Department of Geriatrics, Tianjin Medical University General Hospital, Tianjin Institute of Geriatrics, Tianjin, China (mainland).
Tianjin Medical University, Tianjin, China (mainland).
Med Sci Monit. 2018 Mar 1;24:1258-1267. doi: 10.12659/msm.907718.
BACKGROUND Albumin, as a major urinary protein component, is a risk factor for chronic kidney disease progression. Mitochondrial dysfunction is one of the main causes of albumin-induced proximal tubule cells injury. Mitophagy is considered as a pivotal protective mechanism for the elimination of dysfunctional mitochondria. The objective of this research was to determine whether albumin overload-induced mitochondrial dysfunction can activate PINK1/Parkin-mediated mitophagy in renal tubular epithelial cells (TECs). MATERIAL AND METHODS Immunofluorescence assay and Western blot assay were used to detect the effects of albumin overload on autophagy marker protein LC3. Transmission electron microscopy and Western blot assay were used to investigate the role of albumin in mitochondrial injury. Western blot assay and co-localization of acidic lysosomes and mitochondria assay were employed to detect the activation of mitophagy induced by albumin. Finally, we explored the role of PINK1/Parkin signaling in albumin-induced mitophagy by inhibiting mitophagy by knockdown of PARK2 (Parkin) level. RESULTS Immunofluorescence and Western blot results showed that the expression level of LC3-II increased, and the maximum increase point was observed after 8 h of albumin treatment. Transmission electron microscopy results demonstrated that albumin overload-induced mitochondrial injury and quantity of autophagosomes increased. Additionally, expression of PINK1 and cytosolic cytochrome C increased and mitochondria cytochrome C decreased in the albumin group. The co-localization of acidic lysosomes and mitochondria demonstrated that the number of albumin overload-induced mitophagy-positive dots increased. The transient transfection of PARK2 siRNA result showed knockdown of the expression level of PARK2 can inhibit mitophagy induced by albumin. CONCLUSIONS In conclusion, our study suggests that mitochondrial dysfunction activates the PINK1/Parkin signaling and mitophagy in renal tubular epithelial cells under albumin overload condition.
白蛋白作为主要的尿蛋白成分，是慢性肾脏病进展的危险因素。线粒体功能障碍是白蛋白诱导近端肾小管细胞损伤的主要原因之一。自噬被认为是消除功能失调线粒体的关键保护机制。本研究旨在确定白蛋白过载诱导的线粒体功能障碍是否可以激活 PINK1/Parkin 介导的肾小管上皮细胞（TEC）中的线粒体自噬。
免疫荧光和 Western blot 检测白蛋白过载对自噬标记蛋白 LC3 的影响。透射电镜和 Western blot 检测白蛋白对线粒体损伤的作用。Western blot 和酸性溶酶体与线粒体共定位检测白蛋白诱导的线粒体自噬的激活。最后，通过 PARK2（Parkin）水平的敲低抑制自噬，探讨 PINK1/Parkin 信号在白蛋白诱导的自噬中的作用。
免疫荧光和 Western blot 结果表明，LC3-II 的表达水平增加，白蛋白处理 8 h 时达到最大值。透射电镜结果显示，白蛋白过载诱导线粒体损伤和自噬体数量增加。此外，白蛋白组中 PINK1 和胞质细胞色素 C 的表达增加，线粒体细胞色素 C 减少。酸性溶酶体与线粒体的共定位显示，白蛋白过载诱导的自噬阳性点数量增加。PARK2 siRNA 的瞬时转染结果表明，PARK2 表达水平的敲低可抑制白蛋白诱导的自噬。
综上所述，本研究表明，在白蛋白过载条件下，线粒体功能障碍激活了 PINK1/Parkin 信号和肾小管上皮细胞中的线粒体自噬。
