Submitted in partial fulfillment of the requirements for the degree of Master of Health Sciences in Clinical Technology, Durban University of Technology, Durban, South Africa, 2017. / Introduction The prevalence of infertility is increasing at an alarming rate globally. Many couples are afflicted with infertility due to an array of diseases, trauma and psychological stresses. Renal disease is one such pathophysiological condition which is increasing amongst the younger age group. Often the progression of chronic renal disease leads to end stage renal failure that requires a renal transplantation. Post renal transplant, immunosuppressive agents are routinely prescribed to prevent allograft rejection. Immunosuppressive agents are potent drugs that can have deleterious side effects on semen parameters. However, the effects of the immunosuppressive agents on semen parameters in the literature are unclear and require further investigation. It is, therefore, important to assess the effects of immunosuppressive agents on semen, especially the three vital aspects of sperm concentration, motility and morphology which form the basis of male reproduction. Aims and Objectives of study This was a prospective observational study evaluating the effects of different immunosuppressive regimens on sperm parameters in post renal transplant male patients. The main aspects of semen parameters such as sperm concentration, motility and morphology that determine reproductive potential were assessed in the study patients and compared to the gold standard of semen analysis according to the World Health Organisation (WHO) reference values. Methodology Thirty-four renal transplant patients were recruited from the databases of both private nephrologists in the greater Durban area and the academic renal unit at Inkosi Albert Luthuli Central Hospital. Following bioethical approval and informed consent, patients were required to produce a semen sample by masturbation. A questionnaire documenting the patient’s lifestyle, aetiology of renal disease, transplant date and immunosuppressive duration and regimen were recorded. The semen samples were analysed comprehensively according to the protocol on semen analysis recommended by the WHO. This included the macroscopic investigation (volume, appearance, colour, viscosity, liquefaction time and pH) and microscopic evaluation (sperm concentration, total motility, morphology, IgG/IgA and vitality). Sperm concentration, total motility, morphology and vitality were examined and recorded in duplicate to strengthen the validity of the results. A biostatistician analysed the data and determined the statistical analysis. Descriptive statistics determined values of semen parameters in renal transplanted males and in each race demographic. The one sample t-test analysed the statistical significance between the mean study values and the WHO reference values. The effect of the immunosuppressive agent on semen parameters was determined using multiple linear regressions whilst ROC analysis determined the sensitivity and specificity of sperm concentration, total motility and morphology in predicting pregnancy from the patients that fathered children post renal transplant. Results The mean sperm concentration and morphology in the study patients were 14.0 mill/ml (95% Confidence Interval (CI) 10.2 – 17.7) and 3.3% (95% CI 2.7 – 3.9), respectively. Although values obtained were minimally lower than the WHO reference values, these results were within the 95% CI of the WHO guidelines. Motility evaluation revealed higher values of 43.2% (95% CI 36.6 – 49.7). In contrast, sperm vitality was considerably decreased, 47.5% (95% CI 40.6 – 54.4). All semen parameters exhibited no statistical significance (one sample t-test) when analysed against the WHO reference values except for sperm morphology, (p = 0.025; p< 0.05) which showed decreased morphology irrespective of immunosuppressive regimen. Semen volume 1.7 ml (95% CI 1.3 – 2.0) and pH 7.7 (95% CI 7.6 – 7.9) were both within the WHO guidelines. Descriptive statistics according to racial demographics showed no differences in semen values. An almost perfect linear relationship existed between total sperm motility and vitality (r = 0.967). Multiple linear regressions of duration and dosages of immunosuppressive drugs tacrolimus and mycophenolate mofetil, could not predict the effect of the immunosuppressive agents on sperm concentration, total motility and morphology. There was a significant difference in morphology between those with and without children post renal transplant. Those with children post renal transplant exhibited a higher morphology value, (p = 0.001; p< 0.05). Sensitivity and specificity analysis of the patients with children post renal transplant concluded that morphology is the most optimal indicator and predictor of pregnancy (AUC = 0.854). Tacrolimus was the common immunosuppressive agent used in the four patients that fathered children. This was more evident in patients that underwent therapy with Sirolimus followed by Cyclosporin A (CsA) and changed to Tacrolimus as the last immunosuppressive agent used for maintenance therapy. Conclusion The ability to procreate in renal transplanted males has become increasingly difficult and emotionally challenging. In this study sperm concentration and morphology of renal transplanted males exhibited parameters similar to the general fertile population. Total motility possessed a higher range of values in contrast to sperm vitality which showed a significant decrease from the WHO reference values. The effect of immunosuppressive treatment on semen parameters could not be clearly defined due to the number of immunosuppressive regimens that patients were subjected to intermittently resulting in small sample sizes within each immunosuppressive regimen grouping. The majority of patients underwent a triple maintenance therapy of tacrolimus, MMF and prednisone. The dosage and duration of these tacrolimus and MMF was inconclusive in determining a beneficial or detrimental relationship on semen parameters. Morphology was shown to be the most significant indicator in predicting pregnancy in patients that fathered children. Tacrolimus was a common immunosuppressive agent used in the majority of patients that fathered children. It may have protective effects on sperm parameters as shown in patients that fathered children. This was a study with a small sample size and further investigations are required in a larger cohort of patients to assess individualized effects of the different immunosuppressive agents on sperm parameters. / M

Ferroptosis is a regulated junction between cell death, metabolism, and disease, and it hasbeen implicated in many pathologies. The assorted ferroptosis pharmacology modulators offer valuable means to modulate ferroptosis in multiple diseases, to explore disease etiology, and to develop potential therapeutics. In the first part, the work focuses on inhibiting ferroptosis in a Huntington’s disease model. Ferrostatin-1 (Fer-1) is a potent small-molecule ferroptosis inhibitor that has been adopted to investigate the role of ferroptosis in many disease models. However, its further application is limited by its low potency, poor stability, possible toxicity, and lack of brain penetration. We developed the fourth and fifth generations of ferrostatins and investigated the in vitro and in vivo pharmacokinetics of lead compounds. We identified PHB4082 preferentially accumulating in the kidney as a potential candidate for kidney disease-relevant contexts. Moreover, TH-4-55-2 displayed an excellent brain penetration, preferentially accumulating in the brain at concentrations of magnitude higher than the in vitro IC50 values. In the in vivo toxicity study, it was well-tolerated over 30 days in wild-type and R6/2 mice and exhibited a protective effect against weight loss in a Huntington’s disease model, suggesting it is a strong candidate for application in HD and more neurodegenerative disease models. The second part describes the efforts to explore the therapeutic potential of inducing ferroptosis in a tumor model. Imidazole ketone erastin (IKE) induced ferroptosis by specifically inhibiting system xc– in a subcutaneous xenograft model of Diffuse Large B Cell Lymphoma (DLBCL), suggesting the potential of IKE as a therapeutic strategy for cancer. A biodegradable polyethylene glycol-poly (lactic-co-glycolic acid) nanoparticle formulation was used to aid in delivering IKE to cancer cells in vivo, exhibiting improved tumor accumulation and therapeutic index relative to free IKE, indicating its potential for treating DLBCL. In summary, this work explored the possibility to modulate ferroptosis using small molecule modulators in multiple disease models and identified some potential drug candidates and useful chemical probes.

Real world data have enhanced healthcare research, improving our understanding of disease progression, aiding in diagnosis, and enabling the development of personalized and targeted treatments. In recent years, multi-omics data and electronic health record (EHR) data have become increasingly available, providing researchers with a wealth of information to analyze. The use of machine learning methods with EHR and multi-omics data has emerged as a promising approach to extract valuable insights from these complex data sources. This dissertation focuses on the development of supervised and unsupervised learning methods, as well as their applications to EHR and multi-omics data, with a particular emphasis on early detection of clinical outcomes and identification of novel cancer subtypes. The first part of the dissertation centers on developing a risk prediction tool using EHR data that enables disease early detection so that preventive treatments can be taken to better manage the disease. For this goal, we developed a similarity-based supervised learning method with two applications to predict end-stage kidney disease (ESKD) and aortic stenosis (AS). In the second part of the dissertation, we expanded our goal to a phenome-wide prediction task and developed a patient representation based deep learning method that is able to predict phenotypes across the phenome. Through a weighting scheme, this approach is conducting tailored disease phenotype prediction computationally efficiently with good prediction performance. In the final part of the dissertation, I shifted the focus with the goal to identify clinical meaningful novel disease subtypes with unsupervised learning methods using multi-omics data. We tackled this goal through integrating multiple patient graphs being generated from multiple omics data with molecular level features for an improved disease subtyping. This dissertation has significantly contributed to the development of data-driven approaches to healthcare and biomedical research using EHR data and multi-omics data. The new methodologies developed with applications in multiple diseases using EHR and multi-omics data advanced our knowledge in disease diagnosis, vulnerable groups identification, and ultimately improve patient care.

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目的： 转化生长因子－1（TGF-β1）通过与II型受体结合而引起I型受体活化，进一步激活其下游信号分子蛋白Smad2 和Smad3，它们与Smad4（Co-Smad）结合后形成Smad复合体并发生核转移，从而发挥广泛的生物学效应。同时，整个TGF-β信号通路又受到其抑制因子Smad7的负反馈调节。研究结果显示Smad3是肾脏炎症和纤维化中重要的致病分子，相反，Smad7在多种肾脏疾病中起保护作用。然而，由于转化生长因子II型受体（TβRII），Smad2 或Smad4基因敲除的小鼠无法存活，这些分子在TGF-β1介导的肾脏炎症和纤维化中的功能尚未见报道。因此，本研究旨在剖析TβRII、Smad2 和Smad4 在肾脏疾病发生发展中的作用及机制。 / 方法：本研究利用Cre/LoxP系统分别靶向敲除小鼠肾小管上皮细胞的TβRII、Smad2 或者Smad4，通过结扎小鼠单侧输尿管建立梗阻性肾病模型，观察这些分子对肾脏炎症和纤维化的影响，并用体外实验进行验证。具体实验结果请参见本论文第III,IV, V章。 / 结果：通过分析，本论文取得以下新的发现： / (1) TβRII在TGF-β1介导的肾脏炎症和纤维化的双向调节中起到了决定性的作用：研究结果显示条件性敲除TβRII明显抑制TGF-β/Smad3介导的肾脏纤维化，同时增强NF-κB引起的肾脏炎症反应。由此可见，TRII不仅仅是TGF-β/Smad信号通路的启动因子，更决定了TGF-β1对肾脏炎症和纤维化的双向性调节。(参见第III章) / (2)尽管Smad2和Smad3结构相似并共同介导了TGF-β1的生物学效应，本研究意外发现Smad2可反向调节Smad3引起的纤维化。体内和体外实验共同证实，敲除Smad2基因增强了Smad3的磷酸化，核转位及其转录子活性，并能促进Smad3与I型胶原转录子的结合，进而加重肾脏纤维化（参见第IV章）。 / (3)我们还发现Smad4不仅作为TGF-β/Smad信号通路的共有蛋白，它在TGF-β1介导肾脏炎症和纤维化中起到了重要的双向性调节作用：条件敲除Smad4显著降低了Smad7对NF-κB介导肾脏炎症的抑制作用，同时在转录水平（而非磷酸化水平）抑制Smad3的功能，从而减轻纤维化。（参见第V章） / 结论：TβRII和Smad4 在TGF-β1介导肾脏炎症和纤维化中起到了重要的双向性作用；Smad2通过抑制Smad3信号传导和功能，在肾脏纤维化中起保护作用。 / Objectives: TGF-β1 binds its receptor II (TβRII) and then activates receptor I to initiate the downstream Smad signaling, called Smad2 and Smad3 which bind a common Smad4 to form the Smad complex and then translocate to nucleus to exert its biological activities. This process is negatively regulated by an inhibitory Smad7. While the pathogenic role of Smad3 and the protective role of Smad7 in renal fibrosis and inflammation are clearly understood, the functional role of TβRII, Smad2 and Smad4 in kidney diseases remains largely unexplored due to the lethality of these knockout mice. Therefore, the aim of present study is to dissect the functional role of these TGF-β/Smad signaling molecules in renal inflammation and fibrosis. / Methods: Kidney conditional knockout (KO) mice for TβRII, Smad2 and Smad4 were generated by crossing the FloxFlox mice with the kidney specific promoter driven Cre (KspCre) mice, in which TβRII, Smad2 or Smad4 were specifically deleted from the kidney tubular epithelial cells (TEC) respectively. Then, a well-characterized progressive renal inflammation and fibrosis mouse model of Unilateral ureteral obstructive (UUO) nephropathy was induced in these conditional KO mice and the specific roles for TβRII, Smad2, and Smad4 in renal inflammation and fibrosis were investigated in vivo and in vitro as described in the Chapter III, IV and V of this thesis. / Results: There were several novel findings through this thesis: / 1. TGF-β1 signals through its TβRII to diversely regulate renal fibrosis and inflammation. We found that disrupted TRII suppressed Smad3-dependent renal fibrosis while enhancing NF-κB-driven renal inflammation. Thus, TβRII not only acts as a binding receptor for initiating the TGF-β signaling, but also determines the diverse role of TGF-β1 in inflammation and fibrosis, which was described in the Chapter III. / 2. As shown in the Chapter IV, an unexpected finding from this thesis was that although Smad2 and Smad3 were homologically similar and bound together in response to TGF-β1 stimulation, Smad2 counter-regulated Smad3-mediated renal fibrosis. This was evidenced by the findings that conditional deletion of Smad2 enhanced Smad3 signaling including phosphorylation, nuclear translocation, the Smad3 responsive promoter activity, and the binding of Smad3 to Col1A2 promoter. Thus, disrupted Smad2 from the kidney significantly enhanced Smad3-mediated renal fibrosis in the UUO kidney and in cultured TEC. / 3. Finally, we also showed that that Smad4 acted not only as a common Smad in TGF-β signaling, but exerted its regulatory role in determining the diverse role of TGF-β1 in renal inflammation and fibrosis. Disruption of Smad4 significantly enhanced renal inflammation by impairing inhibitory effect of Smad7 on NF-κB-driven renal inflammation. In contrast, disrupted Smad4 inhibited renal fibrosis by blocking Smad3 functional activity without influencing Smad3 signaling. Because deletion of Smad4 inhibited TGF-β1-induced Smad3 responsive promoter activity and the binding of Smad3 to the Col1A2 promoter without altering the phosphorylation and nuclear translocation of Smad3 (Chapter V). / Conclusions: TβRII and Smad4 may function as key regulators of TGF-β signaling and diversely regulate the renal inflammation and fibrosis. Smad2 plays a protective role in renal fibrosis by counter-regulating Smad3 signaling. / 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. / Meng, Xiaoming. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 202-231). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Abstract --- p.i / Declaration --- p.viii / Acknowledgement --- p.ix / Table of Contents --- p.xii / List of Abbreviations --- p.xxvii / List of Figures/Tables --- p.xxix / Chapter CHAPTER I --- INTRODUCTION --- p.1 / Chapter 1.1 --- TGF-β signaling pathway --- p.2 / Chapter 1.1.1 --- TGF-β superfamily --- p.2 / Chapter 1.1.2 --- TGF-β signaling transduction --- p.3 / Chapter 1.1.2.1 --- Smad-dependent TGF-β signaling --- p.4 / Chapter 1.1.2.2 --- Smad-independent TGF-β signaling --- p.10 / Chapter 1.2 --- Chronic Kideny disease (CKD) --- p.12 / Chapter 1.2.1 --- Epidemiology of CKD --- p.12 / Chapter 1.2.2 --- Pathophysiology of CKD --- p.12 / Chapter 1.3 --- TGF-β signaling in renal diseases --- p.13 / Chapter 1.3.1 --- Role of TGF-β1 in renal diseases --- p.13 / Chapter 1.3.2 --- Potential role of TβRII in renal diseases --- p.15 / Chapter 1.3.3 --- Potential role of Smad2 in renal diseases --- p.17 / Chapter 1.3.4 --- Potential role of Smad4 in renal diseases --- p.20 / Chapter 1.3.5 --- Role of Smad7 in renal diseases --- p.23 / Chapter 1.3.6 --- Role of Smad-independent TGF-β signaling in renal disease --- p.24 / Chapter CHAPTER II --- MATERIALS AND METHODS --- p.26 / Chapter 2.1 --- MATERIALS --- p.27 / Chapter 2.1.1 --- Reagents and Equipments --- p.27 / Chapter 2.1.1.1 --- General reagents and equipments for cell culture --- p.27 / Chapter 2.1.1.2 --- General reagents and equipments for real-time RT-PCR --- p.28 / Chapter 2.1.1.3 --- General reagents and equipments for Masson Trichrome Staining --- p.28 / Chapter 2.1.1.4 --- General reagents and equipments for Immunohistochemistry --- p.29 / Chapter 2.1.1.5 --- General reagents and equipments for Immunofluorescence --- p.29 / Chapter 2.1.1.6 --- General reagents and equipments for Western Blot --- p.29 / Chapter 2.1.1.7 --- General reagents and equipments for Promoter assay --- p.31 / Chapter 2.1.1.8 --- General reagents and equipments for ChIP assay --- p.32 / Chapter 2.1.2 --- Buffers --- p.32 / Chapter 2.1.2.1 --- Buffers for Immunohistochemistry --- p.32 / Chapter 2.1.2.2 --- Buffers for Western blot --- p.35 / Chapter 2.1.3 --- Sequences of Primers and siRNAs --- p.40 / Chapter 2.1.4 --- Antibodies --- p.42 / Chapter 2.2 --- METHODS --- p.44 / Chapter 2.2.1 --- Animal model of Unilateral Ureteral Obstruction (UUO) --- p.44 / Chapter 2.2.2 --- Cell culture --- p.44 / Chapter 2.2.2.1 --- NRK52E cell line --- p.44 / Chapter 2.2.2.2 --- Smad2 WT/KO mouse embryonic fibroblasts (MEFs) --- p.45 / Chapter 2.2.2.3 --- Primary culture of kidney fibroblasts --- p.45 / Chapter 2.2.2.4 --- Primary culture of peritoneal macrophages --- p.46 / Chapter 2.2.3 --- PAS staining --- p.47 / Chapter 2.2.3.1 --- Tissue Handling and Fixation --- p.47 / Chapter 2.2.3.2 --- Tissue embedding and sectioning --- p.47 / Chapter 2.2.3.3 --- Preparation of Paraffin Tissue Sections for PAS staining --- p.48 / Chapter 2.2.3.4 --- PAS staining --- p.48 / Chapter 2.2.4 --- Real-time RT-PCR --- p.48 / Chapter 2.2.4.1 --- Total RNA isolation --- p.48 / Chapter 2.2.4.2 --- Reverse Transcription --- p.49 / Chapter 2.2.4.3 --- Real-time PCR --- p.50 / Chapter 2.2.4.4 --- Analysis of Real-time PCR --- p.50 / Chapter 2.2.5 --- Masson Trichrome Staining --- p.51 / Chapter 2.2.6 --- Immunohistochemistry --- p.52 / Chapter 2.2.6.1 --- Preparation of Paraffin Tissue Sections for IHC --- p.52 / Chapter 2.2.6.2 --- Antigen-Antibody Reaction --- p.52 / Chapter 2.2.6.3 --- Signal Detection --- p.53 / Chapter 2.2.6.4 --- Semi-quantification of Immunohistochemistry --- p.53 / Chapter 2.2.7 --- Immunofluorescence --- p.54 / Chapter 2.2.8 --- Western blot analysis --- p.54 / Chapter 2.2.8.1 --- Protein preparation --- p.55 / Chapter 2.2.8.2 --- SDS-PAGE --- p.56 / Chapter 2.2.8.3 --- Transmembrane of protein --- p.56 / Chapter 2.2.8.4 --- Incubation of first and second antibody --- p.57 / Chapter 2.2.8.5 --- Signal capture and analysis --- p.57 / Chapter 2.2.8.6 --- Stripping --- p.57 / Chapter 2.2.9 --- Promoter assay --- p.58 / Chapter 2.2.10 --- ChIP assay --- p.61 / Chapter 2.2.11 --- Statistical analysis --- p.62 / Chapter CHAPTER III --- THE DIVERSE ROLE OF TGF-BETA RECEPTOR II IN RENAL INFLAMMATION AND FIBROSIS --- p.63 / Chapter 3.1 --- INTRODUCTION --- p.64 / Chapter 3.2 --- AIMS --- p.64 / Chapter 3.3 --- MATERIALS AND METHODS --- p.66 / Chapter 3.3.1 --- Generation and characterization of TβRII conditional Knockout mice --- p.66 / Chapter 3.3.2 --- Generation and characterization of TβRII disrupted tubular epithelial cell line (NRK52E) and kidney interstitial fibroblasts --- p.67 / Chapter 3.3.3 --- Animal model of Unilateral Ureteral Obstruction --- p.67 / Chapter 3.3.4 --- Cell culture --- p.67 / Chapter 3.3.5 --- Real-time RT-PCR --- p.68 / Chapter 3.3.6 --- Masson Trichrome Staining --- p.68 / Chapter 3.3.7 --- Immunohistochemistry --- p.68 / Chapter 3.3.8 --- PAS staining --- p.69 / Chapter 3.3.9 --- Immunofluorescence --- p.69 / Chapter 3.3.10 --- Western blot analysis --- p.70 / Chapter 3.3.11 --- Promoter assay --- p.70 / Chapter 3.3.12 --- Statistical analysis --- p.70 / Chapter 3.4 --- RESULTS --- p.71 / Chapter 3.4.1 --- Characterization of TβRII conditional Knockout mice and TβRII disrupted cells --- p.71 / Chapter 3.4.2 --- Disruption of TβRII suppresses renal interstitial damage in the UUO kidney --- p.72 / Chapter 3.4.3 --- Disruption of TβRII suppresses renal fibrosis in UUO kidney and TGF-β1-induced fibrotic response in vitro --- p.76 / Chapter 3.4.3.1 --- Conditional knockout of TβRII from the kidney decreases the collagen I level in UUO kidney --- p.76 / Chapter 3.4.3.2 --- Disruption of TβRII inhibits TGF-β1 induced collagen I level in vitro --- p.79 / Chapter 3.4.3.3 --- Conditional knockout of TβRII from the kidney decreases the α-SMA positive cells infiltration in vivo --- p.81 / Chapter 3.4.3.4 --- Disruption of TβRII inhibits TGF-β1-induced α-SMA expression in vitro --- p.83 / Chapter 3.4.3.5 --- Conditional knockout of TβRII from the kidney decreases the FN level in UUO nephropathy --- p.85 / Chapter 3.4.3.6 --- Disruption of TβRII decreases TGF-β1-induced FN expression in vitro --- p.87 / Chapter 3.4.4 --- Disruption of TβRII impairs the TGF-β/Smad signaling in vivo in the UUO kidney and in vitro in TGF-β1 treated tubular epithelial cells and kidney fibroblasts --- p.89 / Chapter 3.4.4.1 --- Conditional knockout of TβRII decreases the UUO induced TGF-β1 expression in vivo and the TGF-β1 auto-induction in vitro --- p.89 / Chapter 3.4.4.2 --- Disrupted TβRII decreases CTGF level in the UUO nephropathy in vivo and the TGF-β1 induced CTGF mRNA level in vitro --- p.91 / Chapter 3.4.4.3 --- Conditional knockout of TβRII impairs the Smad3 signaling in the injured kidney --- p.93 / Chapter 3.4.4.4 --- Disrupted TβRII inhibits TGF-β1-induced Smad3 phosphorylation, P-Smad3 nuclear translocation and Smad3 responsive promoter activity in vitro --- p.95 / Chapter 3.4.4.5 --- Conditional knockout of TβRII doesn’t alter the activation of ERK and P38 signaling in the UUO kidney --- p.97 / Chapter 3.4.4.6 --- Disrupted TβRII inhibits TGF-β1-induced ERK and P38 phosphorylation in vitro --- p.99 / Chapter 3.4.5 --- Disruption of TβRII enhances inflammatory cytokines expression in the UUO kidney and impairs the anti-inflammatory effect of TGF-β1 in response to IL-1β triggered inflammatory response in the TEC cells --- p.101 / Chapter 3.4.5.1 --- Conditional knockout of TβRII increases the TNF-α expression in the UUO nephropathy --- p.101 / Chapter 3.4.5.2 --- Conditional knockout of TβRII increases the IL-1β expression in the UUO nephropathy --- p.103 / Chapter 3.4.5.3 --- Conditional knockout of TβRII doesn’t enhance the MCP-1 expression and macrophages infiltration in the UUO nephropathy --- p.104 / Chapter 3.4.5.4 --- Disruption of TβRII in TECs decreases the anti-inflammatory effect of TGF-β1 in response to IL-1β --- p.106 / Chapter 3.4.6 --- Disruption of TβRII enhances NFκB activation in vivo and in vitro --- p.108 / Chapter 3.5 --- DISCUSSION --- p.110 / Chapter 3.6 --- CONCLUSION --- p.114 / Chapter CHAPTER IV --- Smad2 protects against TGF-β/Smad3 mediated renal fibrosis --- p.115 / Chapter 4.1 --- INTRODUCTION --- p.116 / Chapter 4.2 --- AIMS --- p.117 / Chapter 4.3 --- MATERIALS AND METHODS --- p.117 / Chapter 4.3.1 --- Generation and characterization of Smad2 conditional Knockout mice --- p.117 / Chapter 4.3.2 --- Generation and characterization of Smad2 KO MEFs and Smad2 knockdown/overexpression tubular epithelial cell line (NRK52E) --- p.118 / Chapter 4.3.3 --- Animal model of Unilateral Ureteral Obstruction --- p.118 / Chapter 4.3.4 --- Cell culture --- p.118 / Chapter 4.3.5 --- Real-time RT-PCR --- p.119 / Chapter 4.3.6 --- Western blot analysis --- p.119 / Chapter 4.3.7 --- Immunohistochemistry --- p.119 / Chapter 4.3.8 --- Masson Trichrome Staining --- p.119 / Chapter 4.3.9 --- Immunofluorescence --- p.120 / Chapter 4.3.10 --- Promoter assay --- p.120 / Chapter 4.3.11 --- ChIP assay --- p.120 / Chapter 4.3.12 --- Statistical analysis --- p.120 / Chapter 4.4 --- RESULTS --- p.121 / Chapter 4.4.1 --- Characterization of Smad2 disrupted mice and cells --- p.121 / Chapter 4.4.1.1 --- Characterization of Smad2 conditional Knockout mice --- p.121 / Chapter 4.4.1.2 --- Characterization of Smad2 knockout MEFs, Smad2 knockdown/overexpression TECs --- p.123 / Chapter 4.4.2 --- Disruption of Smad2 further enhances renal fibrosis in vivo and in vitro --- p.124 / Chapter 4.4.2.1 --- Conditional knockout of Smad2 increases total collagen deposition and Col.I level in the UUO kidney --- p.124 / Chapter 4.4.2.2 --- Disruption of Smad2 in MEFs and TECs increases Col.I production in a time- and dosage-dependent manner in response to TGF-β1 --- p.126 / Chapter 4.4.2.3 --- Conditional knockout of Smad2 increases Col.III level in the UUO kidney --- p.128 / Chapter 4.4.2.4 --- Disruption of Smad2 in MEFs and TECs increases Col.III production in a time- and dosage-dependent manner in response to TGF-β1 --- p.130 / Chapter 4.4.3 --- Disruption of Smad2 further enhances renal fibrosis by suppressing the collagen degradation system in vivo and in vitro --- p.132 / Chapter 4.4.3.1 --- Conditional knockout of Smad2 inhibits the MMP2 mRNA while enhances TIMP-1 production in UUO kidney --- p.132 / Chapter 4.4.3.2 --- Disruption of Smad2 in MEFs and TECs decreases the MMP2 level while enhances TIMP-1 production in response to TGF-β1 --- p.133 / Chapter 4.4.4 --- Disruption of Smad2 further increases renal fibrosis by increasing TGF-β1 auto-induction and CTGF level in vivo and in vitro --- p.135 / Chapter 4.4.4.1 --- Disruption of Smad2 increases TGF-β1 auto-induction in vivo and in vitro --- p.135 / Chapter 4.4.4.2 --- Disruption of Smad2 increases CTGF synthesis in vivo and in vitro --- p.137 / Chapter 4.4.5 --- Disruption of Smad2 further increases renal fibrosis by enhancing Smad3 signaling in vivo and in vitro --- p.139 / Chapter 4.4.5.1 --- Conditional knockout of Smad2 further enhances Smad3 phosphorylation and nuclear translocation --- p.139 / Chapter 4.4.5.2 --- Disruption of Smad2 in MEFs and TECs further enhances Smad3 phosphorylation, nuclear translocation, Smad3 responsive promoter activity and the binding to the Col1A2 promoter --- p.141 / Chapter 4.4.6 --- Overexpression of Smad2 suppresses Smad3 signaling therefore ameliorates the TGF-β1-induced fibrotic response in TECs --- p.144 / Chapter 4.4.6.1 --- Overexpression of Smad2 ameliorates the TGF-β1- induced fibrotic response in TECs --- p.144 / Chapter 4.4.6.2 --- Overexpression of Smad2 suppresses Smad3 phosphorylation --- p.146 / Chapter 4.5 --- DISCUSSION --- p.147 / Chapter 4.6 --- CONCLUSION --- p.150 / Chapter CHAPTER V --- THE DISTINCT ROLE OF SMAD4 IN RENAL INFLAMMATION AND FIBROSIS --- p.151 / Chapter 5.1 --- INTRODUCTION --- p.152 / Chapter 5.2 --- AIMS --- p.152 / Chapter 5.3 --- MATERIALS AND METHODS --- p.153 / Chapter 5.3.1 --- Generation and characterization of Smad4 conditional Knockout mice --- p.153 / Chapter 5.3.2 --- Generation and characterization of Smad4 disrupted kidney interstitial fibroblasts and peritoneal macrophages --- p.153 / Chapter 5.3.3 --- Animal model of Unilateral Ureteral Obstruction (UUO) --- p.154 / Chapter 5.3.4 --- Cell culture --- p.154 / Chapter 5.3.5 --- Real-time RT-PCR --- p.155 / Chapter 5.3.6 --- Western blot analysis --- p.155 / Chapter 5.3.7 --- Immunohistochemistry --- p.155 / Chapter 5.3.8 --- Masson Trichrome Staining --- p.155 / Chapter 5.3.9 --- Promoter assay --- p.156 / Chapter 5.3.10 --- ChIP assay --- p.156 / Chapter 5.3.11 --- Statistical analysis --- p.156 / Chapter 5.4 --- RESULTS --- p.157 / Chapter 5.4.1 --- Characterization of Smad4 conditional Knockout mice and Smad4 disrupted cells --- p.157 / Chapter 5.4.2 --- Disruption of Smad4 suppresses renal fibrosis in the UUO nephropathy in vivo and TGF-β1-induced fibrotic response in vitro --- p.160 / Chapter 5.4.2.1 --- Conditional knockout of Smad4 from the kidney decreases the total collagen deposition in the UUO nephropathy --- p.160 / Chapter 5.4.2.2 --- Conditional knockout of Smad4 from the kidney decreases the Col.I production in the UUO nephropathy --- p.161 / Chapter 5.4.2.3 --- Disruption of Smad4 inhibits TGF-β1-induced Col.I production in vitro --- p.163 / Chapter 5.4.3 --- Disruption of Smad4 impairs the Smad3 function in vivo and in vitro --- p.164 / Chapter 5.4.3.1 --- Conditional knockout of Smad4 doesn’t decrease Smad3 phosphorylation and P-Smad3 nuclear translocation in vivo and in vitro --- p.164 / Chapter 5.4.3.2 --- Disruption of Smad4 inhibits TGF-β1 induced Smad3 promoter activity and the Smad3 binding to Col1A2 promoter --- p.166 / Chapter 5.4.3.3 --- Disruption of Smad4 has minimal effect on the activation of ERK signaling in vivo and in vitro --- p.167 / Chapter 5.4.4 --- Disruption of Smad4 enhances renal inflammation and impairs the anti-inflammatory effect of TGF-β1 in response to IL-1β triggered inflammatory response in vitro --- p.169 / Chapter 5.4.4.1 --- Conditional knockout of Smad4 increases the inflammatory cells infiltration --- p.169 / Chapter 5.4.4.2 --- Conditional knockout of Smad4 increases the TNFα expression in the UUO nephropathy --- p.171 / Chapter 5.4.4.3 --- Conditional knockout of Smad4 increases the IL-1β expression in the UUO nephropathy --- p.172 / Chapter 5.4.4.4 --- Conditional knockout of Smad4 increases the MCP-1 expression in the UUO nephropathy --- p.173 / Chapter 5.4.4.5 --- Conditional knockout of Smad4 increases the ICAM-1 level in the UUO nephropathy --- p.174 / Chapter 5.4.4.6 --- Time and dosage dependent experiments in response to IL-1β in macrophages --- p.175 / Chapter 5.4.4.7 --- Disruption of Smad4 in macrophages decreases the anti-inflammatory effect of TGF-β1 in response to IL-1β --- p.176 / Chapter 5.4.5 --- Disruption of Smad4 impairs the inhibitory effect of Smad7 on NFκB activation in vivo and in vitro --- p.178 / Chapter 5.4.5.1 --- Conditional knockout of Smad4 largely inhibits Smad7 level in UUO kidney --- p.178 / Chapter 5.4.5.2 --- Conditional knockout of Smad4 suppresses IκBα and further increases NF-κB p65 activation in UUO kidney --- p.180 / Chapter 5.4.5.3 --- Disruption of Smad4 inhibits Smad7 synthesis in macrophages --- p.182 / Chapter 5.4.5.4 --- Conditional knockout of Smad4 impair the inhibition effect of TGF-β1 on the activation of NFκB p65 in macrophages --- p.184 / Chapter 5.5 --- DISCUSSION --- p.186 / Chapter 5.6 --- CONCLUSION --- p.189 / Chapter CHAPTER VI --- SUMMARY AND DISCUSSION OF THE MAJOR FINDINGS --- p.190 / Chapter 6.1 --- SUMMARY AND DISCUSSION --- p.192 / Chapter 6.1.1 --- The diverse role of TβRII in renal inflammation and fibrosis both in vivo and in vitro --- p.192 / Chapter 6.1.2 --- Smad2 protects renal fibrosis by counter-regulating Smad3 signaling --- p.192 / Chapter 6.1.3 --- Disruption of Smad4 increased renal inflammation while suppressed the renal fibrosis in vivo and in vitro --- p.194 / Chapter 6.1.4 --- Comparative analysis of functions and related mechanisms between TβRII and Smad4 in renal disease --- p.195 / Chapter 6.1.5 --- Inadequacies of current work and future plan --- p.197 / Chapter 6.1.6 --- Perspectives (1) : The balance within the TGF-b/Smad signaling may determine the fate of renal diseases --- p.197 / Chapter 6.1.7 --- Perspectives(2)：The balance within the TGF-β/Smad signaling may determine the fate of renal diseases --- p.198 / Chapter 6.2 --- CONCLUSION --- p.201 / REFERENCES --- p.202 / PUBLICATION LIST --- p.232 / HONORS AND AWARDS --- p.237

Submitted in fulfillment of the Master of Technology : Biomedical and Clinical Technology, Durban University of Technology, 2014. / INTRODUCTION End stage renal disease (ESRD) occurs once 90 % of the kidney function is lost. Patients with ESRD must either undergo medical treatments, like haemodialysis, that substitute the function of the kidney, or they must have a kidney transplant. In the 1970s, haemodialysis treatment took 8 to 12 hours, three times per week. As technology advanced, dialyzers were able to handle more dialysate and higher blood flow rates hence treatment times were shortened to between three and five hours per treatment which has remained the norm until present day. One clinic in Tassin, France remained on the longer dialysis program and noticed advantages for patients who were on extended dialysis times. One of the major problems with dialysis done in the traditional sense is that it tries to provide a lot of therapy in a short period of time, and it is difficult to clear toxins and fluid in that time, Nocturnal dialysis provides a greater amount of toxin removal over a long period of time. AIMS AND OBJECTIVES The main aim of this study was to determine if nocturnal dialysis resulted in improved dialysis clearance, better overall patient health and a better quality of life. The primary objective of this study was to compare the clearance of small molecules (for example, urea, phosphate, creatinine and potassium) and large retention products (for example Parathyroid Hormone (PTH) and 2-Microglobulin) between the two haemodialysis procedures. The secondary objective was to compare the quality of life and survival of patients on both nocturnal and daytime dialysis. METHODOLOGY Thirty patients with End Stage Renal Disease (ESRD) presenting to the Sunninghill Hospital Dialysis Unit for treatment, who met the inclusion criteria, were recruited to participate in this study. Blood samples were taken for each participant at a baseline, 3 month and 6 month interval. The Kidney Disease Quality of Life Survey Questionnaire (KDQOL: SF- 36TM) was also given to each participant to complete. This survey consisted of three parts: 1) Physical Component Summary 2) Mental Component Summary and 3) Burden of Kidney Disease. This survey helped to predict the quality of life of the patients in each group. RESULTS In this study, non-significant effects of treatment were found for all small solutes individually. This study showed that there was a statistically significant increase in both dialysis adequacy and the clearance of large molecules (Parathyroid Hormone and Beta-2-Microglobulin) in the nocturnal haemodialysis group. The results of the KDQOL: SF-36 survey showed that the nocturnal dialysis patients scored higher in both the Physical Component Summary and the Mental Component Summary which means that they felt they were in better physical and mental health. The survey also showed that the nocturnal dialysis patients felt the burden of kidney disease less than those patients dialyzing during the day. CONCLUSION Firstly, dialysis adequacy as defined by the formula Kt/V, increased in the nocturnal group while it levelled off in the diurnal group. Secondly, both the Parathyroid Hormone levels and Beta-2-Microglobulin levels decreased more in the nocturnal group therefore resulting in statistically significant effects of treatment. The third and final conclusion drawn was that nocturnal haemodialysis resulted in better physical health, better mental health and a lower burden of kidney disease was felt by patients undergoing nocturnal haemodialysis.

Made available in DSpace on 2015-05-14T16:53:22Z (GMT). No. of bitstreams: 0 Previous issue date: 2014-09-01Bitstream added on 2015-05-14T16:58:56Z : No. of bitstreams: 1 000829061.pdf: 954808 bytes, checksum: 4db24b81e97a0d9789f7fab459997db1 (MD5) / Introdução: O hipercatabolismo é a alteração nutricional mais importante em pacientes com lesão renal aguda (LRA). Balanço nitrogenado (BN) e aparecimento de nitrogênio uréico (UNA) são medidas que permitem estimar a extensão do catabolismo na LRA. Objetivos: descrever e classificar o catabolismo de pacientes com LRA em Hemodiálise (HD) e Diálise Peritoneal de Alto Volume (DPAV) e analisar se BN e UNA são marcadores precoces de infecção e mortalidade nestes pacientes. Metodologia: Estudo tipo coorte prospectiva que avaliou pacientes com LRA dialítica internados no Hospital das Clínicas da Faculdade de Medicina de Botucatu, de março de 2011 a dezembro de 2012. Foi utilizado protocolo de estudo composto por dados clínicos, avaliação nutricional, parâmetros de catabolismo (redução de pelo menos 2g/dia no BN ou aumento de 2g/dia no UNA) e avaliação da presença de quadro infeccioso (presença de febre, leucócitos acima de 12.000/mm³ ou PCR>10mg/dl). Todos os pacientes foram acompanhados em conjunto com a equipe médica até a alta nefrológica hospitalar (na presença de recuperação parcial ou completa da função renal) ou óbito. Este estudo obteve aprovação do Comitê de Ética em Pesquisa da Instituição. Os resultados foram descritos como mediana ou média e desvio padrão. Para comparação das características clínicas, nutricionais e de catabolismo utilizou-se Teste t, Mann-Whitney ou Teste Qui-quadrado. Para a associação do BN e UNA com mortalidade e infecção foi utilizado teste t ou Mann-Whitney e curvas de Kaplan-Meyer, respectivamente. Utilizou-se como nível de significância estatística p<0,05. Resultados: Foram incluídos 124 pacientes, sendo 96 em HD e 28 em DPAV. Os pacientes apresentaram mediana de idade de 63 anos, prevalência de internação em UTI de 85,5%, Índice de Severidade da LRA (ATN-ISS) de 64,85% e taxa de mortalidade de 56,5%. Pacientes em HD e em DPAV apresentaram prevalência ... / Introduction: Hypercatabolism is the most important nutritional alteration in patients with acute kidney injury (AKI). Nitrogen balance (NB) and ureic nitrogen appearance (UNA) are measures that allow to estimate the extent of catabolism in AKI. Objectives: To describe and classify the catabolism of AKI patients on hemodialysis (HD) and high volume peritoneal dialysis (HVPD) and to examine whether NB and UNA are early markers of infection and mortality in these patients. Methodology: Prospective cohort study that evaluated AKI patients requiring dialysis at the Clinical Hospital of Botucatu Medicine College, from March 2011 to December 2012. The protocol study was performed using clinical and nutritional datas and parameters of catabolism. (such as reduction of at least 2g/day in NB or increased 2g/day in UNA) and assessment of presence of infection (fever, leukocytes above 12.000/mm ³ or CRP> 10mg/dl). All patients were followed by the medical staff since admission on unit nephrology until partial or complete recovery of renal function or death. This study was approved by the Ethics Committee of Research. Results are expressed as median or mean and standard deviation. To compare the clinical, nutritional and catabolism characteristics, t test, Mann-Whitney or chi-square were used. To investigate the association of NB and UNA with mortality and infection, t test or Mann-Whitney test and Kaplan-Meyer were used, respectively. The statistical significance level was p <0.05. Results: 124 patients were included, 96 in HD and 28 in HVPD. Patients had a median age of 63 years, prevalence of admission in ICU of 85.5%, AKI Severity Index (ATN-ISS) of 64.85% and mortality rate of 56.5%. HD and HVPD patients showed severe catabolism with NB median of -8.74 gN/day (-14.74;3.93 gN/day) vs. -7.36 gN/day (-13.46;-1.56 gN/day), p=0,73)], respectively. Measures of UNA were [16.74 gN/day (10.38 – 22.76 gN/day) vs. 12.97 gN/day (10.53 – 17.14), ...

Made available in DSpace on 2015-06-17T19:33:40Z (GMT). No. of bitstreams: 0 Previous issue date: 2014-08-26. Added 1 bitstream(s) on 2015-06-18T12:48:14Z : No. of bitstreams: 1 000825977.pdf: 523697 bytes, checksum: d2f8ec7342d5b6db7422e962ecea6b53 (MD5) / Justificativas e objetivos: os anti-inflamatórios não esteroidais (AINEs) têm mostrado sua eficácia na abordagem multimodal da dor, reduzindo o consumo de opioides na dor aguda pós-operatória. Embora os AINEs possam afetar a função renal, existem poucos dados da utilização de AINEs coxibes disponíveis nesse contexto. Os biomarcadores precoces de lesão renal aguda, como o NGAL (neutrophil gelatinase-associated lipocalin), podem ser importantes aliados na determinação de lesão real associada ao uso de AINEs coxibes no período perioperatório. Materiais e métodos: após aprovação pelo Comitê de Ética em Pesquisa Experimental, 40 ratos Wistar foram distribuídos aleatoriamente em quatro grupos. Sob anestesia geral, e dependendo do grupo a qual os animais foram incluídos, eles foram submetidos a isquemia e reperfusão renal. Dois grupos receberam parecoxibe para avaliar a influência deste medicamento na função renal. Dosagem de NGAL e histologia renal bilateral foram realizadas para a avaliação da existência e grau da lesão renal. Resultados: o grupo isquemia (que não recebeu injeção de parecoxibe) apresentou os maiores níveis de NGAL e maior frequência de lesão renal. O grupo que sofreu isquemia e recebeu parecoxibe apresentou níveis de NGAL e frequência de lesão renal similares aos outros grupos que não sofreram tal injúria. Conclusão: neste modelo experimental, parecoxibe exibiu propriedades em promover proteção renal / Background and aims: nonsteroidal anti-inflammatory drugs (NSAIDs) have been proved to be effective, in a multidimensional approach, at reducing the opioid consumption during the postoperative acute pain. Although the NSAIDs may affect the renal function, there are few published data related to the use of coxibs and the kidney function. The early biomarkers of acute renal injury (EBARI) may be important tool to determining the actual risk associated with the use of coxibs NSAIDs in the perioperative period. Methods: after approval by Experimental Ethics Committee, 40 male Wistar rats were randomly assigned into four groups. Under general anesthesia, and depending on the assigned group, rats underwent renal ischemia and reperfusion. Parecoxib was injected in two of the groups to evaluate the coxib intluence on the renal function. Parametric data, serum neutrophil gelatinase-associated lipocalin (NGAL), an EBARI, and right and left renal histology were used to evaluate there renal function. Results: the animals of ischemia group which did not receive parecoxib showed the highest NGAL serum level, as well as the most severe tubular injury. The animals on ischemia group which received parecoxib showed NGAL plasmatic levels and tubular injury similar to the groups not subjected to renal ischemia. Conclusion: on this experimental model, parecoxib showed protective renal properties

Orientador: André Luis Balbi / Banca: Lilian Cuppari / Banca: Aline de Araújo Antunes / Banca: Silvia Papini / Resumo: Introdução: O hipercatabolismo é a alteração nutricional mais importante em pacientes com lesão renal aguda (LRA). Balanço nitrogenado (BN) e aparecimento de nitrogênio uréico (UNA) são medidas que permitem estimar a extensão do catabolismo na LRA. Objetivos: descrever e classificar o catabolismo de pacientes com LRA em Hemodiálise (HD) e Diálise Peritoneal de Alto Volume (DPAV) e analisar se BN e UNA são marcadores precoces de infecção e mortalidade nestes pacientes. Metodologia: Estudo tipo coorte prospectiva que avaliou pacientes com LRA dialítica internados no Hospital das Clínicas da Faculdade de Medicina de Botucatu, de março de 2011 a dezembro de 2012. Foi utilizado protocolo de estudo composto por dados clínicos, avaliação nutricional, parâmetros de catabolismo (redução de pelo menos 2g/dia no BN ou aumento de 2g/dia no UNA) e avaliação da presença de quadro infeccioso (presença de febre, leucócitos acima de 12.000/mm³ ou PCR>10mg/dl). Todos os pacientes foram acompanhados em conjunto com a equipe médica até a alta nefrológica hospitalar (na presença de recuperação parcial ou completa da função renal) ou óbito. Este estudo obteve aprovação do Comitê de Ética em Pesquisa da Instituição. Os resultados foram descritos como mediana ou média e desvio padrão. Para comparação das características clínicas, nutricionais e de catabolismo utilizou-se Teste t, Mann-Whitney ou Teste Qui-quadrado. Para a associação do BN e UNA com mortalidade e infecção foi utilizado teste t ou Mann-Whitney e curvas de Kaplan-Meyer, respectivamente. Utilizou-se como nível de significância estatística p<0,05. Resultados: Foram incluídos 124 pacientes, sendo 96 em HD e 28 em DPAV. Os pacientes apresentaram mediana de idade de 63 anos, prevalência de internação em UTI de 85,5%, Índice de Severidade da LRA (ATN-ISS) de 64,85% e taxa de mortalidade de 56,5%. Pacientes em HD e em DPAV apresentaram prevalência ... / Abstract: Introduction: Hypercatabolism is the most important nutritional alteration in patients with acute kidney injury (AKI). Nitrogen balance (NB) and ureic nitrogen appearance (UNA) are measures that allow to estimate the extent of catabolism in AKI. Objectives: To describe and classify the catabolism of AKI patients on hemodialysis (HD) and high volume peritoneal dialysis (HVPD) and to examine whether NB and UNA are early markers of infection and mortality in these patients. Methodology: Prospective cohort study that evaluated AKI patients requiring dialysis at the Clinical Hospital of Botucatu Medicine College, from March 2011 to December 2012. The protocol study was performed using clinical and nutritional datas and parameters of catabolism. (such as reduction of at least 2g/day in NB or increased 2g/day in UNA) and assessment of presence of infection (fever, leukocytes above 12.000/mm ³ or CRP> 10mg/dl). All patients were followed by the medical staff since admission on unit nephrology until partial or complete recovery of renal function or death. This study was approved by the Ethics Committee of Research. Results are expressed as median or mean and standard deviation. To compare the clinical, nutritional and catabolism characteristics, t test, Mann-Whitney or chi-square were used. To investigate the association of NB and UNA with mortality and infection, t test or Mann-Whitney test and Kaplan-Meyer were used, respectively. The statistical significance level was p <0.05. Results: 124 patients were included, 96 in HD and 28 in HVPD. Patients had a median age of 63 years, prevalence of admission in ICU of 85.5%, AKI Severity Index (ATN-ISS) of 64.85% and mortality rate of 56.5%. HD and HVPD patients showed severe catabolism with NB median of -8.74 gN/day (-14.74;3.93 gN/day) vs. -7.36 gN/day (-13.46;-1.56 gN/day), p=0,73)], respectively. Measures of UNA were [16.74 gN/day (10.38 - 22.76 gN/day) vs. 12.97 gN/day (10.53 - 17.14), ... / Doutor

Orientador: Guilherme Antonio Moreira de Barros / Banca: Eliana Marisa Ganen / Banca: Eunice Sizue Hirata / Resumo: Justificativas e objetivos: os anti-inflamatórios não esteroidais (AINEs) têm mostrado sua eficácia na abordagem multimodal da dor, reduzindo o consumo de opioides na dor aguda pós-operatória. Embora os AINEs possam afetar a função renal, existem poucos dados da utilização de AINEs coxibes disponíveis nesse contexto. Os biomarcadores precoces de lesão renal aguda, como o NGAL (neutrophil gelatinase-associated lipocalin), podem ser importantes aliados na determinação de lesão real associada ao uso de AINEs coxibes no período perioperatório. Materiais e métodos: após aprovação pelo Comitê de Ética em Pesquisa Experimental, 40 ratos Wistar foram distribuídos aleatoriamente em quatro grupos. Sob anestesia geral, e dependendo do grupo a qual os animais foram incluídos, eles foram submetidos a isquemia e reperfusão renal. Dois grupos receberam parecoxibe para avaliar a influência deste medicamento na função renal. Dosagem de NGAL e histologia renal bilateral foram realizadas para a avaliação da existência e grau da lesão renal. Resultados: o grupo isquemia (que não recebeu injeção de parecoxibe) apresentou os maiores níveis de NGAL e maior frequência de lesão renal. O grupo que sofreu isquemia e recebeu parecoxibe apresentou níveis de NGAL e frequência de lesão renal similares aos outros grupos que não sofreram tal injúria. Conclusão: neste modelo experimental, parecoxibe exibiu propriedades em promover proteção renal / Abstract: Background and aims: nonsteroidal anti-inflammatory drugs (NSAIDs) have been proved to be effective, in a multidimensional approach, at reducing the opioid consumption during the postoperative acute pain. Although the NSAIDs may affect the renal function, there are few published data related to the use of coxibs and the kidney function. The early biomarkers of acute renal injury (EBARI) may be important tool to determining the actual risk associated with the use of coxibs NSAIDs in the perioperative period. Methods: after approval by Experimental Ethics Committee, 40 male Wistar rats were randomly assigned into four groups. Under general anesthesia, and depending on the assigned group, rats underwent renal ischemia and reperfusion. Parecoxib was injected in two of the groups to evaluate the coxib intluence on the renal function. Parametric data, serum neutrophil gelatinase-associated lipocalin (NGAL), an EBARI, and right and left renal histology were used to evaluate there renal function. Results: the animals of ischemia group which did not receive parecoxib showed the highest NGAL serum level, as well as the most severe tubular injury. The animals on ischemia group which received parecoxib showed NGAL plasmatic levels and tubular injury similar to the groups not subjected to renal ischemia. Conclusion: on this experimental model, parecoxib showed protective renal properties / Mestre