Role of Tamm-Horsfall Protein in Urinary Track Defense

描述(申请人提供)：这个项目的长期目标是更好地了解尿路结石疾病或尿石症的分子发病机制。维持尿路系统动态平衡的一个核心功能是防止过饱和尿液形成结晶。显然，需要一套强大的防御机制，但到目前为止，这些机制只有部分特征。在上一次授予期间，我们重点研究了Tamm-Horsfall蛋白(THP，或尿调蛋白)在尿路防御中的体内作用。我们发现，THP基因的失活使小鼠易于通过经尿路接种1型菌毛的大肠杆菌进行膀胱定植。此外，缺乏THP的小鼠会出现自发的和化学诱导的肾脏钙结晶。随着肾脏晶体的诱导，骨桥蛋白(OPN)的肾上皮细胞显著增加，骨桥蛋白是一种有效的骨矿化、血管钙化和肾结石形成的抑制剂，这增加了THP和OPN在抑制肾脏结晶方面协同作用的可能性。在下一个授权期，我们将通过重点研究THP在防止肾脏结晶方面的作用，对THP作为泌尿系统关键天然防御因子的作用有更深入的了解。首先，我们将研究THP基因敲除和THP突变是否通过抑制/灭活THP基因敲除小鼠的尿酸酶或在THP启动子的指导下在肾上皮细胞中转基因表达人类相关的THP突变来对肾尿酸处理产生不同的后果。其次，我们将研究THP对肾脏结晶的保护作用是直接由于其对肾脏钙水平的调节，还是间接由于其对肾上皮损伤的保护作用。第三，我们将通过产生THP/OPN双基因敲除并比较肾脏钙化与单一基因敲除的严重程度，来验证THP和OPN是肾脏钙化的共同抑制物的假设。最后，我们将评估THP缺乏对肾上皮细胞基因表达的影响，以更好地了解THP和其他大分子在调节肾脏结晶和其他肾功能中的作用。这些研究的结果将为尿路结石等重要尿路疾病的分子发病机制和干预策略提供新的线索。仅在美国，尿石症每年就困扰着数百万人。

DESCRIPTION (provided by applicant): The long-term goal of this project is to gain a better understanding of the molecular pathogenesis of urinary stone disease or urolithiasis. A centrally important function in maintaining urinary system homeostasis is to prevent supersaturated urine from forming crystals. Clearly a powerful set of defense mechanisms is required, but these mechanisms have been only partially characterized to date. During the last granting period, we have focused on in vivo roles of Tamm-Horsfall protein (THP, or uromodulin) in urinary tract defense. We found that inactivation of the THP gene predisposes mice to bladder colonization by transurethrally inoculated type 1-fimbriated E. coli. In addition, mice lacking THP develop spontaneous and chemically induced renal calcium crystals. The induction of renal crystals is accompanied by a marked increase in renal epithelial cells of osteopontin (OPN), a potent inhibitor of bone mineralization, vascular calcification and renal stone formation, raising the possibility that THP and OPN can act synergistically in inhibiting renal crystallization. In the next granting period, we will gain deeper insights into the role of THP as a critical innate defense factor in the urinary system by focusing on its role in preventing renal crystallization. First, we will examine whether THP knockout and THP mutation have different consequences on renal uric acid handling, by either inhibiting/inactivating uricase in THP knockout mice or by transgenically expressing human-relevant THP mutants in renal epithelial cells under the direction of the THP promoter. Second, we will examine whether the protective role of THP against renal crystallization is due directly to its modulation on renal calcium level or due indirectly to its protection against renal epithelial injury. Third, we will test the hypothesis that THP and OPN are co-inhibitors of renal calcification, by generating THP/OPN double knockouts and comparing the severity of renal calcification with that in the single knockouts. Finally, we will assess gene expression alterations in the renal epithelial cells in response to THP deficiency in order to better understand the roles of THP and other macromolecules in modulating renal crystallization and other renal functions. Results from these studies will shed new light on the molecular pathogenesis and intervention strategies for important urinary tract diseases such as urolithiasis, which afflicts millions of people annually in the United States alone.