Division of Nephrology, Duke University Medical Center, Durham, North Carolina; Center for Human Genetics, Duke University Medical Center, Durham, North Carolina;
Division of Cardiology, Johns Hopkins University School of Medicine, Baltimore, Maryland;
Am J Physiol Renal Physiol. 2014 Jun 15;306(12):F1442-50. doi: 10.1152/ajprenal.00212.2013. Epub 2014 Apr 16.
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.
瞬时受体电位阳离子通道亚型 6（TRPC6）作为影响足细胞的各种病理过程的核心贡献者的新兴作用引起了人们对开发调节其功能的治疗方法的兴趣。最近对 TRPC6 调节的深入了解表明，PKG 通过其在两个高度保守的氨基酸残基上的磷酸化（Thr(69)/Thr(70)（在小鼠中为 Thr(69)，在人类中为 Thr(70)）和 Ser(321)/Ser(322)（在小鼠中为 Ser(321)，在人类中为 Ser(322)），成为 TRPC6 电导和相关信号的有效负调节剂。在这里，我们测试了 PKG 在调节原代和条件性永生化小鼠足细胞中 TRPC6 依赖性反应中的作用。非刺激足细胞中的 TRPC6 在 Thr(69)处磷酸化，但在 ANG II 刺激或组成性激活钙调神经磷酸酶时下降。ANG II 在体外划痕试验中诱导足细胞运动，而过表达磷酸模拟突变体 TRPC6（TRPC6T70E/S322E）或激活的 PKG 的细胞中，这种运动减少了 30-60%（P<0.05）。PKG 激动剂 S-亚硝基-N-乙酰-DL-青霉胺（一氧化氮供体）、8-溴-cGMP、Bay 41-2772（可溶性鸟苷酸环化酶激活剂）或磷酸二酯酶 5（PDE5）抑制剂 4-[[3'，4'-(亚甲基二氧基）苄基]氨基]-[7]-6-甲氧基喹唑啉预处理足细胞可减轻 ANG II 诱导的 Thr(69)去磷酸化，并通过 30-60%抑制 TRPC6 依赖性足细胞运动。这些数据表明，PKG 激活策略，包括 PDE5 抑制，通过靶向足细胞中的 TRPC6 改善 ANG II 诱导的足细胞运动障碍，突出了这些方法治疗高活性 TRPC6 依赖性肾小球疾病的潜在治疗效用。
