Role of Tamm-Horsfall Protein in Urinary Tract Defense

描述（由申请人提供）：在人类尿液中的所有蛋白质中，Tamm-Horsfall蛋白（缩写为THP，也称为尿调素）是迄今为止含量最多的。THP是由肾脏功能特化的上皮细胞产生的，包括肾袢的厚升肢。尽管THP丰富，肾脏特异性和进化保守，但关于THP功能的问题仍然存在。在上一个资助期内，我们不仅在了解THP的体内生物学功能方面取得了重大进展，而且在了解THP与肾脏疾病的关系方面也取得了重大进展。我们的方法是使用基因工程技术产生THP缺失的敲除（KO）小鼠或表达THP点突变的转基因小鼠。我们发现THP KO小鼠极易发生1型纤溶大肠杆菌的实验性尿路感染；它们自发地在肾乳头间隙形成由磷酸钙组成的肾内钙化；而且它们比野生型的同类更容易受到实验性急性肾损伤。此外，我们发现表达THP突变的转基因小鼠在肾小管功能和高尿酸血症方面表现出明显的缺陷。重要的是，在我们的小鼠模型中观察到的肾内钙化和高尿酸血症分别与人类某些类型的特发性肾结石和遗传性高尿酸血症肾病有着惊人的相似之处。这项更新提案的主要目标是显着扩展和深化我们对THP缺陷导致人类相关疾病的分子和细胞机制的理解。具体来说，我们将通过进行超结构、化学和蛋白质组成分析，研究THP KO小鼠间质钙化如何在空间和时间上起源和进化。我们将研究肾上皮细胞如何摄取小管内晶体以及这如何导致细胞毒性。我们将通过产生化合物来确定THP KO小鼠中真正肾结石的形成是否依赖于尿液中磷酸钙或草酸钙的过饱和，通过基因工程小鼠自然发展这些条件。最后，我们将利用我们最近建立的转基因模型，确定特定化学伴侣在缓解人类相关THP突变病理效应方面的体内作用。总之，这四个相互关联的系列研究应该对理解THP的生物学功能和疾病贡献产生重大影响，并为THP相关肾脏疾病的临床治疗提供见解。

DESCRIPTION (provided by applicant): Of all the proteins in human urine, Tamm-Horsfall protein (abbreviated as THP; also named uromodulin) is by far the most abundant. THP is made by kidneys' functionally specialized epithelial cells comprising the thick ascending limb of loop o Henle. Despite its abundance, kidney-specificity and evolutionary conservation, questions about THP's function(s) remain. During the last funding period, we have made major progress in understanding not only the in vivo biological functions of THP, but also its involvement in kidney diseases. Our approach has been to generate, using genetic engineering, knockout (KO) mice deficient for THP or transgenic mice expressing a point mutation of THP. We found that the THP KO mice are highly prone to experimental urinary tract infections by type 1-fimbriated E. coli; that they spontaneously develop intra-renal calcification consisting of calcium phosphate in the interstitial space of the renal papillae; and that they are more susceptible to experimental acute kidney injury than their wild-type counterparts. Additionally, we found that the transgenic mice expressing a THP mutation exhibit marked defects in tubular function and hyperuricemia. Importantly, the intra-renal calcification and hyperuricemia observed in our mouse models bear striking resemblances to certain types of idiopathic kidney stones and hereditary hyperuricemic nephropathies in humans, respectively. The main goal of this renewal proposal is to significantly expand and deepen our understanding of the molecular and cellular mechanisms whereby defects of THP lead to human-relevant disease conditions. Specifically, we will investigate how interstitial calcification in THP KO mice originate and evolve in a spatial and temporal manner by performing ultra-structural and chemical and protein composition analyses. We will examine how renal epithelial cells uptake the intratubular crystals and how this leads to cytotoxicity. We will determine whether formation of bona-fide kidney stones in THP KO mice relies on urinary super saturation of calcium phosphate or calcium oxalate by generating compound, genetically engineered mice that naturally develop these conditions. Finally, we will determine the in vivo effects of specific chemical chaperones in relieving the pathological effects of human-relevant THP mutation, utilizing the transgenic models we recently generated. Together, these four interconnected series of studies should have a major impact on understanding the biological functions and disease contributions of THP and offer insights into how THP-associated kidney diseases can be better managed clinically.