巨噬细胞向肌成纤维细胞转分化通过TGF-β1/Smad3信号通路加重缺血-再灌注损伤后肾纤维化

Macrophage-to-myofibroblast transition exacerbates renal fibrosis after ischemia-reperfusion injury via the TGF-β1/Smad3 signaling pathway

    摘要
  • 摘要:
    目的 探讨巨噬细胞-肌成纤维细胞转分化（MMT）在缺血-再灌注损伤（IRI）所致急性肾损伤（AKI）后肾纤维化过程中的作用及可能的机制。
    方法 采用肾缺血-再灌注构建小鼠AKI模型，随机分为对照组（Con组）、假手术组（Sham组）、IRI术后1 d组（IRI 1d组）、IRI术后3 d组（IRI 3 d组）、IRI术后14 d组（IRI 14 d组），每组5只。检测肾系数、血清肌酐（Scr）和肾损伤分子-1（KIM-1）评估肾损伤情况；过碘酸-雪夫（PAS）染色评估肾小管损伤及炎症细胞浸润情况；Masson染色和免疫组织化学染色评估胶原沉积及α-平滑肌肌动蛋白（α-SMA）、Ⅰ型胶原（COLⅠ）表达情况；流式细胞术分析巨噬细胞浸润及表型；流式细胞术和免疫荧光染色分析MMT；蛋白质印迹法检测转化生长因子（TGF）-β1/Smad3信号通路蛋白。
    结果 与Sham组比较，IRI 1 d组肾系数、Scr和KIM-1水平升高，IRI 3 d组肾系数、KIM-1水平升高；与IRI 1 d组比较，IRI 14 d组肾系数和KIM-1水平下降；与IRI 3 d组比较，IRI 14 d组肾系数、Scr和KIM-1水平下降（均为P<0.05）。PAS染色显示肾小管损伤在IRI 3 d最严重。Masson染色显示胶原沉积随时间延长逐渐增加，免疫组织化学染色显示，α-SMA和COLⅠ于IRI后1 d开始增加，持续到14 d（均为P<0.05）。巨噬细胞浸润自IRI术后1 d开始增加，并持续至14 d（P<0.05）。M1型巨噬细胞在IRI术后1 d迅速升高后下降，而M2型巨噬细胞则在IRI术后3 d增加并持续高水平至14 d（P<0.05）。MMT自IRI术后3 d开始增加，持续到14 d，M2型巨噬细胞是MMT细胞的主要表型（均为P<0.05）。与Sham组比较，IRI 1 d组、IRI 3 d组、IRI 14 d组TGF-β1蛋白表达增加，pSmad3/Smad3升高（均为P<0.05）。
    结论 M2型巨噬细胞可能通过MMT促进IRI-AKI后肾纤维化进展，此过程与TGF-β1/Smad3信号通路的激活密切相关。

     

    Abstract:
    Objective To clarify the role and underlying mechanism of macrophage-to-myofibroblast transition (MMT) in renal fibrosis that develops after acute kidney injury (AKI) induced by ischemia-reperfusion injury (IRI).
    Methods Mouse AKI model was generated by renal ischemia-reperfusion. Animals were randomized into control (Con), sham operated (Sham), and IRI groups sacrificed at 1 d (IRI 1 d), 3 d (IRI 3 d) and 14 d (IRI 14 d) after reperfusion (n = 5). Renal injury was assessed by renal coefficient, serum creatinine (Scr) and kidney injury molecule-1 (KIM-1). Periodic acid-Schiff (PAS) staining was used to evaluate tubular damage and inflammatory infiltration. Masson staining and immunohistochemistry were employed to quantify collagen deposition, α-smooth muscle actin (α-SMA) and type I collagen (COL I). Flow cytometry was used to determine macrophage infiltration and phenotype. MMT was identified by flow cytometry plus immunofluorescence. Transforming growth factor (TGF)-β1/Smad3 pathway proteins were examined by Western blotting.
    Results Compared with Sham group, renal coefficient, Scr and KIM-1 rose in IRI 1 d group, renal coefficient and KIM-1 remained elevated in IRI 3 d group. Compared with the IRI 1 d group, the renal coefficient and KIM-1 decreased in the IRI 14 d group. Compared with the IRI 3 d group, the renal coefficient, Scr and KIM-1 decreased in the IRI 14 d group (all P < 0.05). PAS revealed the most severe tubular injury at IRI 3 d. Masson staining showed progressively increasing collagen deposition, while immunohistochemistry demonstrated α-SMA and COL I rising from day 1 and persisting to day 14 (all P < 0.05). Macrophage infiltration increased from day 1 and lasted to day 14 (P < 0.05). M1 macrophages peaked at day 1 then declined, whereas M2 macrophages increased at day 3 and remained high through day 14 (P < 0.05). MMT began to rise at day 3 and continued to day 14 and M2 macrophages were the predominant source of MMT cells (all P < 0.05). Compared with Sham group, TGF-β1 protein was up-regulated and p-Smad3/Smad3 ratio was elevated in all IRI groups (all P < 0.05).
    Conclusions M2 macrophages promote post-IRI-AKI renal fibrosis via MMT, a process closely linked to activation of the TGF-β1/Smad3 signaling pathway.

     

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