Live imaging analyses of the mechanisms required for coordinated urinary tract peristalsis in lower-order and higher-order mammalian species

摘要 上尿路(UUT)近端至远端蠕动收缩(UUT)平滑肌衣推进排泄 从肾脏到膀胱。蠕动过程中的缺陷非常普遍，具有重要的临床意义。 例如，肾脏的尿流出障碍会引起肾组织的压力调节扩张，或者 肾积水。肾积水是儿童中最常见的异常，在1%的儿童中发现 新生儿，是导致儿童肾功能衰竭的主要原因。这个项目的总体目标是更好地 了解UUT的正常生理学和病理生理学。事实上，尽管发病率很高， 与尿路功能障碍有关，触发肾脏起搏器活动的潜在机制 UUT的蠕动仍然难以捉摸。为了研究这一过程，我们开发了新的实时成像 记录电和收缩兴奋在完整的UUT中传播的技术。结果 我们的研究表明，超极化激活阳离子(HCN)通道高度表达 定位于小鼠UUT的肾起搏组织。HCN通道抑制废除UUT起搏器 活动，并导致失去协调蠕动。而不是近端到远端的收缩和 在对照UUT中观察到的电刺激，HCN抑制的外植体表现出几乎同时的电刺激 激活整个UUT和抽搐样收缩活动。因此，我们已经在体外证明了 UUT的HCN+细胞是设定起始点和协调UUT蠕动的肾脏起搏器。此外，我们 最近发现HCN通道在猪肾起搏器组织中的表达是保守的 和人类的尿路，它们有着独特的解剖学和生理学。在本提案的目标1中，我们将使用 UUT中缺乏HCN+细胞的新型小鼠肾积水模型。我们将使用实时成像 我们已经开发的技术来确定体内HCN+细胞的丢失是否会导致异常的UUT蠕动 是肾积水的原因。目标1还将包括机制研究，以开始阐明转录 管理HCN+起搏器的网络。对于目标2，我们最近开发了一种新的外植体系统来 直观地显示猪UUT中蠕动的电学和收缩特性。我们将使用这个 用于确定协调UUT近距离蠕动是否需要HCN通道电导的植入体系统 与人类同源。这些研究的结果将提供对潜在机制的迫切需要的洞察 低级和高级哺乳动物都存在正常和异常的UUT蠕动。长期 这些研究的翻译含义包括开发新的治疗和诊断方法 尿路疾病，如肾积水。

ABSTRACT Proximal-to-distal peristaltic contractions of the upper urinary tract (UUT) smooth muscle coat propel waste from the kidney to the bladder. Defects in the peristaltic process are highly prevalent and clinically significant. For example, impaired urine outflow from the kidney causes pressure mediated dilation of renal tissues, or hydronephrosis. Hydronephrosis is the most commonly observed abnormality in children, detected in 1% of newborns, and is a leading cause of pediatric kidney failure. The overall goal of this project is to better understand the normal physiology and pathophysiology of the UUT. Indeed, despite the high morbidity associated with urinary tract dysfunctions, the mechanisms underlying renal pacemaker activity that triggers UUT peristalsis have remained elusive. To study this process, we have developed novel live imaging techniques to record the propagation of electrical and contractile excitation throughout the intact UUT. Results of our studies have revealed that hyperpolarization activate cation (HCN) channels are highly expressed and localized to renal pacemaker tissues of the murine UUT. HCN channel inhibition abolishes UUT pacemaker activity, and results in a loss of coordinated peristalsis. Instead of the proximal-to-distal contractile and electrical excitation observed in control UUTs, HCN inhibited explants exhibit near-simultaneous electrical activation throughout the UUT and twitch-like contractile activity. Thus, we have demonstrated ex-vivo that HCN+ cells of the UUT are renal pacemakers that set the origin and coordinate UUT peristalsis. Moreover, we have recently discovered that HCN channel expression is conserved to renal pacemaker tissues of the porcine and human urinary tracts, which share a unique anatomy and physiology. In Aim 1 of this proposal we will use a novel mouse model of hydronephrosis that lacks HCN+ cells in the UUT. We will use the live imaging techniques we have developed to determine if loss of HCN+ cells in vivo results in aberrant UUT peristalsis that underlies hydronephrosis. Aim 1 will also include mechanistic studies to begin to elucidate the transcriptional networks regulating HCN+ pacemakers. For Aim 2, we have recently developed a novel explant system to directly visualize the electrical and contractile properties of peristalsis in the porcine UUT. We will use this explant system to determine if HCN channel conductance is required for coordinated UUT peristalsis in a close homolog to humans. Results of these studies will provide much needed insight into the mechanisms underlying normal and aberrant UUT peristalsis in both lower-order and higher-order mammalian species. Long term translational implications of the studies include the development of novel treatments and diagnostics for uropathies such as hydronephrosis.