INTRODUÇÃO: A obesidade é uma doença crônica com crescimento alarmante no mundo todo. Atualmente, o tratamento cirúrgico, especialmente o Bypass Gástrico em Y de Roux (BGYR), tem se mostrado como a forma mais eficiente para perda de peso e sua manutenção a longo prazo. Contudo, com a formação do neo-estômago e a mudança na conformidade intestinal, há alterações significantes das muitas propriedades físicas e funcionais desses órgãos que levam à deficiência de nutrientes, inclusive de cálcio. Com isso, podem ocorrer modificações no metabolismo ósseo e, conseqüentemente, na estrutura óssea. OBJETIVOS: Avaliar a ingestão de cálcio, as alterações no metabolismo ósseo e mineral; e a ocorrência de osteopenia e osteoporose em mulheres que se submeteram ao BGYR há oito anos. MÉTODO: Neste estudo transversal, foram estudadas 30 mulheres que se submeteram ao BGYR no período de outubro de 1995 a janeiro de 1999, no Hospital das Clínicas da Faculdade de Medicina da USP. Para avaliação da ingestão de cálcio, utilizamos o recordatório de 3 dias (R3D) e o questionário de freqüência alimentar (QFA). Também foram realizados exames laboratoriais referentes ao metabolismo ósseo e mineral e densitometria óssea do seguimento L1-L4, colo femoral (CF) e fêmur proximal (FP). RESULTADOS: Em média, o consumo de cálcio foi de 525,5 ± 250,7 mg/dia pelo R3D e de 542,2 ± 195,6 mg/dia pelo QFA. Houve uma relação estatisticamente significativa entre a ingestão de cálcio por esses dois métodos (p<0,001). Não houve alteração nas determinações de cálcio total e ionizado, magnésio, fósforo e CTX. Os níveis de PTH, Fosfatase alcalina fração óssea (BSAP) e osteocalcina estavam elevados em 53%, 57% e 20% das mulheres, respectivamente; 90% apresentavam deficiência de 25 (OH) vitamina D (40% leve e 50% moderada), e em 70% a calciúria estava abaixo dos valores normais. Observou-se uma correlação positiva entre 25 (OH) vitamina D e a calciúria (p<0,04) e negativa entre 25 (OH) vitamina D e PTH (p<0,017). Com relação à densidade mineral óssea, 13% das mulheres foram diagnosticadas com osteoporose com relação ao CF e FP; 67%, 40% e 27% apresentavam osteopenia em L1-L4, CF e FP, respectivamente. CONCLUSÃO: Na maioria das mulheres estudadas verificou-se um consumo de cálcio cerca de 50% abaixo da recomendação diária para esta faixa etária. Observou-se também, uma deficiência de 25 (OH) vitamina D e elevação de PTH e BSAP. Além disso, houve uma ocorrência de osteopenia superior à esperada indicando que alterações no metabolismo ósseo são provavelmente uma complicação do BGYR. Mais estudos são necessários para definir uma rotina de suplementação de cálcio e vitamina D, e também para a prevenção das alterações ósseas. / INTRODUTION: Obesity is a chronic disease that rises rapidly around the world. Nowadays bariatric surgical procedures, especially Roux-en-Y Gastric Bypass (RYGB) has been shown the most efficient way to lose weight and maintain the weight loss for a long time. However, with the neo-stomach and the modification of intestinal anatomy by the surgery there are significant changes on physiological properties of these organs that lead to a nutrient deficiency, including calcium. Thus, bone metabolism changes may occur leading to a metabolic bone disease. OBJECTIVES: To evaluate calcium intake, bone and mineral metabolism changes and the prevalence of metabolic bone disease in women who were submitted to RYGB after eight years. METHOD: we studied 30 women who were submitted to RYGB during the period between October of 1995 and January of 1999 at Clinical Hospital of Medicine School of São Paulo University. To access calcium intake we used a 3 day dietary recall (3DR) and food frequency questionnaire (FFQ). Laboratory tests of bone metabolism and bone mass density of L1-L4, femoral neck (FN) and proximal femur (PF) were also accessed. RESULTS: calcium intake was 525,5 ± 250,7 mg/day according 3RD and 542,2 ± 195,6 mg/day according FFQ. There was a significantly relation between both methods (p<0,001). Total and ionic calcium, magnesium, phosphorus and CTX were not altered. PTH, bone specific alkaline phosphatase (BSAP) and osteocalcin levels were elevated respectively in 53%, 57% and 20% of women. 90% presented 25 (OH) vitamin D deficiency (40% mild and 50% moderate) and 70% had low urinary calcium. Was observed a positive correlation between 25 (OH) vitamin D and urinary calcium (p<0,04); and a negative correlation between 25 (OH) vitamin D and PTH (p<0,017). 13% of women had osteoporosis in FN and PF; 67%, 40% and 27% had metabolic bone disease in L1-L4, FN and PF respectively. CONCLUSION: Most studied women had a low calcium intake, about 50% of daily recommendation. We also noticed a 25 (OH) vitamin D deficiency and elevated levels of PTH and BSAP. Besides, there was a high prevalence of metabolic bone disease than expected, suggesting that this could be a complication of this surgery. Further studies are needed to define a supplementation routine of calcium and vitamin D to prevent bone metabolic diseases in these patients.

As doenças renais crônicas (DRC) evoluem com distúrbios na homeostase do cálcio e do fósforo, diminuição na produção de vitamina D e aumento na secreção de PTH. Osteodistrofia renal (OR) é o termo usado para definir as alterações ósseas dos pacientes com DRC e classifica-se em doença de alta remodelação representada pela osteíte fibrosa (OF) e doença mista (DM); e de baixa remodelação representada pela osteomalácia (OM) e pela doença adinâmica (DOA). Pacientes com DRC apresentam elevada incidência de fraturas e recentemente demonstrou-se que a hiperfosfatemia leva a diminuição do volume ósseo. Estudamos o efeito isolado do fósforo no tecido ósseo de animais com insuficiência renal mantidos com infusão fixa de PTH variando o conteúdo de fósforo na dieta. Cinqüenta e cinco ratos Wistar foram submetidos à paratireoidectomia (PTX) e nefrectomia (Nx) com reposição de PTH em diferentes concentrações ou foram sham operados e recebiam infusão de veículo. Todos os animais receberam a mesma dieta variando apenas a concentração de P (pobre em P (pP): 0,2% e rico em P (rP):1,2%). Dividimos os grupos em: Sham (N=8); Sham-pP (N=8); Sham-rP (N=7); NxPTHn-pP (N=8); NxPTHn-rP (N=8); NxPTHe-pP (N=9); NxPTHe-rP (N=7). Após 2 meses, realizamos análises bioquímicas e histomorfometria do fêmur proximal. Os animais que ingeriram dieta rica em fósforo apresentaram hiperfosfatemia assim como menor valor de cálcio sérico. A reposição de PTH foi efetiva e proporcional às concentrações infundidas. A histomorfometria óssea mostrou que os ratos que ingeriram dieta rica em fósforo independente da uremia tinham diminuição do volume ósseo (BV/TV), e que este efeito foi amenizado pela reposição do PTH em concentrações elevadas. Nossos resultados demonstram que o fósforo é deletério para o tecido ósseo e que na uremia são necessários níveis mais elevados de PTH para manter a integridade óssea. / Chronic kidney disease (CKD) involves disturbances in calcium and phosphorus metabolism, reduced vitamin D production and increased parathormone (PTH) secretion. Renal osteodistrophy (RO) is a term used to define bone disease complications of patients with CKD, and is classified in high turnover disease represented by osteitis fibrosa (OF) and mixed bone disease; and low turnover disease represented by osteomalacia (OM) and adynamic bone disease (ABD). It is already known that patients with CKD have high incidence of bone fractures, and it has been demonstrated that hyperphosphatemia results in to decreased trabecular bone volume (BV/TV). We evaluated the effect of phosphorus (P) in rats? bone tissue submitted to experimental uremia that received continuous infusion of 1-34 rat PTH in physiologic or five times the normal values. Fifty five Wistar rats were submitted to parathyroidectomy (PTX), nephrectomy (Nx) and received PTH in different concentrations or some were PTX and NX controls (Sham) that received only vehicle. Rats received identical diets, excepted for the P content which was different according to the group [Low P (LP): 0,2% and high P (HP): 1,2%]. Groups were divided as follow: Sham (N=8), Sham LP (N=8), Sham-HP (N=7), NxPTHn-LP (N=8), NxPTHn-HP (N=8), NxPTHh-LP (N=9), NxPTHh-HP (N=7). After two months, animals were sacrificed and biochemical and bone histomorphometry were performed. Rats who received high P diet developed hyperphosphatemia and hypocalcemia. PTH replacement was effective and in accordance with infusion concentration. Bone histomorphometric analysis showed that HP rats presented low trabecular bone volume (BV/TV) independently of the uremia. BV/TV decreased slightly in the group where PTH continuous infusion was five times the physiologic values. Our results demonstrated that P has a deleterious action on bone tissue and in uremia it is necessary high levels of PTH to maintain bone integrity.

Orientador: Anderson Marliere Navarro / Banca: Alcyone Artioli Machado / Banca: Telma Maria Braga Costa / Banca: Paulo Inácio da Costa / Banca: Alceu Afonso Jordão Júnior / Resumo: A história natural da infecção pelo HIV e a terapia antirretroviral (TARV) já estão bem esclarecidos, por proporcionarem aumento da contagem das células T CD4+, redução da viremia e diminuição de riscos para doenças oportunistas. Apesar da eficácia do tratamento antirretroviral, evidenciam-se os efeitos sistêmicos da inflamação mediados pela infecção crônica do HIV e a toxicidade dos antirretrovirais que contribuem para o aumento de riscos de complicações metabólicas. Nós hipotizamos que pacientes HIV positivos em uso ou não da terapia antirretroviral apresentam níveis aumentados nos marcadores inflamatórios, e isto afeta o metabolismo ósseo que leva à perda óssea, sendo mais acometidos os pacientes com maior tempo de exposição ao vírus e ao tratamento antirretroviral. Objetivo: Investigar a influência de citocinas pró-inflamatórias no metabolismo ósseo em pacientes HIV positivos crônicos em uso ou não da terapia antirretroviral. Métodos: Trata-se de um estudo do tipo transversal com 50 homens adultos HIV positivos, em tratamento ou não com drogas antirretrovirais. Os participantes foram subdivididos segundo o uso ou não da TARV, sendo grupo controle (GC): 10 participantes virgens de tratamento; grupo G<2 anos de TARV: 20 participantes abaixo de dois anos de tratamento antirretroviral; grupo G>2 anos de TARV: 20 participantes acima de dois anos de tratamento antirretroviral. Foi realizado dual energy x-ray absorptiometry (DXA) para avaliar a densidade mineral óssea e a compos... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The natural history of HIV infection and antiretroviral treatment (ART) are well evidenced by increase in the CD4+ T cell count, reduction of viral replication, and reduced risk for opportunistic diseases. Despite the efficacy of ART, the current repercussions are the systemic effects of inflammation mediated by chronic HIV infection and toxicity of the antiretroviral drugs that significantly contribute to increase the risk of metabolic complications. We hypothesized that HIV-infected patients treated or not with antiretroviral therapy have increased levels of inflammatory markers that can affect bone metabolism leading to bone loss, and the patients with longer exposure to HIV and ART are more affected. Objective: Investigate the influence of pro-inflammatory cytokines in bone metabolism in patients with chronic HIV infection treated or not with antiretroviral therapy. Methods: A cross-sectional study was conducted on 50 HIV-seropositive men treated or not with ART. The participants were divided, according to the use or not of ART, into the control group (CG): 10 participants not in treatment; the G<2 years of ART group: 20 participants treated with ART for less than 2 years; and the G>2 years of ART group: 20 participants treated with ART for more than 2 years. Dual energy x-ray absorptiometry (DXA) was performed to evaluate bone mineral density and body composition... (Complete abstract click electronic access below) / Doutor

目前，骨形成低下的骨代謝異常在臨床中面臨巨大挑戰。治療這些疾病的途徑之一可通過小干擾核酸沉默骨形成抑制的基因。隨著核酸干擾技術的快速發展，採用核酸干擾策略進行治療的很多問題已被解決。然而，小干擾核酸的安全和有效遞送仍然是核酸干擾治療進行臨床轉化的瓶頸。其主要問題在於促進骨形成治療所需的小干擾核酸劑量較大，其系統給藥後可能對其他非骨組織產生副作用。所以，亟需針對具有促進成骨潛力的小干擾核酸開發安全有效的遞送系統。本研究的目的就是針對具有促進成骨潛力的小干擾核酸開發特定的遞送系統，以便應用於核酸干擾治療中的促進骨形成。策略之一是利用靶向骨形成表面的遞送系統攜載小干擾核酸到富集于骨形成表面的成骨系細胞。策略之二是直接把小干擾核酸遞送到成骨細胞，使其具有高度的細胞選擇性。在該研究中，我們採用具有成骨潛能的酪蛋白激酶2相互作用蛋白1小干擾核酸作為模型小干擾核酸以考察基因沉默效率。 / 靶向骨形成表面的(天門冬氨酸-絲氨酸-絲氨酸)₆-脂質體-小干擾核酸遞送系統：首先對多肽序列(天門冬氨酸-絲氨酸-絲氨酸)₆靶向骨形成表面的特性進行鑒定。進一步將(天門冬氨酸-絲氨酸-絲氨酸)₆作為靶向分子與以DOTAP為主要成分的陽離子脂質體進行連接製備（天門冬氨酸-絲氨酸-絲氨酸)6-脂質體遞送系統。採用凍幹/再水化方法對小干擾核酸進行包裹並對其粒徑，ζ電位，包封率以及穩定性進行考察。最後分別在體外和體內模型對該遞送系統遞送效果以及其攜載小干擾核酸的基因沉默效率進行評價。 / 實驗結果證實(天門冬氨酸-絲氨酸-絲氨酸)₆是一種在體內可以有效靶向骨形成表面的多肽。(天門冬氨酸-絲氨酸-絲氨酸)₆-脂質體的平均粒徑為140 nm左右，其包封率可高達80%。該遞送系統較穩定，可使攜載的小干擾核酸具有較高的基因沉默效率，而且沒有明顯的細胞毒性。體內試驗表明，該遞送系統在促進小干擾核酸在骨組織的分佈同時降低其被肝組織的攝取。該遞送系統所攜帶的酪蛋白激酶2相互作用蛋白1小干擾核酸可選擇性地沉默骨組織中的酪蛋白激酶2相互作用蛋白1基因，且對其他組織並沒有明顯影響。該結果表明(天門冬氨酸-絲氨酸-絲氨酸)₆-脂質體可促進小干擾核酸靶向骨組織並在骨組織沉默攜載小干擾核酸相應的基因。免疫化學分析結果顯示(天門冬氨酸-絲氨酸-絲氨酸)₆-脂質體可攜載小干擾核酸選擇性地到達骨形成表面的成骨系細胞，避免被前破骨細胞/破骨細胞吞噬。大鼠骨髓細胞採用Alp，Stro-1和Oscar抗體分選後的酪蛋白激酶2相互作用蛋白1 mRNA表達水平顯示該遞送系統可選擇性地沉默成骨系細胞。 / 靶向成骨細胞的L6適配子-脂質納米顆粒-小干擾核酸遞送系統：將針對大鼠成骨細胞（ROS 17/2.8細胞系）進行正向篩選，大鼠肝細胞（BRL-3A細胞系）和外周血細胞進行負向篩選的L6適配子與以DLin-KC2-DMA為主要成分的脂質納米顆粒採用膠束形式插入的方法進行連接製備L6適配子-脂質納米顆粒-小干擾核酸遞送系統。並對其粒徑，ζ電位，包封率和形態學進行考察。在體外評價實驗中，考察了該遞送系統的選擇性，細胞毒性，基因沉默效率以及細胞攝取機制。在體內實驗中，對小干擾核酸的組織分佈以及其攜載小干擾核酸在成骨細胞和肝細胞的分佈進行了評價。 / 實驗結果顯示L6適配子-脂質納米顆粒-小干擾核酸的平均粒徑為84.0±5.3 nm，其電勢為-23 ± 2 mV，包封率為80.8 ± 3.4%. 脂質納米顆粒表面的L6適配子可促進小干擾核酸在ROS 17/2.8細胞系（靶向細胞）中的攝取， 然而在BRL-3A 細胞系（非靶向細胞）中攝入很少。該遞送系統沒有明顯細胞毒性，在10 nM小干擾核酸的低濃度下，體外基因沉默效率可高達50 % 以上。由L6適配子引起的巨胞被證實是成骨細胞攝取L6適配子-脂質納米顆粒所攜載小干擾核酸的主要機制。體內實驗顯示該遞送系統可促進小干擾核酸在骨組織的分佈，降低其被肝組織的攝取。在肝组织冰凍切片中，肝血竇和肝細胞中沒有明顯的小干擾核酸分佈，進一步說明該遞送系統可降低對肝組織的影響。免疫化學分析結果顯示L6適配子-脂質納米顆粒-小干擾核酸可攜載小干擾核酸選擇性地到達成骨細胞，避免被前破骨細胞/破骨細胞吞噬。 / 重要意義：本研究中的兩種新型小干擾核酸系統可分別選擇性地遞送小干擾核酸靶向骨形成表面和成骨細胞。 (天門冬氨酸-絲氨酸-絲氨酸)₆-脂質體-小干擾核酸遞送系統開拓了全新的途徑，實現選擇性地遞送小干擾核酸到骨形成表面從而降低對骨吸收的影響。 L6適配子-脂質納米顆粒-小干擾核酸遞送系統在成骨細胞表面特徵蛋白未知的情況下，首次採用適配子技術在細胞水準實現成骨細胞的選擇性遞送。該研究中的兩種遞送系統為核酸干擾治療的促進骨形成策略提供了強而有力的工具，為實現肌肉骨骼疾病相關領域的核酸干擾治療策略從基礎科學向臨床應用的轉化建立了堅實的基礎。 / Metabolic skeletal disorders that are associated with impaired bone formation are a major clinical challenge. One approach to treat these diseases was to silence bone formation-inhibitory genes by small interference RNAs (siRNAs). With the rapid development of RNA interference (RNAi) technology, more issues of RNAi-based therapy strategies have been addressed. However, the safe and effective delivery of siRNAs is still the bottleneck for its translation from bench to bedside. One major concern was that the large therapeutic doses of systemically administered siRNA to stimulate sufficient bone formation may carry a high risk for adverse effects on non-skeletal tissues. Therefore, development of specific siRNA delivery systems for safe and efficient transporting osteogenic siRNAs is highly desirable. The objective of the present study was to explore siRNA delivery systems for osteogenic siRNAs in RNAi-based bone anabolic therapy. One strategy was to develop siRNA delivery system targeting bone formation surfaces to facilitate delivery of siRNAs to osteogenic cells. Another approch was to develop siRNA delivery system targeting osteoblasts directly. Plekho1 siRNA targeting casein kinase-2 interacting protein-1 (Ckip-1) with osteogenic potential was employed as a representative siRNA in our current study. / (AspSerSer)6-liposome-siRNA for targeting bone formation surfaces: (AspSerSer)6 for targeting bone formation surfaces was firstly identified. Then, (AspSerSer)6 was conjugated with DOTAP-based liposome to produce (AspSerSer)6-liposome. (AspSerSer)6-liposome-siNRA was prepared by lyophilization/rehydration method and characterized in terms of particle size, zeta potential, encapsulation efficiency and the stability in serum. Finally, the delivery of siRNA and the corresponding gene silencing mediated by (AspSerSer)6-liposome-siRNA were evaluated in the in vitro and in vivo models. / The results indicated that the novel (AspSerSer)₆ was a promising peptide for targeting bone formation surfaces in vivo. (AspSerSer)₆-liposome with the average particle size of 140 nm encapsulating Plekho1 siRNA exhibited more than 80% encapsulation efficiency and good stability against enzymatic degradation. It demonstrated high knockdown efficiency without obvious cytotoxicity. In in vivo study, the result of tissue distribution experiment indicated that (AspSerSer)6-liposome-siRNA enhanced the distribution of siRNA in bone, meanwhile reduced the uptake of siRNA in liver. The Plekho1 protein and mRNA expression in various tissues demonstrated that (AspSerSer)₆-liposome-siRNA could facilitate gene silencing in a bone-selective manner. The results of immunochemistry analyses indicated (AspSerSer)₆-liposome-siRNA facilitated delivering siRNA to osteogenic cells at bone formation surfaces and avoided siRNA to pre-osteoclast/osteoclast. Plekho1 mRNA expression in rat bone marrow cells sorted by fluorescence activated cell sorting (FACS) using Alp, Stro-1 and Oscar antibody, respectively, further suggested (AspSerSer)₆-liposome-siRNA could silence gene in a cell-selective manner in vivo. / L6-LNPs-siRNA for targeting osteoblasts: L6 aptamer for targeting osteoblasts (ROS 17/2.8 cell line) and using rat hepatocyte (BRL-3A cell line) and peripheral blood cells in negative selection was conjugated to DLin-KC2-DMA-based lipid nanoparticles (LNPs) to generate L6-LNPs-siRNA by post-insertion method in the form of micelles. L6-LNPs-siRNA was characterized with particle size, zeta potential, encapsulation efficiency and morphology. Its selectivity, cytotoxicity and knockdown efficiency were evaluated in vitro. The mechanism of L6-LNPs-mediated siRNA cellular uptake was further investigated. The tissue distribution of the injected siRNA and the localization of the siRNA with osteoblasts as well as hepatocytes were also evaluated in vivo. / The results showed L6-LNPs-siRNA have the average particle size of 84.0 ± 5.3 nm and zeta potential of -23 ± 2 mV. Its encapsulation efficiency was 80.8 ± 3.4%. The L6 aptamer on the surface of LNPs facilitated the cellular uptake of Plekho1 siRNA in ROS 17/2.8 cell line (target cells) but no uptake in BRL-3A cell line (non-target cells) in vitro. L6-LNPs-siRNA with low cytotoxicity exhibited above 50% knockdown efficiency at a low concentration of 10 nM in vitro. Macropinocytosis induced by L6 was demonstrated to be the predominant mechanism of L6-LNPs mediated siRNA uptake in osteoblasts. In in vivo study, it was shown that L6-LNPs-siRNA facilitated the distribution of siRNA in bone and decreased the hepatic uptake. No obvious siRNA fluorescent signals in sinus and hepatocyte was observed in liver cryosection further indicated the reducing influence on liver after administration of L6-LNPs-siRNA. Co-localization of fluorescence-labeled siRNA with Alp-positive cells was dominantly documented, whereas there were no instances of such overlapping staining with Oscar-positive cells after L6-LNPs-siRNA treatment, which suggested L6-LNPs-siRNA facilitated delivering siRNA in a cell-selective manner in vivo. / Significance: These two innovative siRNA delivery systems in the present study selectively targeted bone formation surfaces and osteoblasts, respectively. (AspSerSer)₆-liposome-siRNA opened up a new avenue to specifically deliver therapeutic siRNAs to bone formation surfaces without affecting bone resorption. L6-LNPs-siRNA achieved the osteoblast-specific delivery for siRNA at cellular level by aptamer technology for the first time, even without knowledge of characteristic protein on the surface of osteoblasts. The two delivery systems provided the powerful tools for RNAi-based bone anabolic strategy and established a solid foundation for translating RNAi-based therapies from basic science to clinic applications in the musculoskeletal field. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Wu, Heng. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 130-142). / Abstract also in Chinese. / Acknowledgements --- p.i / Abstract --- p.iii / 論文摘要 --- p.vi / Table of contents --- p.ix / Publications --- p.xiv / List of tables --- p.xvi / List of figures --- p.xvii / List of abbreviations --- p.xxi / Chapter One Introduction --- p.1 / Chapter 1.1 --- Great challenges in skeletal disorders --- p.2 / Chapter 1.2 --- RNA interference (RNAi) as therapeutic strategy --- p.3 / Chapter 1.2.1 --- Mechanism of RNAi --- p.3 / Chapter 1.2.2 --- Potential triggers of RNAi-mediated gene silencing --- p.4 / Chapter 1.2.3 --- Current clinical trials using RNAi as therapeutic strategy --- p.7 / Chapter 1.2.4 --- Current application of therapeutic siRNAs in skeletal disorders --- p.11 / Chapter 1.3 --- Challenges of siRNA in vivo delivery for targeting bone --- p.12 / Chapter 1.3.1 --- General challenges of siRNA delivery in vivo --- p.13 / Chapter 1.3.2 --- Challenges of siRNA delivery to bone --- p.15 / Chapter 1.3.2.1 --- Physiological property --- p.15 / Chapter 1.3.2.2 --- Targeting ligands for approaching bone --- p.16 / Chapter 1.4 --- Strategies of siRNAs in vivo delivery after systemic administration --- p.18 / Chapter 1.4.1 --- Naked siRNA and naked siRNA with chemical conjugation --- p.18 / Chapter 1.4.2 --- Nanoparticle delivery systems --- p.20 / Chapter 1.4.2.1 --- Liposome and lipid-like materials --- p.20 / Chapter 1.4.2.2 --- Polymers --- p.22 / Chapter 1.4.2.3 --- Targeted delivery system --- p.23 / Chapter 1.5 --- Strategies of osteogenic siRNAs delivery for stimulating bone formation --- p.24 / Chapter 1.6 --- Objective of present study --- p.25 / Chapter Chapter Two --- Preparation and characterization of (AspSerSer)₆-liposome-siRNA for targeting bone formation surfaces --- p.26 / Chapter 2.1 --- Introduction --- p.27 / Chapter 2.2 --- Materials and Methods --- p.28 / Chapter 2.2.1 --- Materials --- p.28 / Chapter 2.2.2 --- Identification of (AspSerSer)₆ --- p.29 / Chapter 2.2.3 --- Development of formulation --- p.30 / Chapter 2.2.3.1 --- Selection of the molar ratio of DOTAP --- p.30 / Chapter 2.2.3.2 --- Selection of the molar ratio of siRNA to lipids --- p.30 / Chapter 2.2.4 --- Preparation of (AspSerSer)6-liposome-siRNA --- p.30 / Chapter 2.2.5 --- Characterization of (AspSerSer)₆-liposome --- p.33 / Chapter 2.2.5.1 --- Particle Size and Zeta Potential --- p.33 / Chapter 2.2.5.2 --- Encapsulation Efficiency --- p.33 / Chapter 2.2.5.3 --- Stability in serum --- p.33 / Chapter 2.3 --- Results --- p.34 / Chapter 2.3.1 --- (AspSerSer)₆ as a targeting moiety --- p.34 / Chapter 2.3.2 --- Development of formulation --- p.37 / Chapter 2.3.3 --- Particle size, Zeta Potential and Encapsulation Efficiency --- p.38 / Chapter 2.3.4 --- Stability in serum --- p.38 / Chapter 2.4 --- Discussion --- p.40 / Chapter 2.5 --- Conclusion --- p.42 / Chapter Chapter Three --- Evaluation of (AspSerSer)₆-liposome-siRNA for cell-specific delivery and gene silencing in vitro and in vivo --- p.43 / Chapter 3.1 --- Introduction --- p.44 / Chapter 3.2 --- Materials and Methods --- p.45 / Chapter 3.2.1 --- Materials --- p.45 / Chapter 3.2.2 --- Biological evaluation in vitro --- p.46 / Chapter 3.2.2.1 --- Binding affinity with hydroxyapatite --- p.46 / Chapter 3.2.2.2 --- Cell culture --- p.46 / Chapter 3.2.2.3 --- Cellular uptake --- p.47 / Chapter 3.2.2.4 --- Knockdown efficiency in vitro --- p.47 / Chapter 3.2.2.5 --- Total RNA extraction, reverse transcription and quantitative real-time PCR --- p.48 / Chapter 3.2.3 --- Cytotoxicity --- p.49 / Chapter 3.2.4 --- Tissue distribution --- p.50 / Chapter 3.2.4.1 --- Experimental design --- p.50 / Chapter 3.2.4.2 --- Fluorescence image analysis --- p.50 / Chapter 3.2.4.3 --- Quantitative Analysis --- p.50 / Chapter 3.2.5 --- Localization of siRNA in liver --- p.51 / Chapter 3.2.5.1 --- Experimental design --- p.51 / Chapter 3.2.5.2 --- Histochemisty analysis --- p.51 / Chapter 3.2.6 --- Gene silencing in tissues --- p.52 / Chapter 3.2.6.1 --- Experimental design --- p.52 / Chapter 3.2.6.2 --- Determination of mRNA expression --- p.52 / Chapter 3.2.6.3 --- Western blot analysis --- p.52 / Chapter 3.2.7 --- Localization of siRNA with Osteoblasts/Osteoclasts --- p.53 / Chapter 3.2.7.1 --- Experimental design --- p.53 / Chapter 3.2.7.2 --- Immunohistochemistry analysis --- p.53 / Chapter 3.2.8 --- Gene silencing at cellular levels --- p.54 / Chapter 3.2.8.1 --- Experimental design --- p.54 / Chapter 3.2.8.2 --- Flow cytometry cell sorting --- p.54 / Chapter 3.2.9 --- Statistical analysis --- p.55 / Chapter 3.3 --- Results --- p.56 / Chapter 3.3.1 --- Binding affinity with hydroxyapatite --- p.56 / Chapter 3.3.2 --- Cellular uptake --- p.57 / Chapter 3.3.3 --- Knockdown efficiency in vitro --- p.57 / Chapter 3.3.4 --- Cytotoxicity --- p.59 / Chapter 3.3.5 --- Tissue distribution by imaging analysis --- p.60 / Chapter 3.3.6 --- Quantitative analysis of tissue distribution --- p.62 / Chapter 3.3.7 --- Localization of siRNA in liver --- p.63 / Chapter 3.3.8 --- Plekho1 mRNA and protein expressions --- p.64 / Chapter 3.3.9 --- Immunohistochemistry analysis --- p.65 / Chapter 3.3.10 --- Gene silencing at cellular level --- p.71 / Chapter 3.4 --- Discussion --- p.74 / Chapter 3.5 --- Conclusion --- p.77 / Chapter Chapter Four --- Preparation and characterization of aptamer-functionalized lipid nanoparticle for siRNA cell-specific delivery --- p.78 / Chapter 4.1 --- Introduction --- p.79 / Chapter 4.2 --- Materials and Methods --- p.80 / Chapter 4.2.1 --- Materials --- p.80 / Chapter 4.2.2 --- Synthesis of 2,2-Dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-di- oxolane (DLin-KC2-DMA) --- p.80 / Chapter 4.2.2.1 --- Synthesis of Linoleyl alcohol (1) --- p.81 / Chapter 4.2.2.2 --- Synthesis of Linoleyl bromide (2) --- p.81 / Chapter 4.2.2.3 --- Synthesis of Dilinoleylmethyl formate (3) --- p.82 / Chapter 4.2.2.4 --- Synthesis of Dilinoleyl Methanol (4) --- p.82 / Chapter 4.2.2.5 --- Synthesis of Dilinoleyl Ketone (5) --- p.83 / Chapter 4.2.2.6 --- Synthesis of 2, 2- Dilinoleyl- 4- (2-hydroxyethyl)-[1,3]-dioxolane (6) --- p.83 / Chapter 4.2.2.7 --- Synthesis of DLin-KC2-DMA --- p.83 / Chapter 4.2.3 --- Development of formulation --- p.84 / Chapter 4.2.3.1 --- Selection of the molar ratio of lipids --- p.84 / Chapter 4.2.3.2 --- Selection of the mass ratios of siRNA to lipids --- p.85 / Chapter 4.2.3.3 --- Selection of the molar ratios of L6-PEG2000-DSPE on L6-LNPs-siRNA --- p.85 / Chapter 4.2.4 --- Binding affinity with osteoblasts --- p.86 / Chapter 4.2.5 --- Preparation of L6-LNPs-siRNA --- p.86 / Chapter 4.2.5.1 --- Synthesis of L6-PEG2000-DSPE --- p.87 / Chapter 4.2.5.2 --- Preparation of LNPs-siRNA --- p.87 / Chapter 4.2.5.3 --- Post-insertion of aptamers on the surface of LNPs-siRNA --- p.88 / Chapter 4.2.6 --- Characterization of L6-LNPs-siRNA --- p.88 / Chapter 4.2.6.1 --- Particle size and Zeta Potential --- p.88 / Chapter 4.2.6.2 --- Encapsulation Efficiency (EE) --- p.88 / Chapter 4.2.6.3 --- Cryo-Transmission electron microscope --- p.89 / Chapter 4.3 --- Results --- p.90 / Chapter 4.3.1 --- Synthesis of DLin-KC2-DMA --- p.90 / Chapter 4.3.2 --- Formulation development --- p.93 / Chapter 4.3.3 --- Preparation of L6-LNPs --- p.95 / Chapter 4.3.4 --- Characterization of L6-LNPs-siRNA --- p.96 / Chapter 4.4 --- Discussion --- p.98 / Chapter 4.5 --- Conclusion --- p.101 / Chapter Chapter Five --- Evaluation of L6 aptamer functionalized lipid nanoparticles (L6-LNPs-siRNA) for osteoblast-specific delivery in vitro and in vivo --- p.102 / Chapter 5.1 --- Introduction --- p.103 / Chapter 5.2 --- Materials and Methods --- p.103 / Chapter 5.2.1 --- Materials --- p.103 / Chapter 5.2.2 --- Biological evaluation in vitro --- p.104 / Chapter 5.2.2.1 --- Cell culture --- p.104 / Chapter 5.2.2.2 --- Binding affinity with target/non-target cells --- p.105 / Chapter 5.2.2.3 --- Cellular uptake of siRNA in target/non-target cells --- p.105 / Chapter 5.2.2.4 --- Knockdown efficiency in vitro --- p.105 / Chapter 5.2.3 --- Cytotoxicity --- p.106 / Chapter 5.2.4 --- Mechanism of cellular uptake --- p.106 / Chapter 5.2.4.1 --- Spectral bio-imaging for endocytic pathways --- p.106 / Chapter 5.2.4.2 --- Chemical inhibition for endocytic pathways --- p.107 / Chapter 5.2.4.3 --- Determination of membrane ruffling --- p.107 / Chapter 5.2.5 --- Evaluation of specific delivery in vivo --- p.107 / Chapter 5.2.5.1 --- Experimental design --- p.107 / Chapter 5.2.5.2 --- Tissue distribution --- p.108 / Chapter 5.2.5.3 --- Localization of siRNA in liver --- p.108 / Chapter 5.2.5.4 --- Localization of siRNA with osteoblast/osteoclast --- p.108 / Chapter 5.2.6 --- Statistical analysis --- p.109 / Chapter 5.3 --- Results --- p.109 / Chapter 5.3.1 --- Binding selectivity of L6-LNPs-siRNA --- p.109 / Chapter 5.3.2 --- Selectivity of siRNA cellular uptake --- p.111 / Chapter 5.3.3 --- Knockdown efficiency in vitro --- p.112 / Chapter 5.3.4 --- Cytotoxicity --- p.113 / Chapter 5.3.5 --- Mechanism of cellular uptake --- p.113 / Chapter 5.3.6 --- Tissue distribution --- p.118 / Chapter 5.3.7 --- Localization of siRNA in liver --- p.119 / Chapter 5.3.8 --- Localization of siRNA with Osteoblasts/Osteoclasts --- p.120 / Chapter 5.4 --- Discussion --- p.123 / Chapter 5.5 --- Conclusion --- p.125 / Chapter Chapter Six --- Summary of the study and future research --- p.126 / Chapter 6.1 --- Summary of the study --- p.127 / Chapter 6.2 --- Future research --- p.128 / References --- p.130

Made available in DSpace on 2014-06-11T19:25:37Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-12-08Bitstream added on 2014-06-13T20:14:18Z : No. of bitstreams: 1 apolinario_jc_me_araca.pdf: 190197 bytes, checksum: 1aba6bb35b316b811a8b671145feab29 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / A literatura apresenta que a resposta de reparo ósseo pode ser acentuada pela estimulação física, mecânica ou eletromagnética. Há evidências de que o ultra-som – US – de baixa potência pode acelerar a regeneração óssea. Este trabalho objetivou verificar o efeito do US no defeito ósseo, criado experimentalmente, em tíbias de ratos sob ausência de carga (suspenso pela cauda) por meio de análise densitométrica e biomecânica. Trinta Rattus novergicus albinus, Wistar, adultos, divididos em 3 grupos: G1 (n=10), não suspenso - experimento de 15 dias; G2 (n=10), suspenso pela cauda - experimento de 15 dias e, G3 (n=10), suspensos pela cauda, experimento de 36 dias. Os animais foram submetidos à osteotomia em ambas as tíbias e à aplicação do US (freqüência de 1,5 MHz, ciclo 1:4, 30mW/cm2) na direita (12 sessões de 20 minutos). O G3 somente foi osteotomizado após 21º dia de suspensão. Para análises densitométrica utilizou-se densitômetro DPXLunar ™, sistema digital Digora e o programa computacional Image J ; para ensaio mecânico usou máquina universal de ensaio EMIC . Os resultados do Conteúdo Mineral Ósseo (g), Área (cm²), Densidade Mineral Óssea (g/cm²) e da Densidade Óssea (mmAl) observadas nas tíbias, assim como a Força Máxima (N) e Rigidez (x103N/m) não demonstraram diferenças significantes (tratadas versus controle de cada grupo), possivelmente pelo menor tempo de tratamento com relação aos trabalhos encontrados na literatura. Concluindo que o Ultra-Som de baixa potência não acelerou o processo de consolidação óssea. / Literature shows that bone repair response can be accented by physical, mechanic or electromagnetic stimulation. There are evidences that low power ultrasound – US - can speed up bone regeneration. This work aimed at determining the effect of US in bone defects, experimentally created, in tibia from rats under load absence (suspended by the tail) by densitometric analysis and biomechanics. Thirty Rattus novergicus albinus, Wistar, adult, divided in 3 groups: G1 (n=10), not suspended – a 15 day experiment; G2 (n=10), suspended by the tail – a 15 day experiment and, G3 (n=10), suspended by the tail – a 36 day experiment. , The animals have been submitted to the osteotomy in both tibias and to the US application (1,5 MHz frequency, cycle 1:4, 30mW/cm2), on the right (twelve sessions of 20 minutes). G3 was only osteotomized after the 21st day of suspension. DPX-Lunar™ densitometer, Digora digital system and Image J computer program were used for densitometrical analysis; for the mechanical assay, the universal machine of EMIC assay was used. The results for Bone Mineral Content (g), Area (cm²), Bone Mineral Density (g/cm²) and Bone Density (mmAl) observed in tibias, as well as Maximum Power (N), and Rigidity (x103N/m) did not show any significant differences (treated versus control of each group), possibly due to shorter treatment time as regards the studies found in literature. Concluding that the low power ultrasound not accelerated the process of consolidating bone.

A thesis submitted to the Faculty of Health Sciences, University of the Witwatersrand in fulfilment of the requirements for the degree of Doctor of Philosophy. Johannesburg, 2017. / Background Abnormalities of mineral bone disease have been consistently associated with adverse clinical outcomes in patients with chronic kidney disease (CKD). The consequences of these changes have also been shown to differ across races. However, in Africa the impact of derangements of CKD -mineral and bone disorder (CKD-MBD) on patients with CKD is largely unknown. In addition, studies from the USA have reported racial variations in markers of CKD and it remains unclear whether genetic factors may explain this discrepancy in the levels of biochemical markers of CKD-MBD across ethnic groups. Therefore, this study has been conducted to determine the existence of racial differences in the levels of fibroblast growth factor 23(FGF23) and traditional markers of mineral bone metabolism in a heterogeneous African CKD population, and to provide important insights into the pattern and genetic variability of CKD-MBD in sub-Saharan Africa. Methods This was a cross sectional multicenter study carried out from April 2015 to May 2016, involving two hundred and ninety three CKD patients from three renal units in Johannesburg, South Africa. The retrospective arm of this study involved two hundred and thirteen patients undergoing maintenance haemodialysis (MHD) from two dialysis centers in Johannesburg between January 2009 and March 2016. The first part of this study described the pattern of CKD-MBD in MHD patients using traditional markers of CKD-MBD. The second part of the study looked into the spectrum of CKD-MBD and racial variations in markers of CKD-MBD in pre dialysis and dialysis patients. This was followed by the genetic aspect of the study that examined the influence of vitamin D receptor polymorphisms on biochemical markers of mineral bone disorders. Lastly, the study also evaluated the association between markers of CKD-MBD and mortality in MHD patients. Results The prevalence of hyperparathyroidism (iPTH>150 pg/mL), hyperphosphataemia, hypocalcaemia and 25-hydroxyvitamin D deficiency (<30 ng/mL) was 73.4%, 57.0%, 20.3% and 80.7 % respectively in our MHD patients. The combination of markers of bone turnover (iPTH>150 pg/mL and total alkaline phosphatase > 112 U/L) suggestive of high turnover bone disease, was present in 47.3 % of the study population. The odds ratios for developing secondary hyperparathyroidism with hypocalcaemia and hyperphosphataemia were 5.32 (95% CI 1.10 - 25.9, P =0.03) and 3.06 (95 % CI 1.15 - 8.10, P =0.02) respectively. The 293 CKD patients (208 blacks, 85 whites) had an overall mean age of 51.1±13.6 years, and black patients were significantly younger than the white patients (48.4 ±.13.6 versus 57.1±15.5 years; p<0.001). In comparison to whites, blacks had higher median iPTH (498 [37-1084] versus 274[131-595] pg/ml; P=0.03), alkaline phosphatase (122[89-192] versus 103[74-144] U/L; P=0.03) and mean 25- hydroxyvitamin D (26.8±12.7 versus 22.7 ±12.2 ng/ml, P=0.01) levels, while their median FGF23 (100 [34-639] versus 233[80-1370] pg/ml; P=0.002) and mean serum phosphate (1.3±0.5 versus 1.5±0.5, P =0.001) levels were significantly lower. With the exception of vitamin D receptor (VDR) Taq I polymorphism, the distribution of the VDR polymorphisms differs significantly between blacks and whites. In hemodialysis patients, the BsmI Bb genotype was significantly associated with moderate secondary hyperparathyroidism (OR, 3.88; 95 CI 1.13-13.25, P=0.03) and severe hyperparathyroidism (OR, 2.54; 95 CI 1.08-5.96, P=0.03). Patients with high total alkaline phosphatase (TAP) had significantly higher risk of death compared to patients with TAP <112 U/L (hazard ratio, 2.50; 95% CI 1.24–5.01, P = 0.01). Similarly, serum calcium >2.75 mmol/L was associated with increased risk of death compared to patients within levels of 2.10–2.37 mmol/L (HR 6.34, 95% CI 1.40–28.76; P = 0.02). The HR for death in white patients compared to black patients was 6.88; 95% CI 1.82–25.88; P = 0.004. Conclusions Secondary hyperparathyroidism and 25–hydroxyvitamin D deficiency were common in our haemodialysis patients. The study also highlighted the existence of racial differences in the circulating markers of mineral bone disorders in our African CKD population. In addition, the study showed that both moderate and severe secondary hyperparathyroidism are predicted by the BsmI Bb genotype, and the over expression of this genotype in black patients may partly explain the ethnic variations in the severity of secondary hyperparathyroidism in the CKD population. High levels of serum alkaline phosphatase, hypercalcaemia, and white race are associated with increased risk of death in MHD patients. / LG2018

Distúrbios do metabolismo mineral e ósseo são altamente prevalentes e considerados como causa relevante da morbidade e mortalidade dos pacientes com doença renal crônica. Diversas estratégias diagnósticas e terapêuticas têm sido estudadas nesses doentes; entretanto, pouco valor é dado ao cálcio do dialisato, apesar do impacto que possa exercer sobre o balanço de cálcio durante a hemodiálise. Os fatores determinantes da transferência de cálcio durante o procedimento são ainda controversos. Nesse estudo prospectivo, avaliamos a influência da remodelação óssea sobre o balanço de cálcio em dez pacientes dialíticos em três situações consecutivas: hiperparatireoidismo grave (Pré paratireoidectomia), durante a \"síndrome de fome óssea\" (Fome óssea) imediatamente após a paratireoidectomia e após estabilização clínica (Paratireoidectomia tardia). Durante cada fase os participantes foram submetidos a três sessões randômicas de hemodiálise com diferentes concentrações de cálcio no dialisato: 2,5; 3,0 e 3,5 mEq/L. Todos os pacientes foram submetidos à biópsia óssea para análise histomorfométrica e quantificação de proteínas ósseas no início do estudo. A transferência de cálcio variou grandemente entre os pacientes em cada fase do estudo mesmo usando o mesmo cálcio no dialisato, com valores negativos de medianas no Pré paratireoidectomia e Fome óssea (-161mg e -218mg, respectivamente) e discretamente positivo no Paratireoidectomia tardio (39mg; p < 0,05 versus Pré paratireoidectomia e Fome óssea). Análise de regressão multivariada mostrou que o gradiente de cálcio entre o cálcio iônico sérico inicial e o cálcio do dialisato, a diferença entre o cálcio iônico sérico final e o inicial e a forma não carboxilada da osteocalcina foram preditores independentes da transferencia de cálcio (R2=0.48; p < 0.05). Pelo fato da remodelação óssea também influenciar os níveis séricos de cálcio iônico e suas variações durante a diálise, nesse estudo demonstramos que o esqueleto tem papel fundamental no balanço de cálcio e essas variáveis devem ser consideradas na individualização do cálcio do dialisato de nossos pacientes / Disturbances in mineral and bone metabolism are highly prevalent and are a major cause of morbidity and mortality in chronic kidney disease patients. Different diagnostic and therapeutic strategies have been studied in these patients. However, little attention is paid to the calcium concentration in the dialysate, despite the impact it could exert over calcium balance during dialysis. The variables that determine calcium transfer during hemodialysis are still controversial. In this study, we have prospectively investigated the influence of bone remodeling on calcium balance in ten dialysis patients in three consecutive situations: severe hyperparathyroidism (Pre parathyroidectomy), during \"hungry bone syndrome\" (Hungry bone) right after surgery and after stabilization of clinical status (Late parathyroidectomy). During each phase participants were submitted to 3 random hemodialysis sessions, with different dialysate calcium: 2.5, 3.0 and 3.5 mEq/L. Bone biopsy for hystomorphometric analysis and bone proteins quantification were performed in all patients at baseline. Calcium mass transfer varied widely among patients in each study phase even using the same dialysis calcium with negative median values in Pre parathyroidectomy and Hungry Bone (-161 and -218mg, respectively) and slightly positive in Late parathyroidectomy (39mg; p<0.05 versus Pre parathyroidectomy and Hungry Bone). Multiple regression analysis showed that calcium gradient between initial serum ionic calcium and the dialysate calcium, the difference between final and initial serum ionic calcium and serum undercarboxylated form of osteocalcin were independent predictors of calcium mass transfer (R2=0.48; p<0.05). As bone remodeling also influences the serum levels of ionic calcium and its variance during dialysis, in this study we have added new data by demonstrating that the skeleton plays a key role on calcium balance and these variables must be considered when individualizing calcium dialysate for our patients

A maior parte dos distúrbios metabólicos da doença renal crônica (DRC) é revertida após um transplante renal bem-sucedido. Porém, alterações do metabolismo ósseo podem permanecer e estão associadas ao aumento de fraturas, calcificação vascular, perda de enxerto e mortalidade. A expressão óssea de proteínas osteocíticas está alterada na DRC e parece contribuir negativamente para a homeostase óssea. Há relatos de aumento da expressão óssea de FGF-23 e esclerostina em crianças que receberam transplante de órgãos sólidos em comparação com voluntários normais. Entretanto, análise da expressão destas proteínas em receptores adultos ainda não foi realizada. Avaliação de biopsia óssea em 31 pacientes uma semana antes e 1 ano após o transplante renal. Realizada histomorfometria óssea e avaliação das proteínas ósseas através de imunohistoquimica, multiplex e expressão gênica. Na avaliação das biópsias antes do transplante, houve concordância entre os achados de imunohistoquimica e multiplex para esclerostina e FGF-23. Um ano após o transplante renal bem-sucedido, observamos diminuição dos níveis séricos do PTH, TRAP5b, fosfatase alcalina óssea, FGF-23, OPG e esclerostina. Apesar da diminuição da esclerostina sérica, houve aumento de seu conteúdo ósseo pela imunohistoquimica, multiplex e expressão gênica. Também foi observado aumento do conteúdo proteico e da expressão gênica da beta-catenina fosforilada, confirmando a inibição da via Wnt. Esta inibição foi acompanhada do aumento do conteúdo ósseo de RANKL e diminuição da OPG. Em relação ao FGF-23, houve concordância entre níveis séricos e conteúdo proteico, confirmando sua menor síntese pelos osteócitos, e portanto, menor nível sérico, após o transplante renal. A recuperação da função renal após o transplante é acompanhada de mudanças nas proteínas séricas e ósseas. A esclerostina óssea aumentou, apesar da diminuição do nível sérico, acompanhada de mudanças em outras proteínas que confirmam a inibição da via Wnt. Esse achado pode ajudar a desvendar a fisiopatologia da doença óssea pós transplante e guiar a busca por novas terapias / Most of the metabolic disorders of chronic kidney disease (CKD) improve after kidney transplantation, although bone metabolism might remain compromised, which is evidenced by high rates of bone loss, fractures and vascular calcification. Osteocytic bone protein expression is altered in CKD, and this seems to contribute negatively to bone health in these patients. It has been described that FGF-23 and sclerostin expression is increased in children after solid organ transplantation. However, little is known about bone-related proteins expression in adult recipients, which were analyzed prospectively in this study. Transiliac bone biopsies were obtained from 31 adult patients one week before and one year after transplantation. Bone fragments were used for histomorphometric analysis, as well as for bone proteins expression, measured by immunohistochemistry (IH) and multiplex. At baseline, we observed a significant correlation between IH expression and multiplex concentrations for sclerostin and FGF-23. After a successful transplant, there was a decrease in PTH, TRAP5b, bone alkaline phosphatase, FGF-23, OPG and sclerostin. Although serum sclerostin decreased after the transplant, bone content of this protein increased through immunohistochemistry, multiplex and gene expression. We also observed an increase in the bone content and bone expression of phosphorylated beta-catenin, confirming the Wnt pathway inhibition, which was accompanied by RANKL increase and OPG decrease in the bone. A significant decrease in FGF-23 bone concentration was also seen, compatible with the serum decrease. Kidney function recovery after transplant is accompanied by significant changes in many bone proteins expression. Contradictory to the decrease in levels of serum sclerostin, its bone expression, actually, has increased, accompanied by the change of other proteins that confirm the Wnt pathway inhbition. This findings could help to unveil the patophysiology of post transplant bone disease and help to guide the search to new therapies

A maior parte dos distúrbios metabólicos da doença renal crônica (DRC) é revertida após um transplante renal bem-sucedido. Porém, alterações do metabolismo ósseo podem permanecer e estão associadas ao aumento de fraturas, calcificação vascular, perda de enxerto e mortalidade. A expressão óssea de proteínas osteocíticas está alterada na DRC e parece contribuir negativamente para a homeostase óssea. Há relatos de aumento da expressão óssea de FGF-23 e esclerostina em crianças que receberam transplante de órgãos sólidos em comparação com voluntários normais. Entretanto, análise da expressão destas proteínas em receptores adultos ainda não foi realizada. Avaliação de biopsia óssea em 31 pacientes uma semana antes e 1 ano após o transplante renal. Realizada histomorfometria óssea e avaliação das proteínas ósseas através de imunohistoquimica, multiplex e expressão gênica. Na avaliação das biópsias antes do transplante, houve concordância entre os achados de imunohistoquimica e multiplex para esclerostina e FGF-23. Um ano após o transplante renal bem-sucedido, observamos diminuição dos níveis séricos do PTH, TRAP5b, fosfatase alcalina óssea, FGF-23, OPG e esclerostina. Apesar da diminuição da esclerostina sérica, houve aumento de seu conteúdo ósseo pela imunohistoquimica, multiplex e expressão gênica. Também foi observado aumento do conteúdo proteico e da expressão gênica da beta-catenina fosforilada, confirmando a inibição da via Wnt. Esta inibição foi acompanhada do aumento do conteúdo ósseo de RANKL e diminuição da OPG. Em relação ao FGF-23, houve concordância entre níveis séricos e conteúdo proteico, confirmando sua menor síntese pelos osteócitos, e portanto, menor nível sérico, após o transplante renal. A recuperação da função renal após o transplante é acompanhada de mudanças nas proteínas séricas e ósseas. A esclerostina óssea aumentou, apesar da diminuição do nível sérico, acompanhada de mudanças em outras proteínas que confirmam a inibição da via Wnt. Esse achado pode ajudar a desvendar a fisiopatologia da doença óssea pós transplante e guiar a busca por novas terapias / Most of the metabolic disorders of chronic kidney disease (CKD) improve after kidney transplantation, although bone metabolism might remain compromised, which is evidenced by high rates of bone loss, fractures and vascular calcification. Osteocytic bone protein expression is altered in CKD, and this seems to contribute negatively to bone health in these patients. It has been described that FGF-23 and sclerostin expression is increased in children after solid organ transplantation. However, little is known about bone-related proteins expression in adult recipients, which were analyzed prospectively in this study. Transiliac bone biopsies were obtained from 31 adult patients one week before and one year after transplantation. Bone fragments were used for histomorphometric analysis, as well as for bone proteins expression, measured by immunohistochemistry (IH) and multiplex. At baseline, we observed a significant correlation between IH expression and multiplex concentrations for sclerostin and FGF-23. After a successful transplant, there was a decrease in PTH, TRAP5b, bone alkaline phosphatase, FGF-23, OPG and sclerostin. Although serum sclerostin decreased after the transplant, bone content of this protein increased through immunohistochemistry, multiplex and gene expression. We also observed an increase in the bone content and bone expression of phosphorylated beta-catenin, confirming the Wnt pathway inhibition, which was accompanied by RANKL increase and OPG decrease in the bone. A significant decrease in FGF-23 bone concentration was also seen, compatible with the serum decrease. Kidney function recovery after transplant is accompanied by significant changes in many bone proteins expression. Contradictory to the decrease in levels of serum sclerostin, its bone expression, actually, has increased, accompanied by the change of other proteins that confirm the Wnt pathway inhbition. This findings could help to unveil the patophysiology of post transplant bone disease and help to guide the search to new therapies

As doenças renais crônicas (DRC) evoluem com distúrbios na homeostase do cálcio e do fósforo, diminuição na produção de vitamina D e aumento na secreção de PTH. Osteodistrofia renal (OR) é o termo usado para definir as alterações ósseas dos pacientes com DRC e classifica-se em doença de alta remodelação representada pela osteíte fibrosa (OF) e doença mista (DM); e de baixa remodelação representada pela osteomalácia (OM) e pela doença adinâmica (DOA). Pacientes com DRC apresentam elevada incidência de fraturas e recentemente demonstrou-se que a hiperfosfatemia leva a diminuição do volume ósseo. Estudamos o efeito isolado do fósforo no tecido ósseo de animais com insuficiência renal mantidos com infusão fixa de PTH variando o conteúdo de fósforo na dieta. Cinqüenta e cinco ratos Wistar foram submetidos à paratireoidectomia (PTX) e nefrectomia (Nx) com reposição de PTH em diferentes concentrações ou foram sham operados e recebiam infusão de veículo. Todos os animais receberam a mesma dieta variando apenas a concentração de P (pobre em P (pP): 0,2% e rico em P (rP):1,2%). Dividimos os grupos em: Sham (N=8); Sham-pP (N=8); Sham-rP (N=7); NxPTHn-pP (N=8); NxPTHn-rP (N=8); NxPTHe-pP (N=9); NxPTHe-rP (N=7). Após 2 meses, realizamos análises bioquímicas e histomorfometria do fêmur proximal. Os animais que ingeriram dieta rica em fósforo apresentaram hiperfosfatemia assim como menor valor de cálcio sérico. A reposição de PTH foi efetiva e proporcional às concentrações infundidas. A histomorfometria óssea mostrou que os ratos que ingeriram dieta rica em fósforo independente da uremia tinham diminuição do volume ósseo (BV/TV), e que este efeito foi amenizado pela reposição do PTH em concentrações elevadas. Nossos resultados demonstram que o fósforo é deletério para o tecido ósseo e que na uremia são necessários níveis mais elevados de PTH para manter a integridade óssea. / Chronic kidney disease (CKD) involves disturbances in calcium and phosphorus metabolism, reduced vitamin D production and increased parathormone (PTH) secretion. Renal osteodistrophy (RO) is a term used to define bone disease complications of patients with CKD, and is classified in high turnover disease represented by osteitis fibrosa (OF) and mixed bone disease; and low turnover disease represented by osteomalacia (OM) and adynamic bone disease (ABD). It is already known that patients with CKD have high incidence of bone fractures, and it has been demonstrated that hyperphosphatemia results in to decreased trabecular bone volume (BV/TV). We evaluated the effect of phosphorus (P) in rats? bone tissue submitted to experimental uremia that received continuous infusion of 1-34 rat PTH in physiologic or five times the normal values. Fifty five Wistar rats were submitted to parathyroidectomy (PTX), nephrectomy (Nx) and received PTH in different concentrations or some were PTX and NX controls (Sham) that received only vehicle. Rats received identical diets, excepted for the P content which was different according to the group [Low P (LP): 0,2% and high P (HP): 1,2%]. Groups were divided as follow: Sham (N=8), Sham LP (N=8), Sham-HP (N=7), NxPTHn-LP (N=8), NxPTHn-HP (N=8), NxPTHh-LP (N=9), NxPTHh-HP (N=7). After two months, animals were sacrificed and biochemical and bone histomorphometry were performed. Rats who received high P diet developed hyperphosphatemia and hypocalcemia. PTH replacement was effective and in accordance with infusion concentration. Bone histomorphometric analysis showed that HP rats presented low trabecular bone volume (BV/TV) independently of the uremia. BV/TV decreased slightly in the group where PTH continuous infusion was five times the physiologic values. Our results demonstrated that P has a deleterious action on bone tissue and in uremia it is necessary high levels of PTH to maintain bone integrity.