Elucidating the physiological role of platelet‐derived growth factor receptor‐alpha+ cells and characterization of ANO1 in the murine upper urinary tract.
Authors: Grainger Nathan , Ward Sean M. , Koh Sang Don , Sanders Kenton M. 
Published: June 2021

Spontaneous electrical activity is necessary for driving peristalsis in the upper urinary tract (UUT) and is hypothesized to originate from the proximal renal pelvis. However, the mechanisms by which auto‐rhythmicity is generated remain undetermined. Specialized “atypical” smooth muscle cells have been implicated in pacing the pelvis due to the higher abundance of these cells in the area in which spontaneous contractions are generated. The calcium‐activated chloride channel (CaCC), anoctamin‐1 (ANO1) is essential for slow wave generation and normal gastrointestinal motility. In the pelvis, NFA‐sensitive Cl− currents have been observed in interstitial cells. To investigate the role of ANO1 in the renal pelvis, quantitative RT‐PCR analysis of Ano1 expression in the renal pelvis was assessed in mRNA extracted from single cell populations. Ano1 mRNA was detected in smooth muscle myosin heavy chain‐positive (smMHC+) cells, but was present more abundantly in platelet‐derived growth factor receptor alpha‐positive (PDGFRα+) cells. Furthermore, isolated Ano1eGFP+ cells confirmed the presence of cells co‐expressing both ANO1 and PDGFRα (~95%). Immunohistochemistry further validated this as coronal sections derived from the proximal renal pelvis exhibited dense ANO1 labeling on PDGFRα+ cells. An extensive network of PDGFRα+ cells populated the width of the proximal pelvic wall except the urothelium. Interestingly, smMHC+ “atypical” cells within this proximal region also co‐expressed PDGFRα+, identifying a novel marker of these specialized smooth muscle cells. Next, to determine if Ano1 possessed a physiological role in the pelvis, edge‐tracking and Ca2+ imaging experiments were performed on intact, renal pelvic tissue. Pharmacological inhibition of ANO1 with the CaCC inhibitor, CaCCinh‐A01 (5–10 μM) reduced the frequency of contractions and propagating Ca2+ waves in typical smooth muscle cells, indicating that Ano1 may be responsible for the generation of contractions. To probe underlying intracellular Ca2+ signals in PDGFRα+ and smMHC+ cells, confocal imaging experiments were conducted on the proximal extensions of the renal pelvis from PDGFRα+‐GCaMP6f mice on coronal vibratome sections (~150 μm thick) and flat‐sheet tissue. High frequency, spontaneous intracellular Ca2+ transients were visualized within PDGFRα+/smMHC+ specialized cells, that were abolished upon removal of extracellular Ca2+ but insensitive to L‐Type Ca2+ channel blockade by nicardipine (1 μM). PDGFRα+ cells presented irregular, often long duration Ca2+ transients that occasionally propagated into neighboring cells. Taken together, these data suggest a physiological role for PDGFRα+ cells, whereby activation of ANO1 channels, expressed on their plasma membrane, could contribute to depolarizing neighboring smMHC+ cells and ultimately regulate the propagating contractions in the renal pelvis.Support or Funding InformationR01 DK‐091336 (KMS)P01‐DK041315 (KMS)P30‐GM110767This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.