Role of adenylyl cyclase isoforms in collecting duct physiology & pathophysiology

描述（由申请方提供）：集合管主细胞（PC）负责肾脏Na和水排泄的最终调节。PC Na重吸收的紊乱与大多数形式的单基因高血压相关，而PC水转运的改变伴随着水代谢的紊乱。PC Na和水的转运受多种自分泌、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的信号传导。PC表达AC 3、4和6，这就提出了一个基本问题，即单个AC亚型在PC中发挥哪些生物学作用。我们已经用PC特异性破坏AC 3、4或6的小鼠进行了工程改造，并发现了PC AC亚型AC 3和6的独特作用的令人兴奋的新证据。我们的研究结果表明，AC 6调节PC钠和水的运输，而AC 3选择性地影响PC水的运输。基于这些新的发现，加上目前越来越多的兴趣，在设计抑制剂的具体个别AC亚型作为治疗方式，这一建议将定义的作用，具体的AC亚型，AC 3和6在PC Na和水的运输的调节。此外，PC中的cAMP不仅在调节Na和水的转运中很重要，而且在疾病中也很重要。特别是，在常染色体显性遗传性多囊肾病中，cAMP的产生和作用紊乱在囊肿的发展和扩张中起着重要作用。在初步研究中，我们获得了令人兴奋的新数据，表明靶向AC 6对小鼠模型中囊性肾病的发展具有显着的保护作用。因此，该建议将确定PC中特定AC亚型在多囊肾病发病机制中的作用。该提议的主要假设是：1）PC中的AC 3和6对健康中的PC Na和水运输产生独特的影响;和2）PC中的AC 3和6差异性地调节PC在多囊肾疾病中发展成囊肿。该提案有三个具体目标：1）目标1将确定PC AC亚型（AC 3和6）在肾钠和水处理中的生理作用。每种AC同种型将在体内选择性靶向小鼠PC。将使用完整动物和急性分离的集合管，通过功能丧失评估单个AC亚型在血压调节以及Na和水排泄中的作用。2)目的2将确定与cAMP水平控制和cAMP对Na和水转运的影响相关的PC AC亚型（AC 3和6）的生物化学。将使用具有AC 3、6或两种亚型的PC特异性敲除的小鼠来研究个体AC亚型衍生的cAMP被加压素调节并发挥其生物学效应的机制。特别是，这些研究将确定PC中每个AC亚型的cAMP如何被磷酸二酯酶代谢，以及哪些信号分子（蛋白激酶A和/或由cAMP直接激活的交换蛋白）在PC中转导加压素作用时被每个AC亚型直接激活。3)目的3将确定所有PC AC亚型（AC 3和6）在多囊肾病中的病理生理作用。将采用多囊蛋白-1缺乏的小鼠模型，并评估同时破坏单个AC亚型以及Pkd 1基因对肾结构和功能的影响。这些研究将确定哪些AC亚型在多囊肾病的发病机制中是重要的，并将开始鉴定单个AC亚型在多囊肾病中调节的特异性信号通路。

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.