Extracellular determinants of polycystic kidney disease severity

描述(申请人提供)：对导致常染色体隐性遗传性多囊肾病(ARPKD)患者的一系列并发症的遗传修饰物知之甚少。了解这些遗传修饰物是什么，它们如何破坏器官功能，以及它们如何加重ARPKD，将导致旨在缓解患者痛苦的更有效和量身定制的疗法。实验结果有力地表明，蛋白多糖(PG)的减少是PKD病变严重程度的修饰物。因此，我们研究小组的长期目标是确定负责任的前列腺素，并确定它们在PKD发病机制中的作用，以期开发出降低病变严重程度的靶向治疗方法。在追求这一目标的过程中，我们的研究已经在肝脏中发现了几种PG，当缺乏糖胺多聚糖时，它们是囊变的候选物质。基于大量的初步数据，这项应用的中心假设是PG水平降低是囊性发育的重要调节剂，并可以通过增加典型的Wnt/β-catenin途径活性来促进ARPKD囊性发育和进展。作为迈向我们长期目标的第一步，这项应用的总体目标是通过以下目标来检验这一假说：1)在Fc活性降低和XylT2活性丧失的ARPKD小鼠模型中，确定PG减少对肝和肾囊肿发展的影响。将使用全面的组织学方法和磁共振成像来测量这些小鼠的囊性发育、严重程度和皮损？-连环蛋白水平；2)通过建立显著降低肾脏PGs的成年Xylt2-/-小鼠来确定PGs在肾囊肿发展中所起的作用。将对结果小鼠的肾功能、囊性发育和糖胺多糖含量进行评估，并与对照组进行比较；3)确定最近发现的候选PGs在调节培养的胆道和分离的胆管上皮细胞中Wnt/？-catenin信号的重要性。野生型和Xylt2-/-细胞和胆道将在Wnt/？-catenin活化增加的条件下培养，在核心蛋白存在的情况下，有或没有糖胺聚糖。WNT/？-catenin信号将通过荧光素酶报告分析和免疫组织化学途径中间产物进行评估，并与细胞增殖和囊变相关。这项拟议研究的基本原理是基于这样一个假设，即了解ARPKD病变修饰剂的分子机制将从根本上改善对PKD病变发展的理解。因此，拟议的研究与PA-07-293有关，因为它定义了“引起、加重或改善PKD的分子和途径的功能”，并确定了“在啮齿动物模型中……改变临床表型的特定基因。”这项拟议的研究具有重要意义，因为识别遗传修饰物并了解它们对临床变异的影响有望促进ARPKD患者的个体化治疗和预后。

DESCRIPTION (provided by applicant): Little is understood about the genetic modifiers that are responsible for a wide range of complications that afflict autosomal recessive polycystic kidney disease (ARPKD) patients. Understanding what these genetic modifiers are, how they disrupt organ function, and how they aggravate ARPKD, will lead to more effective and tailored therapies aimed at ameliorating patient suffering. Experimental results strongly suggest that reduced proteoglycans (PGs) are modifiers of PKD lesion severity. Therefore, the broad long-term goal of our research group is to identify the responsible PGs and determine their role in the pathogenesis of PKD with the intent of developing targeted treatments that reduce lesion severity. In pursuit of this goal, our investigations have identified several PGs in liver that when lacking glycosaminoglycans are candidates for cyst development. Based on extensive preliminary data, this application's central hypothesis is that reduced PG levels are important modifiers of cyst development and can augment ARPKD cyst development and progression mediated by increased canonical Wnt/?-catenin pathway activity. The overall objective of this application, as a first step towards our long-term goal, is to test this hypothesis with the following aims: 1) To determine the impact of reduced PG on the development of liver and renal cysts in an ARPKD mouse model with decreased FC activity and loss of XylT2 activity. Cyst development, severity, and lesional ?-catenin levels in these mice will be measured using comprehensive histological methods and magnetic resonance imaging; 2) To determine the role that PGs have in renal cyst development by creating adult Xylt2-/- mice that have significantly reduced renal PGs. Renal function, cystic development, and glycosaminoglycan content will be assessed in the resultant mice and compared to controls; 3) To determine the importance of recently identified candidate PGs in the regulation of Wnt/?- catenin signaling in cultured biliary tracts and in isolated biliary epithelial cells. Wild type and Xylt2-/- cells and biliary tracts will be cultured under conditions of increased Wnt/?-catenin activation in the presence of core protein with and without glycosaminoglycans. Wnt/?-catenin signaling will be assessed by luciferase reporter assays and by immunohistochemistry for pathway intermediates and correlated with cellular proliferation and cyst development. The rationale of the proposed research is based on the assumption that understanding the molecular mechanism of ARPKD lesion modifiers will translate into fundamental improvements in understanding PKD lesion development. Thus the proposed research is relevant to PA-07- 293 in regards to defining "the function of molecules and pathways that cause, aggravate or ameliorate PKD" and identifying "in rodent models...specific genes that modify the clinical phenotype." The proposed research is significant because the identification of genetic modifiers and understanding their impact on clinical variation is expected to advance individualized ARPKD patient treatment and prognostication.