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Avaliação da extração e cinética de solutos em pacientes submetidos à hemodiálise convencional, hemodiafiltração pós-diluição e hemofiltração pré-diluição / Solutes extraction and kinects assessment in patients submitted to a conventional hemodialysis, postdilution hemodiafiltration and predilution hemofiltrationSilva, Paola da Ponte 23 May 2013 (has links)
Introdução: O uso de membranas de alto fluxo tornou-se comum na prática de hemodiálise convencional (HDC), hemodiafiltração (HDF) e hemofiltração (HF) devido ao elevado coeficiente de ultrafiltração e à maior permeabilidade do poro. A produção de fluido de reposição online proporciona maior facilidade na execução de HDF e HF. Estas modalidades aumentam a depuração de solutos maiores por convecção. O objetivo do estudo é comparar a eficácia de três métodos dialíticos: HDC, HDF online (HDF-OL) pós-diluição e HF online (HF-OL) pré-diluição, por meio da quantificação direta da dose de diálise pela extração de solutos. Método: Trata-se de um ensaio clínico, envolvendo 14 pacientes em hemodiálise. Os pacientes iniciaram no estudo na modalidade de HDC com prescrição de quatro horas, fluxo de sangue de 350ml/min e fluxo de dialisato de 800ml/min com dialisador novo de alto fluxo e alta eficiência. Posteriormente, os mesmos pacientes foram submetidos à uma sessão de HDF-OL pós-diluição de quatro horas, fluxo de sangue de 350ml/min, fluxo de reposição de 100ml/min e fluxo de dialisato de 700ml/min. Por último, fizeram uma sessão de HF-OL pré-diluição com mesma duração, fluxo de sangue de 350ml/min e fluxo de reposição de 100% do fluxo de sangue. Foram realizadas 42 sessões de tratamento. A extração de solutos foi avaliada por meio de dosagens plasmáticas e quantificação do efluente. Resultados: As concentrações séricas dos solutos pré-diálise não foram diferentes entre as sessões do estudo. O volume de substituição em HDF-OL foi de 20,6 ± 0,8L/sessão e em HF-OL foi de 81,8 ± 7,1L/sessão. A HDF-OL quando comparada com a HDC não apresentou diferença na extração de moléculas pequenas. Da mesma forma, a extração de ?2-microglobulina foi semelhante nas 3 modalidades: 121,1 ± 46,4 mg em HDC, 130,1 ± 46,5 mg em HDF-OL e 106,0 ± 45,0 mg em HF-OL. A perda de albumina foi significativamente maior em HDF-OL (1360,2 ± 425,0 mg) e HF-OL (1310,3 ± 201,4 mg) Vs. HDC (269,6 ± 15,3 mg) (p<0,0001). A depuração de pequenos solutos foi superior em HDF-OL. A depuração de ?2-microglobulina foi maior em HDF- OL (114,0 ± 15,7 ml/min) e HF-OL (116,1 ± 19,5 ml/min) Vs. HDC (86,5 ± 16,1 ml/min) (p<0,0001). A dose de diálise avaliada pelo singlepool, equilibrated, standard Kt/V e pela quantificação direta da diálise (DDQKt/V) não foi diferente entre HDF-OL e HDC. Na HF-OL a ausência de difusão contribuiu para menor extração e menor Kt/V de solutos pequenos (p<0,0001). Conclusão: A dose de diálise avaliada pela extração de solutos foi semelhante entre HDC e HDF-OL sendo a HF-OL menos favorável na extração de solutos pequenos / Introduction: The use of high-flux membranes became common practice in conventional hemodialysis (CHD), hemodiafiltration (HDF) and hemofiltration (HF) due to the high pore permeability and ultrafiltration coefficient. The online production of substitution volume can make easier HDF and HF execution. These modalities can increase the clearance of larger solutes by convection. The aim of this study is to compare the dialysis efficacy among three methods: CHD, online post-dilution HDF (OL-HDF), and online pre- dilution HF (OL-HF) through the direct quantification of dialysis dose by the solutes extraction. Method: This is a clinical trial involving 14 patients on hemodialysis. The patients began the study in CHD modality with a four-hour prescription, blood flow of 350ml/min and dialysate flow of 800ml/min with new high-flux and high efficiency dialyzer. Subsequently, these patients were submitted to a four-hour post-dilution OL-HDF session, with flow replacement of 100ml/min, blood flow of 350ml/min and dialysate flow of 700ml/min. Finally, patients underwent a pre-dilution OL-HF with the same duration, blood flow of 350ml/min and flow replacement of 100% of the blood flow. Forty-two sessions of treatments were performed. The solutes extraction was assessed by plasma measurements and effluent quantification Results: Pre-treatment serum concentrations of different solutes showed no statistical difference among the modalities. The substitution volume in OL- HDF was 20.6 ± 0.8L/session and in OL-HF was 81.8 ± 7.1 L/session. The OL-HDF compared to CHD showed no difference in the small solutes extraction. Similarly, ?2-microglobulin extraction was similar among the three modalities: 121.1 ± 46.4 mg in CHD, 130.1 ± 46.5 mg in OL-HDF and 106.0 ± 45.0 mg in OL-HF. Albumin loss was significantly higher in OL-HDF (1360.2 ± 425.0 mg) and OL-HF (1310.3 ± 201.4 mg) Vs. CHD (269.6 ± 15.3 mg) (p<0.0001). The small solutes clearance was greater in OL-HDF. ?2- microglobulin clearance was higher in OL-HDF (114.0 ± 15.7 ml/min) and OL- HF (116.1 ± 19.5 ml/min) Vs. CHD (86.5 ± 16.1 ml/min) (p<0.0001). The dialysis dose measured by singlepool, equilibrated, standard Kt/V and by direct dialysis quantification (DDQ Kt/V) was not different between CHD and OL-HDF. In OL-HF the absence of diffusion contributed to lower extraction and lower Kt/V of small solutes. Conclusion: The dialysis dose evaluated by extraction of solutes was similar between CHD and OL-HDF being a OL-HF less favorable in small solutes extraction
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Bone marrow-derived macrophage myofibroblast transition (MMT) in renal fibrosis. / 骨髓来源的巨噬细胞肌纤维母细胞转分化在肾脏纤维化中的作用 / Gu sui lai yuan de ju shi xi bao ji xian wei mu xi bao zhuan fen hua zai shen zang xian wei hua zhong de zuo yongJanuary 2012 (has links)
背景:纤维化是各种因素导致肾脏慢性损伤的最终病理过程,是决定肾功能转归的关键因素。肌纤维母细胞作为构成肾脏纤维化组织的主要细胞成分,其来源尚不清楚。本研究认为骨髓来源的巨噬细胞向肌纤维母细胞转分化(MMT)可能是肾脏纤维化中肌纤维母细胞的主要来源。我们分别在慢性肾脏病患者的肾活检组织和小鼠单侧输料管梗阻模型(UUO)中验证这一假说。 / 方法:我们用激光共聚焦技术和流式细胞染色的方法检测小鼠UUO肾脏和患者肾活检组织中的MMT细胞(F4/80⁺α-SMA⁺或CD68⁺α-SMA⁺)。为了验证骨髓来源的MMT在肾纤维化中的重要作用,UUO模型分别在以下小鼠进行:1)去除骨髓的C57BL/6J小鼠,给予或不给予绿色荧光蛋白(GFP)标记的骨髓细胞移植;2)GFP⁺骨髓的嵌合体小鼠;3)巨噬细胞敲除或不敲除的lysM-Cre/DTR小鼠;4)GFP⁺Smad3⁺/⁺ 或GFP⁺Smad3⁻/⁻骨髓的嵌合体小鼠。我们用实时定量PCR和Western blot检测小鼠肾组织collagen-I和α-SMA水平。另外,我们观察MMT细胞和PDGFR-β⁺ pericytes, CD45⁺collagen I⁺ fibrocytes的关系。最后,通过观察GFP⁺Smad3⁻/⁻骨髓嵌合体小鼠UUO模型肾纤维化程度和TGF-β1刺激下TGF-β受体II或Smad3敲除的骨髓巨噬细胞MMT的不同进一步探索TGF-β/Smad3通路对MMT的影响。 / 结果:去除骨髓后,肾脏collagen-I沉积和α-SMA⁺肌纤维母细胞生成显著受抑制,骨髓细胞移植可以恢复肾脏纤维化,免疫荧光染色显示嵌合体小鼠中多数(80-90%)肌纤维母细胞来自于骨髓巨噬细胞转分化。同时,在白喉霉素诱导的巨噬细胞敲除小鼠中,50-60%巨噬细胞被去除,伴有纤维化明显减少,并且和MMT细胞显著减少相关。进一步验证巨噬细胞通过MMT直接参与肾脏纤维化过程。患者肾活检组织亦可见不同数目MMT细胞,纤维化活跃组织中MMT细胞可占到肌纤维母细胞总数的80%。另外,我们发现无论在小鼠模型还是患者肾活检组织中,多数MMT细胞表达pericyte(PDGFR-β⁺)和fibrocyte(CD45⁺collagen-I⁺)标记物。Smad3⁻/⁻骨髓嵌合体小鼠肾纤维化程度明显低于Smad3⁺/⁺骨髓嵌合体组,TGF-β1刺激下TGF-β受体II或Smad3敲除的骨髓巨噬细胞MMT明显低于不敲除组,提示TGF-β/Smad3通路在MMT过程中起重要作用。 / 结论:骨髓来源的MMT是肾纤维化组织中肌纤维母细胞的主要来源,TGF-β/Smad3 通路在MMT 过程中起重要作用。 / Background: Fibrosis is the ultimate pathological feature and determinant process for chronic kidney disease (CKD) regardless of the underlying etiology. Myofibroblasts are a key cell type in renal fibrosis by producing excessive collagen matrix. However, the origin of myofibroblasts during renal fibrosis remains largely controversial. This thesis tested the hypothesis that bone marrow (BM)-derived macrophage myofibroblast transition (MMT) may be a key pathway leading to renal fibrosis in patients with CKD and in a mouse model of unilateral ureteral obstructive nephropathy (UUO). / Methods: Renal fibrosis was assessed by expression of fibrotic marker collagen I and α-SMA using real-time PCR and western-blot analysis. MMT was determined in both mouse and human kidneys by confocal microscopy and flow cytometry with α-SMA⁺F4/80⁺ (or CD68⁺). The critical role of BM-derived MMT in renal fibrosis was examined in a mouse model of UUO, with various conditions: 1) BM depletion followed by BM transplantation (BMT) with GFP⁺ BM cells; 2) in GFP⁺ BM chimeric mice; 3) in lysM-Cre/DTR mice with or without inducible macrophage deletion; 4) in GFP⁺Smad3⁺/⁺ or GFP⁺Smad3⁻/⁻ BM chimeric mice. In addition, MMT was also validated in renal biopsy tissues from patients with different forms of CKD. Further more, we also studied the relationship between MMT and PDGFR-β⁺ pericytes or CD45⁺collagen I⁺ fibrocytes in both human and mouse fibrotic kidneys. Finally, mechanisms of MMT was examined in the UUO kidney induced in GFP⁺Smad3⁻/⁻ BM chimeric mice and in BM macrophages lacking TGF-β receptor II or Smad3. / Results: As described in Chapter III, mice with BM deletion were protected from renal fibrosis as demonstrated by blocking α-SMA⁺ myofibroblasts and collagen I accumulation. In contrast, BMT restored renal fibrosis in UUO kidney, demonstrating the critical role for BM cells in renal fibrosis. Importantly, the majority (85-90%) of α-SMA⁺ myofibroblasts were derived from BM macrophages as identified by GFP⁺F4/80⁺α-SMA⁺ revealing BM-macrophages given rise to myofibroblasts via MMT during kidney fibrosis. Similarly, MMT appeared as a major pathway of myofibroblast origin in patients with CKD, accounting for up to 80% of total myofibroblasts in the active stage of tissue fibrosis and fibrocellular crescents. To test the function role of macrophages in renal fibrosis via MMT, macrophages were conditionally deleted from the UUO kidneys in lysM-Cre/DTR mice as shown in Chapter IV, deletion (50-60%) of macrophages resulted in inhibition of MMT and renal fibrosis. Unexpectedly, most MMT cells (80-90%) were shown to co-express the pericyte marker (PDGFR-β⁺) and fibrocyte markers (CD45⁺collagen I⁺) in both human CKD and UUO (Chapter V), suggesting a BM macrophage origin for pericytes and fibrocytes during renal fibrosis. Finally, TGF-β/Smad3 appeared to be a mechanism driven MMT because mice and BM macrophages lacking either Smad3 or TβRII were protected against MMT and progressive renal fibrosis in the UUO kidney and in vitro. / Conclusions: MMT is derived from BM macrophages and regulated by TGF-β/Smad3. MMT is a major pathway of myofibroblast origin during renal fibrosis in both human and animal model of 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. / Wang, Shuang. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 161-179). / Abstracts also in Chinese. / Chapter ABSTRACT --- p.ii / Chapter DECLARATION --- p.viii / Chapter ACKNOWLEDGEMENTS --- p.ix / Chapter TABLE OF CONTENTS --- p.xi / Chapter LIST OF ABBREVIATION --- p.xv / Chapter LIST OF FIGURES AND TABLES --- p.xvii / Chapter CHAPTER I --- p.1 / INTRODUCTION --- p.1 / Chapter 1. 1 --- Renal fibrosis and myofibroblasts --- p.2 / Chapter 1. 1. 1 --- Pathology of renal fibrosis --- p.2 / Chapter 1. 1. 2 --- The generation and modulation of myofibroblasts. --- p.3 / Chapter 1. 1. 2. 1 --- EMT and EndMT --- p.5 / Chapter 1. 1. 2. 2 --- Pericytes --- p.8 / Chapter 1. 1. 2. 3 --- Fibrocytes --- p.16 / Chapter 1. 2 --- Role of macrophage in fibrogenesis --- p.21 / Chapter 1. 3 --- TGF-β signaling pathway in renal fibrosis --- p.23 / Chapter 1. 3. 1 --- TGF-β superfamily --- p.23 / Chapter 1. 3. 2 --- TGF-β/Smad signaling pathway --- p.24 / Chapter CHAPTER II --- p.29 / MATERIALS AND METHODS --- p.29 / Chapter 2. 1 --- Materials --- p.30 / Chapter 2. 1. 1 --- Regents and equipments --- p.30 / Chapter 2. 1. 1. 1 --- Regents and equipment for mouse genotyping --- p.30 / Chapter 2. 1. 1. 2 --- Regents and equipments for real-time PCR --- p.30 / Chapter 2. 1. 1. 3 --- Reagents and equipments for immunohistochemistry staining --- p.31 / Chapter 2. 1. 1. 4 --- Reagents and equipment for flow cytometry --- p.32 / Chapter 2. 1. 2 --- Buffer --- p.32 / Chapter 2. 1. 2. 1 --- Buffers for immunohistochemistry and immunofluorescence staining --- p.32 / Chapter 2. 1. 2. 2 --- Buffers for western blot --- p.35 / Chapter 2. 1. 3 --- Sequences of primers for genotyping and real-time PCR --- p.41 / Chapter 2. 1. 4 --- Antibodies --- p.42 / Chapter 2. 2 --- Methods --- p.44 / Chapter 2. 2. 1 --- Generation of gene modified mice --- p.44 / Chapter 2. 2. 2 --- Bone marrow transplantation --- p.45 / Chapter 2. 2. 3 --- Conditional macrophage deletion --- p.45 / Chapter 2. 2. 4 --- Unilateral ureteral obstruction (UUO) mouse model --- p.46 / Chapter 2. 2. 5 --- Histology and immunohistochemistry --- p.46 / Chapter 2. 2. 5. 1 --- Processing paraffin sections --- p.46 / Chapter 2. 2. 5. 2 --- Deparaffinization and hydration --- p.47 / Chapter 2. 2. 5. 3 --- Blocking endogenous peroxidase --- p.47 / Chapter 2. 2. 5. 4 --- Antigen retrieval --- p.48 / Chapter 2. 2. 5. 5 --- Antigen and antibody reaction --- p.48 / Chapter 2. 2. 5. 6 --- Detection of target signals --- p.49 / Chapter 2. 2. 5. 7 --- Quantification of immunohistochemistry staining --- p.49 / Chapter 2. 2. 6 --- Immunofluorescence staining and confocal microscopy analysis --- p.49 / Chapter 2. 2. 6. 1 --- Processing tissue for immune-fluorescent (IF) staining --- p.49 / Chapter 2. 2. 6. 2 --- Serum blocking --- p.50 / Chapter 2. 2. 6. 3 --- Antigen antibody reaction --- p.50 / Chapter 2. 2. 6. 4 --- Signal detection --- p.51 / Chapter 2. 2. 7 --- Flow cytometry --- p.52 / Chapter 2. 2. 7. 1 --- Preparation of single cell suspension --- p.52 / Chapter 2. 2. 7. 2 --- Cell fixation and permeabilization --- p.53 / Chapter 2. 2. 7. 3 --- Staining --- p.53 / Chapter 2. 2. 7. 4 --- Signal detection and analysis --- p.54 / Chapter 2. 2 .8 --- Real time PCR --- p.55 / Chapter 2. 2. 8. 1 --- Total RNA extraction --- p.55 / Chapter 2. 2. 8. 2 --- Reverse transcription --- p.56 / Chapter 2. 2. 8. 3 --- Real-time PCR --- p.57 / Chapter 2. 2. 8. 4 --- Analysis of real-time PCR --- p.57 / Chapter 2. 2. 9 --- Western blot --- p.58 / Chapter 2. 2. 9. 1 --- Protein extraction from tissue --- p.58 / Chapter 2. 2. 9. 2 --- Protein concentration measurement --- p.59 / Chapter 2. 2. 9. 3 --- SDS-PAGE electrophoresis --- p.59 / Chapter 2. 2. 9. 4 --- Protein transfer --- p.60 / Chapter 2. 2. 9. 5 --- Blocking --- p.61 / Chapter 2. 2. 9. 6 --- Antibodies incubation and signal detection --- p.62 / Chapter 2. 2. 9. 7 --- Stripping --- p.62 / Chapter CHAPTER III --- p.63 / EVIDENCE FOR MMT AS A NEW PATHWAY OF MYOFIBROBLAST ORIGIN IN RENAL FIBROSIS --- p.63 / Chapter 3. 1 --- Introduction --- p.64 / Chapter 3. 2 --- Materials and methods --- p.65 / Chapter 3. 2. 1 --- Human renal biopsy tissues --- p.65 / Chapter 3. 2. 2 --- Experimental design --- p.65 / Chapter 3. 2. 3 --- Bone marrow transplantation and GFP⁺ BM chimeric mice --- p.66 / Chapter 3. 2. 4 --- Immunohistochemistry --- p.66 / Chapter 3. 2. 5 --- Immunofluorescence and confocal microscopy analysis --- p.67 / Chapter 3. 2. 6 --- Real-time PCR --- p.68 / Chapter 3. 2. 7 --- Western blot analysis --- p.68 / Chapter 3. 2. 8 --- Flow cytometry --- p.68 / Chapter 3. 3 --- Results --- p.69 / Chapter 3. 3. 1 --- BM-derived myofibroblasts play a key role in renal fibrosis in a mouse model of UUO --- p.69 / Chapter 3. 3. 1. 1 --- α-SMA⁺ myofibroblasts are derived from BM and determine renal fibrosis in a mouse model of UUO --- p.69 / Chapter 3. 3. 1. 2 --- BM as a major source of collagen production in a mouse model of UUO --- p.73 / Chapter 3. 3. --- 2 Evidence for BM derived macrophage-myofibrobalst transition (MMT) in a mouse model of UUO --- p.77 / Chapter 3. 3. 2. 1 --- Characterization of GFP⁺ BM chimeric mice --- p.77 / Chapter 3. 3. 2. 2 --- Evidence for bone marrow-derived MMT is the major source of myofibroblast origin in the UUO kidney --- p.79 / Chapter 3. 3. 3 --- Evidence for MMT in human fibrotic kidney tissues --- p.84 / Chapter 3. 3. 4 --- M2 macrophage is the predomimant phenotype of macrophages in the fibrotic kidney of UUO mouse model. --- p.88 / Chapter 3. 4 --- Discussion --- p.90 / Chapter 3. 5 --- Conclusion --- p.93 / Chapter CHAPTER IV --- p.94 / Chapter GE --- CONDITIONAL MACROPHA DELETION INHIBITS MMT AND RENAL FIBROSIS --- p.94 / Chapter 4. 1 --- Introduction --- p.95 / Chapter 4. 2 --- Materials and methods --- p.98 / Chapter 4. 2. 1 --- Generation of lysM-Cre/DTR mice --- p.98 / Chapter 4. 2. 2 --- Conditional deletion of macrophage --- p.98 / Chapter 4. 2. 3 --- Unilateral Ureteral Obstruction (UUO) mouse model --- p.98 / Chapter 4. 2. 4 --- Real-time PCR --- p.99 / Chapter 4. 2. 5 --- Western blot analysis --- p.99 / Chapter 4. 2. 6 --- Immunohistochemisty --- p.99 / Chapter 4. 2. 7 --- Immunofluorescence --- p.99 / Chapter 4. 3 --- Results --- p.100 / Chapter 4. 3. 1 --- Characterization of lysM-Cre/DTR mice --- p.100 / Chapter 4. 3. 2 --- Conditional deletion of macrophage in a mouse model of UUO --- p.101 / Chapter 4. 3. 3 --- Conditional deletion of macrophage suppresses α-SMA⁺ myofibroblast accumulation in a mouse model of UUO --- p.104 / Chapter 4. 3. 4 --- Conditional deletion of macrophage inhibits collagen I production in a mouse model of UUO --- p.106 / Chapter 4. 3. 5 --- Conditional deletion of macrophage inhibits renal fibrosis through reducing MMT cells in a mouse model of UUO --- p.108 / Chapter 4. 4 --- Discussion --- p.111 / Chapter 4. 5 --- Conclusion --- p.113 / Chapter CHAPTER V --- p.114 / MMT CELLS SHARE PERICYTE AND FIBROCYTE PHENOTYPES --- p.114 / Chapter 5. 1 --- Introduciton --- p.115 / Chapter 5. 2 --- Materials and methods --- p.116 / Chapter 5. 2. 1 --- Human renal biopsy tissues --- p.116 / Chapter 5. 2. 2 --- Animals and UUO mouse model --- p.116 / Chapter 5. 2. 3 --- Immunofluorescence (IF) --- p.116 / Chapter 5. 2. 4 --- Flow cytometry --- p.117 / Chapter 5. 3 --- Results --- p.119 / Chapter 5. 3. 1 --- Evidence for MMT cells co-expressing pericyte marker in the fibrotic kidney of UUO model --- p.119 / Chapter 5. 3. 2 --- Evidence for MMT cells co-expressing pericyte marker in the fibrotic kidney from patients with chronic kidney diseases --- p.124 / Chapter 5. 3. 3 --- Evidence for MMT cells co-expressing fibrocyte marker in the fibrotic kidney of UUO model --- p.126 / Chapter 5. 3. 4 --- Evidence for MMT cells co-expressing fibrocyte marker in the fibrotic kidney from patients with chronic kidney diseases --- p.129 / Chapter 5. 4 --- Dscussion --- p.131 / Chapter 5. 5 --- Conclusion --- p.133 / Chapter CHAPTER VI --- p.134 / SMAD3 MEDIATES MMT DURING RENAL FIBROSIS --- p.134 / Chapter 6. 1 --- Introduction --- p.135 / Chapter 6. 2 --- Materials and methods --- p.137 / Chapter 6. 2. 1 --- Generation of Smad3⁺/⁺ and Smad3⁻/⁻ BM-Chimeric mice --- p.137 / Chapter 6. 2. 2 --- Generation of TbRII disrupted BM macrophages and Smad3⁻/⁻ BM macrophages --- p.137 / Chapter 6. 2. 3 --- UUO mouse model --- p.138 / Chapter 6. 2. 4 --- Cell culture --- p.138 / Chapter 6. 2. 5 --- Real-time PCR --- p.139 / Chapter 6. 2. 6 --- Western blot analysis --- p.139 / Chapter 6. 2. 7 --- Immunohistochemistry (IHC) --- p.139 / Chapter 6. 2. 8 --- Immunofluorescence (IF) --- p.139 / Chapter 6. 2. 9 --- Flow cytometry --- p.140 / Chapter 6. 3 --- Result --- p.141 / Chapter 6. 3. 1 --- Genotyping of Smad3 WT and Smad3 KO mice --- p.141 / Chapter 6. 3. 2 --- Smad3 knockout inhibits TGF-β1 induced MMT in vitro --- p.142 / Chapter 6. 3. 3 --- Disruption of TbRII inhibits TGF-β1 induced MMT in vitro --- p.145 / Chapter 6. 3. 4 --- Deletion of BM Smad3 inhibits α-SMA expression in the UUO kidney --- p.147 / Chapter 6. 3. 5 --- Deletion of BM Smad3 inhibits collagen-I production in the UUO kidney --- p.149 / Chapter 6. 3. 6 --- Inhibition of MMT is a mechanism by which BM Smad3 deficiency inhibits renal fibrosis in a mouse model of UUO --- p.150 / Chapter 6. 4 --- Discussion --- p.153 / Chapter 6. 5 --- Conclusion --- p.154 / Chapter CHAPTER VII --- p.155 / SUMMARY AND DISCUSSION OF THE MAJOR FINDINGS --- p.155 / Chapter 7. 1 --- Summary and discussion --- p.157 / Chapter 7. 1. 1 --- MMT is a major pathway of myofibroblast origin in renal fibrosis --- p.157 / Chapter 7. 1. 2 --- MMT cells shares both pericyte and fibrocyte phenotypes in renal fibrosis --- p.157 / Chapter 7. 1. 3 --- TGF-β/Smad3 is a key mechanism of MMT in renal fibrosis --- p.158 / Chapter 7. 2 --- Conclusion --- p.160 / Chapter REFERENCES --- p.161
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Inducibility and overexpression studies of antiquitin in HEK293 and HepG2 cells. / Inducibility & overexpression studies of antiquitin in HEK293 and HepG2 cellsJanuary 2005 (has links)
Wong Wei-yan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 221-242). / Abstracts in English and Chinese. / Thesis committee --- p.i / Declaration --- p.ii / Acknowledgements --- p.iii / Abstract in Chinese --- p.iv / Abstract in English --- p.vi / List of abbreviations --- p.viii / List of figures --- p.xi / List of tables --- p.xv / Content: --- p.xvi / General introduction --- p.1 / Aldehyde dehydrogenase superfamily --- p.3 / Background of antiquitin --- p.5 / Plant antiqutins (ALDH7B) --- p.5 / Animal antiquitins (ALDH7A) --- p.8 / Human antiquitin information on NCBI --- p.14 / Rationale of studying the inducibility of annquitin and overexpression of it in HEK293 and HepG2 cells --- p.16 / Flowchart 1 Procedure of antiquitin expression studies in the HEK293 and HepG2 cells under stress --- p.19 / Flowchart 2 Procedure to study antiquitin expression in the HEK293 and HepG2 cells after in silico promoter search --- p.20 / Flowchart 3 Procedure to study antiquitin overexpressed HEK293 and HepG2 cells --- p.21 / Chapter Chapter 1 --- Inducibility of antiquitin in the HEK293 and HepG2 cells under hyperosmotic stress / Chapter 1.1 --- Introduction --- p.22 / Chapter 1.1.1 --- Cellular response to hyperosmotic stress --- p.22 / Chapter 1.1.2 --- Methods to study the responses of cells under hyperosmotic stress --- p.24 / Chapter 1.2 --- Materials --- p.26 / Chapter 1.2.1 --- Cell culture media --- p.26 / Chapter 1.2.2 --- Buffers for RNA use --- p.26 / Chapter 1.2.3 --- Buffers for DNA use --- p.27 / Chapter 1.2.4 --- Other chemicals --- p.27 / Chapter 1.3 --- Methods --- p.28 / Chapter 1.3.1 --- Culture of HEK293 and HepG2 cells --- p.28 / Chapter 1.3.2 --- Hyperosmotic stress on HEK293 and HepG2 cells --- p.29 / Chapter 1.3.3 --- MTT assay --- p.29 / Chapter 1.3.4 --- Total RNA extraction --- p.30 / Chapter 1.3.5 --- Reverse transcription polymerase chain reaction (RT-PCR) --- p.30 / Chapter 1.3.6 --- Polymerase chain reaction (PCR) --- p.31 / Chapter 1.3.7 --- Quantification of PCR products --- p.31 / Chapter 1.3.8 --- Statistical analysis --- p.33 / Chapter 1.4 --- Results --- p.34 / Chapter 1.4.1 --- Viability of HEK293 and HepG2 cells under hyperosmotic stress --- p.34 / Chapter 1.4.2 --- Validation of RNA quality --- p.34 / Chapter 1.4.3 --- Validation and determination of PCR conditions --- p.40 / Chapter 1.4.4 --- Inducibility of antiquitin in HEK293 cells under hyperosmotic stress / Chapter 1.4.5 --- Inducibility of antiquitin in HepG2 cells under hyperosmotic stress --- p.43 / Chapter 1.4.6 --- Inducibility of aldose reductase under hyperosmotic stress --- p.43 / Chapter Chapter 2 --- "In silico studies of human antiquitin promoter, genomics sequences and open reading frame" --- p.54 / Chapter 2.1 --- Introduction --- p.54 / Chapter 2.1.1 --- Eukaryotic promoters --- p.55 / Chapter 2.1.2 --- Key events in transcriptional initiation --- p.55 / Chapter 2.1.3 --- Alternative splicing of mRNA --- p.57 / Chapter 2.1.4 --- Bipartite nuclear localization signal (NLS) --- p.57 / Chapter 2.2 --- Methods --- p.60 / Chapter 2.2.1 --- Putative promoter studies of human antiquitin --- p.60 / Chapter 2.2.2 --- Putative promoter studies of Arabidopsis thaliana antiquitin --- p.60 / Chapter 2.2.3 --- Analysis for the alternative splicing of human antiquitin mRNA --- p.60 / Chapter 2.2.4 --- Analysis for the nuclear localization signal (NLS) of human antiquitin amino acid sequence --- p.61 / Chapter 2.2.5 --- Nucleotide / amino acid sequence analyses --- p.61 / Chapter 2.3 --- Results --- p.62 / Chapter 2.3.1 --- Computer search for the putative cis-acting elements on human antiquitin promoter --- p.62 / Chapter 2.3.2 --- Comparison of cis-acting elements found on human antiquitin promoter with those on Arabidopsis thaliana antiquitin promoter --- p.62 / Chapter 2.3.3 --- Possibilities of alternative splicing isoforms of human antiquitin / Chapter 2.3.4 --- Possibilities of bipartite nuclear localization signals on human antiquitin protein --- p.83 / Chapter Chapter 3 --- Overexpression of antiquitin in HEK293 and HepG2 cells and their characterization / Chapter 3.1 --- Introduction --- p.86 / Chapter 3.1.1 --- Cell cycle of a human somatic cell --- p.88 / Chapter 3.1.2 --- Detection of changes in the transcriptome --- p.90 / Chapter 3.1.3 --- Human genome U133 Plus 2.0 array --- p.95 / Chapter 3.1.4 --- Detection of changes in the proteome --- p.96 / Chapter 3.1.5 --- MALDI-TOF MS --- p.97 / Chapter 3.2 --- Materials --- p.99 / Chapter 3.2.1 --- Solutions for cell culture use --- p.99 / Chapter 3.2.2 --- Solutions for cloning --- p.99 / Chapter 3.2.3 --- Buffers for cell cycle analysis --- p.99 / Chapter 3.2.4 --- Buffers for two-dimensional (2D) electrophoresis --- p.100 / Chapter 3.2.5 --- Solutions for silver staining --- p.101 / Chapter 3.2.6 --- Solutions for Coomassie blue protein staining --- p.102 / Chapter 3.2.7 --- Solutions for Western blotting --- p.102 / Chapter 3.2.8 --- Solutions for mass spectrometry --- p.103 / Chapter 3.3 --- Methods --- p.104 / Chapter 3.3.1 --- Hypoosmotic stress --- p.104 / Chapter 3.3.2 --- Heat shock --- p.104 / Chapter 3.3.3 --- Oxidative stress treatment / Chapter 3.3.4 --- Chemical hypoxia --- p.104 / Chapter 3.3.5 --- Treatment of forskolin --- p.106 / Chapter 3.3.6 --- Culture of SHSY5Y cells and its differentiation --- p.106 / Chapter 3.3.7 --- Cloning of pBUDCE4.1/ATQ --- p.106 / Chapter 3.3.8 --- PCR product purification --- p.107 / Chapter 3.3.9 --- Preparation of pEGFP.N1 vector for co-transfection --- p.109 / Chapter 3.3.10 --- Transfection of HEK293 and HepG2 cells --- p.109 / Chapter 3.3.11 --- Assays to characterize transient transfected HEK293 and HepG2 cells --- p.110 / Chapter 3.3.11.1 --- Transfection efficiency monitoring --- p.110 / Chapter 3.3.11.2 --- Cell cycle analysis --- p.112 / Chapter 3.3.11.3 --- Cell doubling time measurement --- p.112 / Chapter 3.3.11.4 --- Stress responsiveness --- p.113 / Chapter 3.3.11.5 --- Oligonucleotide array analysis --- p.113 / Chapter 3.3.11.5.1 --- Total RNA extraction --- p.113 / Chapter 3.3.11.5.2 --- Oligonucleotide array preparations --- p.113 / Chapter 3.3.11.5.3 --- Data analysis --- p.114 / Chapter 3.3.11.6 --- Two-dimensional (2D) electrophoresis --- p.115 / Chapter 3.3.11.6.1 --- Total protein extraction --- p.115 / Chapter 3.3.11.6.2 --- Protein quantification --- p.115 / Chapter 3.3.11.6.3 --- First dimension electrophoresis: isoelectric focusing (IEF) --- p.115 / Chapter 3.3.11.6.4 --- Second dimension electrophoresis: SDS- --- p.116 / Chapter 3.3.11.6.5 --- Silver staining --- p.116 / Chapter 3.3.11.6.6 --- Spots detection --- p.117 / Chapter 3.3.11.7 --- Preparations of samples for MALDI-TOF MS --- p.117 / Chapter 3.3.11.7.1 --- Silver de-staining --- p.117 / Chapter 3.3.11.7.2 --- In-gel tryptic digestion --- p.118 / Chapter 3.3.11.7.3 --- Peptide extraction --- p.118 / Chapter 3.3.11.7.4 --- ZipTip® samples desalting and concentrating --- p.119 / Chapter 3.3.11.7.5 --- MALDI-TOF MS --- p.119 / Chapter 3.3.11.8 --- Western blotting --- p.119 / Chapter 3.3.11.8.1 --- Antibodies probing --- p.120 / Chapter 3.3.11.8.2 --- Enhanced chemiluminescence's (ECL) assay --- p.121 / Chapter 3.4 --- Results --- p.122 / Chapter 3.4.1 --- Inducibility of antiquitin in HEK293 cells under xenobiotic stimulus --- p.122 / Chapter 3.4.2 --- Inducibility of antiquitin in HEK293 and HepG2 cells under chemical hypoxia --- p.122 / Chapter 3.4.3 --- Inducibility of antiquitin in HEK293 and HepG2 cells under hypoosmotic stress --- p.122 / Chapter 3.4.4 --- Inducibility of antiquitin in HEK293 and HepG2 cells under heat shock --- p.122 / Chapter 3.4.5 --- Inducibility of antiquitin in HEK293 and HepG2 cells under forskolin challenge --- p.128 / Chapter 3.4.6 --- Expression of antiquitin in differentiating SHSY5Y cells by retinoic acid and N2 supplement --- p.128 / Chapter 3.4.7 --- Overexpression of antiquitin in HEK293 and HepG2 cells --- p.128 / Chapter 3.4.8 --- Viability of transfected HEK293 and HepG2 cells under hyperosmotic stress --- p.136 / Chapter 3.4.9 --- Cell doubling times of transfected HEK293 and HepG2 cells --- p.143 / Chapter 3.4.10 --- Cell cycle analysis of transfected HEK293 and HepG2 cells --- p.143 / Chapter 3.4.11 --- "Western blot analysis of cyclin D, cyclin A and cyclin B of transfected HEK293 and HepG2 cells" --- p.148 / Chapter 3.4.12 --- RNA quality control tests for oligonucleotide array analysis --- p.148 / Chapter 3.4.13 --- Oligonucleotide array analysis on transfected HEK293 and HepG2 cells --- p.155 / Chapter 3.4.14 --- Two-dimensional electrophoresis of transfected HEK293 and HepG2 cells --- p.169 / Chapter 3.4.15 --- MALDI-TOF MS of transfected HEK293 and HepG2 cells --- p.169 / Chapter 3.4.16 --- Genes and proteins upregulnted in the antiquitin transfected HEK293 and HepG2 cells --- p.190 / Discussion --- p.197 / Reference --- p.221 / Appendix Materials used in the project --- p.243
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Patient participation in end-stage renal disease care: a grounded theory approach.January 1999 (has links)
by Tong Lai Wah, Christina. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1999. / Includes bibliographical references (leaves 101-112). / Abstracts in English and Chinese. / Title Page --- p.i / Authorization Page --- p.ii / Signature Page --- p.iii / Acknowledgements --- p.iv / Table of Contents --- p.v-viii / List of Figures --- p.ix / List of Tables --- p.x / List of Append --- p.ix xi / Title Page --- p.xii / Abstract --- p.xiii / Chapter 1 --- Introduction --- p.14-15 / Chapter 2 --- Literature Review --- p.16-24 / Chapter 2.1 --- Introduction / Chapter 2.2 --- End-stage renal disease / Chapter 2.3 --- Continuous ambulatory peritoneal dialysis / Chapter 2.4 --- Patient participation / Chapter 2.4.1 --- Definition of participation / Chapter 2.4.2 --- Benefits of participation / Chapter 2.4.3 --- Problems of patient participation / Chapter 2.4.4 --- Application of patient participation / Chapter 2.5 --- Conclusion / Chapter 3 --- Methodology --- p.25-43 / Chapter 3.1 --- Introduction / Chapter 3.2 --- Overview of grounded theory / Chapter 3.3 --- Procedures / Chapter 3.3.1 --- Data generation / Chapter - --- Sampling / Chapter - --- Data gathering / Chapter - --- Data recording / Chapter 3.3.2 --- Data analysis / Chapter - --- Open coding / Chapter - --- Constant comparative analysis / Chapter - --- Categorization / Chapter - --- Axial coding / Chapter - --- Theoretical sensitivity / Chapter - --- Memoing / Chapter 3.3.3 --- Theory construction / Chapter - --- Core category / Chapter 3.4 --- Method application / Chapter 3.4.1 --- Data collection / Chapter - --- Sampling / Chapter - --- Interview / Chapter - --- Recording / Chapter 3.4.2 --- Data analysis / Chapter - --- Open coding / Chapter - --- Constant comparative analysis / Chapter - --- Categorization and Axial coding / Chapter - --- Theoretical sensitivity / Chapter - --- Memoing / Chapter 3.4.3 --- Theoretical construction / Chapter - --- Concept formation / Chapter - --- Concept development / Chapter 3.5 --- Credibility & Trustworthiness / Chapter 3.6 --- Conclusion / Chapter 4 --- Findings --- p.44-72 / Chapter 4.1 --- Introduction / Chapter 4.2 --- Core category: Integrative Restructuring / Chapter 4.3 --- Emotional Labour / Chapter 4.3.1 --- Entering the active zone / Chapter (a) --- Conditions to go into active zone / Chapter (b) --- Outcomes of emotional labour / Chapter (c) --- Strategies used for emotional labour / Chapter - --- Letting go of emotions / Chapter - --- Aligning cognitive consistency / Chapter - --- Maximizing ego / Chapter - --- Locating self / Chapter - --- Boosting power / Chapter i. --- Active control / Chapter ii. --- Building positive expectancies / Chapter iii. --- Covariance to positive expectancies / Chapter 4.3.2 --- Retreating into comfort zone / Chapter (a) --- Contexts of comfort zone / Chapter (b) --- Conditions to build comfort zone / Chapter (c) --- Strategies used within comfort zone / Chapter - --- Defending / Chapter - --- Relinquishing / Chapter - --- Anchoring / Chapter 4.3.3 --- Migrating between the two zones / Chapter (a) --- Conditions to initiate the move / Chapter (b) --- Covariance to the movement / Chapter (c) --- Strategies to make progress / Chapter 4.4 --- Conclusion / Chapter 5 --- Discussion --- p.73-92 / Chapter 5.1 --- Introduction / Chapter 5.2 --- Theoretical framework / Chapter 5.3 --- Core category: Integrative Restructuring / Chapter 5.4 --- Variables affecting the move to active zone / Chapter 5.4.1 --- Preparations / Chapter 5.4.2 --- Support / Chapter (a) --- Source of support / Chapter (b) --- Context of support / Chapter (c) --- Effects of support / Chapter (i) --- Effects upon support-seekers / Chapter (ii) --- Supporter's reaction to support-giving relationship / Chapter 5.4.3 --- Commitment / Chapter (a) --- Perception of the situation / Chapter (b) --- Cultural influences / Chapter 5.4.4 --- Control / Chapter 5.5 --- Conclusion / Chapter 6 --- Concluding Chapter --- p.93-100 / Chapter 6.1 --- Limitations / Chapter 6.2 --- Implications / Chapter 6.2.1 --- Practice / Chapter 6.2.2 --- Research / Chapter 6.2.3 --- Teaching / Chapter 6.2.4 --- Policy Making / Chapter 6.2.5 --- Summary / Chapter 6.3 --- Future research / Chapter 6.4 --- Reflections upon the study / Chapter 6.5 --- Conclusion / References --- p.101-112
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Should glomerular filtration rate (GFR) be affected by the amount of viable, functioning tubular cells which in turn reflected by absolute renal uptake of Tc-99m DMSA.January 1998 (has links)
Wong Wai Lun. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (leaves 119-125). / Abstract also in Chinese. / Acknowledgments --- p.i / Legend for Figures --- p.ii / Legend for Tables --- p.iv / Abstract --- p.v / Abstract in Chinese --- p.ix / Chapter Chapter I --- Introduction --- p.1 / Objective --- p.5 / Chapter Chapter II --- Literature Review / Chapter II.1. --- Anatomy of the urinary system --- p.6 / Chapter II.2. --- Physiology of the urinary system --- p.10 / Chapter II.3. --- Methods for investigating the urinary system --- p.12 / Chapter II.3.1. --- Plain film radiography --- p.12 / Chapter II.3.2. --- Excretory Urogram --- p.12 / Chapter II.3.3. --- Ultrasound --- p.13 / Chapter II.3.4. --- Computed Tomography --- p.15 / Chapter II.3.5. --- Renal Angiography --- p.16 / Chapter II.3.6. --- Magnetic Resonance Imaging (MRI) --- p.16 / Chapter II.3.7. --- Radionuclide Imaging --- p.17 / Chapter II.4. --- Radiopharmaceuticals for renal parenchyma imaging --- p.17 / Chapter II.4.1. --- Tc-99m GHA --- p.18 / Chapter II.4.1.1. --- Chemistry of Tc-99m GHA --- p.18 / Chapter II.4.1.2. --- Preparation --- p.18 / Chapter II.4.1.3. --- Doses --- p.18 / Chapter II.4.1.4. --- Biological behavior --- p.19 / Chapter II.4.2. --- Tc-99m DMSA / Chapter II.4.2.1. --- Chemistry of Technetium-99m Dimercaptosuccinic Acid (Tc-99m DMSA) --- p.20 / Chapter II.4.2.2. --- Chemical property of Tc-99m DMSA --- p.21 / Chapter II.4.2.3. --- Preparation --- p.22 / Chapter II.4.2.4. --- Radiochemical purity measurement --- p.22 / Chapter II.4.2.5. --- Doses --- p.23 / Chapter II.4.2.6. --- Pharmacokinetic of Tc-99m DMSA --- p.23 / Chapter II.4.2.7. --- Renal handling of injected Tc-99m DMSA --- p.25 / Chapter II.5. --- General consideration for quantitative uptake measurement in organs --- p.26 / Chapter II.5.1. --- Clinical significance of renal Tc-99m DMSA uptake --- p.28 / Chapter II.5.2. --- Special consideration and problems for quantitative renal Tc-99m uptake measurement --- p.29 / Chapter II.5.3. --- Suggestions and solutions for quantitative renal Tc-99m uptake measurement --- p.29 / Chapter II.5.3.1. --- Planar images Vs SPECT images for quantification --- p.29 / Chapter II.5.3.2. --- Background subtraction --- p.31 / Chapter II.5.3.3. --- Choice of location for background ROI --- p.32 / Chapter II.5.3.4. --- Attenuation --- p.35 / Chapter II.5.3.5. --- Principle of the conjugate view method --- p.36 / Chapter II.5.3.6. --- Body thickness and kidney depth measurement --- p.37 / Chapter II.6. --- Glomerular Filtration / Chapter II.6.1. --- Introduction --- p.39 / Chapter II.6.2. --- Gold standard for GFR measurement --- p.40 / Chapter II.6.3. --- Laboratory studies for the measurement of glomerular filtration : Serum Creatinine and Blood Urea Nitrogen (BUN) levels --- p.41 / Chapter II.6.3.1. --- Calculation of Creatinine Clearance Rate --- p.43 / Chapter II.6.3.2. --- Critique for using creatinine clearance as a measurement of renal function --- p.44 / Chapter II.6.3.3. --- Limitation of the serum creatinine concentration used alone as a measurement of renal function --- p.46 / Chapter II.6.4. --- Radionuclide technique for the assessment of the glomerular function --- p.48 / Chapter II.6.4.1. --- Diethylene Triamine Penta Acetic acid (DTPA) --- p.49 / Chapter II.6.4.2. --- Methods / Chapter II.6.4.2.1. --- Measurement of Glomerular Filtration Rate using Tc-99m DTPA with single injection techniques --- p.51 / Chapter II.6.4.2.2. --- Compartment model --- p.52 / Chapter II.6.4.2.2a. --- Two-compartment model --- p.52 / Chapter II.6.4.2.2b. --- Single-compartment model --- p.54 / Chapter II.6.4.2.3. --- Single blood sample technique: a modification of Tauxe's OIH method in which counts in a single plasma sample correlated with a GFR nomogram --- p.56 / Chapter II.6.4.2.4. --- Gamma camera based method --- p.58 / Chapter II.6.4.2.4a. --- Gates-modification of Schlegel's OIH technique --- p.58 / Chapter II.6.4.2.4b. --- Critique for the Gamma camera technique for measuring GFR --- p.62 / Chapter II.7. --- The relationship between the Tc-99m DMSA uptake and GFR --- p.67 / Chapter Chapter III --- Material and Methods --- p.69 / Chapter III.1. --- Subjects and Sampling Methods --- p.69 / Chapter III.2. --- Quantitation of Absolute DMSA uptake --- p.70 / Chapter III.2.1. --- Parameters for Tc-99m DMSA uptake study --- p.70 / Chapter III.2.1.1. --- Materials and methods --- p.70 / Chapter III.2.1.1.1. --- Instrumentation --- p.70 / Chapter III.2.1.1.2. --- Dosage --- p.70 / Chapter III.2.1.1.3. --- Optimum acquisition start time --- p.70 / Chapter III.2.1.1.4. --- Length of acquisition time --- p.71 / Chapter III.2.1.1.5. --- Acquisition parameter --- p.71 / Chapter III.3. --- Calculation of absolute renal DMSA uptake --- p.72 / Chapter III.3.1. --- Attenuation Coefficient factor(μ) --- p.73 / Chapter III.3.2. --- Table attenuation --- p.75 / Chapter III.3.3. --- Body thickness measurement --- p.77 / Chapter III.3.4. --- Decay correction --- p.78 / Chapter III.3.5. --- Calculation of DMSA uptake --- p.78 / Chapter III.3.6. --- Counting dose injected --- p.80 / Chapter III.3.7. --- Calculation of absolute quantitation of Tc-99m DMSA uptake --- p.80 / Chapter III.3.8. --- Dose infiltration --- p.81 / Chapter III.4. --- GFR measurement --- p.82 / Chapter III.4.1. --- Instrumentation --- p.82 / Chapter III.4.2. --- Methods --- p.82 / Chapter III.5. --- Statistical and analytical methods --- p.84 / Chapter Chapter IV --- Results --- p.87 / Chapter IV. 1. --- Characteristics of experimental subjects and their serum creatinine profile --- p.88 / Chapter IV.2. --- Absolute Tc-99m DMSA uptake / Chapter IV.2.1. --- The change of absolute Tc-99m uptake with time --- p.89 / Chapter IV.2.2. --- Absolute Tc-99m DMSA uptake measurement at 6 and 24 hours --- p.90 / Chapter IV.2.3. --- Gender difference in absolute Tc-99m uptake measurement at 6 hour --- p.92 / Chapter IV.3. --- GFR measurement --- p.93 / Chapter IV.3.1. --- GFR measurement by single (3hr) and double (1&3 hrs) plasma sampling --- p.93 / Chapter IV.3.2. --- Gender difference in GFR measurement using single plasma sampling --- p.96 / Chapter IV.4. --- Univariate Correlation --- p.97 / Chapter IV.4.1. --- Correlation between GFR using single plasma sampling and absolute Tc-99m uptake --- p.97 / Chapter IV.4.2. --- Correlation between GFR using single plasma sampling and plasma creatinine levels --- p.98 / Chapter IV.4.3. --- Correlation between anthropometric variables on GFR(3 hr) --- p.99 / Chapter IV.4.4. --- Correlation between anthropometric variables and serum creatinine plasma level on absolute Tc-99m DMSA uptake measurement at 6 hour --- p.101 / Chapter IV.4.5. --- Multiple linear stepwise regression --- p.103 / Chapter Chapter V. --- Discussion / Chapter V. 1 --- . Review of the study --- p.104 / Chapter V.1.1. --- Experimental subjects and their absolute Tc-99m DMSA uptake (%) at 6 hr --- p.104 / Chapter V.1.2. --- Experimental subjects and their GFR(3 hr) --- p.105 / Chapter V.2. --- Discussion on subject --- p.105 / Chapter V.2.1. --- Subject preparation --- p.106 / Chapter V.3. --- Discussion of method --- p.106 / Chapter V.3.1. --- Equipment --- p.106 / Chapter (a) --- Dose calibrator --- p.106 / Chapter (b) --- The sensitivity of the head 1 and 2 of the gamma camera --- p.106 / Chapter (c) --- Validation of quantification of injected activity by gamma camera method--------constancy of performance for gamma camera --- p.110 / Chapter (d) --- LEHR Collimator --- p.112 / Chapter (f) --- Dead time loss --- p.112 / Chapter V.4. --- Discussion on measurement --- p.113 / Chapter (a) --- Length of acquisition time --- p.113 / Chapter (b) --- Attenuation Coefficient factor (\x) --- p.113 / Chapter (c) --- "Body thickness, L, measurement" --- p.113 / Chapter (d) --- Optimum acquisition time for data collection --- p.115 / Chapter v.5. --- Discussion on overall error estimation --- p.115 / Chapter (a) --- Tc-99m DMSA uptake measurement at 6 hr --- p.115 / Chapter (b) --- GFR measurement by single (3 hr) sample --- p.116 / Chapter Chapter VI --- Conclusion --- p.117 / Reference --- p.119 / Appendix I --- p.126 / Appendix II --- p.128 / Appendix III --- p.134
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Novel therapies in acute kidney injuryMemon, Shoab Ahmed January 2015 (has links)
Renal ischaemia-reperfusion injury (IRI) is a major cause of acute kidney injury (AKI) which is in turn the leading cause of morbidity and mortality in hospitalized patients. The principle aim of this thesis was to evaluate potential new therapies that might afford protection against IRI in both in vitro and in vivo settings. Recent evidence suggests that nitrite (NO2-) may play an important role in protecting the myocardium from IRI. Our initial work into the role of NO2- in an in vitro model of renal IRI in proximal tubular epithelial cells provided evidence that NO2- can prevent apoptosis and preserve cell viability. This lead to an in vivo study where high NO2- concentrations (50 mg/L) were given orally to rats for 7 days prior to inducing renal IRI but no beneficial effects of this treatment were observed. Another potential treatment identified was thiamine (vitamin B1) and this, like NO2-was investigated to see if it had the potential to protect rats from AKI injury. It has been previously recognized that in renal IRI the high energy phosphate ATP is found to be severely depleted whilst is is known that thiamine can play a pivotal role in generating ATP. Furthermore, thiamine has previously been demonstrated to protect against myocardial ischaemic injury and has the ability to reduce myocardial infarct size. In vitro, thiamine was found to reduce the degree of apoptosis in cultured HK-2 cells caused by ischaemia whilst in vivo it afforded protection against AKI caused by renal IRI by anti-apoptotic, anti-inflammatory and anti-oxidant mechanisms. Finally, a study into the possible therapeutic role of gene therapy with bone morphogenic protein 7 (BMP-7) in renal IRI was undertaken. Previous work has established that i.v. BMP-7 is able to protect against renal IRI but it has also been associated with ectopic bone formation at the site of injection. Therefore another method to increase circulating BMP-7 was sought. We initially found that BMP-7 gene therapy could attenuate apoptosis and preserves cell viability in an in vitro model of renal IRI. However, whilst in vivo gene therapy with electroporation of BMP-7 plasmid DNA increased BMP-7 expression in mice serum 2 days post electroporation, it was unable to protect the animals against IRI induced AKI. In rats the direct injection of naked DNA BMP-7 plasmid systematic 2 days prior to renal IRI was able to upregulate BMP-7 expression 4 days later in kidney tissue. Despite this it was unable to afford protection against renal IRI. Apoptosis and necrosis play a crucial role in the pathogenesis of renal IRI induced AKI. In this thesis we investigated the role of three putative therapeutic agents and their role in apoptosis and necrosis in vitro in PTECs and in vivo against renal IRI induced AKI. All three therapeutic drugs were able to attenuate apoptosis in PTECs but were unable to protect against necrosis, whilst against renal IRI induced AKI only thiamine was found to be protective. Thiamine appears to hold the most promise and more work needs to be undertaken so that its potential benefit in AKI can be realised.
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Vitamin D and endothelial function in chronic kidney diseaseDreyer, Gavin January 2014 (has links)
Vitamin D deficiency in patients with chronic kidney disease, measured by reduced serum concentrations of 25 hydroxy vitamin D, is highly prevalent and associated with both endothelial dysfunction and an increased risk of cardiovascular disease. Observational studies in chronic kidney disease have demonstrated that vitamin D therapy reduces the risk of cardiovascular disease. In patients with chronic kidney disease and concomitant vitamin D deficiency, the effect of vitamin D therapy on endothelial function, which is associated with cardiovascular disease, is poorly understood. The mechanism by which vitamin D affects endothelial function is unclear. Methods Presented in this thesis, two studies have addressed these issues: 1. A double blind, randomized controlled trial evaluating the effect of ergocalciferol compared to placebo on microcirculatory endothelial function in patients with non-dialysis chronic kidney disease and concomitant vitamin D deficiency 2. In vitro and in vivo experiments to determine the mechanistic effect of ergocalciferol on endothelial function in an experimental model of uraemia. Results In the clinical study, ergocalciferol increased vitamin D serum concentrations and improved microcirculatory endothelial function measured by laser Doppler flowmetry after iontophoresis of acetylcholine. Oxidative stress measured by skin autofluorescence for advanced glycation end products did not change in the ergocalciferol group but increased significantly in the placebo group. Ergocalciferol increased endothelial nitric oxide synthase expression and activity in cultured human endothelial cells and improved endothelial function in an in vivo model of mild uraemia. The findings from the in vivo and clinical studies occurred independently of changes in blood pressure, conduit artery function, serum calcium, phosphate and parathyroid hormone supporting in vitro findings that ergocalciferol acts directly on the endothelium. Conclusion Ergocalciferol improved endothelial function in both rodent and human subjects with chronic kidney disease. Experimental evidence suggests this effect occurs through an endothelium dependent mechanism involving changes in the upregulation and function of endothelial nitric oxide synthase.
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Avaliação de terapias imunossupressoras em transplantes renais com uma abordagem metabolômica / Assessment of immunosuppressive therapies with a metabolomic approachPedro Luis Rocha da Cruz 23 June 2017 (has links)
O aprimoramento das técnicas analíticas viabilizou a metabolômica, uma área da ciência que busca compreender, de forma comparativa, os metabólitos envolvidos nas vias bioquímicas. A metabolômica está inserida no contexto das \"ômicas\", que teve início na \"Era Genômica\", a qual permitiu a identificação de diversos genes. Em seguida, o interesse dos pesquisadores centrou no estudo dos metabólitos (metabolômica) mostrando ser uma ferramenta valiosa na pesquisa do transplante renal, que exige um tratamento medicamentoso por meio de imunossupressores. A combinação destes imunossupressores pode minimizar a rejeição do órgão transplantado, reforçando a necessidade de um estudo metabolômico, a fim de avaliar e comparar as mudanças ocorridas após o transplante em nível molecular, melhorando o conhecimento sobre a influência destes regimes e dando subsídios sobre prognósticos possíveis na área de transplante renal. Nesta tese foram avaliadas 2 terapias: Everolimo/ Prednisona/Tacrolimo (grupo 1) e Micofenolato mofetil/Prednisona/Tacrolimo (grupo 2) a partir de uma abordagem untargeted. No presente trabalho foram coletadas amostras de urina de pacientes ao longo de 6 meses. Foi necessário determinar a melhor condição para análise das amostras de urina dos pacientes. Desta forma, foram realizados estudos sobre alguns parâmetros que impactam no preparo de amostra abordando a influência da urease, tipos e proporção de solventes para precipitação de proteína, seleção do melhor agente derivatizante e tratamento de dados. A avaliação da medida de qualidade dos tratamentos com urease foi feita a partir do desvio padrão relativo (RSD) dos valores de intensidade de pico. A concentração de 10 mg mL-1 apresentou o melhor resultado. O estudo mostrou também que o teor de ureia na urina pode influenciar na identificação dos compostos. O número de compostos identificados foi menor quando a urina não foi tratada com urease, com aproximadamente 10 compostos a menos em relação à amostra tratada com a enzima, na mesma concentração de ureia adicionada. Dos solventes orgânicos testados para precipitação de proteínas nas amostras de urina, o isopropanol mostrou ser o solvente mais adequado na proporção 1:6 urina:solvente (v/v), utilizando-se 100 µL de urina. Foram testados dois protocolos de derivatização para análises por GC-MS: metoximação e sililação utilizando BSTFA e cloroformiato de metila. A comparação mostrou que o procedimento por BSTFA, com 40 metabólitos identificados, foi superior ao cloroformiato de metila, com 13 compostos identificados. No tratamento de dados com o software XCMS, os seguintes parâmetros foram avaliados: largura a meia altura do pico (fwhm), largura da banda (bw) e threshold (sntresh). Para avaliar a melhor combinação destes parâmetros, foi feita uma variação univariada destes valores. A qualidade do resultado de cada combinação foi monitorada pelos valores gerados de número de missing values, quantidade de picos com RSD <15% e número de valores duplicados. Os valores ótimos foram obtidos para a combinação: fwhm=4, bw=2 e threshold=5. A abordagem do estudo dos dois grupos de pacientes baseou-se inicialmente na comparação entre o dia 7 da terapia com os demais períodos (dia 14, mês 1, mês 3 e mês 6) e posteriormente avaliou-se a evolução temporal. A partir do mês 3 os valores de correlação e predição dos modelos de PLS-DA são melhores e já é eficaz na diferenciação entre os dois grupos. Foram observadas perturbações no metabolismo de carboidratos em ambos os grupos, como açúcares, glicerol e N-acetil-D-manosamina. No grupo 1, foram observados metabólitos discriminantes da classe dos poliois e das vias do ciclo do ácido cítrico e degradação de xenobióticos, enquanto que, no grupo 2, foi observada alteração do hidroxibutirato, um corpo cetônico. Neste grupo, foi observado também um aumento do ácido hipúrico, ácido acetamido butírico, ácido benzoico, entre outros. Nesta tese foi possível demonstrar que a metabolômica é uma ferramenta importante para comparar metabólitos discriminantes entre dois regimes imunossupressores, sendo um estudo piloto que visa fornecer subsídios para avaliação da influência destas terapias no prognóstico de transplante renal. / The improvement of analytical techniques enabled the emergence of metabolomics, which aims to compare the metabolites involved in biochemical pathways, in certain biological conditions. Metabolomics is inserted in the \"omics\" context, which began in the \"Genomic Age\", and allowed the identification of several genes. After that, the researchers focused on the study of metabolites. Among several applications, metabolomics can be a valuable tool in renal transplant research, which requires a drug treatment through immunosuppressants. The combination of these immunosuppressants can minimize toxicity and rejection of the transplanted organ, reinforcing the need for a metabolomic study, in order to evaluate and compare changes after transplantation at the molecular level, improving knowledge about the influence of these regimens and giving subsidies on prognosis in the area of renal transplantation. In this thesis two immunosuppressive therapies were evaluated by an untargeted approach: Everolimus/Prednisone/Tacrolimus (group 1) and Mycophenolate mofetil/Prednisone/Tacrolimus (group 2). In this study, urine samples were collected from patients over 6 months. It was necessary to determine the best condition for analysis of patients\' urine samples. Thus, studies were carried out on some parameters that impact on sample preparation, evaluating the influence of urease, types and proportion of solvents for protein precipitation, selection of the best derivatizing agent, and data treatment. The evaluation of the quality measure of the urease treatments was made from the relative standard deviation (RSD) of the peak intensity values. The concentration of 10 mg mL-1 presented the best result. The study also showed that urine urea content may influence the identification of the compounds. The number of identified compounds was lower when urine was not treated with urease, with approximately 10 compounds less than the enzyme-treated sample, at the same concentration of urea added. In the evaluation of the organic solvents tested for protein precipitation in the urine samples, isopropanol was the most suitable solvent in the ratio 1: 6 urine:solvent (v/v), using 100 µL of urine. Two derivatization protocols were tested for GC-MS analysis: metoximation and silylation with BSTFA and methyl chloroformate. The comparison between the two derivatization protocols showed that the BSTFA procedure, with 40 identified metabolites, was superior to methyl chloroformate with 13 compounds identified. In data processing with the XCMS software, the following parameters were evaluated: full width at half maximum of the peak (fwhm), bandwidth (bw) and threshold (sntresh). To evaluate the best combination of these parameters, a univariate variation of these values was made. The quality of the result of each combination was monitored by the number of missing values, number of peaks with RSD <15%, and number of duplicate values. The optimal values were obtained for the combination: fwhm=4, bw=2 and threshold =5. The study of the two groups of patients was initially based on the comparison between day 7 of the therapy with the other periods (day 14, month 1, month 3 and month 6) and later the temporal evolution was evaluated. From month 3 the values of correlation and prediction of the PLS-DA models are better and already effective in the differentiation between the two groups. Disorders in carbohydrate metabolism were observed in both groups with sugars and glycerol and N-acetyl-D-mannosamine as discriminant metabolites. In group 1, discriminant metabolites of the class of polyols and citric acid cycle pathways and degradation of xenobiotics were observed, and in group 2 alteration of hydroxybutyrate, a ketone body, was observed. In this group an increase of hippuric acid, acetamido butyric acid, benzoic acid, among others, was also observed. In this thesis it was possible to demonstrate that metabolomics is an important tool to compare discriminant metabolites between two immunosuppressive regimens, being a pilot study that aims to provide future subsidies to evaluate the influence of these therapies on the renal transplant prognosis
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Effet des composés phénoliques sur le vieillissement cardiaque et rénal : étude expérimentale chez le rat / Effect of phenolic compounds on cardiac and renal aging : experimental study on a rat modelChacar, Stéphanie 06 July 2018 (has links)
Le vieillissement est un processus physiologique au cours duquel l’ensemble de l’organisme voit son fonctionnement modifié. Il s’agit d’un remodelage génotypique et phénotypique, en lien avec le stress oxydatif. Les molécules antioxydantes comme les composés phénoliques (CP) ont pris une place importante dans la diète humaine, à titre de compléments alimentaires et/ou à titre thérapeutique. Toutefois, les conséquences d’un usage à long terme de ces molécules visant à reverser les effets du vieillissement sur les fonctions organiques, restent encore mal élucidées. Dans ce contexte, et compte tenu de notre intérêt pour le cœur, le rein et le microbiote intestinal, l’objectif de cette thèse est d’évaluer, sur de jeunes rats mâles, les effets des CP administrés à différentes concentrations, pendant quatorze mois. Les groupes traités par les CP ont montré une préservation, avec l’âge, de la performance cardiaque par rapport aux témoins non traités. De plus, les myocardes de rats âgés traités ont présenté de moindres signes d’inflammation, de fibrose et d’apoptose que les témoins. Ces modifications sont soutenues par un remodelage du niveau d’expression protéique des marqueurs de l’hypertrophie et du stress oxydatif, et des résultats préliminaires suggèrent une activation du courant potassique KATP en présence des CP sur les myofibroblastes. Le tissu rénal conserve son architecture normale avec l’âge chez tous les groupes. Enfin, les métabolites issus des CP ont montré une modulation sélective du microbiote intestinal vers un phénotype sain. Nos travaux montrent dans un modèle murin qu’une consommation régulière de CP permet de préserver le cœur, les reins et le microbiote du remodelage lié au vieillissement. / Aging is a physiological process in which the entire body sees its normal functional capacities modified. It is associated with a genotypic and phenotypic remodeling, related to oxidative stress. Antioxidant molecules such as phenolic compounds (PC), have taken an important place in the human diet, as food supplements and/or as therapeutics. However, the consequences of long-term use of these molecules to reverse the effects of aging on organic functions, remain poorly understood. In this context, and considering our interest for the heart, the kidney and the intestinal microbiota, the aim of this thesis is to evaluate, in young male rats, the effects of PC administered at different concentrations for a period of fourteen months. PC treated groups showed a dose-dependent preservation of cardiac morphology and performance compared to control untreated ones. Additionally, myocardia from treated aged rats exhibited less inflammation, fibrosis and cardiomyocyte apoptosis than controls. These modifications were supported by a remodeling of the proteins level expression of the markers of hypertrophy and oxidative stress, and preliminary data suggest a concomitant activation of potassium current KATP on myofibroblasts in the presence of PC. Renal tissues retained their normal architecture with age in all groups. Finally, derived-metabolites from PC showed a selective modulation of intestinal microbiota towards a healthy phenotype. Our work shows that regular consumption of PC may preserve the heart, kidneys and microbiota of age-related remodeling.
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Fatores de personalidade, autoeficácia e depressão em pacientes em lista de espera para transplante renalThomas, Caroline Venzon 10 December 2009 (has links)
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Previous issue date: 10 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Os aspectos psicológicos de pacientes em lista de transplante renal são importantes, pois podem ter reflexo na sua adaptação pós-transplante. Diante disto, os fatores de personalidade, a autoeficácia e os sintomas depressivos são variáveis destacadas na literatura que podem ter relação com os resultados do tratamento nestes pacientes. Assim, o objetivo deste estudo foi avaliar fatores de personalidade, autoeficácia e depressão em pacientes em lista de espera para transplante renal. Participaram 65 pacientes, homens e mulheres adultos, em lista de espera para transplante renal em tratamento de hemodiálise na cidade de Porto Alegre, com idade média de 49,11 anos (DP=11,58). Instrumentos: Ficha de Dados sóciodemográficos e clínicos, Bateria Fatorial de Personalidade (BFP), Escala de Autoeficácia Geral Percebida e Inventário de Depressão de Beck (BDI). Resultados: Análise de correlação de Pearson mostrou associações positivas entre os fatores Extroversão (r=-0,35, p<0,01), Realização (r=0,41, p<0,01) e auto- efic
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