM chimeric mice. In addition, MMT was also validated in renal biopsy tissues from patients with different forms of CKD. Further more, we also studied the relationship between MMT and PDGFR-β⁺ pericytes or CD45⁺collagen I⁺ fibrocytes in both human and mouse fibrotic kidneys. Finally, mechanisms of MMT was examined in the UUO kidney induced in GFP⁺Smad3⁻/⁻ BM chimeric mice and in BM macrophages lacking TGF-β receptor II or Smad3. / Results: As described in Chapter III, mice with BM deletion were protected from renal fibrosis as demonstrated by blocking α-SMA⁺ myofibroblasts and collagen I accumulation. In contrast, BMT restored renal fibrosis in UUO kidney, demonstrating the critical role for BM cells in renal fibrosis. Importantly, the majority (85-90%) of α-SMA⁺ myofibroblasts were derived from BM macrophages as identified by GFP⁺F4/80⁺α-SMA⁺ revealing BM-macrophages given rise to myofibroblasts via MMT during kidney fibrosis. Similarly, MMT appeared as a major pathway of myofibroblast origin in patients with CKD, accounting for up to 80% of total myofibroblasts in the active stage of tissue fibrosis and fibrocellular crescents. To test the function role of macrophages in renal fibrosis via MMT, macrophages were conditionally deleted from the UUO kidneys in lysM-Cre/DTR mice as shown in Chapter IV, deletion (50-60%) of macrophages resulted in inhibition of MMT and renal fibrosis. Unexpectedly, most MMT cells (80-90%) were shown to co-express the pericyte marker (PDGFR-β⁺) and fibrocyte markers (CD45⁺collagen I⁺) in both human CKD and UUO (Chapter V), suggesting a BM macrophage origin for pericytes and fibrocytes during renal fibrosis. Finally, TGF-β/Smad3 appeared to be a mechanism driven MMT because mice and BM macrophages lacking either Smad3 or TβRII were protected against MMT and progressive renal fibrosis in the UUO kidney and in vitro. / Conclusions: MMT is derived from BM macrophages and regulated by TGF-β/Smad3. MMT is a major pathway of myofibroblast origin during renal fibrosis in both human and animal model of CKD. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Wang, Shuang. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 161-179). / Abstracts also in Chinese. / Chapter ABSTRACT --- p.ii / Chapter DECLARATION --- p.viii / Chapter ACKNOWLEDGEMENTS --- p.ix / Chapter TABLE OF CONTENTS --- p.xi / Chapter LIST OF ABBREVIATION --- p.xv / Chapter LIST OF FIGURES AND TABLES --- p.xvii / Chapter CHAPTER I --- p.1 / INTRODUCTION --- p.1 / Chapter 1. 1 --- Renal fibrosis and myofibroblasts --- p.2 / Chapter 1. 1. 1 --- Pathology of renal fibrosis --- p.2 / Chapter 1. 1. 2 --- The generation and modulation of myofibroblasts. --- p.3 / Chapter 1. 1. 2. 1 --- EMT and EndMT --- p.5 / Chapter 1. 1. 2. 2 --- Pericytes --- p.8 / Chapter 1. 1. 2. 3 --- Fibrocytes --- p.16 / Chapter 1. 2 --- Role of macrophage in fibrogenesis --- p.21 / Chapter 1. 3 --- TGF-β signaling pathway in renal fibrosis --- p.23 / Chapter 1. 3. 1 --- TGF-β superfamily --- p.23 / Chapter 1. 3. 2 --- TGF-β/Smad signaling pathway --- p.24 / Chapter CHAPTER II --- p.29 / MATERIALS AND METHODS --- p.29 / Chapter 2. 1 --- Materials --- p.30 / Chapter 2. 1. 1 --- Regents and equipments --- p.30 / Chapter 2. 1. 1. 1 --- Regents and equipment for mouse genotyping --- p.30 / Chapter 2. 1. 1. 2 --- Regents and equipments for real-time PCR --- p.30 / Chapter 2. 1. 1. 3 --- Reagents and equipments for immunohistochemistry staining --- p.31 / Chapter 2. 1. 1. 4 --- Reagents and equipment for flow cytometry --- p.32 / Chapter 2. 1. 2 --- Buffer --- p.32 / Chapter 2. 1. 2. 1 --- Buffers for immunohistochemistry and immunofluorescence staining --- p.32 / Chapter 2. 1. 2. 2 --- Buffers for western blot --- p.35 / Chapter 2. 1. 3 --- Sequences of primers for genotyping and real-time PCR --- p.41 / Chapter 2. 1. 4 --- Antibodies --- p.42 / Chapter 2. 2 --- Methods --- p.44 / Chapter 2. 2. 1 --- Generation of gene modified mice --- p.44 / Chapter 2. 2. 2 --- Bone marrow transplantation --- p.45 / Chapter 2. 2. 3 --- Conditional macrophage deletion --- p.45 / Chapter 2. 2. 4 --- Unilateral ureteral obstruction (UUO) mouse model --- p.46 / Chapter 2. 2. 5 --- Histology and immunohistochemistry --- p.46 / Chapter 2. 2. 5. 1 --- Processing paraffin sections --- p.46 / Chapter 2. 2. 5. 2 --- Deparaffinization and hydration --- p.47 / Chapter 2. 2. 5. 3 --- Blocking endogenous peroxidase --- p.47 / Chapter 2. 2. 5. 4 --- Antigen retrieval --- p.48 / Chapter 2. 2. 5. 5 --- Antigen and antibody reaction --- p.48 / Chapter 2. 2. 5. 6 --- Detection of target signals --- p.49 / Chapter 2. 2. 5. 7 --- Quantification of immunohistochemistry staining --- p.49 / Chapter 2. 2. 6 --- Immunofluorescence staining and confocal microscopy analysis --- p.49 / Chapter 2. 2. 6. 1 --- Processing tissue for immune-fluorescent (IF) staining --- p.49 / Chapter 2. 2. 6. 2 --- Serum blocking --- p.50 / Chapter 2. 2. 6. 3 --- Antigen antibody reaction --- p.50 / Chapter 2. 2. 6. 4 --- Signal detection --- p.51 / Chapter 2. 2. 7 --- Flow cytometry --- p.52 / Chapter 2. 2. 7. 1 --- Preparation of single cell suspension --- p.52 / Chapter 2. 2. 7. 2 --- Cell fixation and permeabilization --- p.53 / Chapter 2. 2. 7. 3 --- Staining --- p.53 / Chapter 2. 2. 7. 4 --- Signal detection and analysis --- p.54 / Chapter 2. 2 .8 --- Real time PCR --- p.55 / Chapter 2. 2. 8. 1 --- Total RNA extraction --- p.55 / Chapter 2. 2. 8. 2 --- Reverse transcription --- p.56 / Chapter 2. 2. 8. 3 --- Real-time PCR --- p.57 / Chapter 2. 2. 8. 4 --- Analysis of real-time PCR --- p.57 / Chapter 2. 2. 9 --- Western blot --- p.58 / Chapter 2. 2. 9. 1 --- Protein extraction from tissue --- p.58 / Chapter 2. 2. 9. 2 --- Protein concentration measurement --- p.59 / Chapter 2. 2. 9. 3 --- SDS-PAGE electrophoresis --- p.59 / Chapter 2. 2. 9. 4 --- Protein transfer --- p.60 / Chapter 2. 2. 9. 5 --- Blocking --- p.61 / Chapter 2. 2. 9. 6 --- Antibodies incubation and signal detection --- p.62 / Chapter 2. 2. 9. 7 --- Stripping --- p.62 / Chapter CHAPTER III --- p.63 / EVIDENCE FOR MMT AS A NEW PATHWAY OF MYOFIBROBLAST ORIGIN IN RENAL FIBROSIS --- p.63 / Chapter 3. 1 --- Introduction --- p.64 / Chapter 3. 2 --- Materials and methods --- p.65 / Chapter 3. 2. 1 --- Human renal biopsy tissues --- p.65 / Chapter 3. 2. 2 --- Experimental design --- p.65 / Chapter 3. 2. 3 --- Bone marrow transplantation and GFP⁺ BM chimeric mice --- p.66 / Chapter 3. 2. 4 --- Immunohistochemistry --- p.66 / Chapter 3. 2. 5 --- Immunofluorescence and confocal microscopy analysis --- p.67 / Chapter 3. 2. 6 --- Real-time PCR --- p.68 / Chapter 3. 2. 7 --- Western blot analysis --- p.68 / Chapter 3. 2. 8 --- Flow cytometry --- p.68 / Chapter 3. 3 --- Results --- p.69 / Chapter 3. 3. 1 --- BM-derived myofibroblasts play a key role in renal fibrosis in a mouse model of UUO --- p.69 / Chapter 3. 3. 1. 1 --- α-SMA⁺ myofibroblasts are derived from BM and determine renal fibrosis in a mouse model of UUO --- p.69 / Chapter 3. 3. 1. 2 --- BM as a major source of collagen production in a mouse model of UUO --- p.73 / Chapter 3. 3. --- 2 Evidence for BM derived macrophage-myofibrobalst transition (MMT) in a mouse model of UUO --- p.77 / Chapter 3. 3. 2. 1 --- Characterization of GFP⁺ BM chimeric mice --- p.77 / Chapter 3. 3. 2. 2 --- Evidence for bone marrow-derived MMT is the major source of myofibroblast origin in the UUO kidney --- p.79 / Chapter 3. 3. 3 --- Evidence for MMT in human fibrotic kidney tissues --- p.84 / Chapter 3. 3. 4 --- M2 macrophage is the predomimant phenotype of macrophages in the fibrotic kidney of UUO mouse model. --- p.88 / Chapter 3. 4 --- Discussion --- p.90 / Chapter 3. 5 --- Conclusion --- p.93 / Chapter CHAPTER IV --- p.94 / Chapter GE --- CONDITIONAL MACROPHA DELETION INHIBITS MMT AND RENAL FIBROSIS --- p.94 / Chapter 4. 1 --- Introduction --- p.95 / Chapter 4. 2 --- Materials and methods --- p.98 / Chapter 4. 2. 1 --- Generation of lysM-Cre/DTR mice --- p.98 / Chapter 4. 2. 2 --- Conditional deletion of macrophage --- p.98 / Chapter 4. 2. 3 --- Unilateral Ureteral Obstruction (UUO) mouse model --- p.98 / Chapter 4. 2. 4 --- Real-time PCR --- p.99 / Chapter 4. 2. 5 --- Western blot analysis --- p.99 / Chapter 4. 2. 6 --- Immunohistochemisty --- p.99 / Chapter 4. 2. 7 --- Immunofluorescence --- p.99 / Chapter 4. 3 --- Results --- p.100 / Chapter 4. 3. 1 --- Characterization of lysM-Cre/DTR mice --- p.100 / Chapter 4. 3. 2 --- Conditional deletion of macrophage in a mouse model of UUO --- p.101 / Chapter 4. 3. 3 --- Conditional deletion of macrophage suppresses α-SMA⁺ myofibroblast accumulation in a mouse model of UUO --- p.104 / Chapter 4. 3. 4 --- Conditional deletion of macrophage inhibits collagen I production in a mouse model of UUO --- p.106 / Chapter 4. 3. 5 --- Conditional deletion of macrophage inhibits renal fibrosis through reducing MMT cells in a mouse model of UUO --- p.108 / Chapter 4. 4 --- Discussion --- p.111 / Chapter 4. 5 --- Conclusion --- p.113 / Chapter CHAPTER V --- p.114 / MMT CELLS SHARE PERICYTE AND FIBROCYTE PHENOTYPES --- p.114 / Chapter 5. 1 --- Introduciton --- p.115 / Chapter 5. 2 --- Materials and methods --- p.116 / Chapter 5. 2. 1 --- Human renal biopsy tissues --- p.116 / Chapter 5. 2. 2 --- Animals and UUO mouse model --- p.116 / Chapter 5. 2. 3 --- Immunofluorescence (IF) --- p.116 / Chapter 5. 2. 4 --- Flow cytometry --- p.117 / Chapter 5. 3 --- Results --- p.119 / Chapter 5. 3. 1 --- Evidence for MMT cells co-expressing pericyte marker in the fibrotic kidney of UUO model --- p.119 / Chapter 5. 3. 2 --- Evidence for MMT cells co-expressing pericyte marker in the fibrotic kidney from patients with chronic kidney diseases --- p.124 / Chapter 5. 3. 3 --- Evidence for MMT cells co-expressing fibrocyte marker in the fibrotic kidney of UUO model --- p.126 / Chapter 5. 3. 4 --- Evidence for MMT cells co-expressing fibrocyte marker in the fibrotic kidney from patients with chronic kidney diseases --- p.129 / Chapter 5. 4 --- Dscussion --- p.131 / Chapter 5. 5 --- Conclusion --- p.133 / Chapter CHAPTER VI --- p.134 / SMAD3 MEDIATES MMT DURING RENAL FIBROSIS --- p.134 / Chapter 6. 1 --- Introduction --- p.135 / Chapter 6. 2 --- Materials and methods --- p.137 / Chapter 6. 2. 1 --- Generation of Smad3⁺/⁺ and Smad3⁻/⁻ BM-Chimeric mice --- p.137 / Chapter 6. 2. 2 --- Generation of TbRII disrupted BM macrophages and Smad3⁻/⁻ BM macrophages --- p.137 / Chapter 6. 2. 3 --- UUO mouse model --- p.138 / Chapter 6. 2. 4 --- Cell culture --- p.138 / Chapter 6. 2. 5 --- Real-time PCR --- p.139 / Chapter 6. 2. 6 --- Western blot analysis --- p.139 / Chapter 6. 2. 7 --- Immunohistochemistry (IHC) --- p.139 / Chapter 6. 2. 8 --- Immunofluorescence (IF) --- p.139 / Chapter 6. 2. 9 --- Flow cytometry --- p.140 / Chapter 6. 3 --- Result --- p.141 / Chapter 6. 3. 1 --- Genotyping of Smad3 WT and Smad3 KO mice --- p.141 / Chapter 6. 3. 2 --- Smad3 knockout inhibits TGF-β1 induced MMT in vitro --- p.142 / Chapter 6. 3. 3 --- Disruption of TbRII inhibits TGF-β1 induced MMT in vitro --- p.145 / Chapter 6. 3. 4 --- Deletion of BM Smad3 inhibits α-SMA expression in the UUO kidney --- p.147 / Chapter 6. 3. 5 --- Deletion of BM Smad3 inhibits collagen-I production in the UUO kidney --- p.149 / Chapter 6. 3. 6 --- Inhibition of MMT is a mechanism by which BM Smad3 deficiency inhibits renal fibrosis in a mouse model of UUO --- p.150 / Chapter 6. 4 --- Discussion --- p.153 / Chapter 6. 5 --- Conclusion --- p.154 / Chapter CHAPTER VII --- p.155 / SUMMARY AND DISCUSSION OF THE MAJOR FINDINGS --- p.155 / Chapter 7. 1 --- Summary and discussion --- p.157 / Chapter 7. 1. 1 --- MMT is a major pathway of myofibroblast origin in renal fibrosis --- p.157 / Chapter 7. 1. 2 --- MMT cells shares both pericyte and fibrocyte phenotypes in renal fibrosis --- p.157 / Chapter 7. 1. 3 --- TGF-β/Smad3 is a key mechanism of MMT in renal fibrosis --- p.158 / Chapter 7. 2 --- Conclusion --- p.160 / Chapter REFERENCES --- p.161