The incidence and mortality from cardiovascular disease (CVD) in patients with chronic kidney disease (CKD) far exceeds that seen in the general population. Whilst a number of risk factors and associations have been identified in patients with CKD that may contribute to the increased risk of CVD, our understanding of the underlying pathophysiology remains poor. It has previously been reported that uraemic animals sustain larger myocardial infarcts and that this ‘reduced ischaemia tolerance’ may in part explain the excess mortality from CVD seen in CKD patients. The aim of this work was to establish an in vivo model of uraemic myocardial infarction in order to further explore the pathophysiology of uraemic CVD with particular focus on ameliorating myocardial ischaemia-reperfusion injury using ischaemic and pharmacological preconditioning. An increase in myocardial infarct size was demonstrated in the sub-total nephrectomy model of chronic uraemia, confirming previous reports in the literature. However, infarct size was not found to be increased in adenine diet induced renal failure. In addition, it was demonstrated for the first time, that the techniques of ischaemic preconditioning (IPC) and remote ischaemic preconditioning (RIPC) are both efficacious and not attenuated by chronic uraemia induced by sub-total nephrectomy or adenine diet (IPC only). Investigations were undertaken using an agent (a HIF stabiliser, FG4497) to induce pharmacological preconditioning in both animals with renal insufficiency and those without. These studies demonstrate that stabilisation of hypoxia inducible factor (HIF) may be a promising strategy to induce pharmacological preconditioning. It is hoped that this work may lay the foundations for future investigations to determine why sub-totally nephrectomised rats have larger infarcts whilst those with adenine induced renal failure, with a substantially greater degree of renal dysfunction, do not. Moreover, it is hoped that; by demonstrating that uraemia 3 does not prevent or attenuate the myocardial protection afforded by ischaemic preconditioning, the recruitment of patients with CKD will be encouraged to clinical trials of both ischaemic preconditioning and other therapies to limit myocardial infarction.

Cardiovascular disease is the leading cause of death in patients with underlying chronic kidney disease (CKD). Up to one third of patients presenting with an acute coronary syndrome have CKD stage 3-5. Outcomes following acute myocardial infarction in patients with underlying CKD remain poor. CKD patients are routinely excluded from clinical trials in novel cardioprotective strategies resulting in a paucity of prospective data on which to base guidelines for clinical practice. The aims of this work were to: • Establish and characterise two models of chronic uraemia in rodents: the subtotal nephrectomy model and the adenine diet model. • Determine the effects of underlying chronic uraemia on myocardial ischaemia tolerance. • Examine pharmacological cardioprotective strategies in the context of underlying uraemia using a PARP inhibitor • Investigate the cardioprotective effects of ischaemic conditioning in the context of uraemia. Ischaemic preconditioning and postconditioning protocols were used in both uraemic and non-uraemic animals in a model of acute myocardial infarction. • Preliminary work, using standard molecular biological techniques, was carried out in order to confirm the putative survival pathways responsible for the effect of preconditioning. • Investigate the effect of combining early and late remote ischaemic preconditioning to identify whether summation of these strategies could provide additional tissue protection in a model of acute kidney injury. The results demonstrate that both models develop a uraemic phenotype. Subtotal nephrectomy animals exhibit reduced ischaemia tolerance. PARP inhibition as a pharmacological post conditioning agent was shown to be ineffective at conferring tissue protection, whereas both ischaemic preconditioning and postconditioning were effective cytoprotective strategies in both non-uraemic and uraemic animals. Furthermore, additional benefit was seen when early and late remote preconditioning were summated in a rodent model of acute kidney injury. This work provides a basis for future clinical trials in cardioprotection in the context of underlying CKD.

Mutations of the gene encoding the inositol polyphosphate 5-phosphatase OCRL1 are responsible for causing two disorders in humans: Lowe syndrome and type 2 Dent's disease (Dent-2). Lowe syndrome (oculocerebrorenal syndrome of Lowe) is an X-linked genetic disorder that causes multisystem defects affecting predominantly the eyes, brain and kidneys. Dent-2 disease is very similar to Lowe syndrome, but it affects primarily the kidneys with little or no symptoms in the brain and eyes. The enzymatic activity, structure and binding partners of the OCRL1 protein have been described and progress on the cellular functions of OCRL1 has been made. However the studies to date have not provided the necessary insight to explain the tissue-specific defects observed in Lowe syndrome and Dent-2 patients. In order to investigate the role of OCRL1 and the consequences of its deficiency in a physiological context an animal model is required. We have chosen the zebrafish for this study due to its suitability for investigating vertebrate early development and the abundance of research techniques available for this model organism. We have studied the expression of OCRL1 in zebrafish and its role in the early embryonic development. We have also investigated its role in the endocytic function of the zebrafish larval pronephric kidney. Finally we have investigated its role in ciliogenesis and function of pronephric cilia. Our studies show that OCRL1 depletion does not cause gross developmental defects, nor affects the development of pronephros, but impairs their endocytic activity. We have also shown, that efficient pronephric uptake requires OCRL1 interactions with clathrin, Rab GTPase family proteins, APPL1 and IPIP27A/B. Our studies link the reduced uptake with lowered levels of megalin receptor, which is responsible for the bulk of protein reabsorption in the kidney. Together our results strongly suggest that defects in this process are responsible for low molecular weight proteinuria present in Lowe syndrome and Dent-2 patients and zebrafish is a suitable model to study the renal aspect of these diseases.

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

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / O acidente causado pela serpente Lachesis muta muta à grave e o seu veneno à responsÃvel por uma sÃrie de alteraÃÃes sistÃmicas, como a hipotensÃo arterial. Neste trabalho, foram investigados os efeitos renais do veneno total desta serpente em sistema de perfusÃo renal e em cultura de cÃlulas tubulares renais do tipo MDCK (Madin-Darby Canine Kidney). Foram utilizados ratos Wistar machos pesando entre 250 e 300g, cujos rins foram isolados e perfundidos com SoluÃÃo de Krebs-Hanseleit contendo 6%p/v de albumina bovina previamente dialisada. Foram investigados os efeitos do veneno (10 Âg/mL; n=6) sobre a PressÃo de PerfusÃo (PP), ResistÃncia Vascular Renal (RVR), Fluxo UrinÃrio (FU), Ritmo de FiltraÃÃo Glomerular (RFG), Percentual de Transporte Tubular de SÃdio (%TNa+), de PotÃssio (%TK+) e de Cloreto (%TCl-). O veneno da Lachesis muta muta foi adicionado apÃs 30 minutos de controle interno. As cÃlulas MDCK foram cultivadas em meio de cultura RPMI 1640 suplementado com 10% v/v de Soro Bovino Fetal e entÃo avaliadas na presenÃa do veneno total da serpente Lachesis muta muta nas concentraÃÃes 1&#956;g/mL, 10&#956;g/mL e 100&#956;g/mL. ApÃs 24 horas de experimento, foram realizados ensaios de viabilidade e citotoxicidade celular. O veneno total, na dose de 10&#956;g/mL, causou reduÃÃo transitÃria na pressÃo de perfusÃo (C60= 108,27  4,88 mmHg; VT L. m. muta60= 88,17  5,27# mmHg; C90= 108,69  5,08 mmHg; VT L. m. muta90= 78,71  6,94#* mmHg) e na resistÃncia vascular renal (C60= 5,57  0,49 mmHg/mL.g-1.min-1; VT L. m. muta60= 3,50  0,22# mmHg/mL.g-1.min-1; C90= 5,32  0,57 mmHg/mL.g-1.min-1; VT L. m. muta90= 3,11  0,26#* mmHg/mL.g-1.min-1) alÃm de aumento do fluxo urinÃrio (C60= 0,158  0,015 mL.g-1.min-1; VT L. m. muta60= 0,100  0,012# mL.g-1. min-1; C120= 0,160  0,020 mL.g-1.min-1; VT L. m. muta120= 0,777 0,157#*) e do ritmo de filtraÃÃo glomerular (C60= 0,707  0,051 mL.g-1.min-1; VT L. m. muta60= 0,232  0,042# mL.g-1.min-1; C120= 0,697  0,084 mL.g-1.min-1; VT L. m. muta120= 1,478  0,278#* mL.g-1.min-1). A dose estudada tambÃm promoveu reduÃÃo significativa do percentual de transporte tubular de sÃdio (%TNa+), potÃssio (%TK+) e cloreto (%TCl-) nos trÃs perÃodos analisados (30, 60 e 90 minutos), com conseqÃente aumento do clearence osmÃtico(Cosm) (C90= 0,141  0,011; VT L. m. muta90= 0,309  0,090; C120= 0,125  0,016; VT L. m. muta120= 0,839  0,184#*). A avaliaÃÃo histopatolÃgica revelou a presenÃa de Ãreas focais com cÃlulas com nÃcleos picnÃticos nos tÃbulos renais dos rins perfundidos com veneno. O veneno tambÃm apresentou efeito citotÃxico sobre as cÃlulas MDCK apenas nas concentraÃÃes de 10Âg/mL e 100Âg/mL reduzindo a viabilidade destas cÃlulas a 38,60  17,9% e 10,62  2,9%, respectivamente. Estes resultados demonstram que o veneno total da Lachesis muta muta alterou todos os parÃmetros renais avaliados na perfusÃo renal, induzindo hipotensÃo transitÃria e intensa diurese. O veneno tambÃm possui aÃÃo citotÃxica sobre as cÃlulas MDCK apÃs 24 horas de incubaÃÃo. / The accident caused by the snake Lachesis muta muta is serious and its venom is responsible for many systemic changes, such as hypotension. In this work, the renal effects of the total venom of this snake in the renal perfusion system and in cultured renal tubular cells of the type MDCK (Madin-Darby Canine Kidney) are investigated. Isolated kidneys from Wistar rats, weighing 250 to 300g, were perfused with previously dialysed, Krebs-Henseleit solution containing 6% w/v bovine albumin. The effects of the venom (10 mg / mL, n = 6) on the perfusion pressure (PP), renal vascular resistance (RVR), urinary flow (UF), glomerular filtration rate (GFR), sodium tubular transport (%TNa+), potassium tubular transport (%TK+) and chloride tubular transport (%TCl-) were submitted to analysis. Lachesis muta muta venom was added to the system after 30 minutes of internal control. MDCK cells were cultured in RPMI 1640 medium supplemented with 10% v/v fetal bovine serum and then assessed in the presence of the total venom of the snake Lachesis muta muta in the concentrations of 1&#956;g/mL, 10&#956;g/mL and 100&#956;g/mL 24 hours afterwards, tests concerning viability and cellular cytotoxicity were brought about. The total venom (10&#956;g/mL) promoted a transient reduction in perfusion pressure (C60= 108.27  4.88; VT L. m. muta60= 88.17  5.27#; C90= 108.69  5.08; VT L. m. muta90= 78.71  6.94#*) and renal vascular resistance (C60= 5.57  0.49; VT L. m. muta60= 3.50  0.22#; C90= 5.32  0.57; VT L. m. muta90= 3.11  0.26#*); increase in urinary flow (C60= 0.158  0.015; VT L. m. muta60= 0.100  0.012#; C120= 0.160  0.020; VT L. m. muta120= 0.777 0.157#*) and in glomerular filtration rate (C60= 0.707  0.051; VT L. m. muta60= 0.232  0.042#; C120= 0.697  0.084; VT L. m. muta120= 1.478  0.278#*). Such a concentration also reduced significantly sodium tubular transport (%TNa+), potassium tubular transport (%TK+) and chloride tubular transport (%TCl-) in the three periods analyzed (30, 60 and 90 minutes), with a consequent increase in the osmotic clearance (Cosm) (C90= 0.141  0.011; VT L. m. muta90= 0.309  0.090; C120= 0.125  0.016; VT L. m. muta120= 0.839  0,.184#*). Histological analysis of the kidneys perfused with the poison revealed focal areas of renal tubular cells with nuclear pyknosis. The venom also promoted a cytotoxic effect on MDCK cells at concentrations 10&#956;g/mL and 100&#956;g/mL, thus reducing the viability of these cells to 38.60  17.9% and 10.62  2.9%, respectively. These results demonstrate that the venom of Lachesis muta muta altered all the renal parameters assessed in renal perfusion, inducing transient hypotension and intense diuresis. The venom also exhibits cytotoxic activity on MDCK cells after 24 hours of incubation.

Nos mamíferos, o fígado e o rim são órgãos fundamentais para uma adequada homeostase. Nos cetáceos, são de especial importância frente aos desafios da vida no ambiente marinho. O presente estudo teve como objetivo investigar as principais lesões hepáticas e renais de cetáceos do Brasil, utilizando-se amostras mantidas junto ao Banco de Tecidos de Mamíferos Marinhos (BTMM), Laboratório de Patologia Comparada de Animais Selvagens. Para a caracterização das lesões foram utilizadas técnicas anatomopatológicas, imuno-histoquímicas e ultraestrutural. Foram estudados 197 cetáceos de 18 espécies, encontrados mortos em decorrência de captura incidental em apetrecho de pesca, encalhe ou após tentativas de reabilitação. A principal espécie amostrada foi toninha (Pontoporia blainvillei) com 65,9% (130/197) dos casos. Quanto à distribuição geográfica as amostras provieram principalmente do estado de São Paulo (41,6%, 82/197), seguido do Rio Grande do Sul (36,5%, 72/192) e Ceará (11,7%, 23/197). Entre as principais lesões hepáticas diagnosticadas, as inclusões hialinas citoplasmáticas (IHC) apresentaram maior frequência (46,3%, 88/190), seguidas pelas hepatites portais linfoplasmocíticas crônicas observadas em 36,5% (69/190), esteatose, em 14,2% (27/190), hepatite necrótica, em 4,7% (9/190), e colangiohepatite parasitária, em 2,6% (5/190) dos casos. A ocorrência de IHC foi mais frequente em animais capturados do que encalhados. Entre as principais lesões renais diagnosticadas, a glomerulonefrite membranosa apresentou maior frequência (14,5%, 28/192). Foram observadas também glomerulonefrine membranoproliferativa, em 10,4% (20/192), nefrite intersticial, em 10,9% (21/192), cistos simples, em 4,16% (8/192), doença glomerulocística primária, em 4,6% (9/192), doença glomerulocística secundária (DGCS), em 8,3% (16/192), e doença renal policística e adenoma tubular, com 0,5% (1/192) de ocorrência cada. A incidência de DGCS apresentou diferença entre as espécies, sendo menos frequente em toninhas do que nos demais cetáceos. Um boto-cinza (Sotalia guianensis) morto em decorrência de captura incidental na baia de Paranaguá, Paraná, foi diagnosticado com toxoplasmose e devido à sua importância, fragmentos de todos seus órgãos, disponíveis no BTMM, foram avaliados. O presente estudo reflete a relevância em manter o BTMM, o qual consiste em uma fonte de informação ímpar, que possibilita a realização de estudos retroativos em tecidos de cetáceos e outras espécies de mamíferos aquáticos. O presente trabalho traz contribuições sobre as enfermidades em cetáceos, e aborda de maneira sistemática as lesões hepáticas e renais nestas espécies. Futuros estudos são necessários para elucidar aspectos sobre o impacto das lesões renais e hepáticas e sua relação com as condições mórbidas dos cetáceos, bem como para avaliar o impacto da toxoplasmose, nos cetáceos e outros mamíferos marinhos brasileiros. / In mammals, the main organs for an adequate homeostase are the liver and the kidney. These organs in Cetaceans have especial importance because of the challenges of life in a marine environment. This study had as main objective find the principal hepatic and renal lesions in Cetaceans from Brazil. Samples from the Marine Mammal Tissue Bank (BTMM) of the Laboratory of Comparative Pathology of Wild Animals were used. Anatomopathological, immunohistochemical and ultrastructural studies were performed. A total of 197 cetaceans belonging to 18 species were studied. They were found dead because of incidental capture or after attempts of rehabilitation for the stranded ones. Franciscana (Pontoporia blainvillei) was the principal specie sampled with a 65,9% (130/197) of the cases. Related to geographic distribution, samples were more frequent in São Paulo state (41,6%, 82/197), then Rio Grande do Sul (36,5%, 72/192) and Ceará (11,7%, 23/197). The hepatic lesions found include: hyaline cytoplasmatic inclusions (IHC) (46,3%, 88/190), lymphoplasmacytic chronic portal hepatitis (36,5%, 69/190), steatosis (14,2%, 27/190), necrotic hepatitis (4,7%, 9/190) and parasitic colangiohepatitis (2,6% , 5/190). The occurrences of IHC were more frequent in captured animals than stranded. The main kidney lesion found was the membranous glomerulonephritis (14,5%, 28/192). Additionally, there were observed membranoproliferative glomerulonephritis (10,4%, 20/192), intersticial nephritis (10,9%, 21/192), simple cysts 4,16% (8/192), glomerulocystic primary disease (4,6%, 9/192), glomerulocystic secondary disease (DGCS) (8,3% ,16/192) and polycystic kidney disease and tubular adenome (0,5%, 1/192). The incidence of DGCS differ among species, in Fransiscanas it was less frequent than in other cetaceans. A Guiana Dolphin (Sotalia guianensis) dead by incidental capture in the bay of Paranaguá, Paraná, was diagnosed with toxoplasmosis and because of its importance, fragments of all its organs available on BTMM, were evaluated. This study reflects the relevance to maintain the BTMM as an important primary source of information, enabling the realization of future reprospective studies in tissues of whales and other species of aquatic mammals. Furthermore, this study presents contributions on cetacean diseases and addresses in a systematic way lesions in the liver and kidney in these species. Future studies are necessary to elucidate aspects of the impact of renal and hepatic lesions and their relation to the morbid conditions of cetaceans, as well as to evaluate the impact of toxoplasmosis in cetaceans and other marine mammals in Brazil.

Lack of the enzyme 11β-hydroxysteroid dehydrogenase type 2 (11βHSD2) causes the syndrome of apparent mineralocorticoid excess (AME): low-renin hypertension, renal sodium (Na +) retention, hypokalaemic alkalosis and polyuria. This rare autosomal recessive disorder is observed in human kindreds carrying mutations in the HSD11B2 gene. Genetically modified mice, in which the homologue Hsd11b2 is rendered non-functional, have been used to study the pathogenesis of AME. Hitherto, data obtained from humans and mice have suggested that the physiological phenotype is a consequence of enhanced reabsorption of Na + through the epithelial sodium channel (ENaC) in the renal connecting tubule (CNT) and collecting duct. However, Hsd11b2 null mice exhibit epithelial hypertrophy in a different nephron segment, namely the distal convoluted tubule (DCT). The studies described herein aimed to characterise this structural phenotype and to examine the consequences for renal Na + reabsorption in AME. Hsd11b2 null mice exhibited hypertrophy and hyperplasia in the DCT, with an elevated rate of epithelial cell proliferation in this nephron segment at 60 days of age. Hsd11b2 null kidneys contained greater quantities of the thiazide-sensitive NaCl co-transporter (NCC), the dominant Na + transporter protein in the DCT. They also contained greater quantities of the phosphorylated forms of NCC that are associated with NaCl transport activity. Despite this, there was no increase in the proportion of filtered Na + that was reabsorbed in the DCT. This was assessed in anaesthetised mice, using clearance methodology to measure the thiazide-induced increment in the fractional excretion of Na + (FENa) during continuous ENaC blockade. Wild-type DCTs did not express 11βHSD2; therefore the structural and molecular changes were not a direct consequence of the loss of 11βHSD2 in affected cells. The discussion examines the likely mechanisms causing structural remodelling in the distal renal tubule of Hsd11b2 null kidneys and potential explanations for the dissociation between structural and functional phenotypes in the DCT. There are implications for our understanding of the cellular and molecular mechanisms underlying various renal phenomena including structural remodelling in the distal tubule, resolution of the ‘aldosterone paradox’ and escape from chronic aldosterone excess.

Cardiovascular disease (CVD) is highly prevalent in chronic kidney disease (CKD) patients. Whilst this can in part be explained by the high incidence of traditional CVD risk factors such as hypertension and diabetes evident in CKD patients, recent focus has been on non-traditional risk factors and their role in CVD progression. These include endothelial dysfunction, arterial stiffness, inflammation and oxidative stress. The potent vasoconstrictor endothelin-1 (ET-1) has been implicated in the pathogenesis of CKD and the CVD associated with it. Further understanding of the mechanisms by which it contributes to CKD and CVD pathogenesis, specifically its interactions with non-traditional risk factors are still required. Additionally, the potential applications of ET antagonists in renal disease have not been fully explored. This thesis aims to investigate the role of ET-1 in the development of renal disease and the associated inflammation, hypertension and vascular dysfunction through a series of in vitro, in vivo and clinical studies. I have demonstrated using in vitro techniques that murine macrophages (Mϕ) express both endothelin A (ETA) and endothelin B (ETB) receptors but that ET-1 does not elicit either a classical pro-inflammatory or alternative anti-inflammatory phenotype in Mϕ. I was however, able to show that M display chemokinesis towards ET-1 and M ETB receptors provide a novel clearance mechanism for ET-1 through receptor mediated dynamin-dependent endocytosis In an in vivo study I investigated whether ET-1 mediates the progressive renal injury after renal ischaemia reperfusion injury (IRI) that leads to the development of CKD. I demonstrated that endothelin A receptor antagonism provided long term beneficial effects reducing blood pressure and preventing progressive kidney injury, inflammation, and the development of fibrosis resulting from an episode of acute kidney injury (AKI). Similar benefits were observed with calcium channel blockade, suggesting hypertension may mediate some of the long term effects of renal IRI and anti-hypertensive treatments could prevent the development of CKD after AKI. Finally, in a clinical study I showed for the first time that CKD patients lack the diurnal variation in arterial stiffness that is seen in matched subjects without CKD. Alteration in the circadian variation of the ET-1 system may contribute to this. In summary, my studies have furthered our understanding of the role of ET-1 in CKD progression and the cardiovascular risk associated with it. Mϕ were shown to express both ET receptors and a novel mechanism of ET-1 clearance was observed in Mϕ. Using an in vivo model of AKI I was able to identify ETA receptor antagonism as a novel therapeutic agent in preventing the development of CKD caused by AKI where data are limited. Finally, alterations in the circadian rhythm of the cardiovascular system is emerging as an important factor in disease pathogenesis. Here the diurnal variation in arterial stiffness was described for the first time in a group of CKD patients and matched controls.

Sushi, von Willebrand factor type A, epidermal growth factor and pentraxin domain containing 1 (SVEP1) is an extracellular matrix protein which may bind to cell surface molecules such as integrins. A non-synonymous single amino acid polymorphism in the Svep1 gene is associated with a 14% increased risk of coronary heart disease, a 13% higher risk of type 2 diabetes and a 1mmHg increase in systolic blood pressure. Expression of the SVEP1 gene is increased in the kidney in the Cyp1a1mRen2 rat model of diabetes and hypertension previously developed in our lab. SVEP1 is also known to be upregulated in human diabetic nephropathy and is upregulated in rodent models of renal fibrosis. I hypothesized that Svep1 played a role in renal fibrosis, diabetes and blood pressure. Hence, the primary goal of this thesis was to investigate the role of SVEP1 and in the pathogenesis of diabetes, hypertension and renal fibrosis. Svep1 gene expression is increased in the kidney in the DOCA-salt-angII-uninephrectomy model of hypertension and following UUO. SVEP1 hemizygous mice showed no differences in expression of pro-fibrotic genes after UUO compared to wildtype littermates. No overt metabolic phenotype was exhibited by the Svep1 hemizygous mice, however there was a significant decrease in fat depot weights after high fat diet (HFD) and a significant increase in blood glucose concentrations during the glucose tolerance test at the 12 week time point in hemizygous Svep1 mice compared with wild-type controls. After telemetry analysis of blood pressure no difference was seen in blood pressure but SVEP1+/-animals had an increased heart rate of 100 beats per minute compared to wildtype animals. Svep1 expression is increased in the kidney in models of hypertension and fibrosis, however loss of one Svep1 allele did not alter the severity of fibrosis in the UUO model or significantly alter glucose tolerance after high fat diet. However, the high fat diet experiment was a pilot study and should be repeated with a larger number of animals. In addition, generation of a mouse with the human point mutation could determine the mechanisms by which this extracellular matrix protein confers risk of diabetes and hypertension.