ABSTRACT

Recently, we identified a novel mechanism of lipotoxicity in the kidney proximal tubular cells (PTECs); lipid overload stimulates macroautophagy/autophagy for the renovation of plasma and organelle membranes to maintain the integrity of the PTECs. However, this autophagic activation places a burden on the lysosomal system, leading to a downstream suppression of autophagy, which manifests as phospholipid accumulation and inadequate acidification in lysosomes. Here, we investigated whether pharmacological correction by eicosapentaenoic acid (EPA) supplementation could restore autophagic flux and alleviate renal lipotoxicity. EPA supplementation to high-fat diet (HFD)-fed mice reduced several hallmarks of lipotoxicity in the PTECs, such as phospholipid accumulation in the lysosome, mitochondrial dysfunction, inflammation, and fibrosis. In addition to improving the metabolic syndrome, EPA alleviated renal lipotoxicity via several mechanisms. EPA supplementation to HFD-fed mice or the isolated PTECs cultured in palmitic acid (PA) restored lysosomal function with significant improvements in the autophagic flux. The PA-induced redistribution of phospholipids from cellular membranes into lysosomes and the HFD-induced accumulation of SQSTM1/p62 (sequestosome 1), an autophagy substrate, during the temporal and genetic ablation of autophagy were significantly reduced by EPA, indicating that EPA attenuated the HFD-mediated increases in autophagy demand. Moreover, a fatty acid pulse-chase assay revealed that EPA promoted lipid droplet (LD) formation and transfer from LDs to the mitochondria for beta-oxidation. Noteworthy, the efficacy of EPA on lipotoxicity is autophagy-dependent and cell-intrinsic. In conclusion, EPA counteracts lipotoxicity in the proximal tubule by alleviating autophagic numbness, making it potentially suitable as a novel treatment for obesity-related kidney diseases.

Abbreviations: 4-HNE: 4-hydroxy-2-nonenal; ACTB: actin beta; ADGRE1/F4/80: adhesion G protein-coupled receptor E1; ATG: autophagy-related; ATP: adenosine triphosphate; BODIPY: boron-dipyrromethene; BSA: bovine serum albumin; cKO: conditional knockout; CML: N-carboxymethyllysine; COL1A1: collagen type I alpha 1 chain; COX: cytochrome c oxidase; CTRL: control; DGAT: diacylglycerol O-acyltransferase; EPA: eicosapentaenoic acid; FA: fatty acid; FFA: free fatty acid; GFP: green fluorescent protein; HFD: high-fat diet; iKO: inducible knockout; IRI: ischemia-reperfusion injury; LAMP1: lysosomal-associated membrane protein 1; LD: lipid droplet; LRP2: low density lipoprotein receptor-related protein 2; MAP1LC3: microtubule-associated protein 1 light chain 3; MTORC1: mechanistic target of rapamycin kinase complex 1; OA: oleic acid; PAS: periodic-acid Schiff; PPAR: peroxisome proliferator activated receptor; PPARGC1/PGC1: peroxisome proliferator activated receptor, gamma, coactivator 1; PTEC: proximal tubular epithelial cell; ROS: reactive oxygen species; RPS6: ribosomal protein S6; SDH: succinate dehydrogenase complex; SFC/MS/MS: supercritical fluid chromatography triple quadrupole mass spectrometry; SQSTM1/p62: sequestosome 1; TFEB: transcription factor EB; TG: triglyceride; TUNEL: terminal deoxynucleotidyl transferase dUTP nick end labeling.

摘要

最近，我们在肾近端肾小管细胞 (PTECs) 中发现了一种新的脂毒性机制。脂质超载刺激巨自噬/自噬，以修复血浆和细胞器膜，以维持 PTEC 的完整性。然而，这种自噬激活给溶酶体系统带来了负担，导致下游自噬受到抑制，表现为磷脂积累和溶酶体酸化不足。在这里，我们研究了补充二十碳五烯酸 (EPA) 的药理学校正是否可以恢复自噬通量并减轻肾脂毒性。向高脂饮食 (HFD) 喂养的小鼠补充 EPA 可降低 PTEC 中的几个脂毒性标志，例如溶酶体中的磷脂积累、线粒体功能障碍、炎症和纤维化。除了改善代谢综合征外，EPA 还通过多种机制减轻了肾脂毒性。向 HFD 喂养的小鼠补充 EPA 或在棕榈酸 (PA) 中培养的分离的 PTEC 可恢复溶酶体功能，并显着改善自噬通量。在自噬的时间和基因消融过程中，PA 诱导的磷脂从细胞膜重新分布到溶酶体中，HFD 诱导的自噬底物 SQSTM1/p62（sequestosome 1）的积累显着降低，表明 EPA 减弱了HFD 介导的自噬需求增加。此外，脂肪酸脉冲追踪分析表明，EPA 促进了脂滴 (LD) 的形成并从 LD 转移到线粒体进行 β 氧化。值得注意的是，EPA对脂毒性的功效是自噬依赖性和细胞内在的。总之，EPA 通过减轻自噬性麻木来抵消近端肾小管的脂毒性，使其可能适合作为肥胖相关肾脏疾病的新疗法。

缩写：4-HNE：4-羟基-2-壬烯醛；ACTB：肌动蛋白β；ADGRE1/F4/80：粘附 G 蛋白偶联受体 E1；ATG：自噬相关；ATP：三磷酸腺苷；BODIPY：硼-二吡咯亚甲基；BSA：牛血清白蛋白；cKO：有条件淘汰赛；CML：N-羧甲基赖氨酸；COL1A1：I型胶原蛋白α1链；COX：细胞色素 c 氧化酶；CTRL：控制；DGAT：二酰基甘油O-酰基转移酶；EPA：二十碳五烯酸；FA：脂肪酸；FFA：游离脂肪酸；GFP：绿色荧光蛋白；HFD：高脂肪饮食；iKO：诱导型敲除；IRI：缺血再灌注损伤；LAMP1：溶酶体相关膜蛋白 1；LD：脂滴；LRP2：低密度脂蛋白受体相关蛋白2；MAP1LC3：微管相关蛋白 1 轻链 3；MTORC1：雷帕霉素激酶复合物 1 的机械靶点；OA：油酸；PAS：高碘酸席夫；PPAR：过氧化物酶体增殖物激活受体；PPARGC1/PGC1：过氧化物酶体增殖物激活受体，γ，共激活因子 1；PTEC：近端肾小管上皮细胞；ROS：活性氧；RPS6：核糖体蛋白 S6；SDH：琥珀酸脱氢酶复合物；SFC/MS/MS：超临界流体色谱三重四极杆质谱；SQSTM1/p62：隔离体 1；TFEB：转录因子EB；TG：甘油三酯；TUNEL：末端脱氧核苷酸转移酶 dUTP 缺口末端标记。转录因子EB；TG：甘油三酯；TUNEL：末端脱氧核苷酸转移酶 dUTP 缺口末端标记。转录因子EB；TG：甘油三酯；TUNEL：末端脱氧核苷酸转移酶 dUTP 缺口末端标记。