Thesis--Aarhus. / Summary in English and Danish. Bibliography: p. [139]-145.

Thesis--Aarhus. / Summary in English and Danish. Bibliography: p. [139]-145.

Thesis (doctoral)--Lund University, 1994.

Thesis (doctoral)--Lund University, 1994.

Thesis (doctoral)--Rijksuniversiteit te Groningen.

A thesis submitted to the Faculty of Health Sciences, University of the Witwatersrand, in fulfilment of the requirements for the degree of Doctor of Philosophy Johannesburg, 2016 / Background: Despite preventive guidelines, iatrogenic contrast-induced nephropathy (CIN) ranks third as a cause of hospital acquired acute kidney injury (AKI), and impacts significantly on morbidity and mortality and is associated with high hospital costs. In Sub-Saharan Africa, the rates and risk factors for CIN remain unexplored. Despite the positive association of genetic polymorphisms in the TNFα and IL10 genes with CIN in Asian populations, the CIN genetic susceptibility in other races is unknown. Serum creatinine is a sub-optimal biomarker for the early diagnosis of CIN resulting in delayed interventions. This study investigated rates, risk factors and outcomes of CIN, the influence of genetic susceptibility to CIN in the black population and lastly, the accuracy of novel biomarkers in the early diagnosis of CIN and prognosticating patient outcomes. Methods: This was a prospective case-controlled study conducted at Charlotte Maxeke Johannesburg Academic Hospital, in South Africa from January 1, 2014 to December 30, 2015.Hospitalized patients undergoing enhanced computed tomography and angiography were consecutively recruited to the study and followed up for development of CIN. CIN was defined as an increase in serum creatinine >25% or an absolute increase of >44 μmol/l from baseline at 48-72 hours after exposure to contrast media. In the second part of the study, a nested case-controlled cohort that included 30 CIN patients and 60 controls (those undergoing contrast administrations and not meeting CIN criteria) were ethnically matched for gender, and age in a case: control ratio of 1:2 at all-time intervals. Sera for neutrophil gelatinaseassociated lipocalin-2 (NGAL), cystatin C, beta-2 microglobulin (β2M), interleukin 18 (IL18), IL10, and tumor necrosis factor alpha (TNFα) were collected at four time points: baseline (pre-contrast), 24 hours, 48 hours and ≥5-7 days after contrast administration and their concentrations were determined using luminex assays and an enzyme linked immunosorbent assay for β2M as per manufacturer’s instructions. The areas under receiver operating characteristic curves (AUROC) were generated to determine accuracy of novel biomarkers to diagnose CIN and CIN mortality. Genomic DNA was extracted from peripheral blood samples of 208 black South Africans using the Maxwell DNA purification kit (Promega AS1010, USA) and their genotypes for - 308(rs1800629) and -857(rs1799724) in the TNFα gene and -592(rs1800872), - 819(rs1800871), -1082 (rs1800896) and +1582(rs1554286) in the IL10 gene were determined by restriction fragment length polymorphism (RFLP). Results: We recruited 371 hospitalized patients (mean age 49.3±15.9); the rates of CIN were4.6% and 16.4% respectively, using an absolute or relative increase in serum creatinine from baseline. Anaemia was an independent predictor for the development of CIN (RR 1.71, 95% 1.01-2.87; p=0.04). The median serum albumin was 34 g/l (IQR: 29-39.5) vs. 38 g/l (IQR: 31-42), p=0.01 in the CIN and control groups respectively.Mortality was significantly increased in the CIN group (22.4% vs. 6.8%; p<0.001), and CIN together with anaemia predicted mortality with a 2-fold (p=0.01) and a 3-fold (RR p=0.003) riskrespectively. The median cystatin C at 24 hours (p<0.001) and β2M(at all-time points)levels were significantly higher in the CIN group compared to controls. The median cystatin C at 24 hours and β2Mlevels at 48 hours were 856.59 ng/ml (IQR 620.75-1002.96) vs. 617.42 ng/ml (IQR 533.11-805.20); p<0.001 and 5.3 μg/ml (IQR 3.8-6.9) vs. 3.3 μg/ml (IQR 2.7-4.5); p<0.001 with AUROCs of 0.75 and 0.78 respectively for early CIN discrimination.Pre-contrast IL18 (p <0.001), β2M (p=0.04) and TNFα (p<0.001) levels were significantly higher in the nonsurviving group and their AUROC were 0.83, 0.82 and 0.94 for CIN+ mortality. Baseline NGAL was a better marker for excluding patients at higher risk of developing CIN with negative predictive and positive predictive values of 0.81 and 0.50 respectively. The frequency of TNFα -308 AA genotype was significantly increased in the CIN group compared to controls (13.3% vs.1.82%, p=0.016) and the presence of the TNFα-308 AA (high producer) vs. GA genotypes was associated with a 9-fold CIN risk (9.24, 95% CI, 1.88-45, p=0.006). The IL10-1082 AA-allele (low producer) was significantly higher in the non-surviving CIN+ patients compared to controls (p=0.01). Conclusions:CIN occurred at a relatively high rate in our study and predicted poorer clinical outcomes. The presence of CIN and anaemia positively predicted mortality. Caution should be exercised in patients with anaemia and hypoalbuminaemia undergoing contrast studies. Serumcystatin C was the best novel biomarker for the early diagnosis of CIN and while baseline NGAL is superior as a biomarker for excluding patients at higher risk for CIN. IL18, β2M and TNFα are the best novel biomarkers for predicting the prognosis of patients with CIN. Increased frequency of the TNFα-308 AA genotype is a predisposing factor for CIN development. The low producer IL10-1082 AA genotype decreases survival in patient with CIN. / MT2017

This document only includes an excerpt of the corresponding thesis or dissertation. To request a digital scan of the full text, please contact the Ruth Lilly Medical Library's Interlibrary Loan Department (rlmlill@iu.edu).

The aim of this thesis was to assess whether platelet, endothelial and coagulation biomarkers of thrombotic risk are increased in ECKD-HD patients. Five individual studies were performed (1) venous blood samples between controls and resting HD patients, (2) simultaneous blood samples between vascular access (VA) and venous samples in HD patients (3) pre and post dialysis from the VA, (4) samples pre and post dialysis in venous samples, (5) a retrospective study of VA thrombosis in HD patients. Venous blood samples were taken from 78 resting healthy volunteers and from 78 HD patients immediately before and 30 minutes after dialysis. We also took blood samples from the VA of 55 patients immediately before and after dialysis. In 26 patients venous and VA samples were taken simultaneously. Our results have shown HD patients potentially have evidence of a prothrombotic state compared to controls. This is further increased by each session of dialysis and is present in both VA and venous samples distant from the site of haemodialysis. We have shown some differences in platelet activation and inflammatory markers between simultaneous VA and venous samples. Furthermore, some of these biomarkers may be associated with a retrospective history of VA occlusion. Our study has shown that in patients with ECKD on HD there may be evidence of an underlying prothrombotic tendency. There is a need to determine the optimal anti-platelet and anti-coagulation therapy in these patients.

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