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.

PROJECT NARRATIVE: Cannabis consumption for recreational and medicinal purposes has increased worldwide over the last decade. Emerging evidence suggests that the kidney expresses cannabinoid receptors and produces endogenous cannabinoids. Activation of these receptors acutely increases urine output with variable effects on sodium and potassium excretion in mice, rats, and rabbits. A body of very few studies in humans suggest that cannabinoids can act as diuretics to promote sodium bicarbonate excretion. Given the emerging evidence suggesting that cannabinoids regulate urine output and ion transport in the kidney, and rapidly increasing widespread usage of cannabis, the proposed investigation promises to elucidate the functional role of cannabinoids in the kidney epithelial cells that regulate total body electrolyte, acid/base and fluid homeostasis, and will allow us to identify new therapeutic agents for the treatment of disorders common among people living with kidney disease such as hypertension, volume overload, metabolic acidosis, and hyponatremia.