The regulation of renal tubular transport by cannabinoid receptor type 1 (CB1R) and its endogenous lipid ligands

项目总结/摘要： 大麻是美国联邦政府最常用的非法娱乐毒品，和法律的娱乐 在过去十年中，药物的使用有所增加。大麻含有不同亲和力的植物大麻素 广泛表达的G蛋白偶联大麻素受体CB 1 R和CB 2 R。肾脏产生 内源性大麻素（EC）增加尿量，对Na+和K+排泄有不同的影响， 啮齿动物很少有人类研究表明大麻素可以作为利尿剂，不仅促进尿Na+， 还有HCO 3-损失。我们已经初步表明，在小鼠中，CB 1 R在pHi调节中表达并损害pHi调节， 在闰细胞（IC）中，皮质集合管（CCD）中负责H+/HCO 3-分泌的细胞 和Na+（和K+）运输。我们还发现，大麻素受体激动剂急性增加尿 小鼠的水排泄。因此，我们推测IC可能参与了CB 1 R介导的利尿作用， 通过与主要细胞的交互作用，主要细胞负责Na+和水的重吸收。 这个VA职业发展奖申请旨在定义EC在CCD中的作用。三个具体 将研究目的（SA）以评价CB 1 R信号传导对功能的存在和生理意义 鼠标的CCD。SA 1将定义EC系统组分在该段中的细胞特异性表达; SA 2将确定CB 1 R信号传导对CCD中细胞特异性功能的作用， CB 1 R的激活剂/抑制剂;和SA 3将确定IC中CB 1 R的靶向缺失是否改变细胞特异性免疫应答。 功能协调发展的该结果有望为大麻素对肾脏的影响提供新的见解，并允许 用户识别目标（例如，CB 1 R激动剂）用于开发用于治疗以下疾病的新型治疗剂 例如高血压、容量超负荷、代谢性酸中毒和低钠血症的疾病。 拟议的培训计划包括职业发展活动、课程和讲习班， PI（约书亚雷恩博士，委员会认证的肾病学家和认证的高血压专家）在以下方面的熟练程度 单小管显微切割，离体小管微灌注，功能荧光显微镜，脂质 生物化学和药理学，细胞生物学，电生理学，细胞分选和动物模型，包括 转基因小鼠模型的开发。这些技能将得到一个由导师，顾问和 合作者，他们都有必要的专业知识，知识和指导跟踪记录。 总而言之，这一VA CDA拨款提案为Rein博士提供了一个强大的职业发展计划， 他的职业目标是发展作为肾脏EC系统专家的全国声誉，旨在发现新的 肾脏调节电解质和酸/碱平衡的分子途径。他的长期目标是 将临床前研究的关键观察结果转化为对人类疾病的更好理解， 治疗学导师的专业知识和支持对他的职业发展至关重要。詹姆斯J。 Peters VAMC和西奈山伊坎医学院为学生提供了出色的学术环境， 博士Rein的职业发展，领导层致力于他的成功，75%的时间受到保护， research.

PROJECT SUMMARY/ABSTRACT: Cannabis is the most commonly used federally illicit recreational drug in the U.S., and legal recreational and medicinal use has increased over the last decade. Cannabis contains phytocannabinoids with differing affinities for the ubiquitously expressed G-protein coupled cannabinoid receptors CB1R and CB2R. The kidney produces endogenous cannabinoids (ECs) that increase urine output with variable effects on Na+ and K+ excretion in rodents. Few studies in humans suggest that cannabinoids can act as diuretics, promoting not only urinary Na+ but alsoHCO3- losses. We have preliminarily shown in mice that CB1R is expressed in and impairs pHi regulation in intercalated cells (ICs), cells in the cortical collecting duct (CCD) that are responsible for H+/HCO3- secretion and Na+ (and K+) transport. We have also shown that cannabinoid receptor agonists acutely increase urinary water excretion in mice. Therefore, we hypothesize that ICs participate in CB1R mediated diuresis possibly through cross-talk with principal cells, cells responsible for the reabsorption of Na+ and water. This VA Career Development Award application aims to define the role of ECs in the CCD. Three Specific Aims (SA) will be studied to evaluate the presence and physiological significance of CB1R signaling on function of the mouse CCD. SA1 will define cell-specific expression of components of the EC system in this segment; SA2 will determine the role of CB1R signaling on cell-specific function in the CCD, utilizing pharmacologic activators/inhibitors of CB1R; and SA3 will determine whether targeted deletion of CB1R in ICs alters cell-specific functions. The results promise to provide new insight into the effects of cannabinoids on the kidney and allows us to identify targets (e.g., CB1R agonists) for development of novel therapeutic agents for the treatment of disorders such as hypertension, volume overload, metabolic acidosis, and hyponatremia. The proposed training plan includes career development activities, courses, and workshops to enhance the proficiency of the PI (Dr. Joshua Rein, a board-certified nephrologist and certified hypertension specialist) in single tubule microdissection, isolated tubule in vitro microperfusion, functional fluorescent microscopy, lipid biochemistry and pharmacology, cell biology, electrophysiology, cell sorting, and animal models including the development of transgenic mouse models. These skills will be reinforced by a team of mentors, advisors, and collaborators, all of whom have the requisite expertise, knowledge, and mentorship track-records. In sum, this VA CDA grant proposal provides a robust career development plan for Dr. Rein to accomplish his career goals to develop a national reputation as an expert in the renal EC system, aiming to uncover novel molecular pathways underlying the renal regulation of electrolyte and acid/base balance. His long-term goal is to translate key observations from preclinical studies into an enhanced understanding of human disease and therapeutics. The expertise and support of his mentors will be critical to his career development. The James J. Peters VAMC and the Icahn School of Medicine at Mount Sinai provide outstanding academic environments for Dr. Rein’s career development, with leadership committed to his success and 75% protected time conduct his research.