Modifying kidney injury through p53 signaling.

描述(由申请人提供)：在我们对急性肾损伤(AKI)中决定功能损害的机制的理解上存在着根本的差距。这一差距的持续存在带来了一个重要的临床问题，因为预防、治疗或加速AKI康复的治疗干预没有也不能完全实现，直到这一差距被 满员了。长期目标是了解肾脏中P53蛋白在健康和疾病下的细胞和分子调节功能，并针对这些机制开发新的和特定的治疗干预措施。这项应用的目的是为了选择性地确定AKI期间P53如何调节肾脏基础代谢和调节炎症，这是朝着实现长期目标迈出的一步。这一应用的中心假设是，P53是肾脏葡萄糖代谢的重要调节器，以及AKI期间的先天免疫反应。这一假设是在现有文献和申请者实验室强大的初步数据的基础上提出的。这项拟议研究的基本原理是，一旦确定了P53如何调节AKI期间的肾脏新陈代谢和炎症反应，它就允许对P53进行战略性调节，作为预防和治疗AKI的一种新的创新药理学方法。中心假说将通过追求两个具体目标来检验：1)通过先进的成像和生化技术，描述通过抑制P53将肾脏的葡萄糖代谢转变为保护性糖酵解表型的能力；2)确定肾小管和白细胞P53在AKI过程中调节炎症反应的相对作用。根据第一个目标，体内对p53的抑制将通过药物抑制(匹非菊酯α)、siRNA沉默技术和有针对性的基因缺失来实现。细胞水平的糖酵解和氧化磷酸化的转变将通过活体动物肾脏的活体多光子显微镜通过使用针对这两个主要代谢途径的荧光探针来确定，肾脏代谢的全球转变将通过PET扫描技术进行量化。这些研究将得到测量已知受p53调控的关键代谢酶变化的技术的补充。这种代谢转变的保护能力将在AKI模型中进行检验。在第二个目标下，将使用类似的策略在体内抑制P53。流式细胞术结合先进的分子和细胞生物学技术将用于确定急性心肌梗死期间炎症反应的变化。这些研究将得到嵌合小鼠模型的补充，以进一步弄清其中涉及的机制。这种方法是创新的，因为我们操纵p53信号以改变肾脏新陈代谢和先天免疫的方法代表了与现状的新的和实质性的背离。这项拟议的研究意义重大，因为它是一系列研究中的下一步，有望导致针对p53的更具体和有针对性的治疗干预措施的开发，从而预防和限制AKI的后续功能障碍。

DESCRIPTION (provided by applicant): There is a fundamental gap in our understanding of the mechanisms that determine functional impairment in acute kidney injury (AKI). Continued existence of this gap presents an important clinical problem because therapeutic interventions to prevent, treat or hasten recovery in AKI have not and cannot be fully realized until this gap is filled. The long term goal is to understand the cellular and molecular regulatory functions of the protein p53 in the kidney under health and disease and to develop novel and specific therapeutic interventions targeting these mechanisms. The objective of this application, which is a step toward attainment of the long term goal, is to selectively determine how p53 regulates basal kidney metabolism and modulates inflammation during AKI. The central hypothesis of this application is that p53 is an important modulator of glucose metabolism in the kidney, as well as the innate immune response during AKI. This hypothesis has been formulated on the basis of existing literature and strong preliminary data from the applicants' laboratories. The rationale fo the proposed research is that once it is determined how p53 regulates kidney metabolism and inflammatory responses during AKI, it then permits the strategic modulation of p53 as a new and innovative pharmacological approach towards preventing and treating AKI. The central hypothesis will be tested by pursuing two specific aims: 1) Delineate, by way of advanced imaging and biochemical techniques, the capacity to shift kidney glucose metabolism towards a protective glycolytic phenotype through inhibition of p53; and 2) Determine the relative roles of tubular and leukocyte p53 in regulating the inflammatory response during AKI. Under the first aim, in vivo inhibition of p53 will be accomplished by pharmacologic inhibition (pifithrin alpha), siRNA silencing techniques, and targeted genetic deletion. Shifts in glycolysis and oxidative phosphorylation at the cellular level will be determined with intravital multiphoton microscopy of the kidney in living animals by employing fluorescent probes specific for the two major metabolic pathways and global shifts in kidney metabolism will be quantified with PET scanning techniques. These studies will be complemented by techniques to measure changes in key metabolic enzymes known to be modulated by p53. The protective capacity of this metabolic shift will be examined in models of AKI. Under the second aim, similar strategies will be used to inhibit p53 in vivo. Flow cytometry coupled with advanced molecular and cell biological techniques will be used to define alterations in the inflammatory response during AKI. These studies will be complemented by chimeric mouse models to further discern the mechanisms involved. The approach is innovative, because our approach to manipulate p53 signaling in order to modify kidney metabolism and innate immunity represents a new and substantial departure from the status quo. The proposed research is significant, because it is the next step in a continuum of research that is expected to lead to the development of more specific and targeted therapeutic interventions aimed at p53 that will prevent and limit subsequent dysfunction in AKI.