Regulation of Detrusor Overactivity by Cellular Stress in Bladder Ischemia

这项更新研究提案的总体目标是确定缺血在脑缺血发展中的作用， 非梗阻膀胱逼尿肌过度活动另一个目标是检查靶向细胞的功效， 应激反应途径，以预防或逆转膀胱功能障碍。下尿路症状（LUTS） 对生活质量有显著影响的一系列排尿症状。之时尚 退伍军人中膀胱功能障碍和LUTS的发病率几乎是退伍军人中的五倍。 普通民众。结果表明，退伍军人与LUTS有一个更差的生活质量评分比退伍军人 没有LUTS。在大多数情况下，特别是在老年退伍军人中，LUTS导致失眠，焦虑，疲劳， 萧条大多数男性LUTS病例归因于膀胱出口梗阻（BOO）， 前列腺肥大（BPE）。然而，已经表明，在大约三分之一至一半以上的时间里， 例，LUTS与BPE或BOO无关。这些观察结果表明，除了BOO， 膀胱中与年龄相关的局部变化导致LUTS。越来越多的基础和临床证据 研究表明，与年龄相关的膀胱缺血起关键作用。人膀胱血损伤 流动和膀胱缺血的发展已经在患有LUTS的老年患者中得到证实。但 在膀胱缺血中导致逼尿肌过度活动的潜在机制仍然很大程度上未知。我们 初步数据表明，缺血引起细胞应激并损害细胞防御能力 通过损害细胞能量传感器腺苷一磷酸激活的蛋白激酶α-2（AMPK-2）， α 2）。细胞应激和AMPK-α 2缺陷引起一种独特的应激反应RNA， 链，即双链RNA（dsRNA），导致AMPK-α 2/dsRNA应激反应的激活 通路AMPK-α 2/dsRNA途径似乎损害了毒蕈碱M2和M3受体，引起了 收缩蛋白的翻译后修饰，增加平滑肌收缩， 逼尿肌过度活动我们假设"慢性缺血是发展中的一个独立因素， 非梗阻膀胱逼尿肌过度活动其机制涉及激活细胞应激 通过AMPK-α 2/dsRNA途径的反应，通过修饰平滑肌细胞来触发膀胱过度活动症收缩。 肌肉收缩成分"。使用完善的膀胱缺血模型沿着敲除小鼠， 细胞培养转染和基因缺失技术，我们提出了三个具体目标。在目标I中，我们将定义 AMPK-α 2/dsRNA应激反应通路在膀胱缺血中的分子调控我们将确定 AMPK-α 2损伤机制、膀胱缺血中dsRNA的定量、克隆及串扰的定义 AMPK-α 2和dsRNA之间的作用机制。在目标II中，我们将通过以下方式定义过度活跃收缩的调节： AMPK-α 2/dsRNA通路与膀胱缺血我们将确定AMPK-α 2/dsRNA通路如何引起 通过毒蕈碱M2和M3的转录和翻译后修饰引起膀胱收缩过度 受体。我们将研究治疗策略，以防止M2和M3的应激调节修饰， 逆转逼尿肌过度活动在目标III中，我们将定义AMPK-α 2/dsRNA下游的信号传导机制 在膀胱缺血中修饰收缩蛋白的途径。我们将确定AMPK-α 2/dsRNA- 在肌动蛋白-α 1和肌球蛋白重链（MHC）的修饰中调节氧化还原和PKR信号传导， 平滑肌钙吸收的能力我们将研究治疗策略，以防止氧化还原和PKR介导的 肌动蛋白-α 1和MHC修饰与膀胱缺血中逼尿肌过度活动的逆转结束时 这些研究，我们将：（1）提供了新的见解逼尿肌过度活动的病理生理学在 （2）阐明AMPK-α 2/dsRNA通路与膀胱过度活动的分子联系 膀胱收缩和3）在过度活动的膀胱收缩中定义的应激致敏机制。我们 建议的研究可能会导致新的诊断和治疗策略对逼尿肌过度活动/下尿路症状。

The overall goal of this renewal research proposal is to determine the role of ischemia in the development of detrusor overactivity in the non-obstructed bladder. Another goal is to examine the efficacy of targeting cellular stress response pathways to prevent or reverse bladder dysfunction. Lower urinary tract symptoms (LUTS) are bothersome constellation of voiding symptoms with significant impact on quality of life. The prevalence of bladder dysfunction and LUTS among Veterans is almost five folds higher in comparison with its incidence in the general population. It was shown that Veterans with LUTS had a worse quality of life score than Veterans without LUTS. In most cases, particularly in elderly Veterans, LUTS resulted in insomnia, anxiety, fatigue, and depression. Most cases of LUTS in male are attributed to bladder outlet obstruction (BOO) due to benign prostatic enlargement (BPE). However, it has been shown that in approximately one third to more than one half of cases, LUTS are not associated with BPE or BOO. These observations suggest that, in addition to BOO, aging-related local changes in the bladder contribute to LUTS. Growing evidence from basic and clinical research suggests that aging-associated bladder ischemia play a key role. Impairment of human bladder blood flow and the development of bladder ischemia have been verified in elderly patients with LUTS. However, the underlying mechanisms contributing to detrusor overactivity in bladder ischemia remain largely unknown. Our preliminary data suggest that ischemia provokes cellular stress and compromises cellular defensive capacity by impairing the cellular energy sensor adenosine monophosphate-activated protein kinase alpha-2 (AMPK- α2). Cell stress and defective AMPK-α2 give rise to a unique stress response RNA with two complementary strands namely double-stranded RNA (dsRNA) leading to activation of the AMPK-α2/dsRNA stress response pathway. The AMPK-α2/dsRNA pathway seems to compromise muscarinic M2 and M3 receptors, provoke post-translational modifications of contractile proteins, increase smooth muscle contractions and engender detrusor overactivity. We hypothesize that “chronic ischemia is an independent factor in the development of detrusor overactivity in the non-obstructed bladder. The mechanism involves activation of cellular stress response via AMPK-α2/dsRNA pathway that triggers overactive bladder contractions by modification of smooth muscle contractile elements”. Using a well-established bladder ischemia model along with knockout mice and cell culture transfection and gene deletion technologies, we propose three specific aims. In aim I, we will define molecular regulation of the AMPK-α2/dsRNA stress response pathway in bladder ischemia. We will determine the mechanism of AMPK-α2 impairment, quantify and clone dsRNA in bladder ischemia and define crosstalk mechanisms between AMPK-α2 and dsRNA. In aim II, we will define regulation of overactive contractions by AMPK-α2/dsRNA pathway in bladder ischemia. We will determine how AMPK-α2/dsRNA pathway provokes overactive bladder contractions by transcriptional and post-translational modifications of muscarinic M2 and M3 receptors. We will examine therapeutic strategies to prevent stress-regulated modifications of M2 and M3 and reverse detrusor overactivity. In aim III, we will define signaling mechanisms downstream of AMPK-α2/dsRNA pathway that modify contractile proteins in bladder ischemia. We will determine the role of AMPK-α2/dsRNA- regulated redox and PKR signaling in modifications of actin-α1 and myosin heavy chain (MHC) and regulation of smooth muscle calcium uptake. We will examine therapeutic strategies to prevent redox- and PKR-mediated actin-α1 and MHC modifications and reverse detrusor overactivity in bladder ischemia. At the conclusion of these studies, we will have: (1) provided new insights into the pathophysiology of detrusor overactivity in the non-obstructed bladder; (2) elucidated the molecular link between AMPK-α2/dsRNA pathway and overactive bladder contractions and 3) defined stress sensitization mechanisms in overactive bladder contractions. Our proposed research may lead to novel diagnostic and therapeutic strategies against detrusor overactivity/LUTS.