Protein Kinase C in the Repair of Cellular Functions

描述(申请人提供)：我们研究的长期目标是阐明涉及以下方面的信号事件和调控机制：1)与缺血/再灌流引起的急性肾功能衰竭(ARF)相关的肾功能丧失；2)ARF后的肾功能修复。肾脏是缺血/再灌注和氧化应激损伤的主要靶器官之一。肾近端小管细胞(RPTC)因其依赖线粒体功能和氧气(ATP)的产生以及大量的外源物质的生物转化能力而成为肾脏内这些损伤的主要靶点。蛋白激酶信号转导正逐渐成为调节线粒体功能的主要机制。本研究的目的是确定两种蛋白激酶C同工酶(PKC-？和PKC-？)的信号传导机制。在缺氧后RPTC的损伤和修复过程中，调节线粒体功能障碍、氧化应激和细胞存活。我们最近的研究表明，在受损的RPTC修复过程中，两种PKC亚型在调节线粒体功能方面发挥了关键作用。我们的初步数据表明，新的观察结果表明，在RPTC线粒体中存在PKC-，PKC-？，并且PKC可以磷酸化一些尚未确定的线粒体蛋白。我们证明了PKC？？激活介导了线粒体损伤后的功能障碍。抑制PKC的激活可促进线粒体功能的恢复，减少能量缺乏，减少缺氧和氧化剂损伤后RPTC的坏死。相反，在低氧和氧化剂诱导的损伤后，PKC-β的激活减少了线粒体功能障碍和RPTC的坏死。这一假设的中心假设是，PKC和PKC通过磷酸化线粒体氧化磷酸化装置和/或线粒体通透性转换孔的关键蛋白来不同地调节ATP的合成。以下具体目标将检验这一假设。特异性目标1将确定在RPTC损伤和修复过程中受PKC？和PKC？调节的特定的线粒体能量代谢途径。特异性目标2将确定PKC？和PKC？调节受损RPTC中线粒体能量代谢的蛋白。特异性目标3将确定PKC和/或PKC是否调节缺血肾脏线粒体的呼吸和ATP的产生，以及参与氧化磷酸化的蛋白质复合体是否是体内肾脏中PKC和/或PKC的靶点。这个项目完成后，我们将获得重要的新信息，帮助我们了解PKC？和PKC？？在损伤后肾脏修复中的重要性，并将为使用这些激酶作为新的治疗干预措施治疗肾功能衰竭的靶点提供见解。与公共卫生相关：目前治疗肾脏和其他器官缺血(氧气可获得性降低)造成的损伤的疗法是有限的，因为调节肾脏损伤和恢复的机制尚不清楚。这个项目将研究三种不同的酶(蛋白激酶)如何调节受损肾脏中能量的产生，以及肾脏从缺血造成的损伤中恢复。因此，在这个项目完成后，我们将有一个重要的新信息，将帮助我们了解这些激酶在肾脏修复中的重要性，并将为将这些激酶用作治疗肾功能衰竭的新疗法的可能性提供洞察力。

DESCRIPTION (provided by applicant): The long-term goal of our research is to elucidate the signaling events and regulatory mechanisms involved in: 1) the loss of renal functions associated with acute renal failure (ARF) as caused by ischemia/reperfusion and 2) the repair of renal functions following ARF. The kidney is one of the major target organs for ischemia/reperfusion and oxidative stress-induced damage. Renal proximal tubular cells (RPTC) are a major target of these insults within the kidney due to their dependence on mitochondrial function and oxygen for energy (ATP) generation and large capacity for biotransformation of xenobiotics. Protein kinase signaling is emerging as a major mechanism regulating mitochondrial function. The goal of this proposal is to determine signaling mechanisms through which two protein kinase C isozymes (PKC-??and PKC-?) regulate mitochondrial dysfunction, oxidative stress, and cell survival during injury and repair of RPTC following hypoxia. Our recent studies demonstrated a key role of two isoforms of PKC in regulating mitochondrial functions during repair of injured RPTC. Our preliminary data demonstrate the novel observation that PKC-?, PKC-??are present in RPTC mitochondria and that PKC phosphorylates a number of yet unidentified mitochondrial proteins. We demonstrated that PKC-??activation mediates mitochondrial dysfunction following injury. Inhibition of PKC-??activation promotes recovery of mitochondrial function, diminishes energy deficits, and decreases RPTC necrosis following hypoxia and oxidant-induced injury. In contrast, the activation of PKC-??reduces mitochondrial dysfunction and RPTC necrosis following hypoxia and oxidant-induced injury. The central hypothesis of this proposal is that PKC-??and PKC-??differentially regulate ATP synthesis by phosphorylating key proteins of mitochondrial oxidative phosphorylation apparatus and/or the mitochondrial permeability transition pore. The following specific aims will test this hypothesis. Specific Aim 1 will determine the specific mitochondrial pathways of energy metabolism that are regulated by PKC-??and PKC-??during RPTC injury and repair. Specific Aim 2 will identify proteins through which PKC-??and PKC- ??regulate mitochondrial energy metabolism in injured RPTC. Specific Aim 3 will determine whether PKC-??and/or PKC-??regulate mitochondrial respiration and ATP production in ischemic kidney and whether protein complexes involved in oxidative phosphorylation are mitochondrial targets for PKC-??and/or PKC-??in the kidney in vivo. Upon completion of this project, we will have important novel information that will help us understand the significance of PKC-??and PKC-??in kidney repair following injury and will provide insights into using these kinases as targets for new therapeutic interventions to treat renal failure. PUBLIC HEALTH RELEVANCE: Current therapies to treat injury caused by ischemia (reduced availability of oxygen) in the kidney and other organs are limited because the mechanisms that regulate renal injury and recovery are not well understood. This project will examine how three different enzymes (protein kinases) regulate production of energy in the injured kidney and the recovery of the kidney from injury caused by ischemia. Therefore, upon completion of this project we will have an important novel information that will help us understand the significance of these kinases in renal repair and will provide insights into the possibility of using these kinases as targets for new therapies against renal failure.