Data from: Downregulation of autophagy is associated with severe ischemia-reperfusion-induced acute kidney injury in overexpressing C-reactive protein mice

C-reactive protein (CRP), was recently reported to be closely associated with poor renal function in patients with acute kidney injury (AKI), but whether CRP is pathogenic or a mere biomarker in AKI remains largely unclear. Impaired autophagy is known to exacerbate renal ischemia-reperfusion injury (IRI). We examined whether the pathogenic role of CRP in AKI is associated with reduction of autophagy. We mated transgenic rabbit CRP over-expressing mice (Tg-CRP) with two autophagy reporter mouse lines, Tg-GFP-LC3 mice (LC3) and Tg-RFP-GFP-LC3 mice (RG-LC3) respectively to generate Tg-CRP-GFP-LC3 mice (PLC3) and Tg-CRP-RFP-GFP-LC3 mice (PRG-LC3). AKI was induced by IRI. Compared with LC3 mice, PLC3 mice developed more severe kidney damage after IRI. Renal tubules were isolated from LC3 mice at baseline for primary culture. OKP cells were transiently transfected with GFP-LC3 plasmid. CRP addition exacerbated lactate dehydrogenase release from both cell types. Immunoblots showed lower LC-3 II/I ratios and higher levels of p62, markers of reduced autophagy flux, in the kidneys of PLC3 mice compared to LC3 mice after IRI, and in primary cultured renal tubules and OKP cells treated with CRP and H2O2 compared to H2O2 alone. Immunohistochemistry showed much fewer LC-3 punctae, and electron microscopy showed fewer autophagosomes in kidneys of PLC3 mice compared to LC3 mice after IRI. Similarly, CRP addition reduced GFP-LC3 punctae induced by H2O2 in primary cultured proximal tubules and in GFP-LC3 plasmid transfected OKP cells. Rapamycin, an autophagy inducer, rescued impaired autophagy and reduced renal injury in vivo. In summary, it was suggested that CRP be more than mere biomarker in AKI, and render the kidney more susceptible to ischemic/oxidative injury, which is associated with down-regulating autophagy flux.

近期有研究报道，C反应蛋白（C-reactive protein, CRP）与急性肾损伤（acute kidney injury, AKI）患者的肾功能不良密切相关，但CRP在AKI中究竟发挥致病作用，还是仅作为一种生物标志物，目前仍基本尚不明确。已知自噬功能受损会加重肾缺血再灌注损伤（renal ischemia-reperfusion injury, IRI）。本研究旨在探讨CRP在AKI中的致病作用是否与自噬水平下调有关。我们将过表达兔CRP的转基因小鼠（Tg-CRP）分别与两种自噬报告基因小鼠株——Tg-GFP-LC3小鼠（LC3）及Tg-RFP-GFP-LC3小鼠（RG-LC3）交配，成功构建了Tg-CRP-GFP-LC3小鼠（PLC3）与Tg-CRP-RFP-GFP-LC3小鼠（PRG-LC3）。本研究通过IRI构建AKI模型。与LC3小鼠相比，PLC3小鼠在IRI造模后出现更严重的肾损伤。我们从基线状态下的LC3小鼠体内分离肾小管进行原代培养。将GFP-LC3质粒瞬时转染至OKP细胞中。加入CRP可加重两种细胞的乳酸脱氢酶（lactate dehydrogenase, LDH）释放。免疫印迹结果显示，与IRI造模后的LC3小鼠肾脏相比，PLC3小鼠肾脏中LC3Ⅱ/Ⅰ比值更低，而p62蛋白水平更高，二者均为自噬流（autophagy flux）受损的标志物；同时，在CRP联合H₂O₂处理的原代肾小管细胞及OKP细胞中，相较于仅用H₂O₂处理的对照组，同样观察到上述自噬流受损的分子表型。免疫组化结果显示，IRI造模后PLC3小鼠肾脏内的LC3斑点数量显著少于LC3小鼠；电子显微镜观察则发现，PLC3小鼠肾脏内的自噬体数量同样更少。类似地，在H₂O₂诱导的原代近端肾小管细胞及转染了GFP-LC3质粒的OKP细胞中，加入CRP均可减少GFP-LC3斑点的形成。自噬诱导剂雷帕霉素（rapamycin）可在体内挽救受损的自噬功能，并减轻肾损伤。综上，本研究结果提示，CRP在AKI中并非仅作为一种生物标志物，而是可使肾脏更易受到缺血/氧化应激损伤，这一作用与其下调自噬流密切相关。