Dual role of the TRPV4 channel as a sensor of flow and osmolality in renal epithelial cells

Abstract

Gain/loss of function studies were utilized to assess the potential role of the endogenous vanilloid receptor TRPV4 as a sensor of flow and osmolality in M-1 collecting duct cells (CCD). TRPV4 mRNA and protein were detectable in M-1 cells and stably transfected HEK-293 cells, where the protein occurred as a glycosylated doublet on Western blots. Immunofluorescence imaging demonstrated expression of TRPV4 at the cell membranes of TRPV4-transfected HEK and M-1 cells and at the luminal membrane of mouse kidney CCD. By using intracellular calcium imaging techniques, calcium influx was monitored in cells grown on coverslips. Application of known activators of TRPV4, including 4alpha-PDD and hypotonic medium, induced strong calcium influx in M-1 cells and TRPV4-transfected HEK-293 cells but not in nontransfected cells. Applying increased flow/shear stress in a parallel plate chamber induced calcium influx in both M-1 and TRPV4-transfected HEK cells but not in nontransfected HEK cells. Furthermore, in loss-of-function studies employing small interference (si)RNA knockdown techniques, transfection of both M-1 and TRPV4-transfected HEK cells with siRNA specific for TRPV4, but not an inappropriate siRNA, led to a time-dependent decrease in TRPV4 expression that was accompanied by a loss of stimuli-induced calcium influx to flow and hypotonicity. It is concluded that TRPV4 displays a mechanosensitive nature with activation properties consistent with a molecular sensor of both fluid flow (or shear stress) and osmolality, or a component of a sensor complex, in flow-sensitive renal CCD.

Angiotensin II Increases Activity of the Epithelial Na+ Channel (ENaC) in Distal Nephron Additively to Aldosterone

“…[Ca 2ϩ ] i Measurements-Intracellular calcium levels were measured in cells of the split-opened ASDNs using Fura-2 fluorescence ratiometric imaging as described previously (32,33). Split-opened ASDNs were loaded with Fura-2 by incubation with 2 M Fura-2/AM in a bath solution for 40 min at room temperature.…”

Function of Transient Receptor Potential Cation Channel Subfamily V Member 4 (TRPV4) as a Mechanical Transducer in Flow-sensitive Segments of Renal Collecting Duct System

“…Although the TRPV4 channel can be activated by a wide range of stimuli, it has been shown to be particularly sensitive to physical alterations of the cellular microenvironment as it can be activated by variations in osmolality (1,2), fluid shear stress/flow (3)(4)(5)(6), pressure (7), and moderate heat (8,9). Hence, it is becoming evident that the channel may play a critical role in sensing and/or transducing changes in the microenvironmental physical state, at least in some cells.…”

“…Recent studies, however, point to an apparent role of TRPV4 as a sensor of fluid shear stress or fluid flow in renal cells and tissues. Indeed, our laboratory recently demonstrated that endogenous TRPV4 in M-1 collecting duct cells is activated by increases in shear stress/fluid flow, a response that is abolished following siRNA knockdown of TRPV4 (3,4). Furthermore, Suzuki and co-workers (14) have shown that flow-dependent K ϩ secretion, a Ca 2ϩ -dependent process utilizing the maxi-K channel (11)(12)(13), is essentially abolished in TRPV4 knock-out animals.…”

Vascular Responses to Shear Stress: The Involvement of Mechanosensors in Endothelial Cells

“…It revealed a temperature-induced potentiation or switching on of a channel signaling pathway. It has also been demonstrated that TRPV4 is activated by shear stress in native collecting duct cells [26].…”