Physiol Rep:FXa因子抑制剂艾多沙班可通过减少上皮间质转化和炎症反应来改善肾大部分切除术后的肾损伤

2022-03-27 “ 心关注”公众号 “ 心关注”公众号

最近的证据表明,CKD患者普遍存在凝血障碍,随CKD进展,凝血因子增加,从而导致CKD程度进一步恶化。然而,目前关于凝血因子在CKD发展中的作用尚不明确。

随着人口老龄化的加剧,慢性肾脏病(CKD)的患病率也不断增加。据估计,全世界的CKD患者占成年人口的8%~16%。即使是没有相关病因(如2型糖尿病和慢性肾小球肾炎)的健康人群,肾功能也会随年龄的增长而下降。最近的证据表明,CKD患者普遍存在凝血障碍,随CKD进展,凝血因子增加,从而导致CKD程度进一步恶化。然而,目前关于凝血因子在CKD发展中的作用尚不明确。

活化凝血因子X(FXa)是调节内源性和外源性凝血级联的关键因素。FXa及其受体蛋白酶活化受体-2(PAR2)信号传导在炎症、纤维化和动脉粥样硬化等疾病中发挥重要作用。FXa抑制剂,例如直接口服抗凝剂(DOAC),被广泛用于预防卒中和其他非瓣膜性心房颤动的血栓并发症。最近,Horinouchi等人报道艾多沙班(一种FXa抑制剂)可改善单侧尿路梗阻小鼠模型的肾纤维化。在本研究中,研究者使用5/6肾切除小鼠模型观察艾多沙班对CKD的治疗作用。此外,还研究了FXa对近端小管上皮细胞纤维化、炎症反应和氧化应激的影响。

研究方法

对8周龄WT小鼠进行造模;切除左肾的上极和下极,1周后结扎右肾动脉、静脉和输尿管,通过右正中切口取出剩余的右肾。消融后7天,将WT小鼠随机分为两组,艾多沙班(25 mg/kg/d)混合饮食组和正常饮食组。消融后7周,采集WT小鼠尿液和血液标本,并进行分析。

研究结果

● 艾多沙班可减轻肾大部分切除术后的肾损伤和肾小球内纤维蛋白沉积

5/6肾切除术后显着提高了WT小鼠的血浆FXa水平;与对照组WT小鼠相比,艾多沙班组的WT小鼠血浆FXa水平显着降低。肾大部分切除术后,WT小鼠的尿白蛋白排泄和循环中的尿素氮(UN)和肌酐(Cr)水平显着增加(图1a)。与对照组相比,艾多沙班组的WT小鼠在肾大部分切除术后尿白蛋白排泄和血浆UN水平降低(图1a)。相比之下,两组血浆Cr水平无显着差异。

与正常饮食组的WT小鼠相比,艾多沙班组的WT小鼠在肾大部分切除术后肾小球球体截面积和肾小球内细胞数量减少(图1b)。

与假手术组相比,肾大部分切除术后肾小球内纤维蛋白沉积增加;与对照组治疗相比,艾多沙班治疗显着降低了术后肾小球内微栓塞(图1c)。

图1. 艾多沙班可减轻5/6肾切除术后的肾损伤和肾小球内纤维蛋白沉积

● 艾多沙班通过减少上皮间质转化(EMT)、炎症反应和氧化应激改善肾小管间质纤维化

5/6肾切除术增加了WT小鼠的肾纤维化面积。与对照组治疗相比,艾多沙班治疗减少了WT小鼠的肾纤维化面积(图2a);同样,艾多沙班组的WT小鼠在肾大部分切除术后,包括胶原I、胶原III和转化生长因子(TGF-β1)在内的纤维化标志物的表达水平降低(图2b);同时,包括α-平滑肌肌动蛋白(α-SMA)、n-钙粘蛋白(N-cadherin)和波形蛋白(Vimentin)在内的EMT表达水平出现降低(图2c);此外,WT小鼠残肾部分的炎症介质(包括肿瘤坏死因子TNF-α和单核细胞趋化蛋白-1MCP-1)和氧化应激标志物(包括 gp91phox、p47phox 和 p67phox)表达水平也出现降低。

图2. 艾多沙班通过减少EMT、炎症反应和氧化应激改善肾小管间质纤维化

● 在培养的肾小管上皮细胞中,艾多沙班可减轻FXa诱导的EMT、炎症反应和氧化应激的增加

为探讨艾多沙班降低肾大部分切除术后残肾EMT、炎症反应和氧化应激的确切机制,研究者先用艾多沙班或对照药预处理MT小鼠近端肾小管上皮细胞(HK-2),再用FXa治疗。用艾多沙班预处理的HK-2细胞显着降低了FXa刺激的EMT表达,包括α-SMA、N-Cadherin和Vimentin的表达(图3a)。此外,艾多沙班预处理的HK-2细胞显着降低了FXa诱导的炎症介质表达和氧化应激表达(图3b,c)。这些结果表明,艾多沙班消除了FXa诱导的近端肾小管细胞EMT、炎症反应和氧化应激的增加。

因为近端小管上皮细胞的EMT和炎症反应分别是由ERK和NF-κB信号通路介导,研究者评估了FXa对HK-2细胞中ERK和NF-κB磷酸化的影响。FXa处理HK-2细胞后,ERK和NF-κB的磷酸化水平呈时间依赖性增加(图4a)。用艾多沙班预处理HK-2细胞可消除FXa诱导的ERK和NF-κB磷酸化水平的增加(图4b)。肾大部分切除术增加了MT小鼠残肾ERK和NF-κB磷酸化水平;与对照组相比,艾多沙班组治疗的MT小鼠残肾RK和NF-κB磷酸化水平降低(图4c)。

为研究PAR2对FXa诱导的ERK和NF-κB磷酸化的影响,研究者发现通过靶向PAR2的siRNA转染HK-2细胞后,PAR2mRNA的表达降低71.4±0.7%;FXa诱导的ERK和NF-κB磷酸化减弱(图4d)。这些结果表明,FXa通过PAR2依赖途径诱导ERK和NF-κB活化。

图4. 艾多沙班抑制FXa诱导的HK-2细胞中ERK和NF-κB的磷酸化

研究结论

FXa抑制剂艾多沙班可以通过减少EMT和炎症反应来改善肾大部分切除术后的肾损伤,这提示FXa可能是CKD合并房颤患者的一个新的治疗靶点。

原始出处:

Fang L, Ohashi K, Ogawa H, et al. Factor Xa inhibitor, edoxaban ameliorates renal injury after subtotal nephrectomy by reducing epithelial-mesenchymal transition and inflammatory response. Physiol Rep. 2022 Mar;10(5):e15218. doi: 10.14814/phy2.15218. PMID: 35262272; PMCID: PMC8905573.

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    2023-03-04 jklm09
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    2022-04-01 仇敏瑜

    学习了

    0

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