Understanding the Function of Tamm-Horsfall Protein in Acute Kidney Injury

描述(由申请人提供)： 急性肾损伤(AKI)是住院退伍军人死亡率和发病率的主要决定因素。了解AKI的病理生理学对于治疗的发展至关重要。本申请建议研究Tamm-Horsfall蛋白(THP)在AKI中的作用。该蛋白功能不明，仅在肾脏粗大的升支(TAL)中表达。这项研究的直接目标是确定THP在肾脏损伤和炎症中的一种新的保护作用。PI的长期目标是了解THP在AKI过程中的作用如何被调节，以达到预防和治疗的目的。中心假说是Tamm-Horsfall蛋白调节外髓缺血损伤引发的炎症反应；这种作用需要THP与周围的小管元件相互作用。这一假说是建立在PI产生的强有力的初步数据的基础上的，该数据表明THP在AKI中具有保护作用，并且与S3节段的炎症和损伤较少相关，S3节段是肾脏外髓中与TAL相邻的小管。以下具体目标将用于研究这一假说。目的1将定义THP和近端小管在缺血损伤中的相互作用，从而支持THP-S3的直接相互作用。这一目标将确定THP从TAL释放的基侧路径，以及其与S3节段受体相互作用的意义。目标2将确定THP调控的肾脏损伤中的特定炎症途径。目的是确定THP的保护作用部分是通过抑制特定的炎性趋化因子途径实现的。这项研究将使用THP基因敲除和野生型小鼠。AKI的动物模型是通过夹闭肾蒂造成的肾缺血再灌注损伤。体外研究将涉及近端小管和粗大上肢的细胞系。这项工作中使用的其他技术包括：免疫荧光共聚焦显微镜、免疫金电子显微镜、流式细胞术、实时荧光聚合酶链式反应、沉默RNA、蛋白质印迹、激光捕获显微切割和原位杂交。这项研究具有创新性，因为它将揭示THP的新调节功能，这些功能可能不仅与肾脏损伤有关，而且与其他形式的急性或慢性肾脏疾病有关。未来加强THP在肾脏中作用的策略将具有重要的治疗意义。

DESCRIPTION (provided by applicant): Acute kidney injury (AKI) is a major determinant of mortality and morbidity in hospitalized veterans. Understanding the pathophysiology of AKI is essential for the development of therapy. This application proposes to study the role of Tamm-Horsfall protein (THP) in AKI. This protein has unclear functions and is expressed exclusively in thick ascending limbs (TAL) of the kidney. The immediate goal of this research is to characterize a novel protective role of THP in kidney injury and inflammation. The PI's long term goal is to understand how the role of THP can be modulated during AKI for preventative and therapeutic purposes. The central hypothesis is that Tamm-Horsfall protein regulates the inflammatory response triggered by ischemic injury in the outer medulla; an effect that requires an interaction of THP with the surrounding tubular elements. This hypothesis has been formulated on the basis of strong preliminary data produced by the PI showing that THP is protective in AKI and is associated with less inflammation and injury to S3 segments, which are neighboring tubules to TAL in the kidney outer medulla. The following specific aims will be used to investigate this hypothesis. Aim 1 will define the interaction between THP and proximal tubules in ischemic injury, thereby supporting a direct THP-S3 interaction. This aim will define the basolateral path for THP release from TAL and the significance of its interaction with receptors on S3 segments Aim 2 will identify specific inflammatory pathways in kidney injury that are modulated by THP. The goal is to establish that the protective effect of THP is in part through the inhibition of a specific inflammatory chemokine pathway. This research will involve the use of THP knockout and wild type mice. The animal model used for AKI is renal ischemia-reperfusion injury achieved through renal pedicel clamping. In vitro studies will involve cell lines for proximal tubules and thick ascending limbs. Other techniques used in this work include: Immunofluoresence confocal microscopy, immuno-gold electron microscopy, flow cytometry, real time-PCR, silencing RNA, western blot, laser capture microdissection and in-situ hybridization. This research is innovative, because it will uncover novel regulatory functions for THP that may be relevant not only in kidney injury but also in other forms of acute or chronic renal disease. Future strategies that enhance the role of THP in the kidney will be of important therapeutic significance.