Dual role of the TRPV4 channel as a sensor of flow and osmolality in renal epithelial cells.
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.
Wu
Ling
L
Department of Integrative Biology and Pharmacology, The University of Texas Health Science Center at Houston, 6431 Fannin, Houston, TX 77030, USA.
Gao
Xiaochong
X
Brown
Rachel C
RC
Heller
Stefan
S
O'Neil
Roger G
RG
D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003470:Culture Media / Q000494:pharmacology; D004847:Epithelial Cells / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D006031:Glycosylation; D006801:Humans; D007038:Hypotonic Solutions / Q000494:pharmacology; D007672:Kidney Cortex; D007685:Kidney Tubules, Collecting / Q000166:cytology* / Q000378:metabolism*; D008465:Mechanoreceptors / Q000502:physiology*; D051379:Mice; D008810:Mice, Inbred C57BL; D009994:Osmolar Concentration; D010703:Phorbol Esters / Q000494:pharmacology; D012333:RNA, Messenger / Q000037:antagonists & inhibitors / Q000378:metabolism; D034741:RNA, Small Interfering / Q000494:pharmacology; D013314:Stress, Mechanical; D050916:TRPV Cation Channels / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology*; D014018:Tissue Distribution; D014162:Transfection
D003470:Culture Media; D007038:Hypotonic Solutions; D010703:Phorbol Esters; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D050916:TRPV Cation Channels; C486576:Trpv4 protein, mouse; C008876:phorbol-12,13-didecanoate; D002118:Calcium