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）平滑肌被膜的近端至远端蠕动收缩推动废物 从肾脏到膀胱蠕动过程中的缺陷非常普遍且具有临床意义。 例如，从肾脏流出的尿液受损导致肾组织的压力介导的扩张，或 肾积水肾积水是儿童最常见的异常， 新生儿，并且是儿科肾衰竭的主要原因。该项目的总体目标是更好地 了解UUT的正常生理和病理生理。事实上，尽管发病率很高， 与尿路功能障碍相关，肾起搏器活动的潜在机制， UUT的入侵者仍然难以捉摸。为了研究这一过程，我们开发了新的实时成像技术， 记录完整UUT中电兴奋和收缩兴奋传播的技术。结果 我们的研究表明，超极化激活阳离子（HCN）通道是高表达的， 定位于鼠UUT的肾起搏器组织。HCN通道抑制废除了UUT起搏器 活动，并导致失去协调的关节炎。而不是近端到远端的收缩和 在对照UUT中观察到电激发，HCN抑制的外植体表现出几乎同时的电激发， 整个UUT的激活和抽搐样收缩活动。因此，我们已经证明了离体， UUT的HCN+细胞是设置原点并协调UUT起搏的肾脏起搏器。而且我们 最近发现HCN通道表达在猪的肾起搏组织中是保守的 和人类泌尿道，它们共享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+细胞的新型肾积水小鼠模型。我们将使用实时成像 我们已经开发了技术来确定体内HCN+细胞的损失是否导致异常的UUT增殖， 导致了肾积水目标1还将包括机制研究，开始阐明转录 调节HCN+起搏器的网络。对于Aim 2，我们最近开发了一种新的外植体系统， 直接可视化猪UUT中的电特性和收缩特性。我们将使用这个 外植系统，以确定是否需要HCN通道电导以在闭合时协调UUT外植 与人类同源。这些研究的结果将提供急需的深入了解的机制， 正常和异常的UUT在低级和高级哺乳动物物种中的分布。长期 这些研究的转化意义包括开发新的治疗和诊断方法， 尿路病如肾积水。

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.