Background. The Vanilloid subfamily of transient receptor potential (TRPV) ion channels has been widely implicated in detecting\r\nosmotic and mechanical stress. In the current study, we examine the functional expression of TRPV4 channels in cell volume\r\nregulation in cells of the human collecting duct. Methods. Western blot analysis, siRNA knockdown, and microfluorimetry were\r\nused to assess the expression and function of TRPV4 in mediating Ca2+-dependent mechanical stimulation within a novel system\r\nof the human collecting duct (HCD). Results. Native and siRNA knockdown of TRPV4 protein expression was confirmed by\r\nwestern blot analysis. Touch was used as a cell-directed surrogate for osmotic stress.Mechanical stimulation of HCD cells evoked a\r\ntransient increase in [Ca2+]i that was dependent upon thapsigargin-sensitive store release and Ca2+ influx. At 48 hrs, high glucose\r\nand mannitol (25mM) reduced TRPV4 expression by 54% and 24%, respectively. Similar treatment doubled SGK1 expression.\r\nTouch-evoked changes were negated following TRPV4 knockdown. Conclusion. Our data confirm expression of Ca2+-dependent\r\nTRPV4 channels in HCDcells and suggest that a loss of expression in response to high glucose attenuates the ability of the collecting\r\nduct to exhibit regulatory volume decreases, an effect that may contribute to the pathology of fluid and electrolyte imbalance as\r\nobserved in diabetic nephropathy.
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