Lee Hak Joo, Feliers Denis, Mariappan Meenalakshmi M, Sataranatarajan Kavithalakshmi, Choudhury Goutam Ghosh, Gorin Yves, Kasinath Balakuntalam S
From the Department of Medicine, University of Texas Health Science Center and South Texas Veterans Healthcare System, San Antonio, Texas 78229.
From the Department of Medicine, University of Texas Health Science Center and.
J Biol Chem. 2015 May 8;290(19):12014-26. doi: 10.1074/jbc.M114.615377. Epub 2015 Mar 9.
Diabetes-induced kidney cell injury involves an increase in matrix protein expression that is only partly alleviated by current treatment, prompting a search for new modalities. We have previously shown that hydrogen sulfide (H2S) inhibits high glucose-induced protein synthesis in kidney podocytes. We tested whether tadalafil, a phosphodiesterase 5 inhibitor used to treat erectile dysfunction, ameliorates high glucose stimulation of matrix proteins by generating H2S in podocytes. Tadalafil abrogated high glucose stimulation of global protein synthesis and matrix protein laminin γ1. Tadalafil inhibited high glucose-induced activation of mechanistic target of rapamycin complex 1 and laminin γ1 accumulation in an AMP-activated protein kinase (AMPK)-dependent manner. Tadalafil increased AMPK phosphorylation by stimulating calcium-calmodulin kinase kinase β. Tadalafil rapidly increased the expression and activity of the H2S-generating enzyme cystathionine γ-lyase (CSE) by promoting its translation. dl-Propargylglycine, a CSE inhibitor, and siRNA against CSE inhibited tadalafil-induced AMPK phosphorylation and abrogated the tadalafil effect on high glucose stimulation of laminin γ1. In tadalafil-treated podocytes, we examined the interaction between H2S and nitric oxide (NO). N(ω)-Nitro-L-arginine methyl ester and 1H-[1,2,4]-oxadiazolo-[4,3-a]-quinoxalin-1-one, inhibitors of NO synthase (NOS) and soluble guanylyl cyclase, respectively, abolished tadalafil induction of H2S and AMPK phosphorylation. Tadalafil rapidly augmented inducible NOS (iNOS) expression by increasing its mRNA, and siRNA for iNOS and 1400W, an iNOS blocker, inhibited tadalafil stimulation of CSE expression and AMPK phosphorylation. We conclude that tadalafil amelioration of high glucose stimulation of synthesis of proteins including matrix proteins in podocytes requires integration of the NO-H2S-AMPK axis leading to the inhibition of high glucose-induced mechanistic target of rapamycin complex 1 activity and mRNA translation.
糖尿病诱导的肾细胞损伤涉及基质蛋白表达增加，而目前的治疗仅能部分缓解这种情况，这促使人们寻找新的治疗方法。我们之前已经表明，硫化氢（H2S）可抑制高糖诱导的肾足细胞中的蛋白质合成。我们测试了用于治疗勃起功能障碍的磷酸二酯酶5抑制剂他达拉非是否通过在足细胞中产生H2S来改善高糖对基质蛋白的刺激。他达拉非消除了高糖对整体蛋白质合成和基质蛋白层粘连蛋白γ1的刺激。他达拉非以AMP激活蛋白激酶（AMPK）依赖的方式抑制高糖诱导的雷帕霉素复合物1的机制靶点激活和层粘连蛋白γ1积累。他达拉非通过刺激钙调蛋白激酶激酶β增加AMPK磷酸化。他达拉非通过促进其翻译迅速增加产生H2S的酶胱硫醚γ-裂解酶（CSE）的表达和活性。CSE抑制剂dl-炔丙基甘氨酸和针对CSE的小干扰RNA（siRNA）抑制他达拉非诱导的AMPK磷酸化，并消除他达拉非对高糖刺激层粘连蛋白γ1的作用。在他达拉非处理的足细胞中，我们研究了H2S与一氧化氮（NO）之间的相互作用。分别作为NO合酶（NOS）和可溶性鸟苷酸环化酶抑制剂的N（ω）-硝基-L-精氨酸甲酯和1H-[1,2,4]-恶二唑并-[4,3-a]-喹喔啉-1-酮消除了他达拉非诱导的H2S和AMPK磷酸化。他达拉非通过增加其信使核糖核酸（mRNA）迅速增强诱导型NOS（iNOS）表达，针对iNOS的siRNA和iNOS阻滞剂1400W抑制他达拉非对CSE表达和AMPK磷酸化的刺激。我们得出结论，他达拉非改善高糖对足细胞中包括基质蛋白在内的蛋白质合成的刺激需要整合NO-H2S-AMPK轴，从而抑制高糖诱导的雷帕霉素复合物1活性和mRNA翻译。
