The renal pelvis (RP), a smooth muscle organ that transports urine from the kidney to the ureter, generates regular rhythmic contractions that are vital for urine transport and bladder filling. Propagating contractions originate in the most proximal region of the RP, where specialized cells, called ?atypical? smooth muscle cells (ASMCs)?, have been proposed to serve as the pacemaker cells. However, identification of ASMCs has been imprecise, and thus experimental findings regarding their phenotype and functions are controversial. Ambiguities arise from the lack of specific markers for ASMCs and have prevented understanding the role of ASMC in driving peristaltic contractions or the mechanism of pacemaker activity in the RP. Newer technologies can now identify specific cell-types within tissues composed of heterogeneous populations of cells. This study will employ strains of mice with cell-specific reporters and optogenetic sensors to clarify how the pacemaker and responder cells of the RP generate peristaltic contractions. Our preliminary data show that the specialized cells, ASMCs, express platelet-derived-growth-factor-receptor-alpha (PDGFR?), and using transgenic mice that express a histone 2B- eGFP fusion protein driven off the endogenous PDGFR?+ promoter, we can identify these cells unequivocally in intact tissues or in enzymatic dispersions of the tissues. We hypothesize that PDGFR?+ cells are pacemaker cells in the renal pelvis. The following Specific Aims will be addressed: 1. Test the hypothesis that PDGFR?+ are the primary pacemaker cells of the RP; 2. Investigate the dynamics of Ca2+ signaling in PDGFR?+ cells; 3. Elucidate the specific mechanisms of pacemaker generation and propagation. Because of their specialized function as pacemaker cells, PDGFR?+ cells are likely to have specialized gene expression patterns that encode essential ionic conductances and other signaling molecules to facilitate pacemaker activity. This will be investigated by analysis of gene expression in FACS-enriched populations PDGFR?+ cells isolated from the proximal RP of the reporter strain of mice. Mice expressing a genetically encoded Ca2+ indicator (GECI) in PDGFR? + cells will be used to image Ca2+ signaling in PDGFR?+cells in situ. The ion channels activated by Ca2+ dynamics will be determined and the consequences of this conductance in RP peristalsis will be evaluated. Preliminary data show that PDGFR?+ cells exhibit dynamic Ca2+ signaling in situ and express the Ca2+-activated- Cl- channel, Ano1, making this a prime candidate for the pacemaker conductance. This project will serve as a basis for future studies to understand developmental or pathological problems affecting RP function. OMB No. 0925-0001/0002 (Rev. 01/18 Approved Through 03/31/2020) Page Continuation Format Page
The renal pelvis is a smooth muscle organ that pumps urine collected from the kidney to the ureter, and defects in this organ can cause urinary tract dysfunction and kidney damage. This project will investigate how specific cells in the proximal renal pelvis generate the rhythmic contractions that facilitate normal urine transport. This pre-clinical investigation will provide a novel concept for renal pelvis pacemaker activity and identify the specific cells, cellular signaling pathways and ionic conductances responsible for generation and propagation of pacemaker activity, providing the potential for more targeted therapeutic interventions for upper urinary tract dysfunction. OMB No. 0925-0001/0002 (Rev. 01/18 Approved Through 03/31/2020) Page Continuation Format Page
