Diabetes causes early structural changes to the glomerular basement membrane (GBM), which alters its function and leads to loss of protein in urine. Formation of advanced glycation endproducts (AGEs) is one mechanism proposed to be responsible for the structural changes to the GBM. AGEs are thought to affect blood flow i.e. glomerular filtration rate (GFR) and vascular permeability which over time manifests as overt proteinuria. The gradual loss of minute amounts of protein (albumin) is referred to as microalbuminuria (MA). Microalbuminuria is a dynamic process, with patients regressing to normoalbuminuria more often than progressing to overt proteinuria. Microalbuminuria is not specific to patients with diabetic nephropathy (DN) and new markers specific to DN are being sought. A prospective study was undertaken at the Inkosi Albert Luthuli Central Hospital (IALCH) to evaluate the relationship of serum glycated albumin, urinary and serum components of capillary basement membrane and DN in South African Black and Indian patients with type 1 diabetes. The study was undertaken with sampling of blood and urine at baseline, 6-months, 1 year and 2-year follow-up. Serum glycated albumin, urinary type IV collagen and plasma fibronectin were measured at each visit. Since correlations could be performed only at each time point individually, generalised estimating equation (GEE) regression models were constructed in SPSS (15.0) with time specified as a factor in order to take account of relationships among variables over time. The results of this study showed that serum percentage glycated albumin (PGA), plasma fibronectin (FN) and urinary type IV collagen were not better predictors of incipient impaired renal function than MA. Although previous authors have variously reported serum GA, plasma FN and urinary type IV collagen to be predictive of impaired renal function, these studies were conducted mainly in patients with overt DN. The present study suggest that markers of overt renal dysfunction are not necessarily useful predictors of incipient DN. Differences in predictive relationships point to a different disease processes in the two ethnic groups. Of particular note was the lack of a predictive relationship of either fasting plasma glucose (FPG) or glycated haemoglobin (HbA1c) with any of isotope GFR, estimated GFR and proteinuria in Black patients. The most significant finding of this study showed that combination of serum creatinine and MA provided broadest range of predictors of isotope GFR, estimated GFR and proteinuria. / Thesis (M.Sc.)-University of KwaZulu-Natal, Westville, 2010.

Thesis (M.A.)--Northern Kentucky University, 2007. / Made available through ProQuest. Publication number: AAT 1447083. ProQuest document ID: 1436380211. Includes bibliographical references (p. 25-26)

Diss. (sammanfattning) Uppsala : Univ., 2004. / Härtill 5 uppsatser.

研究介紹：炎症與纖維化是糖尿病腎病（DN）的主要特徵。研究發現生長轉化因子TGF-β/Smad信號在糖尿病所致炎症與纖維化中均起重要作用。我們認為TGF-β/Smad信號通路失調是導致糖尿病腎損傷的主要機制，恢復信號通路或有治療價值。為此我們通過以下研究證實：（1）研究Smad7基因在DN中的作用，及評估Smad7基因治療效果；（2）研究miR-29在DN中的作用，及評估miR-29基因治療效果；（3）研究C反應蛋白（CRP）在DN中的作用及機制。 / 研究方法：（1）利用Smad7基因敲除（KO）小鼠建立糖尿病小鼠，並研究Smad7基因在DN的作用，並在链脲佐菌素（STZ）誘導的糖尿病大鼠上利用微泡導入Smad7基因治療觀察其療效；（2）在10週齡db/db小鼠上利用微泡導入可誘導的miR-29b基因，觀察miR-29b在糖尿病腎病中的作用，並用miR-29敲除或高表達細胞株研究其機制；（3）利用CRP轉基因小鼠誘導糖尿病，觀察CRP在DN中的作用，及以高糖和/或CRP刺激腎小管細胞研究CRP的致病機制。 / 研究結果：我們發現（1）糖尿病Smad7 KO小鼠出現更嚴重的腎損傷，包括蛋白尿增加，腎臟炎症及纖維化加重。進一步研究發現Smad7下調所致TGF-β/Smad和NF-kB信號過度活化是導致腎臟炎症及纖維化加重的重要原因。運用基因治療恢復糖尿病大鼠的Smad7水平，發現能夠減輕蛋白尿增加，及抑制TGF-β/Smad引起的纖維化和NF-kB所致炎症反應；（2）我們發現miR-29b在20週齡db/db小鼠比10週齡的顯著降低，並伴隨有蛋白尿加重，腎臟纖維化和炎症反應增加，及TGF-β/Smad，NF-kB，T-bet信號上調，而miR-29b基因治療能減輕蛋白尿，及減輕腎臟纖維化和炎症反應增加，及TGF-β/Smad，NF-kB，T-bet信號上調。體外實驗證實AGEs刺激miR-29敲除細胞株增加纖維化，伴隨有TGF-β/Smad3及炎症因子上調，而刺激高表達細胞株能抑制纖維化，及TGF-β/Smad和炎症因子下調；（3）糖尿病CRP轉基因小鼠出現更嚴重的腎損傷，出現蛋白尿和腎損傷分子-1上升、巨噬細胞和T細胞侵潤、炎症和纖維化增加，並伴有CRP受體（CD32a）上調、TGF-β/Smads及NFκB/p65信號過度活化。體外實驗進一步證實CRP通過其受體CD32a/CD64增加炎症和纖維化。另外證實CRP與高糖有協同作用。 / 結論：TGF-β/Smad信號通路是糖尿病腎病的重要致病機制。糖尿病腎病導致Smad7、miR-29b下調，運用基因治療恢復其表達能減輕糖尿病腎損傷。 / Diabetic nephropathy (DN) is characterized by renal fibrosis and inflammation. Increasing evidence shows that TGF-β/Smad signaling plays a critical role in DN. This thesis tested a hypothesis that TGF-β/Smad signaling may play a central role in diabetic kidney injury and targeting this pathway may represent a novel therapy for DN. The hypothesis was tested in a type-1 model of diabetes induced in Smad7 knockout (KO) or CRP transgene, and the therapeutic potential for DN was examined by overexpressing renal Smad7 or miR-29b in both type-1 or type-2 models of diabetes. / As described in Chapter Three, the protective role and therapeutic potential of Smad7 in diabetic kidney disease was investigated in streptozotocin-induced diabetic model in Smad7 KO mice and in diabetic rats given Smad7 gene transfer using an ultrasound-microbubble-mediated technique. Results showed that Smad7 KO mice developed more severe diabetic kidney injury than wild type (WT) mice as evidenced by a signicant increase in microalbuminuria, renal brosis, and renal inammation, which was mediated by enhanced activation of both TGF-β/Smads and NF-κB signaling pathways. To develop a therapeutic potential for diabetic kidney disease, Smad7 gene was transferred into the kidney, which results in high levels renal Smad7, thereby blocking microalbuminuria, TGF-β/Smad3-mediated renal brosis and NF-κB/p65-driven renal inammation in diabetic rats. / To test a novel hypothesis that TGF-β/Smad3-mediated DN via the Smad3-dependent miR-29, in Chapter Four, the role and mechanisms of miR-29b in DN were examined in vitro in a stable mesangial cell line with overexpression or knockdown of miR-29b and the therapeutic effect of miR-29b on DN was developed by delivering a Dox-inducible miR-29b into 10-week db/db mice. Results showed that addition of AGEs induced a loss of miR-29b with increased fibrosis and inflammation in mesangial cells, which was further enhanced with miR-29b knockdown, but inhibited by overexpressing miR-29b. In db/db mice, reduction of renal miR-29b over the 20 week time was associated with a marked increase in microabluminuria, renal fibrosis and inflammation. Restoring miR-29b resulted in inhibition of kidney injuries by blocking TGF-β/Smad3-mediated renal fibrosis, NF-kB/p65-driven renal inflammation, and importantly, the Th1-dependent immune response, revealing a critical role and therapeutic potential for miR-29b in the pathogenesis of DN. / Finally, diabetic kidney injury was also assessed in under high inflammation conditions in CRP transgenic (Tg) mice. As shown in Chapter Five, CRP Tg mice developed more severe diabetic kidney injury than WT mice, as evidenced by a significant increase in microalbuminuria and kidney injury molecule-1, macrophages and T cells, and upregulation of pro-inflammatory cytokines and extracellular matrix. CRP-mediated DN was associated with upregulation of CRP receptor, CD32a, and over-activation of the TGF-β/Smads and NFκB/p65 signaling pathways. These findings were further confirmed in vitro under high levels of CRP. In addition, CRP was induced by high glucose, which synergistically promoted high glucose-mediated renal inflammation and fibrosis, suggesting a positive feedback-loop between CRP and high glucose under diabetic conditions. / In conclusion, TGF-β/Smads play critical roles in the pathogenesis of DN. Loss of renal Smad7 and miR-29b may be a key mechanism of DN. Thus, over-expression of Smad7 or miR-29b may represent novel therapeutic strategies for diabetic kidney complications. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Chen, Haiyong. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 202-236). / 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.ii / DECLARATION --- p.vi / ACKNOWLEDGEMENT --- p.vii / PUBLICATIONS --- p.ix / PRESENTATIONS/AWARDS --- p.xi / TABLE OF CONTENTS --- p.xii / LIST OF ABBREVIATIONS --- p.xxii / LIST OF FIGURES/TABLES --- p.xxiv / Chapter CHAPTER ONE --- INTRODUCTION --- p.1 / Chapter 1.1 --- TGF-β superfamily --- p.2 / Chapter 1.2 --- TGF-β/Smad signaling pathway --- p.3 / Chapter 1.2.1 --- TGF-β --- p.3 / Chapter 1.2.1.1 --- TGF-β structure --- p.3 / Chapter 1.2.1.2 --- Activation of latent TGF-β --- p.4 / Chapter 1.2.1.3 --- Latent TGF-β receptors --- p.6 / Chapter 1.2.2 --- TGF-β signaling pathway --- p.7 / Chapter 1.2.2.1 --- Receptors --- p.7 / Chapter 1.2.2.2 --- Smads --- p.10 / Chapter 1.2.2.3 --- Smad-dependent TGF-β signaling pathways --- p.13 / Chapter 1.2.2.4 --- Smad-independent TGF-β signaling pathways --- p.14 / Chapter 1.3 --- Diabetes nephropathy --- p.15 / Chapter 1.3.1 --- Diabetes Mellitus --- p.15 / Chapter 1.3.2 --- Type 1 and type 2 diabetes --- p.16 / Chapter 1.3.3 --- Diabetic complications --- p.16 / Chapter 1.3.4 --- Cellular and molecular mechanisms in diabetic complications --- p.17 / Chapter 1.3.4.1 --- Increased polyol pathway flux --- p.17 / Chapter 1.3.4.2 --- Increased advanced glycation end-products (AGEs) formation --- p.18 / Chapter 1.3.4.3 --- Activation of protein kinase C (PKC) isoforms --- p.20 / Chapter 1.3.4.4 --- Increased hexosamine pathway flux --- p.22 / Chapter 1.3.4.5 --- Increased Reactive Oxygen Species --- p.23 / Chapter 1.3.5 --- Diabetic kidney injuries --- p.24 / Chapter 1.3.5.1 --- Exacerbation of renal structure and function --- p.24 / Chapter 1.3.5.2 --- Fibrosis in diabetic nephropathy --- p.25 / Chapter 1.3.5.3 --- Inflammation in diabetic nephropathy --- p.26 / Chapter 1.4 --- Role of TGF-β/Smad signaling pathway in diabetic nephropathy --- p.28 / Chapter 1.4.1 --- Smad-depedent signaling in diabetic nephropathy --- p.28 / Chapter 1.4.2 --- Cross talk between Smads and other signaling pathways in diabetic nephropathy --- p.30 / Chapter 1.4.3 --- TGF-β/Smads and MicroRNA regulation in diabetic nephropathy --- p.32 / Chapter CHAPTER TWO --- MATERIALS AND METHODS --- p.35 / Chapter 2.1 --- Materials --- p.36 / Chapter 2.1.1 --- Regents and equipment --- p.36 / Chapter 2.1.1.1 --- Reagents and equipment for cell culture --- p.36 / Chapter 2.1.1.2 --- Reagents and equipment for real-time RT-PCR --- p.37 / Chapter 2.1.1.3 --- Reagents and equipment for western blotting --- p.38 / Chapter 2.1.1.4 --- Reagents and equipment for immunohistochemistry --- p.39 / Chapter 2.1.1.5 --- Reagents and equipment for in situ hybridization --- p.40 / Chapter 2.1.1.6 --- Reagents and equipment for plasmid purification --- p.40 / Chapter 2.1.1.7 --- Reagents and equipment for genotyping --- p.41 / Chapter 2.1.1.8 --- Other reagents --- p.41 / Chapter 2.1.2 --- Buffers --- p.42 / Chapter 2.1.2.1 --- Western blotting buffer --- p.42 / Chapter 2.1.2.2 --- Immunohistochemistry buffer --- p.45 / Chapter 2.1.2.3 --- ELISA buffers --- p.47 / Chapter 2.1.2.4 --- In Situ hybridization buffer --- p.48 / Chapter 2.2.2 --- Antibodies --- p.49 / Chapter 2.2.3 --- Primer sequences --- p.49 / Chapter 2.2 --- Methods --- p.56 / Chapter 2.2.1 --- Animal model --- p.56 / Chapter 2.2.1.1 --- Animals --- p.56 / Chapter 2.2.1.2 --- Diabetic animal models --- p.57 / Chapter 2.2.2 --- Sample Collection --- p.59 / Chapter 2.2.2.1 --- Urine collection --- p.59 / Chapter 2.2.2.2 --- Plasma collection --- p.59 / Chapter 2.2.2.3 --- Tissue collection --- p.60 / Chapter 2.2.2.4 --- Paraffin embedding --- p.60 / Chapter 2.2.3 --- Ultrasound-microbubble-mediated gene transfer system --- p.61 / Chapter 2.2.3.1 --- Smad7 gene therapy --- p.61 / Chapter 2.2.3.2 --- miR-29 gene therapy --- p.62 / Chapter 2.2.4 --- Microalbumin and renal function --- p.63 / Chapter 2.2.4.1 --- Microalbuminuria --- p.63 / Chapter 2.2.4.2 --- Creatinine measurement --- p.63 / Chapter 2.2.5 --- Enzyme-Linked Immunosorbent Assay (ELISA) --- p.64 / Chapter 2.2.6 --- Histology and immunohistochemistry --- p.64 / Chapter 2.2.6.1 --- Tissue process --- p.64 / Chapter 2.2.6.2 --- Periodic Acid-Schiff Staining (PAS) --- p.64 / Chapter 2.2.6.3. --- Immunohistochemistry (IHC) --- p.65 / Chapter 2.2.6.4 --- In Situ hybridization --- p.66 / Chapter 2.2.6.5 --- Quantitation of histology and IHC --- p.67 / Chapter 2.2.7 --- Cell culture --- p.67 / Chapter 2.2.8 --- Real-time polymerase chain reaction (PCR) --- p.69 / Chapter 2.2.9 --- Western Blotting --- p.70 / Chapter 2.3 --- Statistical analysis --- p.71 / Chapter CHAPTER THREE --- THE PROTECTIVE ROLE OF SMAD7 IN DIABETIC NEPHROPATHY --- p.72 / Chapter 3.1 --- Introduction --- p.73 / Chapter 3.2 --- Materials and methods --- p.74 / Chapter 3.2.1 --- Animal models --- p.74 / Chapter 3.2.2 --- Ultrasound-mediated gene transfer of inducible Smad7 gene-bearing microbubbles into the kidney --- p.74 / Chapter 3.2.3 --- Real-time PCR --- p.75 / Chapter 3.2.4 --- Western blotting --- p.75 / Chapter 3.2.5 --- Microalbuminuria and urinary creatinine analysis --- p.76 / Chapter 3.2.6 --- Histology and immunohistochemistry --- p.76 / Chapter 3.2.7 --- Statistical analysis --- p.77 / Chapter 3.3 --- Results --- p.77 / Chapter 3.3.1 --- Genotyping for Smad7 KO and WT mice --- p.77 / Chapter 3.3.2 --- Disruption of Smad7 enhances diabetic kidney injury --- p.78 / Chapter 3.3.3 --- Disruption of Smad7 enhanced fibrosis in diabetic kidney --- p.80 / Chapter 3.3.3.1 --- Collagen I is enhanced in diabetic Smad7 KO mice --- p.81 / Chapter 3.3.3.2 --- Collagen IV is enhanced in diabetic Smad7 KO mice --- p.82 / Chapter 3.3.3.3 --- Fibronectin is enhanced in diabetic Smad7 KO mice --- p.84 / Chapter 3.3.4 --- Disruption of Smad7 exacerbates inflammation in diabetic kidney --- p.85 / Chapter 3.3.4.1 --- Disruption of Smad7 increases IL-1β in diabetic kidney --- p.85 / Chapter 3.3.4.2 --- Disruption of Smad7 increases TNF-α in diabetic kidney --- p.86 / Chapter 3.3.4.3 --- Disruption of Smad7 Increases MCP-1 in diabetic kidney --- p.87 / Chapter 3.3.4.4 --- Disruption of Smad7 increases ICAM-1 in diabetic kidney --- p.88 / Chapter 3.3.4.5 --- Disruption of Smad7 increases macrophage infiltration in diabetic kidney --- p.90 / Chapter 3.3.5 --- Enhanced activation of TGF-β/Smad3 and NF-κB Signaling is a central mechanism by which disruption of Smad7 promotes diabetic renal fibrosis and inflammation --- p.91 / Chapter 3.3.5.1 --- Smad7 decreases in diabetic kidney --- p.91 / Chapter 3.3.5.2 --- Enhanced activation of TGF-β/Smad3 signaling pathway contributes to fibrosis in diabetic kidney --- p.92 / Chapter 3.3.5.3 --- Enhanced activation of NF-κB/p65 signaling pathway contributes to inflammation in diabetic kidney --- p.93 / Chapter 3.3.6 --- Smad7 transfection rate by gene therapy in diabetic rats --- p.94 / Chapter 3.3.7 --- Restoring Smad7 attenuates kidney injury in diabetic rats --- p.96 / Chapter 3.3.8 --- Restoring Smad7 attenuates renal fibrosis in diabetic rats --- p.98 / Chapter 3.3.8.1 --- Restoring Smad7 attenuates collagen I in diabetic kidney --- p.98 / Chapter 3.3.8.2 --- Restoring Smad7 attenuates collagen IV in diabetic kidney --- p.100 / Chapter 3.3.8.3 --- Restoring Smad7 attenuates collagen III in diabetic kidney --- p.101 / Chapter 3.3.9 --- Restoring Smad7 attenuates renal inflammation in diabetic rats --- p.104 / Chapter 3.3.9.1 --- Restoring Smad7 attenuates IL-1b in diabetic kidney --- p.104 / Chapter 3.3.9.2 --- Restoring Smad7 attenuates TNF-α in diabetic kidney --- p.106 / Chapter 3.3.9.3 --- Restoring Smad7 Attenuates MCP-1 in diabetic kidney --- p.107 / Chapter 3.3.9.4 --- Restoring Smad7 attenuates ICAM-1 in diabetic kidney --- p.109 / Chapter 3.3.9.5 --- Restoring Smad7 attenuates macrophage infiltration in diabetic kidney --- p.111 / Chapter 3.3.10 --- Blockade of activation of TGF-β/Smad3 and NF-κB signaling is a key mechanism by which overexpression of smad7 inhibits diabetic renal injury --- p.113 / Chapter 3.3.10.1 --- Restoring Smad7 inhibits activation of TGF-β/Smad3 signaling --- p.113 / Chapter 3.3.10.2 --- Restoring Smad7 inhibits activation of NF-κB signaling --- p.115 / Chapter 3.3 --- Discussion --- p.117 / Chapter CHAPTER FOUR --- THE PROTECTIVE ROLE OF MICRORNA-29B IN DIABETIC NEPHROPATHY --- p.121 / Chapter 4.1 --- Introduction --- p.122 / Chapter 4.2 --- Materials and methods --- p.123 / Chapter 4.2.1 --- Animal model --- p.123 / Chapter 4.2.2 --- Ultrasound-microbubble-mediated-miR-29 gene transfer --- p.124 / Chapter 4.2.3 --- Real-time polymerase chain reaction (PCR) --- p.124 / Chapter 4.2.4 --- Western Blotting --- p.125 / Chapter 4.2.5 --- Albumin excretion measurement --- p.126 / Chapter 4.2.6 --- ELISA --- p.126 / Chapter 4.2.7 --- Histology and immunohistochemistry --- p.126 / Chapter 4.2.8 --- In Situ hybridization --- p.127 / Chapter 4.2.9 --- Cell culture --- p.128 / Chapter 4.2.10 --- Statistical analysis --- p.129 / Chapter 4.3 --- Results --- p.129 / Chapter 4.3.1 --- Over-expression of miR-29b attenuates, but knockdown of miR-29b enhances fibrosis in vitro --- p.129 / Chapter 4.3.1.1 --- Over-expression of miR-29b attenuates fibrosis --- p.129 / Chapter 4.3.1.2 --- Knockdown of miR-29b enhances fibrosis --- p.132 / Chapter 4.3.2 --- Restoring miR-29b attenuates kidney injury in db/db mice --- p.134 / Chapter 4.3.3 --- Restoring miR-29b attenuates renal fibrosis in db/db mice --- p.139 / Chapter 4.3.3.1 --- Restoring miR-29b attenuates collagen IV in db/db mice --- p.139 / Chapter 4.3.3.2 --- Restoring miR-29b attenuates collagen I in db/db mice --- p.141 / Chapter 4.3.3.3 --- Restoring miR-29b attenuates fibronectin in db/db mice --- p.144 / Chapter 4.3.4 --- Restoring miR-29b inhibits renal fibrosis via TGF-β/Smad3 dependent pathway --- p.146 / Chapter 4.3.5 --- Restoring miR-29b inhibits th1 immune response in diabetic kidney --- p.148 / Chapter 4.3.6 --- Restoring miR-29b inhibits inflammation in diabetic kidney --- p.151 / Chapter 4.4 --- Discussion --- p.154 / Chapter 4.5 --- Conclusion --- p.161 / Chapter CHAPTER FIVE --- THE PATHOGENIC ROLE OF C-REACTIVE PROTEIN IN DIABETIC NEPHROPATHY --- p.162 / Chapter 5.1 --- Introduction --- p.163 / Chapter 5.2 --- Materials and methods --- p.164 / Chapter 5.2.1 --- Mouse model of STZ induced diabetes --- p.164 / Chapter 5.2.2 --- Measurement of blood glucose, urinary albumin excretion, and creatinine clearance --- p.165 / Chapter 5.2.3 --- Histology and immunohistochemistry --- p.165 / Chapter 5.2.4 --- Cell culture --- p.166 / Chapter 5.2.5 --- Real-time PCR --- p.166 / Chapter 5.2.6 --- Western blotting --- p.167 / Chapter 5.2.7 --- Statistical analyses --- p.168 / Chapter 5.3 --- Results --- p.168 / Chapter 5.3.1 --- Diabetic renal injury is exacerbated in CRP Tg mice --- p.168 / Chapter 5.3.2 --- Renal inflammation is exacerbated in diabetic CRP Tg mice --- p.172 / Chapter 5.3.2.1 --- F4/80+ macrophage infiltration is enhanced in diabetic CRP Tg mice --- p.172 / Chapter 5.3.2.2 --- CD3+ T cell infiltration is enhanced in diabetic CRP Tg mice --- p.173 / Chapter 5.3.2.3 --- TNF-α expression is enhanced in diabetic CRP Tg mice --- p.173 / Chapter 5.3.2.4 --- IL-1β expression is enhanced in diabetic CRP Tg mice --- p.174 / Chapter 5.3.3 --- Renal fibrosis is enhanced in diabetic CRP Tg mice --- p.175 / Chapter 5.3.3.1 --- Collagen I is enhanced in Diabetic CRP Tg mice --- p.175 / Chapter 5.3.3.2 --- Collagen IV is enhanced in diabetic CRP Tg mice --- p.176 / Chapter 5.3.4 --- Enhanced CRP signaling and activation of NF-κB and TGF-β/Smad3 signaling are key mechanism by which CRP promotes diabetic renal injury --- p.177 / Chapter 5.3.4.1 --- Enhanced CRP signaling via upregulation of CD32a expression --- p.177 / Chapter 5.3.4.2 --- enhanced activation of NF-κB signaling is key mechanism by which CRP promotes renal inflammation --- p.179 / Chapter 5.3.4.3 --- Enhanced activation of TGF-β/Smad3 signaling is key mechanism by which CRP promotes renal inflammation --- p.181 / Chapter 5.4 --- Discussion --- p.194 / Chapter 5.5 --- Conclusion --- p.197 / Chapter CHAPTER SIX --- SUMMARY AND CONCLUSION --- p.198 / REFERENCES --- p.202

Ho, Wing-Yin. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 115-131). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abbreviations --- p.iii / Abstract --- p.v / 摘要 --- p.ix / Publications --- p.xii / Table of Contents --- p.xiv / Chapter 1. --- General Introduction / Chapter 1.1. --- Diabetes Mellitus (DM) and Diabetic Nephropathy --- p.1 / Chapter 1.1.1. --- "Prevalence, Diagnosis and Classification of DM" --- p.1 / Chapter 1.1.2. --- Type 2 DM and its Complications: Diabetic Nephropathy --- p.5 / Chapter 1.1.3. --- Diagnosis and Impacts of Diabetic Nephropathy --- p.7 / Chapter 1.1.4. --- Current Treatment of Type 2 DM and Diabetic Nephropathy --- p.8 / Chapter 1.2. --- Cytokines and Chemokines --- p.9 / Chapter 1.2.1. --- Types and Properties --- p.9 / Chapter 1.2.2. --- Cytokines and chemokines in Type 2 DM and Diabetic Nephropathy --- p.13 / Chapter 1.3. --- T Lymphocyte Costimulatory Molecules --- p.15 / Chapter 1.3.1. --- Types and Properties --- p.15 / Chapter 1.3.2. --- T Lymphocyte Costimulatory Molecules in Type 2 DM and Diabetic Nephropathy --- p.16 / Chapter 1.4. --- Adhesion Molecules --- p.18 / Chapter 1.4.1. --- Types and Properties --- p.18 / Chapter 1.4.2. --- Adhesion Molecules in Type 2 DM and Diabetic Nephropathy --- p.20 / Chapter 1.5. --- Intracellular Signaling Pathways --- p.21 / Chapter 1.5.1. --- Types and Properties --- p.21 / Chapter 1.5.2. --- Intracellular Signaling Pathways in Type 2 DM and Diabetic Nephropathy --- p.23 / Chapter 1.6. --- Objectives of Our Study --- p.24 / Chapter 2. --- Materials and Methods / Chapter 2.1. --- Materials --- p.26 / Chapter 2.1.1. --- "Patients, Control Subjects and Blood Samples" --- p.26 / Chapter 2.1.2. --- Cell Line --- p.27 / Chapter 2.1.3. --- "Cell Culture Media, Buffers and Other Reagents" --- p.28 / Chapter 2.1.4. --- "Recombinant Human Cytokines, Inhibitors and Other Stimulators" --- p.30 / Chapter 2.1.5. --- Reagents and Buffers for Flow Cytometric Analysis --- p.31 / Chapter 2.1.5.1. --- Cytometric Bead Array (CBA) of Cytokines and Chemokines --- p.33 / Chapter 2.1.5.2. --- Multiplex Fluorescent Bead Immunoassay (FBI) of Soluble Adhesion Molecules --- p.33 / Chapter 2.1.5.3. --- Phosphorylation State Analysis of Signaling Molecules --- p.34 / Chapter 2.1.5.4. --- Immunofluorescent Staining of Cell Surface Molecules --- p.36 / Chapter 2.1.6. --- Reagents and Buffers for Protein Array Analysis --- p.37 / Chapter 2.1.7. --- "Reagents and Buffers for 3-(4,5-Dimethylthiazol-2-yl)-2,5- diphenylytetrazolium Bromide (MTT) Assay" --- p.37 / Chapter 2.1.8. --- Reagents for Human Enzyme-Linked Immunosorbent Assay (ELISA) --- p.37 / Chapter 2.2. --- Methods --- p.38 / Chapter 2.2.1. --- Whole Blood Culture Experiments --- p.38 / Chapter 2.2.2. --- "Collection of Serum and Plasma, and Purification of PBMC from EDTA-Blood" --- p.39 / Chapter 2.2.3. --- HK-2 Cell Cultures --- p.39 / Chapter 2.2.4. --- HK-2 Cell Treatments --- p.40 / Chapter 2.2.5. --- Flow Cytometric Analysis --- p.41 / Chapter 2.2.5.1. --- CBA of Cytokines and Chemokines --- p.41 / Chapter 2.2.5.2. --- Multiplex FBI of Soluble Adhesion Molecules --- p.41 / Chapter 2.2.5.3. --- Phosphorylation State Analysis of Signaling Molecules --- p.42 / Chapter 2.2.5.4. --- Immunofluorescent Staining of Cell Surface Molecules --- p.43 / Chapter 2.2.6. --- Protein Array Analysis --- p.44 / Chapter 2.2.7. --- MTT Assay --- p.44 / Chapter 2.2.8. --- ELISA --- p.45 / Chapter 2.2.9. --- Statistical Analysis --- p.46 / Chapter 3. --- "Clinical Study on the Expressions of Cytokines, Chemokines, Co-stimulatory Molecules, Phosphorylated Signaling Molecules in Patients with Diabetic Nephropathy" / Chapter 3.1. --- Introduction --- p.47 / Chapter 3.2. --- Results --- p.49 / Chapter 3.2.1. --- Demographic Data of Participants --- p.49 / Chapter 3.2.2. --- Expression Profile in Plasma of Patients --- p.49 / Chapter 3.2.2.1. --- Cytokines and Chemokines --- p.49 / Chapter 3.2.2.2. --- Soluble Costimulatory Molecules --- p.55 / Chapter 3.2.2.3. --- Soluble Adhesion Molecules --- p.55 / Chapter 3.2.2.4. --- "Correlations between Plasma Concentrations of Cytokines, Chemokines, soluble Costimulatory Molecules and soluble Adhesion Molecules and UACR in Patients" --- p.60 / Chapter 3.2.3. --- Effects ofTNF-α and IL-18 on the ex vivo Production from Whole Blood of Patients --- p.65 / Chapter 3.2.3.1. --- Ex vivo Production of Cytokines and Chemokines --- p.65 / Chapter 3.2.3.2. --- Ex vivo Production of Soluble Costimulatory Molecules --- p.70 / Chapter 3.2.4. --- "Expression of Phosphorylated p38 MAPK, JNK and ERK in PBMC of Patients" --- p.73 / Chapter 3.3. --- Discussion --- p.77 / Chapter 3.3.1. --- "Cytokines, Chemokines and Diabetic Nephropathy" --- p.77 / Chapter 3.3.2. --- Soluble Costimulatory Molecules and Diabetic Nephropathy --- p.80 / Chapter 3.3.3. --- Soluble Adhesion Molecules and Diabetic Nephropathy --- p.83 / Chapter 3.3.4. --- Intracellular Signaling and Diabetic Nephropathy --- p.87 / Chapter 4. --- In vitro Study on the Signal Transduction Mechanism Regulating the Expression of CCL2 and Cell Surface Adhesion Molecules in Tumour Necrosis Factor (TNF)-α-Stimulated HK-2 Cells / Chapter 4.1. --- Introduction --- p.90 / Chapter 4.2. --- Results --- p.93 / Chapter 4.2.1. --- Expression Profile of Cytokines and Chemokines of TNF-α-activated HK-2 Cells --- p.93 / Chapter 4.2.2. --- "TNF-α Upregulated CCL2, ICAM-1 and VCAM-1 Expression in HK-2 Cells" --- p.95 / Chapter 4.2.3. --- "TNF-α Activated the p38 MAPK, JNK and ERK Signaling Pathways in HK-2 Cells" --- p.96 / Chapter 4.2.4. --- Cytotoxicity of MAPK Inhibitors --- p.96 / Chapter 4.2.5. --- "Effects of p38 MAPK, JNK and ERK Inhibitors on TNF-α-induced Expressions of CCL2, ICAM-1 and VCAM-1" --- p.100 / Chapter 4.3. --- Discussion --- p.102 / Chapter 5. --- Conclusion and Future Prospects / Chapter 5.1. --- Conclusion --- p.107 / Chapter 5.2. --- Future Prospects --- p.111 / References --- p.115

published_or_final_version / Community Medicine / Master / Master of Public Health

Submitted in fulfillment of the requirements for the degree of Master of Clinical Technology (Nephrology), Durban University of Technology, Durban, South Africa, 2013. / Diabetic nephropathy is a serious complication of diabetes that can lead to end stage renal failure (ESRF). It is now the most common cause of ESRF in patients accepted onto renal replacement therapy (RRT) programmes. Kidney disease is common in South Africa. 60-65% is due to inherited hypertension and 20-25% due to Type 2 diabetes (National Kidney Foundation of South Africa, 2002). The renal replacement therapies include haemodialysis, peritoneal dialysis and transplantation. Successful long-term haemodialysis in patients with end stage renal disease (ESRD) depends to a large extent upon a trouble- free vascular access. Achieving a successful vascular access remains a challenge especially in the diabetic population. Current Kidney Dialysis Outcome Quality Initiative (KDOQI) guidelines encourage placing Arterio-Venous Fistula (AVF) in more haemodialysis patients. While the upper limb is the preferred site for AVF creation, researchers are undecided on which is the ideal location (distal or proximal arm) in the diabetic population. Many new fistulae fail to mature sufficiently to be usable for haemodialysis. Pre-insertion work-up with regard to haemodialysis access is important in maintaining the most appropriate access in the growing diabetic population requiring haemodialysis. Pre-operative vascular mapping to identify suitable vessels has been reported to improve vascular access outcomes . In South Africa, duplex scanning is not routinely done, and a clinical judgement by the surgeon remains in most instances the deciding factor on the site of the AVF. Whilst conducting this research, it has been found that while diabetic patients may have AVF created, the maturation time is of a much extended period, and a challenge to achieve the desired dose of dialysis. This is a prospective, quantitative and qualitative study of 21 diabetic patients. These included patients that were starting on the chronic haemodialysis program and limited to patients that were having first attempt of AVF creation and aims to establish if sonogram testing provides a more accurate measure of the ideal location for the AVF, or if a clinical evaluation alone by the surgeon is sufficient. Surgical techniques are different amongst surgeons and clinical evaluation is more a subjective decision. By limiting the surgeons performing the AVF, a standardized surgical procedure was established. If an ideal AVF access for the patient is created, haemodialysis efficiency is increased and ultimately patient outcome improved. The AVF was created according to the clinical evaluation as is the current process, and the surgeons were not aware of the duplex sonogram results. Failure and success of AVF were analysed according to primary patency and functional success. A primary patency success of the AVF does not guarantee functional success. If an AVF is not able to complete an entire haemodialysis session trouble free at the prescribed dialysis dose, the AVF is considered a failure irrespective of primary patency success. This was evident with 10% of patients who had primary patency but functional success was not achieved. With a 55% functional success in this study with AVF created on clinical evaluation, there was no significance difference (p=0.795) if AVFs were based on duplex sonogram findings. However, there was evidence of increased AVF success in 33% of the failed AVFs when the new AVFs were created at the duplex sonogram site. 95% of patients in this study had commenced haemodialysis with a Central Venous Catheter (CVC). AVF success could be increased if early referral of diabetic patients for permanent access to the surgeon occurred. Maturation rate of AVF differed from KDOQI guidelines with AVF first cannulation only after 17 weeks, and not after the recommended time of 6 weeks. Blood flow rates on dialysis also varied with international standards, with only maximum of 400mls/min reached after one year. With distal arm AVF, diameter of radial artery of less than 2mm and cephalic vein less than 3mm was associated with AVF failure. This research study represents the first of its kind in Kwazulu Natal looking at vascular access sites in diabetic patients with End Stage Renal Disease on haemodialysis. / PDF Full-text unavailable. Please refer to hard copy for Full-text / M

Introdução: O quelante de fósforo (P) Osvaren® foi lançado recentemente no mercado internacional para tratamento da hiperfosfatemia nos pacientes com Doença Renal Crônica (DRC). Não existem estudos experimentais com esta medicação. Objetivo: Avaliar a ação deste quelante no tratamento do Distúrbio Mineral e Ósseo (DMO) de ratos com DRC induzida por adenina e por nefrectomia 5/6 (NX5/6). Métodos: Experimento 1: Durante 4 semanas, ratos Wistar receberam dieta com adenina [2 semanas (0,75%); 2 semanas (0,50%)] e então foram divididos em 3 grupos para receber dieta padrão (grupo DRC); dieta padrão com 3% de Acetato de Cálcio (grupo Ca); e dieta padrão com 3% de Osvaren® (grupo CaMg). Um grupo Controle (função renal normal) recebeu a dieta padrão durante todo o estudo. Ao final de cinco semanas de tratamento os animais foram sacrificados. Experimento 2: ratos da mesma espécie foram submetidos à NX5/6, divididos em grupos como no experimento 1 para receberem os quelantes de P imediatamente após a nefrectomia, e sacrificados após 9 semanas. Nos dois experimentos foram coletados soro, fêmures, aortas e paratireóides para análise bioquímica, histomorfometria óssea, pesquisa de calcificação vascular (CV) e imunohistoquímica de paratireóides, respectivamente. Resultados: Experimento 1: Os animais que receberam adenina apresentaram níveis maiores de creatinina, PTH, P e magnésio (Mg) que os do grupo Controle. Os quelantes de P reduziram a fração de excreção de P (FeP), mas não o P sérico, enquanto os níveis de PTH, FGF23 e calcitriol diminuíram de forma não significativa nos grupos tratados. O Mg sérico foi significativamente maior no grupo CaMg, enquanto maior calciúria, mais CV e menor marcação pelo PCNA nas paratireóides foram observadas no grupo Ca. Os animais que receberam adenina apresentaram doença óssea mista e ambos os quelantes de P melhoraram a remodelação, mas favoreceram a um acúmulo ainda maior de osteóide. Experimento 2: Da mesma forma que no experimento 1, o animais submetidos à NX5/6 evoluíram com uremia, e os efeitos dos quelantes no PTH, na CV e nas paratireóides foram similares. No entanto, o grupo CaMg apresentou mortalidade de 54% (vs 20% no grupo Ca), além de retardo na mineralização óssea. Conclusão: O tipo de nefropatia pode ter influência no efeito do quelante de P Osvaren®, já que o acúmulo de Mg provavelmente foi o responsável pela maior mortalidade e déficit de mineralização observados no experimento 2. Em relação ao efeito como quelante de P, o Osvaren® foi equivalente ao acetato de cálcio, mas este último está associado a maior sobrecarga de cálcio e a CV. O acúmulo de osteóide observado em ambos os experimentos parece ser o preço pago pela reduzida absorção intestinal de P favorecida pelos quelantes no hiperpatireoidismo secundário severo e esse efeito deveria ser avaliado em pacientes em diálise / Introduction: Osvaren® is a phosphate (P) binder that has been recently introduced for Chronic Kidney Disease (CKD) patients therapy. However, it has not been tested in experimental models yet. Aims: To study Osvaren® effects on mineral and bone metabolism in CKD experiment models. Methods: Experiment 1: For 4 weeks (wk), rats were fed normal chow or an adenine-enriched diet (0,75% for 2 wks; 0,50% for 2 wks). After that, adenine was discontinued and rats were divided into 3 groups: Ca (3% calcium acetate); CaMg (3% Osvaren®) and CKD (normal diet/untreated). A control group, with normal renal function was also studied. After 5 wks of therapy, animals were sacrificed. Experiment 2: Animals underwent 5/6 nephrectomy, and were submitted to different P binders treatment as in experiment 1 and then sacrificed 9 weeks after surgery. We performed biochemical and histomorphometric analyses, parathyroid PCNA imunohistochemistry, as well as Von Kossa staining and calcium content of aortic sections. Results: Experiment 1: Higher creatinine, PTH, FGF-23, P and Mg were found in all adenine-fed rats. P binders did not reduce serum P but decreased fractional excretion of P (FeP) and tended to reduce PTH, FGF23 and calcitriol levels. In the CaMg group, Mg was slightly elevated, whereas Ca group had greater urinary calcium as well as vascular calcification and smaller parathyroid PCNA staining. Adenine-fed rats presented features of mixed bone disease and P binders therapy was able to decrease bone resorption, but was associated with an osteoid accumulation. Experiment 2: As in experiment 1, nephrectomized rats were uremic and P binder effects on PTH, vascular calcification and parathyroid were very similar, except for the mortality rate, that was higher in CaMg group (54% vs 20% in Ca group). An increase in mineralization lag time was also found in CaMg group. Conclusions: Osvaren® may cause different effects on different types of nephropathy, as in experiment 2 the Mg was probably the responsible for higher mortality as well as for bone mineralization defect. In terms of P binding efficacy, Osvaren® was equivalent to calcium acetate, but the last one caused calcium overload and vascular calcification. The osteoid accumulation is probably related to the decrease in intestinal P absorption caused by the P binders in these models of secondary hyperparathyroidism. These effects should also be evaluated in dialysis patients

Tez (Tıpta Uzmanlık) - Süleyman Demirel Üniversitesi, Tıp Fakültesi, İç Hastalıkları Anabilim Dalı, 2003. / Kaynakça var.

Thesis (M. Res. (Med.))--University of Hong Kong, 2005.