title: Functional expression of TRPV4 channels in human collecting duct cells: implications for secondary hypertension in diabetic nephropathy.
abstract: BACKGROUND
  The Vanilloid subfamily of transient receptor potential (TRPV) ion channels has been widely implicated in detecting osmotic and mechanical stress. In the current study, we examine the functional expression of TRPV4 channels in cell volume regulation in cells of the human collecting duct.
  
  
  METHODS
  Western blot analysis, siRNA knockdown, and microfluorimetry were used to assess the expression and function of TRPV4 in mediating Ca²⁺-dependent mechanical stimulation within a novel system of the human collecting duct (HCD).
  
  
  RESULTS
  Native and siRNA knockdown of TRPV4 protein expression was confirmed by western blot analysis. Touch was used as a cell-directed surrogate for osmotic stress. Mechanical stimulation of HCD cells evoked a transient increase in [Ca²⁺](i) that was dependent upon thapsigargin-sensitive store release and Ca²⁺ influx. At 48 hrs, high glucose and mannitol (25 mM) reduced TRPV4 expression by 54% and 24%, respectively. Similar treatment doubled SGK1 expression. Touch-evoked changes were negated following TRPV4 knockdown.
  
  
  CONCLUSIONS
  Our data confirm expression of Ca²⁺-dependent TRPV4 channels in HCD cells and suggest that a loss of expression in response to high glucose attenuates the ability of the collecting duct to exhibit regulatory volume decreases, an effect that may contribute to the pathology of fluid and electrolyte imbalance as observed in diabetic nephropathy.
authors.affiliation: School of Life Sciences, The University of Warwick, Coventry CV4 7AL, UK. c.hills@warwick.ac.uk
authors.forename: Claire E
authors.initials: CE
authors.lastname: Hills
authors.forename: Rosemary
authors.initials: R
authors.lastname: Bland
authors.forename: Paul E
authors.initials: PE
authors.lastname: Squires
mesh_terms: D020013:Calcium Signaling; D002461:Cell Line, Transformed; D048429:Cell Size; D002999:Clone Cells; D003928:Diabetic Nephropathies / Q000378:metabolism* / Q000473:pathology; D015536:Down-Regulation; D005947:Glucose / Q000378:metabolism; D006801:Humans; D006973:Hypertension / Q000150:complications / Q000378:metabolism* / Q000473:pathology; D017874:Immediate-Early Proteins / Q000235:genetics / Q000378:metabolism; D007685:Kidney Tubules, Collecting / Q000166:cytology / Q000378:metabolism* / Q000473:pathology; D008353:Mannitol / Q000378:metabolism; D009997:Osmotic Pressure; D010812:Physical Stimulation; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering; D050916:TRPV Cation Channels / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D015854:Up-Regulation; D014883:Water-Electrolyte Imbalance; D000099184:Serum-Glucocorticoid Regulated Kinases
chemical_list: D005947:Glucose; D017874:Immediate-Early Proteins; D008353:Mannitol; D017346:Protein Serine-Threonine Kinases; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D050916:TRPV Cation Channels; D000099184:Serum-Glucocorticoid Regulated Kinases; C416986:TRPV4 protein, human