Role of adenylyl cyclase isoforms in collecting duct physiology & pathophysiology

描述（由申请人提供）：收集管主细胞（PC）负责最终调节肾钠和水的排泄。大多数形式的单基因高血压与PC钠重吸收紊乱有关，而PC水转运的改变伴随着水代谢紊乱。pcna和水运受多种自分泌、旁分泌和内分泌因素的影响。介导许多这些作用的关键机制涉及通过腺苷酸环化酶（AC）衍生的cAMP信号传导。PC表达AC3、4和6，这就提出了一个基本问题，即单个AC同工异构体在PC中发挥何种生物效应。我们设计了具有PC特异性AC3、4或6破坏的小鼠，并发现了令人兴奋的新证据，证明PC AC异构体AC3和6的独特作用。研究结果表明，AC6调节PC Na和水分转运，而AC3选择性地影响PC的水分转运。基于这些新发现，加上目前对设计针对单个AC异构体的抑制剂作为治疗方式的兴趣日益增加，本提案将确定特定AC异构体，AC3和6在调节PC Na和水运输中的作用。此外，PC中的cAMP不仅在调节Na和水的运输中很重要，而且在疾病中也很重要。特别是，常染色体显性多囊肾病中cAMP的产生和作用紊乱在囊肿的发展和扩大中起着重要作用。在初步研究中，我们获得了令人兴奋的新数据，表明靶向AC6对小鼠模型中囊性肾病的发展具有显着的保护作用。因此，本研究将明确在多囊肾病发病机制中特异性AC亚型在PC中的作用。本研究的主要假设是：1)PC中的AC3和ac6在健康状态下对PC Na和水分运输有显著影响；2)在多囊肾病中，PC中的AC3和6对PC向囊肿的发展有差异调节。该提案有三个具体目标：1)目的1将确定PC AC异构体（AC3和6）在肾钠和水处理中的生理作用。每个AC异构体将在小鼠PC中选择性靶向。完整的动物和急性分离的收集管将被用来评估单个AC异构体在血压、钠和水排泄调节中的作用。2)目的2将确定与cAMP水平控制和cAMP对Na和水运输的影响相关的PC AC异构体（AC3和6）的生物化学。我们将使用pc特异性敲除AC3、6或两种异构体的小鼠，研究抗利尿激素调节单个AC异构体衍生的cAMP的机制，并发挥其生物学效应。特别是，这些研究将确定PC中每个AC异构体衍生的cAMP是如何被磷酸二酯酶代谢的，以及哪些信号分子（蛋白激酶A和/或cAMP直接激活的交换蛋白）在转导PC中的抗利尿激素作用时被每个AC异构体直接激活。3)目的3将确定所有PC AC亚型（AC3和6）在多囊肾病中的病理生理作用。将采用多囊素-1缺乏症小鼠模型，并评估同时破坏单个AC亚型和Pkd1基因对肾脏结构和功能的影响。这些研究将确定哪些AC异构体在多囊肾病的发病机制中是重要的，并将开始确定多囊肾病中单个AC异构体调节的特定信号通路。

DESCRIPTION (provided by applicant): The collecting duct principal cell (PC) is responsible for the final adjustment of renal Na and water excretion. Derangements in PC Na reabsorption are associated with most forms of monogenic hypertension, while altered PC water transport accompanies disorders of water metabolism. PC Na and water transport are subject to the influences of multiple autocrine, paracrine and endocrine factors. A key mechanism for mediating many of these effects involves signaling through adenylyl cyclase (AC)-derived cAMP. The PC expresses AC3, 4 and 6, raising the fundamental question as to which biologic effects individual AC isoforms exert in the PC. We have engineered mice with PC-specific disruption of AC3, 4 or 6, and have found exciting new evidence for unique roles of the PC AC isoforms, AC3 and 6. Our findings suggest that AC6 regulates PC Na and water transport, while AC3 selectively affects PC water transport. Based on these novel findings, together with the currently increasing interest in designing inhibitors specific for individual AC isoforms as therapeutic modalities, this proposal will define the role of the specific AC isoforms, AC3 and 6 in the regulation of PC Na and water transport. In addition, cAMP in the PC is not only important in regulating Na and water transport, but it can be of primary importance in disease. In particular, disordered cAMP production and actions in autosomal dominant polycystic kidney disease play a fundamental role in cyst development and expansion. In preliminary studies, we have obtained exciting new data suggesting that targeting AC6 is markedly protective against the development of cystic kidney disease in a mouse model. Consequently, this proposal will define the role of specific AC isoforms in the PC in the pathogenesis of polycystic kidney disease. The major hypotheses of this proposal are: 1) AC3 and 6 in the PC exert distinctive effects on PC Na and water transport in health; and 2) AC3 and 6 in the PC differentially modulate PC development into cysts in polycystic kidney disease. The proposal has three specific aims: 1) Aim 1 will determine the physiologic role of PC AC isoforms (AC3 and 6) in renal Na and water handling. Each AC isoform will be selectively targeted in mouse PC in vivo. Intact animals and acutely isolated collecting ducts will be used to assess the role of the individual AC isoforms by loss of function in the regulation of blood pressure and Na and water excretion. 2) Aim 2 will determine the biochemistry of PC AC isoforms (AC3 and 6) related to control of cAMP levels and cAMP effects on Na and water transport. The mechanisms by which individual AC isoform-derived cAMP is modulated by vasopressin and exerts its biologic effects will be studied using mice with PC-specific knockout of AC3, 6 or both isoforms. In particular, these studies will define how cAMP derived from each AC isoform in the PC is metabolized by phosphodiesterases and which signaling molecules (protein kinase A and/or exchange protein directly activated by cAMP) are directly activated by each AC isoform in transducing vasopressin actions in the PC. 3) Aim 3 will determine the pathophysiologic effects of all PC AC isoforms (AC3 and 6) in polycystic kidney disease. A mouse model of polycystin-1 deficiency will be employed and the effect of simultaneously disrupting individual AC isoforms together with the Pkd1 gene on renal structure and function will be assessed. These studies will define which AC isoform(s) is/are important in the pathogenesis of polycystic kidney disease and will begin to identify the specific signaling pathways that individual AC isoforms modulate in polycystic kidney disease.