Adenylyl cyclase isoforms in collecting duct physiology and pathophysiology

描述(由申请人提供)： 集合管主细胞(PC)负责肾脏钠和水排泄的最终调节。PC钠重吸收的紊乱与大多数形式的单基因高血压有关，而PC水运输的改变伴随着水代谢的紊乱。PC钠和水的转运受多种自分泌、旁分泌和内分泌因素的影响。介导这些效应的一个关键机制是通过腺酰环化酶(AC)衍生的cAMP进行信号传递。PC表达AC3、4和6，提出了一个基本问题，即各个AC亚型在PC中发挥哪些生物效应。我们用PC特异性的AC3、4或6干扰小鼠，并发现了令人兴奋的新证据，证明了PC AC亚型AC3和6的独特作用。我们的发现表明，AC6调节PC钠和水的运输，而AC3选择性地影响PC的水运输。基于这些新的发现，再加上目前人们对设计针对单个AC异构体的抑制剂作为治疗方法的兴趣日益增加，该提案将定义特定的AC异构体AC3和AC6在调节PC Na和水运输中的作用。此外，PC中的cAMP不仅在调节钠和水的运输方面很重要，而且在疾病中也可能是首要的。尤其是在常染色体显性遗传性多囊肾病中，cAMP的异常产生和作用在囊变的发展和扩大中起着基础性的作用。在初步研究中，我们获得了令人兴奋的新数据，表明靶向AC6在小鼠模型中对囊性肾脏疾病的发展具有显著的保护作用。因此，这项提议将确定PC中特定的AC亚型在多囊肾病发病机制中的作用。这一设想的主要假设是：1)PC中的AC3和6对PC钠和水的运输有不同的影响；2)PC中的AC3和6在多囊肾病中不同地调控PC发展为囊性病变。该提案有三个具体目标：1)目标1将确定PC AC异构体(AC3和6)在肾脏钠和水处理中的生理作用。每种AC异构体都将在体内选择性地靶向于小鼠PC。完整的动物和急性分离的收集管道将被用来评估单个AC亚型在调节血压、钠和水排泄方面的功能丧失的作用。2)目的2将确定PC AC亚型(AC3和6)的生化与cAMP水平的控制以及cAMP对钠和水运输的影响。单个AC异构体衍生的cAMP受加压素调节并发挥其生物学效应的机制将在PC特异性敲除AC3、6或两者的小鼠身上进行研究。特别是，这些研究将确定来自PC中每个AC亚型的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/或由cAMP直接激活的交换蛋白)在转导PC中的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(AC3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，并将评估同时干扰单个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亚型(S)在多囊肾病的发病机制中起重要作用，并将开始确定单个AC亚型在多囊肾病中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 o 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.