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.

Phosphodiesterase Type 5 Inhibition Reduces Albuminuria in Subjects with Overt Diabetic Nephropathy

“…These effects have been suggested to be primarily mediated by cGMP-mediated protein kinase-G activation, secondary to phosphodiesterase type 5 inhibitor (PDE5i)-induced elevation of the NO-derived cGMP pool. 19,27,28 PF-00489791 was safe and generally well-tolerated when administered at a dose of 20 mg daily for 12 weeks in T2DM subjects with overt DN. The most commonly observed AEs in the present study were consistent with those reported in subjects with mild-to-moderate hypertension treated with PF-00489791 for 28 days, 29 including headaches and upper gastrointestinal events.…”

“…[15][16][17] Specifically, PDE5 inhibition-mediated elevation of cGMP in podocytes attenuates podocyte damage in diabetic rats, resulting in decreased albuminuria, 18 likely through correction of podocyte dysmotility as suggested by in vitro data. 19 The first clinical study to suggest translation of the PDE5-related preclinical findings was conducted by Grover-Páez et al who demonstrated that administration of a PDE5 inhibitor once daily for 30 days to microalbuminuric T2DM patients significantly reduced albuminuria. 20 To further assess the renoprotective effect of chronic PDE5 inhibition in DN, the present study evaluated the safety and efficacy of the novel, selective, and long-acting PDE5 inhibitor, PF-00489791, in subjects with T2DM and overt DN receiving ACEi or ARB background therapy.…”

Prevention of PKG1α oxidation augments cardioprotection in the stressed heart

“…Reduced channel suppression by PKG1α oxidation fits this observation. Redox sensitivity of PKG1α suppression of TRPC6 may also have therapeutic implications beyond the heart, including in dystrophinopathies (24) and glomerulosclerosis (26,27), where ROS and TRPC6 play a pathophysiological role. Future studies are needed to identify the protein partners that control PKG1α translocation and develop strategies to selectively sustain its reduced state to maximize the efficacy of its activation against myocardial disease.…”

Translating genetic findings in hereditary nephrotic syndrome: the missing loops

“…Furthermore, Canaud et al (7) described a potential mechanism for sirolimus-induced proteinuria via the mTORC2-dependent inhibition of AKT2 phosphorylation in podocytes. Most recently, Hall et al (22) demonstrated that phosphodiesterase-V inhibitors ameliorated ANG II-induced podocyte dysmotility via the PKG-mediated downregulation of transient receptor potential cation channel 6 (TRPC6) activity. Finally, using a reverse genetics approach, angiopoietin-like 4 (ANGPT4) was identified as a podocyte-secreted inducer of GBM injury and NS, making ANGPT4 a promising novel therapeutic target for minimal-change disease (MCD) (9).…”

“…However, improvements in our understanding of the mechanisms of TRPC6-mediated podocyte injury in FSGS have not been matched by therapeutic advances. While pharmacological therapies have shown promise in modulating the activity of TRPC6 in vitro (22), these findings have not translated into clinically useful therapeutic tools. Some of the factors responsible for this are 1) the lack of disease-specific cell lines for in vitro modeling and 2) unavailability of suitable animal models for NS.…”