title: Phosphodiesterase 5 inhibition ameliorates angiontensin II-induced podocyte dysmotility via the protein kinase G-mediated downregulation of TRPC6 activity.
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.
authors.affiliation: Division of Nephrology, Duke University Medical Center, Durham, North Carolina; Center for Human Genetics, Duke University Medical Center, Durham, North Carolina;
authors.forename: Gentzon
authors.initials: G
authors.lastname: Hall
authors.affiliation: Division of Cardiology, Johns Hopkins University School of Medicine, Baltimore, Maryland;
authors.forename: Janelle
authors.initials: J
authors.lastname: Rowell
authors.affiliation: Division of Cardiology, Johns Hopkins University School of Medicine, Baltimore, Maryland;
authors.forename: Federica
authors.initials: F
authors.lastname: Farinelli
authors.affiliation: Division of Nephrology, Duke University Medical Center, Durham, North Carolina; Center for Human Genetics, Duke University Medical Center, Durham, North Carolina; Department of Pediatrics, Duke University Medical Center, Durham, North Carolina; and.
authors.forename: Rasheed A
authors.initials: RA
authors.lastname: Gbadegesin
authors.affiliation: Trinity Health Kidney Centre, Tallaght Hospital, Trinity College, Dublin, Ireland.
authors.forename: Peter
authors.initials: P
authors.lastname: Lavin
authors.affiliation: Center for Human Genetics, Duke University Medical Center, Durham, North Carolina;
authors.forename: Guanghong
authors.initials: G
authors.lastname: Wu
authors.affiliation: Center for Human Genetics, Duke University Medical Center, Durham, North Carolina;
authors.forename: Alison
authors.initials: A
authors.lastname: Homstad
authors.affiliation: Division of Nephrology, Duke University Medical Center, Durham, North Carolina; Center for Human Genetics, Duke University Medical Center, Durham, North Carolina;
authors.forename: Andrew
authors.initials: A
authors.lastname: Malone
authors.affiliation: Center for Human Genetics, Duke University Medical Center, Durham, North Carolina;
authors.forename: Thomas
authors.initials: T
authors.lastname: Lindsey
authors.affiliation: Center for Human Genetics, Duke University Medical Center, Durham, North Carolina;
authors.forename: Ruiji
authors.initials: R
authors.lastname: Jiang
authors.affiliation: Division of Nephrology, Duke University Medical Center, Durham, North Carolina;
authors.forename: Robert
authors.initials: R
authors.lastname: Spurney
authors.affiliation: Division of Cardiology, Johns Hopkins University School of Medicine, Baltimore, Maryland;
authors.forename: Gordon F
authors.initials: GF
authors.lastname: Tomaselli
authors.affiliation: Division of Cardiology, Johns Hopkins University School of Medicine, Baltimore, Maryland;
authors.forename: David A
authors.initials: DA
authors.lastname: Kass
authors.affiliation: Division of Nephrology, Duke University Medical Center, Durham, North Carolina; Center for Human Genetics, Duke University Medical Center, Durham, North Carolina; michelle.winn@duke.edu.
authors.forename: Michelle P
authors.initials: MP
authors.lastname: Winn
mesh_terms: D000804:Angiotensin II / Q000494:pharmacology*; D000818:Animals; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology; D002465:Cell Movement / Q000187:drug effects*; D002478:Cells, Cultured; D017869:Cyclic GMP-Dependent Protein Kinases / Q000378:metabolism*; D015536:Down-Regulation / Q000187:drug effects* / Q000502:physiology; D057809:HEK293 Cells; D006801:Humans; D066298:In Vitro Techniques; D051379:Mice; D008815:Mice, Inbred Strains; D023421:Models, Animal; D050778:NFATC Transcription Factors / Q000378:metabolism; D058986:Phosphodiesterase 5 Inhibitors / Q000494:pharmacology*; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects / Q000502:physiology; D050199:Podocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D050052:TRPC Cation Channels / Q000378:metabolism*; D000074287:TRPC6 Cation Channel
chemical_list: D050778:NFATC Transcription Factors; D058986:Phosphodiesterase 5 Inhibitors; D050052:TRPC Cation Channels; D000074287:TRPC6 Cation Channel; C492146:Trpc6 protein, mouse; D000804:Angiotensin II; D017869:Cyclic GMP-Dependent Protein Kinases; C120817:calcineurin phosphatase; D010744:Phosphoric Monoester Hydrolases
keywords: calcineurin; phosphodiesterase 5; podocyte; protein kinase G; transient receptor potential cation channel isotype 6
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