New Autophagy Reporter Mice Reveal Dynamics of Proximal Tubular Autophagy
New Autophagy Reporter Mice Reveal Dynamics of Proximal Tubular Autophagy
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DOI：
10.1681/asn.2013040374
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发表时间：
2014-02-01
影响因子：
13.6
通讯作者：
Lin, Fangming
中科院分区：
文献类型：
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作者：
Li, Ling;Wang, Zhao V.;Lin, Fangming
自噬体在缺血后肾脏中的积累可能具有肾脏保护作用，但这种积累是由于自噬的诱导还是由于自噬过程中的阻塞尚不清楚。利用红色荧光蛋白（RFP; pK（a）4.5）和增强型绿色荧光蛋白（EGFP; pK（a）5.9）的不同pH敏感性，我们产生了CAG-RFP-EGFP-LC 3小鼠以区分早期自噬空泡和自溶体。体外和体内研究证实，为了响应营养剥夺，CAG-RFP-EGFP-LC 3小鼠的肾上皮细胞产生表达RFP和EGFP斑点的自噬空泡。EGFP荧光大幅减少在酸性环境中的自溶酶体，而明亮的RFP信号仍然存在。在正常条件下，肾单位表达很少的EGFP和RFP斑点，但缺血再灌注损伤（IRI）导致近端小管的动态变化，RFP和EGFP斑点的数量增加，在IRI后1天达到峰值。IRI后3天，EGFP斑点的数量恢复到对照水平，而RFP斑点的高水平持续存在，表明自噬在第1天开始，自噬体在第3天肾恢复期间清除。值得注意的是，在含有RFP斑点的细胞中增殖减少，表明自噬细胞不太可能分裂用于肾小管修复。此外，87%的近端肾小管细胞具有激活的雷帕霉素（mTOR）机制靶点，可防止自噬，不含RFP斑点。相反，抑制mTOR复合物1诱导RFP和EGFP表达并降低细胞增殖。总之，我们的研究结果强调了缺血后肾脏自噬的动态调节，并表明mTOR在肾脏修复过程中自噬消退中的作用。
The accumulation of autophagosomes in postischemic kidneys may be renoprotective, but whether this accumulation results from the induction of autophagy or from obstruction within the autophagic process is unknown. Utilizing the differential pH sensitivities of red fluorescent protein (RFP; pK(a) 4.5) and enhanced green fluorescent protein (EGFP; pK(a) 5.9), we generated CAG-RFP-EGFP-LC3 mice to distinguish early autophagic vacuoles from autolysosomes. In vitro and in vivo studies confirmed that in response to nutrient deprivation, renal epithelial cells in CAG-RFP-EGFP-LC3 mice produce autophagic vacuoles expressing RFP and EGFP puncta. EGFP fluorescence diminished substantially in the acidic environment of the autolysosomes, whereas bright RFP signals remained. Under normal conditions, nephrons expressed few EGFP and RFP puncta, but ischemia-reperfusion injury (IRI) led to dynamic changes in the proximal tubules, with increased numbers of RFP and EGFP puncta that peaked at 1 day after IRI. The number of EGFP puncta returned to control levels at 3 days after IRI, whereas the high levels of RFP puncta persisted, indicating autophagy initiation at day 1 and autophagosome clearance during renal recovery at day 3. Notably, proliferation decreased in cells containing RFP puncta, suggesting that autophagic cells are less likely to divide for tubular repair. Furthermore, 87% of proximal tubular cells with activated mechanistic target of rapamycin (mTOR), which prevents autophagy, contained no RFP puncta. Conversely, inhibition of mTOR complex 1 induced RFP and EGFP expression and decreased cell proliferation. In summary, our results highlight the dynamic regulation of autophagy in postischemic kidneys and suggest a role of mTOR in autophagy resolution during renal repair.