Role of Tamm-Horsfall Protein in Urinary Tract Defense

描述（由申请人提供）：该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。维持泌尿系统内稳态的一个中心重要功能是防止过饱和尿液形成晶体。显然，需要一套强大的防御机制，但迄今为止，这些机制只得到了部分描述。在上一个批准期内，我们专注于Tamm-Horsfall蛋白（THP，或尿调素）在尿道防御中的体内作用。我们发现THP基因的失活使经尿道接种的1型纤维化大肠杆菌更易在小鼠膀胱中定植。此外，缺乏THP的小鼠产生自发的和化学诱导的肾钙晶体。肾结晶的诱导伴随着骨桥蛋白（OPN）的肾上皮细胞的显著增加，骨桥蛋白是骨矿化、血管钙化和肾结石形成的有效抑制剂，这提高了THP和OPN协同抑制肾结晶的可能性。在下一个授权期内，我们将通过关注THP在防止肾脏结晶中的作用，深入了解THP作为泌尿系统中关键的先天防御因子的作用。首先，我们将研究THP敲除和THP突变是否对肾尿酸处理有不同的影响，通过抑制/灭活THP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与人类相关的THP突变体。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钙水平的调节，还是间接由于其对肾上皮损伤的保护。第三，我们将验证THP和OPN是肾脏钙化的共同抑制剂的假设，通过产生THP/OPN双敲除，并将肾脏钙化的严重程度与单敲除进行比较。最后，我们将评估肾上皮细胞对THP缺乏的基因表达改变，以便更好地了解THP和其他大分子在调节肾脏结晶和其他肾脏功能中的作用。这些研究的结果将为重要的尿路疾病（如尿石症）的分子发病机制和干预策略提供新的线索，尿石症每年仅在美国就折磨数百万人。

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.