Phosphodiesterase 5 inhibition ameliorates angiontensin II-induced podocyte dysmotility via the protein kinase G-mediated downregulation of TRPC6 activity

Am J Physiol Renal Physiol. 2014 Jun 15;306(12):F1442-50. doi: 10.1152/ajprenal.00212.2013. Epub 2014 Apr 16.

Abstract

The emerging role of the transient receptor potential cation channel isotype 6 (TRPC6) as a central contributor to various pathological processes affecting podocytes has generated interest in the development of therapeutics to modulate its function. Recent insights into the regulation of TRPC6 have revealed PKG as a potent negative modulator of TRPC6 conductance and associated signaling via its phosphorylation at two highly conserved amino acid residues: Thr(69)/Thr(70) (Thr(69) in mice and Thr(70) in humans) and Ser(321)/Ser(322) (Ser(321) in mice and Ser(322) in humans). Here, we tested the role of PKG in modulating TRPC6-dependent responses in primary and conditionally immortalized mouse podocytes. TRPC6 was phosphorylated at Thr(69) in nonstimulated podocytes, but this declined upon ANG II stimulation or overexpression of constitutively active calcineurin phosphatase. ANG II induced podocyte motility in an in vitro wound assay, and this was reduced 30-60% in cells overexpressing a phosphomimetic mutant TRPC6 (TRPC6T70E/S322E) or activated PKG (P < 0.05). Pretreatment of podocytes with the PKG agonists S-nitroso-N-acetyl-dl-penicillamine (nitric oxide donor), 8-bromo-cGMP, Bay 41-2772 (soluble guanylate cyclase activator), or phosphodiesterase 5 (PDE5) inhibitor 4-{[3',4'-(methylenedioxy)benzyl]amino}[7]-6-methoxyquinazoline attenuated ANG II-induced Thr(69) dephosphorylation and also inhibited TRPC6-dependent podocyte motility by 30-60%. These data reveal that PKG activation strategies, including PDE5 inhibition, ameliorate ANG II-induced podocyte dysmotility by targeting TRPC6 in podocytes, highlighting the potential therapeutic utility of these approaches to treat hyperactive TRPC6-dependent glomerular disease.

Keywords: calcineurin; phosphodiesterase 5; podocyte; protein kinase G; transient receptor potential cation channel isotype 6.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Angiotensin II / pharmacology*
  • Animals
  • Calcium Signaling / drug effects
  • Calcium Signaling / physiology
  • Cell Movement / drug effects*
  • Cells, Cultured
  • Cyclic GMP-Dependent Protein Kinases / metabolism*
  • Down-Regulation / drug effects*
  • Down-Regulation / physiology
  • HEK293 Cells
  • Humans
  • In Vitro Techniques
  • Mice
  • Mice, Inbred Strains
  • Models, Animal
  • NFATC Transcription Factors / metabolism
  • Phosphodiesterase 5 Inhibitors / pharmacology*
  • Phosphoric Monoester Hydrolases / metabolism
  • Phosphorylation / drug effects
  • Phosphorylation / physiology
  • Podocytes / cytology
  • Podocytes / drug effects
  • Podocytes / metabolism*
  • Signal Transduction / drug effects
  • Signal Transduction / physiology
  • TRPC Cation Channels / metabolism*

Substances

  • NFATC Transcription Factors
  • Phosphodiesterase 5 Inhibitors
  • TRPC Cation Channels
  • Trpc6 protein, mouse
  • Angiotensin II
  • Cyclic GMP-Dependent Protein Kinases
  • calcineurin phosphatase
  • Phosphoric Monoester Hydrolases