Mia Sobuj, Federico Giuseppina, Feger Martina, Pakladok Tatsiana, Meissner Adrian, Voelkl Jakob, Groene Hermann-Josef, Alesutan Ioana, Lang Florian
Department of Physiology, University of Tübingen, Gmelinstr. 5, D-72076, Tübingen, Germany.
Department of Cellular and Molecular Pathology, German Cancer Research Center, Im Neuenheimer Feld, 280, 69120, Heidelberg, Germany.
PLoS One. 2015 Aug 18;10(8):e0135235. doi: 10.1371/journal.pone.0135235. eCollection 2015.
AMP-activated protein kinase (Ampk) is a sensor of the cellular energy status and a powerful regulator of metabolism. Activation of Ampk was previously shown to participate in monocyte-to-fibroblast transition and matrix protein production in renal tissue. Thus, the present study explored whether the catalytic Ampkα1 isoform participates in the regulation of the renal fibrotic response following unilateral ureteral obstruction (UUO).
UUO was induced in gene-targeted mice lacking functional Ampkα1 (Ampkα1-/-) and in corresponding wild-type mice (Ampkα1+/+). In the obstructed kidney and, for comparison, in the non-obstructed control kidney, quantitative RT-PCR, Western blotting and immunostaining were employed to determine transcript levels and protein abundance, respectively.
In Ampkα1+/+ mice, UUO significantly up-regulated the protein abundance of the Ampkα1 isoform, but significantly down-regulated the Ampkα2 isoform in renal tissue. Phosphorylated Ampkα protein levels were significantly increased in obstructed kidney tissue of Ampkα1+/+ mice but not of Ampkα1-/- mice. Renal expression of α-smooth muscle actin was increased following UUO, an effect again less pronounced in Ampkα1-/- mice than in Ampkα1+/+ mice. Histological analysis did not reveal a profound effect of Ampkα1 deficiency on collagen 1 protein deposition. UUO significantly increased phosphorylated and total Tgf-ß-activated kinase 1 (Tak1) protein, as well as transcript levels of Tak1-downstream targets c-Fos, Il6, Pai1 and Snai1 in Ampkα1+/+ mice, effects again significantly ameliorated in Ampkα1-/- mice. Moreover, Ampkα1 deficiency inhibited the UUO-induced mRNA expression of Cd206, a marker of M2 macrophages and of Cxcl16, a pro-fibrotic chemokine associated with myeloid fibroblast formation. The effects of Ampkα1 deficiency during UUO were, however, paralleled by increased tubular injury and apoptosis.
Renal obstruction induces an isoform shift from Ampkα2 towards Ampkα1, which contributes to the signaling involved in cell survival and fibrosis.
AMP激活的蛋白激酶（Ampk）是细胞能量状态的传感器，也是新陈代谢的强大调节因子。先前的研究表明，Ampk的激活参与肾组织中单核细胞向成纤维细胞的转变和基质蛋白的产生。因此，本研究探讨了催化性Ampkα1亚型是否参与单侧输尿管梗阻（UUO）后肾纤维化反应的调节。
在缺乏功能性Ampkα1的基因靶向小鼠（Ampkα1-/-）和相应的野生型小鼠（Ampkα1+/+）中诱导UUO。在梗阻肾脏以及作为对照的非梗阻对照肾脏中，分别采用定量RT-PCR、蛋白质印迹法和免疫染色来确定转录水平和蛋白质丰度。
在Ampkα1+/+小鼠中，UUO显著上调了肾组织中Ampkα1亚型的蛋白质丰度，但显著下调了Ampkα2亚型。Ampkα1+/+小鼠梗阻肾脏组织中磷酸化Ampkα蛋白水平显著升高，而Ampkα1-/-小鼠则未升高。UUO后，α-平滑肌肌动蛋白的肾表达增加，Ampkα1-/-小鼠的这种效应再次不如Ampkα1+/+小鼠明显。组织学分析未发现Ampkα1缺乏对I型胶原蛋白沉积有显著影响。UUO显著增加了Ampkα1+/+小鼠中磷酸化和总Tgf-β激活激酶1（Tak1）蛋白以及Tak1下游靶点c-Fos、Il6、Pai1和Snai1的转录水平，Ampkα1-/-小鼠的这些效应再次显著改善。此外，Ampkα1缺乏抑制了UUO诱导的Cd206（M2巨噬细胞标志物）和Cxcl16（与髓样成纤维细胞形成相关的促纤维化趋化因子）的mRNA表达。然而，UUO期间Ampkα1缺乏的效应伴随着肾小管损伤和凋亡增加。
肾梗阻诱导了从Ampkα2向Ampkα1的亚型转变，这有助于细胞存活和纤维化相关的信号传导。
