ABSTRACT

While constant basal levels of macroautophagy/autophagy are a prerequisite to preserve long-lived podocytes at the filtration barrier, MTOR regulates at the same time podocyte size and compensatory hypertrophy. Since MTOR is known to generally suppress autophagy, the apparently independent regulation of these two key pathways of glomerular maintenance remained puzzling. We now report that long-term genetic manipulation of MTOR activity does in fact not influence high basal levels of autophagy in podocytes either in vitro or in vivo. Instead we present data showing that autophagy in podocytes is mainly controlled by AMP-activated protein kinase (AMPK) and ULK1 (unc-51 like kinase 1). Pharmacological inhibition of MTOR further shows that the uncoupling of MTOR activity and autophagy is time dependent. Together, our data reveal a novel and unexpected cell-specific mechanism, which permits concurrent MTOR activity as well as high basal autophagy rates in podocytes. Thus, these data indicate manipulation of the AMPK-ULK1 axis rather than inhibition of MTOR as a promising therapeutic intervention to enhance autophagy and preserve podocyte homeostasis in glomerular diseases.

Abbreviations: AICAR: 5-aminoimidazole-4-carboxamide ribonucleotide; AMPK: AMP-activated protein kinase; ATG: autophagy related; BW: body weight; Cq: chloroquine; ER: endoplasmic reticulum; ESRD: end stage renal disease; FACS: fluorescence activated cell sorting; GFP: green fluorescent protein; i.p.: intra peritoneal; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MTOR: mechanistic target of rapamycin kinase; NPHS1: nephrosis 1, nephrin; NPHS2: nephrosis 2, podocin; PLA: proximity-ligation assay; PRKAA: 5ʹ-AMP-activated protein kinase catalytic subunit alpha; RPTOR/RAPTOR: regulatory associated protein of MTOR, complex 1; RFP: red fluorescent protein; TSC1: tuberous sclerosis 1; ULK1: unc-51 like kinase 1

摘要

虽然巨自噬/自噬的恒定基础水平是在过滤屏障处保存长寿足细胞的先决条件，但 MTOR 同时调节足细胞大小和代偿性肥大。由于已知 MTOR 通常会抑制自噬，因此这两个肾小球维持关键途径的明显独立调节仍然令人费解。我们现在报告说，MTOR 活性的长期遗传操作实际上并不影响体外或体内足细胞中自噬的高基础水平. 相反，我们提供的数据显示足细胞中的自噬主要受 AMP 活化蛋白激酶 (AMPK) 和 ULK1（unc-51 类激酶 1）控制。MTOR 的药理学抑制进一步表明 MTOR 活性和自噬的解偶联是时间依赖性的。总之，我们的数据揭示了一种新的和意想不到的细胞特异性机制，它允许同时发生 MTOR 活性以及足细胞中的高基础自噬率。因此，这些数据表明操纵 AMPK-ULK1 轴而不是抑制 MTOR 作为一种有前途的治疗干预措施，以增强自噬和保持肾小球疾病中的足细胞稳态。

缩写： AICAR：5-氨基咪唑-4-甲酰胺核糖核苷酸；AMPK：AMP活化蛋白激酶；ATG：自噬相关；BW：体重；Cq：氯喹；ER：内质网；ESRD：终末期肾病；FACS：荧光激活细胞分选；GFP：绿色荧光蛋白；ip：腹膜内；MAP1LC3/LC3：微管相关蛋白1轻链3；MTOR：雷帕霉素激酶的机制靶点；NPHS1：肾病 1，肾素；NPHS2: 肾病 2, podocin; PLA：邻近连接试验；PRKAA：5ʹ-AMP 活化蛋白激酶催化亚基 α；RPTOR/RAPTOR：MTOR 的调节相关蛋白，复合体 1；RFP：红色荧光蛋白；TSC1：结节性硬化症 1；ULK1：unc-51 像激酶 1