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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