Regulation of Detrusor Overactivity by Cellular Stress in Bladder Ischemia

这项更新研究方案的总体目标是确定缺血在脑缺血发展中的作用。 非梗阻性膀胱的逼尿肌过度活动。另一个目标是检查靶向细胞的有效性。 预防或逆转膀胱功能障碍的应激反应途径。下尿路症状(LUT)是 令人讨厌的排尿症状，对生活质量有重大影响。疾病的流行 退伍军人中的膀胱功能障碍和LUTS的发病率几乎是#年的五倍 普通民众。结果表明，退伍军人LUTS患者的生活质量得分低于退伍军人 没有LUTS。在大多数情况下，尤其是在老年退伍军人中，LUT会导致失眠、焦虑、疲劳和 抑郁症。男性LUTS多为良性膀胱出口梗阻(BOO) 前列腺增生症(BPE)然而，研究表明，在大约三分之一到一半以上的情况下 在病例中，LUT与BPE或BOO无关。这些观察表明，除了Boo之外， 与衰老相关的膀胱局部变化有助于LUTS。越来越多的基础和临床证据 研究表明，与衰老相关的膀胱缺血起着关键作用。人体膀胱血的损伤 在老年LUTS患者中，血流和膀胱缺血的发展已得到证实。然而， 导致逼尿肌过度活动的潜在机制在很大程度上仍不清楚。我们的 初步数据表明，缺血会引发细胞应激，损害细胞防御能力。 通过损害细胞能量感受器一磷酸腺苷激活的蛋白激酶α-2(AMPK-2) α2)。细胞应激和AMPK-α2缺陷引起一个独特的应激反应rna，具有两个互补 链即导致AMPK-α2/dsRNA应激反应的双链RNA 路径。AMPK-α2/dsRNA通路似乎损害了M_2和M_3受体，刺激 收缩蛋白的翻译后修饰，增加平滑肌收缩并产生 逼尿肌过度活动。我们假设“慢性缺血是血管紧张性心脏病发生的一个独立因素。 非梗阻性膀胱的逼尿肌过度活动。其机制涉及激活细胞应激。 通过AMPK-α2/dsRNA途径的反应，通过修改Smooth来触发过度活跃的膀胱收缩 肌肉收缩元素“。使用已建立的膀胱缺血模型以及基因敲除小鼠和 针对细胞培养、转基因和基因缺失技术，我们提出了三个具体目标。在Aim 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决定 膀胱缺血时AMPK-α-2损伤机制、双链RNA定量和克隆及串扰定义 AMPK-α-2与双链RNA的作用机制。在AIM II中，我们将通过以下方式定义对过度活动收缩的调节 AMPK-α2/dsRNA通路在膀胱缺血中的作用我们将确定AMPK-α2/dsRNA通路是如何激发 毒扁豆碱M2和M3的转录和翻译后修饰引起的过度活动性膀胱收缩 感受器。我们将研究治疗策略，以防止M2和M3的应激调节修饰和 逆转逼尿肌过度活动。在AIM III中，我们将定义AMPK-α2/dsRNA下游的信号机制 膀胱缺血时调节收缩蛋白的途径。我们将确定AMPK-α2/双链RNA- 肌动蛋白-α1和肌球蛋白重链修饰中的氧化还原和蛋白酪氨酸激酶受体信号调控 平滑肌的钙摄取。我们将研究防止氧化还原和PKR介导的治疗策略 肌动蛋白-α1和MHC修饰与膀胱缺血时逼尿肌过度活动的逆转。在……结束时 这些研究，我们将有：(1)为逼尿肌过度活动的病理生理学提供了新的见解。 (2)阐明了AMPK-α-2/dsRNA通路与过度活跃的分子联系 膀胱收缩和3)定义了过度活跃的膀胱收缩的应激敏化机制。我们的 拟议的研究可能导致针对逼尿肌过度活动/下尿路综合征的新的诊断和治疗策略。

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.