Добірка наукової літератури з теми "Kidney Pathophysiology"

There is a strong association between both acute and chronic dysfunction of the heart and kidneys with respect to morbidity and mortality. The complex interrelationships of longitudinal changes in both organ systems have been difficult to describe and fully understand due to a lack of categorization of the common clinical scenarios where these phenomena are encountered. Thus, cardiorenal syndromes (CRSs) have been subdivided into five syndromes which represent clinical vignettes in which both the heart and the kidney are involved in bidirectional injury and dysfunction via a final common pathway of cell-to-cell death and accelerated apoptosis mediated by oxidative stress. Types 1 and 2 involve acute and chronic cardiovascular disease (CVD) scenarios leading to acute kidney injury (AKI) or accelerated chronic kidney disease (CKD). Types 3 and 4 describe AKI and CKD, respectively, leading primarily to heart failure, although it is possible that acute coronary syndromes, stroke, and arrhythmias could be CVD outcomes in these forms of CRS. Finally, CRSs type 5 describe a systemic insult to both heart and the kidneys, such as sepsis, where both organs are injured simultaneously in persons with previously normal heart and kidney function at baseline. Both blood and urine biomarkers, including the assessment of catalytic iron, a critical element to the generation of oxygen-free radicals and oxidative stress, are reviewed in this paper.

The kidney is the central organ involved in maintaining water and sodium balance. In human kidneys, nine aquaporins (AQPs), including AQP1–8 and AQP11, have been found and are differentially expressed along the renal tubules and collecting ducts with distinct and critical roles in the regulation of body water homeostasis and urine concentration. Dysfunction and dysregulation of these AQPs result in various water balance disorders. This review summarizes current understanding of physiological and pathophysiological roles of AQPs in the kidney, with a focus on recent progress on AQP2 regulation by the nuclear receptor transcriptional factors. This review also provides an overview of AQPs as clinical biomarkers and therapeutic targets for renal diseases.

Mitochondrial dysfunction has gained recognition as a contributing factor in many diseases. The kidney is a kind of organ with high energy demand, rich in mitochondria. As such, mitochondrial dysfunction in the kidney plays a critical role in the pathogenesis of kidney diseases. Despite the recognized importance mitochondria play in the pathogenesis of the diseases, there is limited understanding of various aspects of mitochondrial biology. This review examines the physiology and pathophysiology of mitochondria. It begins by discussing mitochondrial structure, mitochondrial DNA, mitochondrial reactive oxygen species production, mitochondrial dynamics, and mitophagy, before turning to inherited mitochondrial cytopathies in kidneys (inherited or sporadic mitochondrial DNA or nuclear DNA mutations in genes that affect mitochondrial function). Glomerular diseases, tubular defects, and other renal diseases are then discussed. Next, acquired mitochondrial dysfunction in kidney diseases is discussed, emphasizing the role of mitochondrial dysfunction in the pathogenesis of chronic kidney disease and acute kidney injury, as their prevalence is increasing. Finally, it summarizes the possible beneficial effects of mitochondrial-targeted therapeutic agents for treatment of mitochondrial dysfunction-mediated kidney injury-genetic therapies, antioxidants, thiazolidinediones, sirtuins, and resveratrol-as mitochondrial-based drugs may offer potential treatments for renal diseases.

The measurement of the cardiac troponins has produced a paradigm shift in the management of cardiac disease. Elevation of cTn without acute myocardial infarction (AMI) also occurs in non-cardiac patients including those with chronic kidney disease (CKD). Cardiovascular disease accounts for 50% of mortality in CKD. In this thesis, the prognostic value of cTn elevation in CKD was investigated by meta-analysis of published data and recruitment of a CKD cohort. The relationship between elevated cTn and cardiac imaging; the involvement of inflammation, oxidative stress and platelet activation were investigated. The difference in cTn pre and post haemodialysis was investigated. The forms of cTn released into the circulation in CKD was investigated and compared to the forms released following AMI. CKD patients positive for cTn are three times more likely to die than cTn negative patients. Elevated cTn was not associated with extent of cardiac pathology but rather the presence of diffuse global ischemia. Elevated cTn in CKD is associated with increased C-reactive protein but not other markers of inflammation, oxidative stress or platelet activation. cTn, CRP and interleukin-6 were predictive of all-cause mortality. Following dialysis, cTnl but not cTnT adsorbs to the membrane within the vascular compartment. Intact cTnT and cTnl were observed in CKD patient serum by Western blotting, which is similar to the cTn forms of observed following myocardial infarction. Some lower molecular weight fragments are demonstrable but their presence is method dependent and heterogeneous between patients. Elevated cTn is of prognostic value in CKD and reflects the high incidence of cardiovascular disease and cardiac death. Elevated cTn is not a false positive. The mechanism of cTn release in CKD remains to be understood. The clinical challenge is for the renal physician to translate the potential for cardiovascular disease monitoring conferred by cTn into improved patient management.

Malaria remains one of the world's major health problems, especially in developing countries. A better understanding of the pathology and pathophysiology of severe malaria is key to develop new treatments. Different approaches have been used in malaria research including the in vitro co-culture models with endothelial cells and both murine and simian animal models. However these are open to controversy due to disagreement on their representativeness of human disease. Using human post-mortem tissue in malaria research is another important approach but is practically challenging, limiting the availability of post mortem samples from malaria patients. The work in this thesis had two main themes. First I examined the role of the endothelial signalling Angiopoetin-Tie-2 receptor pathway in malaria. Ang-2 has been shown to be a significant biomarker of severe and fatal malaria. I examined the tissue specific expression of proteins from this pathway in post-mortem brain tissues from fatal malaria cases, but found no difference between cerebral malaria and non-cerebral malaria cases. Ang-2 correlated with the severity of malaria in these patients. An attempt to examine the interaction of hypoxia and the Ang-Tie-2 pathway in vitro using a co-culture model of human brain endothelial cells was unsuccessful due to contamination of the cell line. The second part of the thesis aimed to utilise molecular pathology techniques including miRNA and whole-genome microarrays. I have shown for the first time that these can be successfully applied to human post-mortem tissue in malaria. First I used archival tissues to examine the microRNA signature in the kidney of patients with malaria associated renal failure. Second I optimised a protocol to preserve post mortem tissue for molecular pathology, from an autopsy study in Mozambique. Using the subsequent total mRNA transcriptomic data and bioinformatics analysis this work has expanded our knowledge of differential gene expression and the families of genes which are dysregulated in the brain in response to malaria infection.

Includes bibliographical references (leaves 226-273.) Investigates the regulation of the expression of CYP27B1, CYP24 and vitamin D receptor (VDR) mRNA, both in the bone and in the kidney, with the aim to determine whether the regulation of the vitamin D metabolism in the bone is independent from that in the kidney. The effects of age, dietary calcium and vitamin D status on the expression of these genes in both the kidney and the bone, as well as on a number of biochemical factors known to regulate the renal metabolism of 1,25D, such as PTH, calcium and 1,25D itself, were examined. CYP27B1 mRNA expression was also studied in histological sections of rat femoral bone.

Reperfusion injury may mediate renal dysfunction following ischaemia. A murine model was developed to investigate the role of the tissue factor-thrombin-protease activated receptor pathway in renal ischaemia reperfusion injury (IRI). In this model, mice received 25 minutes of ischaemia and subsequent periods of reperfusion. C57BL6, protease activated receptor-1 (PAR-1) knockout mice, and tissue factor (TF) deficient mice were used. Following 24 hours IRI, PAR-1 deficiency resulted in protection against severe renal failure compared to the C57BL6 mice (creatinine, 118.2 ?? 6.3 vs 203 ?? 12 ??mol/l, p<0.001). This was confirmed by lesser tubular injury. By 48 hours IRI, this resulted in a survival benefit (survival, 87.5% vs 0%, p<0.001). Treatment of C57BL6 mice with hirudin, a specific thrombin inhibitor, offered renoprotection at 24 hours IRI (creatinine, 107 ?? 10 ??mol/l, p<0.001), leading to a 60% survival rate at 48 hours IRI (p<0.001). TF deficient mice expressing less than 1% of C57BL6 mouse TF were also protected (creatinine, 113.6 ?? 7 ??mol/l, p<0.001), with a survival benefit of 75% (p<0.001). The PAR-1 knockout, hirudin treated C57BL6 and TF deficient mice had reduced myeloperoxidase activity and tissue neutrophil counts compared to the C57BL6 mice, along with reduced KC and MIP-2 chemokine mRNA and protein expression. Hirudin treatment of PAR-1 knockout mice had no additional benefit over PAR-1 absence alone, suggesting no further contribution by activation of other protease activated receptors (creatinine at 24 hours IRI, 106.5 ?? 10.5 ??mol/l, p>0.05). Furthermore, immunofluoresence staining for fibrin(ogen) showed no difference between C57BL6 and PAR-1 knockout mice, suggesting no major contribution by fibrin in this model. Renal IRI resulted in increased levels of TF mRNA expression in the C57BL6, PAR-1 knockout, and hirudin treated C57BL6 mice compared to normal controls, suggesting that TF mRNA expression was upregulated in this model. This resulted in increased TF functional activity in the C57BL6 and PAR-1 knockout mice, but TF activity was negligible in hirudin treated C57BL6 and TF deficient mice. The data therefore suggests that the TF-thrombin cascade contributes to renal IRI by signalling via PAR-1 that then regulates chemokine gene expression and subsequent neutrophil recruitment.

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

Indiana University-Purdue University Indianapolis (IUPUI)
Advancements in human genomics have simultaneously enhanced our basic understanding of the human body and ability to combat debilitating diseases. Historically, research has shown that there have been many hindrances to realizing this medicinal revolution. One hindrance, with particular regard to the kidney, has been our inability to effectively and routinely delivery genes to various loci, without inducing significant injury. However, we have recently developed a method using hydrodynamic fluid delivery that has shown substantial promise in addressing aforesaid issues. We optimized our approach and designed a method that utilizes retrograde renal vein injections to facilitate widespread and persistent plasmid and adenoviral based transgene expression in rat kidneys. Exogenous gene expression extended throughout the cortex and medulla, lasting over 1 month within comparable expression profiles, in various renal cell types without considerably impacting normal organ function. As a proof of its utility we by attempted to prevent ischemic acute kidney injury (AKI), which is a leading cause of morbidity and mortality across among global populations, by altering the mitochondrial proteome. Specifically, our hydrodynamic delivery process facilitated an upregulated expression of mitochondrial enzymes that have been suggested to provide mediation from renal ischemic injury. Remarkably, this protein upregulation significantly enhanced mitochondrial membrane potential activity, comparable to that observed from ischemic preconditioning, and provided protection against moderate ischemia-reperfusion injury, based on serum creatinine and histology analyses. Strikingly, we also determined that hydrodynamic delivery of isotonic fluid alone, given as long as 24 hours after AKI is induced, is similarly capable of blunting the extent of injury. Altogether, these results indicate the development of novel and exciting platform for the future study and management of renal injury.

Arteriovenous fistula (AVF) is one type of vascular access which is a surgically created vein used to remove and return blood during hemodialysis [1]. It is a long-term treatment for kidney failure. Although clinical treatment and technology have both achieved great improvements in recent years, the vascular access for hemodialysis still has significant early failure rates after the insertion of AVF in patients [2]. Studies have shown that stenosis in the vascular access circuit is the single major cause for access morbidity. Majority of efforts to understand the mechanisms of stenosis formation, and its prevention and management have largely focused on understanding and managing this complication based on the pathophysiology, tissue histology and molecular biology; however these efforts have not resulted in significant progress to date. We believe that the major impact in this area will come from continued and accurate understanding of the hemodynamics, and by development of techniques of intervention to modulate factors such as flow rates, pressures and compliance of the circuit. The goal of this paper is to study anastomotic models of AV access using Computational Fluid Dynamics (CFD) and optimize them to minimize the wall shear stress (WSS). In order to achieve this goal, the commercial CFD software FLUENT [3] is employed in conjunction with a single objective genetic algorithm [4]. Computations for two types of AVF currently in use in clinical practice are performed. AVF with 25° angle/3–4mm diameter and 90° angle/3–5mm diameter are selected to conduct the optimization. A single-objective genetic algorithm is employed in the optimization process and a k-kl-ω turbulence model is employed in CFD simulations; this model can accurately compute transitional/turbulent flows. In order to optimize for the same flow conditions, a fixed boundary condition is used during the optimization process. Computations for 16 to 20 generations of the selected AVFs are obtained from the genetic algorithm solver. The maximum WSS in the two AVFs considered are 6997.8 and 7750 dynes/cm2; however, the maximum WSS in the shape-optimized AVFs are reduced to 3511.2 and 4293.9 dynes/cm2 respectively, which have decreased by 49.82% and 44.59% respectively. Thus, the probability of the formation of stenosis in AVFs and early failure rates of vascular access are reduced by using the optimized AVFs.

Arteriovenous fistula (AVF) is one type of vascular access which is a surgically created vein used to remove and return blood during hemodialysis [1]. It is a long-term treatment for kidney failure. Although clinical treatment and technology have both achieved great improvements in recent years, the vascular access for hemodialysis still has significant early failure rates after the insertion of AVF in patients [2]. Studies have shown that stenosis in the vascular access circuit is the single major cause for access morbidity. Majority of efforts to understand the mechanisms of stenosis formation, and its prevention and management have largely focused on understanding and managing this complication based on the pathophysiology, tissue histology and molecular biology; however these efforts have not resulted in significant progress to date. We believe that the major impact in this area will come from continued and accurate understanding of the hemodynamics, and by development of techniques of intervention to modulate factors such as flow rates, pressures and compliance of the circuit. The goal of this paper is to study anastomotic models of AV access using Computational Fluid Dynamics (CFD) and optimize them to minimize the wall shear stress (WSS). In order to achieve this goal, the commercial CFD software FLUENT [3] is employed in conjunction with a single objective genetic algorithm [4]. Computations for two types of AVF currently in use in clinical practice are performed. AVF with 25° angle/3–4mm diameter and 90° angle/3–5mm diameter are selected to conduct the optimization. A single-objective genetic algorithm is employed in the optimization process and a k-kl-ω turbulence model is employed in CFD simulations; this model can accurately compute transitional/turbulent flows. In order to optimize for the same flow conditions, a fixed boundary condition is used during the optimization process. Computations for 16 to 20 generations of the selected AVFs are obtained from the genetic algorithm solver. The maximum WSS in the two AVFs considered are 6997.8 and 7750 dynes/cm2; however, the maximum WSS in the shape-optimized AVFs are reduced to 3511.2 and 4293.9 dynes/cm2 respectively, which have decreased by 49.82% and 44.59% respectively. Thus, the probability of the formation of stenosis in AVFs and early failure rates of vascular access are reduced by using the optimized AVFs.