Department of Pharmacology and Toxicology, Virginia Commonwealth University School of Medicine, Richmond, VA, USA.
J Cell Mol Med. 2013 Dec;17(12):1598-607. doi: 10.1111/jcmm.12173. Epub 2013 Nov 17.
Podocytes are highly differentiated glomerular epithelial cells that contribute to the glomerular barrier function of kidney. A role for autophagy has been proposed in maintenance of their cellular integrity, but the mechanisms controlling autophagy in podocytes are not clear. The present study tested whether CD38-mediated regulation of lysosome function contributes to autophagic flux or autophagy maturation in podocytes. Podocytes were found to exhibit a high constitutive level of LC3-II, a robust marker of autophagosomes (APs), suggesting a high basal level of autophagic activity. Treatment with the mTOR inhibitor, rapamycin, increased LC3-II and the content of both APs detected by Cyto-ID Green staining and autophagolysosomes (APLs) measured by acridine orange staining and colocalization of LC3 and Lamp1. Lysosome function inhibitor bafilomycin A1 increased APs, but decreased APLs content under both basal and rapamycin-induced conditions. Inhibition of CD38 activity by nicotinamide or silencing of CD38 gene produced the similar effects to that bafilomycin A1 did in podocytes. To explore the possibility that CD38 may control podocyte autophagy through its regulation of lysosome function, the fusion of APs with lysosomes in living podocytes was observed by co-transfection of GFP-LC3B and RFP-Lamp1 expression vectors. A colocalization of GFP-LC3B and RFP-Lamp1 upon stimulation of rapamycin became obvious in transfected podocytes, which could be substantially blocked by nicotinamide, CD38 shRNA, and bafilomycin. Moreover, blockade of the CD38-mediated regulation by PPADS completely abolished rapamycin-induced fusion of APs with lysosomes. These results indicate that CD38 importantly control lysosomal function and influence autophagy at the maturation step in podocytes.
足细胞是高度分化的肾小球上皮细胞，有助于维持肾脏的肾小球屏障功能。自噬在维持其细胞完整性方面的作用已被提出，但控制足细胞自噬的机制尚不清楚。本研究检测了 CD38 介导的溶酶体功能调节是否有助于足细胞中的自噬流或自噬成熟。研究发现，足细胞表现出高水平的 LC3-II，这是自噬体 (APs) 的一个强有力的标志物，这表明它们具有高水平的基础自噬活性。用 mTOR 抑制剂雷帕霉素处理会增加 LC3-II 的含量，并通过 Cyto-ID Green 染色检测到 APs 的含量增加，通过吖啶橙染色检测到自噬溶酶体 (APLs) 的含量增加，同时 LC3 和 Lamp1 的共定位增加。溶酶体功能抑制剂巴弗洛霉素 A1 增加 APs，但在基础和雷帕霉素诱导条件下均减少 APLs 的含量。用烟酰胺抑制 CD38 活性或沉默 CD38 基因，会产生与巴弗洛霉素 A1 相似的效果。为了探索 CD38 可能通过调节溶酶体功能来控制足细胞自噬的可能性，通过共转染 GFP-LC3B 和 RFP-Lamp1 表达载体观察活足细胞中 APs 与溶酶体的融合。雷帕霉素刺激后 GFP-LC3B 和 RFP-Lamp1 的共定位在转染的足细胞中变得明显，这一现象可以被烟酰胺、CD38 shRNA 和巴弗洛霉素显著阻断。此外，PPADS 阻断 CD38 介导的调节作用可完全消除雷帕霉素诱导的 APs 与溶酶体的融合。这些结果表明，CD38 重要地控制溶酶体功能，并在足细胞的自噬成熟阶段影响自噬。
