Impaired Osteoblast and Osteocyte Maturation in the Pathogenesis of Renal Osteodystrophy

肾性骨营养不良发病机制中成骨细胞和骨细胞成熟受损

• 批准号: 9761460
• 负责人: KATHERINE WESSELING-PERRY
• 金额: $ 17.21万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-08-09 至 2022-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9761460
• 关键词: Adult; Affect; Apoptosis; Biopsy Specimen; Bone Diseases; Bone Matrix; Calcium; Cardiovascular Diseases; Cell Maturation; Cells; Child; Childhood; Chronic Kidney Failure; Clinical; Comorbidity; Complex; Cross-Sectional Studies; DNA Nucleotidylexotransferase; Data; Defect; Deformity; Disabled Persons; Electrons; Extracellular Matrix; Failure; Fracture; Genetic Diseases; Goals; Growth; High Prevalence; Hormones; Human; Image; Immunofluorescence Immunologic; Immunohistochemistry; Impairment; In Vitro; Integrins; Kidney Diseases; Label; Libraries; Link; Location; MEPE gene; Mass Spectrum Analysis; Measures; Mediating; Minerals; Modeling; Morbidity - disease rate; Nature; Osteoblasts; Osteocytes; PTH gene; Pathogenesis; Pathway interactions; Patients; Phase; Phenotype; Phosphorus; Physiologic calcification; Plague; Population; Process; Proliferating; Renal Osteodystrophy; Reporting; Research; Resistance; Role; Signal Pathway; Signal Transduction; Signaling Protein; Small Interfering RNA; Testing; Western Blotting; beta catenin; bone; bone cell; bone health; bone turnover; dentin matrix protein 1; disease phenotype; fibroblast growth factor 23; handicapping condition; improved; in vivo; innovation; insight; kidney dysfunction; knock-down; mineralization; novel; response; skeletal; skeletal disorder; stem; therapeutic target; therapy development

ABSTRACT Bone disease in chronic kidney disease (CKD) has been traditionally defined by changes in bone turnover stemming from altered circulating parathyroid hormone (PTH) concentrations. However, skeletal mineralization defects and increased numbers of fibroblast growth factor 23 (FGF23)-expressing osteocytes are present in children with very early CKD; these abnormalities are unrelated to circulating calcium, phosphorus, and PTH concentrations and contribute to bone deformities, fractures, poor growth, and cardiovascular disease that are not treated by current therapies. Our preliminary data suggest that osteoblasts and osteocytes from CKD patients have impaired maturation. Our data demonstrate that locally secreted signals regulate this maturation failure in primary osteoblast in vitro, likely via the Wingless/Integrin 1 (Wnt) signaling pathway. Local deficiencies in extracellular matrix concentrations dentin matrix protein 1 (DMP1), with subsequent alterations in Wnt signaling, may contribute to impaired osteocyte maturation, excess FGF23 expression, and skeletal mineralization defects in vivo. To test the hypothesis that CKD-mediated skeletal mineralization defects occur in response to abnormal secretion of local factors critical to osteoblast and osteocyte maturation, we will first establish that osteocyte maturation defects co-occur with or precede skeletal mineralization defects in vivo. We will evaluate markers of osteocyte maturity by immunohistochemistry and immunofluorescence in intact human iliac-crest sections from patients with early and late CKD. We will correlate expression of these markers with histomorphometric and quantitative backscatter electron imaging (qBEI) measures of bone mineralization. We will evaluate expression and location of DMP1 and Wnt signaling proteins in whole bone and in extracellular matrix by immunohistochemistry, immunofluorescence, and Western blot analysis. Next, we will characterize CKD osteoblast maturation and determine which secreted factor(s) modulate osteoblast maturation and Wnt signaling in vitro. We will characterize markers of osteoblast maturity in control and CKD osteoblasts during proliferative, matrix-generating, and mineralizing phases of maturation. We will rescue the CKD phenotype in the presence of healthy extracellular matrix and conditioned media and we will characterize signaling pathways affected by phenotype rescue. We will identify specific factors missing from CKD extracellular matrix that rescue the CKD phenotype and normalize Wnt signaling in vitro. We will attempt to recreate the CKD phenotype by siRNA knockdown of these same factors in control cells. At the completion of this study, we anticipate insight into the secreted factors and mechanistic pathways that contribute to osteoblast and osteocyte maturation failure in CKD. We anticipate that these findings will propel further research into understanding the mechanisms of defective skeletal mineralization in CKD and in defining therapeutic targets for improving bone health in the CKD population.

摘要 慢性肾脏病（CKD）中的骨疾病传统上定义为骨转换的变化 源于循环甲状旁腺激素（PTH）浓度的改变。然而，骨骼矿化 缺陷和增加的成纤维细胞生长因子23（FGF 23）表达的骨细胞的数量存在于 患有极早期CKD的儿童;这些异常与循环钙、磷和PTH无关 浓度和有助于骨畸形，骨折，生长不良和心血管疾病， 目前的治疗方法无法治疗。我们的初步数据表明，来自CKD的成骨细胞和骨细胞 患者的成熟度受损。我们的数据表明，局部分泌的信号调节这种成熟 体外原代成骨细胞的失败，可能是通过无翼/整合素1（Wnt）信号通路。当地 牙本质基质蛋白1（DMP 1）细胞外基质浓度不足，随后发生改变 在Wnt信号传导中，可能导致骨细胞成熟受损、FGF 23过度表达和骨骼肌损伤。 体内矿化缺陷。 为了检验CKD介导的骨骼矿化缺陷是由于异常的 分泌对成骨细胞和骨细胞成熟至关重要的局部因子，我们将首先建立骨细胞 成熟缺陷在体内与骨骼矿化缺陷同时发生或先于骨骼矿化缺陷。我们将评估 正常人髂嵴骨细胞成熟标志物的免疫组织化学和免疫荧光研究 早期和晚期CKD患者的切片。我们将这些标记物的表达与 骨矿化的组织形态学和定量背散射电子成像（qBEI）测量。我们 将评估DMP 1和Wnt信号蛋白在整个骨和细胞外的表达和位置， 免疫组化、免疫荧光和Western印迹分析。接下来，我们将描述 CKD成骨细胞成熟，并确定哪些分泌因子调节成骨细胞成熟 和体外Wnt信号转导。我们将描述对照组和CKD组成骨细胞成熟度的标志物 成骨细胞在增殖、基质生成和矿化成熟阶段。我们将拯救 CKD表型在健康的细胞外基质和条件培养基的存在下，我们将表征 受表型拯救影响的信号通路。我们将确定CKD缺失的特定因素 细胞外基质，拯救CKD表型和正常化Wnt信号在体外。我们将尝试 通过在对照细胞中siRNA敲低这些相同的因子来重建CKD表型。 在这项研究完成后，我们预计深入了解分泌因子和机制途径， 导致CKD中成骨细胞和骨细胞成熟失败。我们预计，这些发现将推动 进一步研究了解慢性肾病骨骼矿化缺陷的机制并定义 改善CKD人群骨健康的治疗目标。