Podocyte Purinergic P2X4 Channels Are Mechanotransducers That Mediate Cytoskeletal Disorganization

J Am Soc Nephrol. 2016 Mar;27(3):848-62. doi: 10.1681/ASN.2014111144. Epub 2015 Jul 9.

Abstract

Podocytes are specialized, highly differentiated epithelial cells in the kidney glomerulus that are exposed to glomerular capillary pressure and possible increases in mechanical load. The proteins sensing mechanical forces in podocytes are unconfirmed, but the classic transient receptor potential channel 6 (TRPC6) interacting with the MEC-2 homolog podocin may form a mechanosensitive ion channel complex in podocytes. Here, we observed that podocytes respond to mechanical stimulation with increased intracellular calcium concentrations and increased inward cation currents. However, TRPC6-deficient podocytes responded in a manner similar to that of control podocytes, and mechanically induced currents were unaffected by genetic inactivation of TRPC1/3/6 or administration of the broad-range TRPC blocker SKF-96365. Instead, mechanically induced currents were significantly decreased by the specific P2X purinoceptor 4 (P2X4) blocker 5-BDBD. Moreover, mechanical P2X4 channel activation depended on cholesterol and podocin and was inhibited by stabilization of the actin cytoskeleton. Because P2X4 channels are not intrinsically mechanosensitive, we investigated whether podocytes release ATP upon mechanical stimulation using a fluorometric approach. Indeed, mechanically induced ATP release from podocytes was observed. Furthermore, 5-BDBD attenuated mechanically induced reorganization of the actin cytoskeleton. Altogether, our findings reveal a TRPC channel-independent role of P2X4 channels as mechanotransducers in podocytes.

Keywords: cell and transport physiology; cell signaling; glomerulosclerosis.

MeSH terms

  • Adenosine Triphosphate / metabolism*
  • Adenosine Triphosphate / pharmacology
  • Animals
  • Benzodiazepinones / pharmacology
  • Calcium / metabolism*
  • Cells, Cultured
  • Cholesterol / metabolism
  • Cytoskeleton / ultrastructure
  • Intracellular Signaling Peptides and Proteins / genetics
  • Intracellular Signaling Peptides and Proteins / metabolism
  • Mechanotransduction, Cellular* / drug effects
  • Membrane Proteins / genetics
  • Membrane Proteins / metabolism
  • Mice
  • Mice, Inbred C57BL
  • Mice, Knockout
  • Podocytes / metabolism*
  • Purinergic P2X Receptor Antagonists / pharmacology
  • Receptors, Purinergic P2X4 / genetics
  • Receptors, Purinergic P2X4 / metabolism
  • Receptors, Purinergic P2X4 / physiology*
  • Stress, Mechanical
  • TRPC Cation Channels / deficiency
  • TRPC Cation Channels / genetics

Substances

  • 5-(3-bromophenyl)-1,3-dihydro-2H-benzofuro(3,2-e)-1,4-diazepin-2-one
  • Benzodiazepinones
  • Intracellular Signaling Peptides and Proteins
  • Membrane Proteins
  • NPHS2 protein
  • Purinergic P2X Receptor Antagonists
  • Receptors, Purinergic P2X4
  • TRPC Cation Channels
  • TRPC3 cation channel
  • Trpc6 protein, mouse
  • transient receptor potential cation channel, subfamily C, member 1
  • Adenosine Triphosphate
  • Cholesterol
  • Calcium