MR体素内不相干运动成像评价兔肾动脉狭窄肾纤维化的研究

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• 基础研究 •

查婷婷陈杰俞胜男刘国强陈婧邢伟

Cite this article as: ZHA T T, CHEN J, YU S N, et al. Value of intravoxel incoherent motion magnetic resonance imaging in evaluating renal fibrosis in rabbits with renal artery stenosis[J]. Chin J Magn Reson Imaging, 2023, 14(11): 128-135.本文引用格式：查婷婷, 陈杰, 俞胜男, 等. MR体素内不相干运动成像评价兔肾动脉狭窄肾纤维化的研究[J]. 磁共振成像, 2023, 14(11): 128-135. DOI:10.12015/issn.1674-8034.2023.11.021.

摘要

[摘要] 目的探讨MR体素内不相干运动（intravoxel incoherent motion, IVIM）成像评价兔肾动脉狭窄肾纤维化（renal fibrosis, RF）的价值。材料与方法 将78只健康新西兰大白兔随机分为对照组（8例）和RF组（70例），RF组再随机分为6个亚组，分别为建模前pre-RF亚组、建模后RF-1W、RF-2W、RF-3W、RF-4W亚组（每个亚组12例），以及动态观察（RF-longitude, RF-L）亚组（10例）。RF组实验兔行左肾动脉狭窄建立RF模型。pre-RF、RF-1W、RF-2W、RF-3W和RF-4W亚组实验兔分别于术前、术后1、2、3和4周行MR轴位T2WI和IVIM扫描，扫描后立即切除左肾并行病理组织学检查。RF-L亚组和对照组实验兔每个时间点均进行MR扫描并在术后4 W扫描结束后行组织病理学检查。在IVIM图像上分别测量计算双肾皮、髓质的真扩散系数（true diffusion coefficient, D）、伪扩散系数（pseudo-diffusion coefficient, D^*）和灌注分数（perfusion fraction, f）。不同时间点左、右肾皮质和髓质D、D^*、f值差异的比较采用重复测量方差分析。同一时间点组间双肾皮质、髓质D、D^*、f值的差异比较采用独立样本t检验。采用Spearman相关性分析比较左肾皮质、髓质D、D^*、f值与RF程度的相关性。采用受试者工作特征（receiver operating characteristic, ROC）曲线判断左肾皮质、髓质D、D^*、f值对RF诊断和分期的效能。结果 RF组左肾皮质和髓质的D值和f值随RF程度加深均逐渐减低。左肾皮质的D^*值RF-4W和pre-RF、RF-1W、RF-2W亚组比较差异有统计学意义（P均＜0.05）。而左肾髓质的D^*值随时间差异无统计学意义。RF组右肾皮质和髓质的D^*值和f值随RF程度加深均逐渐升高。而右肾皮质和髓质的D值随时间差异无统计学意义。对照组左、右肾皮质、髓质的IVIM参数值随时间差异均无统计学意义。RF-4W亚组左肾皮质与髓质的D值、D^*值差异均有统计学意义（P均＜0.05）；pre-RF、RF-3W和RF-4W亚组左肾皮质与髓质的f值差异均有统计学意义（P均＜0.05）。pre-RF、RF-4W亚组右肾皮质与髓质的f值差异有统计学意义（P均＜0.05）。Spearman相关性检验显示左肾皮质D、f值及髓质f值与RF程度呈中等或强负相关（r值分别为-0.595、-0.717、-0.412，P均＜0.01）；而左肾皮质D^*值、髓质D、D^*值与纤维化程度均无明显相关性（P均＞0.05）。f值对鉴别pre-RF和RF-1W-RF-4W、pre-RF-RF-1W和RF-2W-RF-4W的效能最优；D值对鉴别pre-RF-RF-2W和RF-3W-RF-4W、pre-RF-RF-3W和RF-4W的效能最优。结论 MR-IVIM可以分别从灌注和扩散两方面反映RF发生发展的过程，可以为RF的分期诊断提供信息，对RF的评价具有较大的应用前景。

[Abstract] Objective To investigate the value of MR intravoxel incoherent motion imaging (IVIM) in evaluating renal fibrosis (RF) in rabbits with renal artery stenosis.Materials and Methods Seventy-eight healthy New Zealand white rabbits were randomly divided into control group (n=8) and RF group (n=70). The RF group was further randomly divided into six subgroups: pre-RF、RF-1W、RF-2W、RF-3W、RF-4W subgroup (n=12 in each subgroup), and RF-longitude (RF-L) subgroup (n=10) for dynamic observation. The rabbits in the RF group underwent left renal artery stenosis to establish the RF model. MR axial T2WI and IVIM scans were performed before operation and at 1, 2, 3 and 4 weeks after operation, respectively. After the last MR scan, the left kidney was resected and histopathological examination was performed. The rabbits in the RF-L subgroup and the control group underwent MR scanning at each time point and histopathological examinations at the end of the scanning at 4 weeks after operation. The true diffusion coefficient (D)、pseudo-diffusion coefficient (D^*) and perfusion fraction (f) values of bilateral renal cortex and medulla were measured and calculated on IVIM images. Repeated measures analysis of variance was used to compare the differences of D, D^* and f values in the left and right renal cortex and medulla over time in each subgroup. Independent sample t test was used to compare the differences of D, D^* and f values of bilateral renal cortex and medulla at each time point. Spearman correlation analysis was used to compare the correlation between the D, D^*, f values of the left renal cortex and medulla and the degree of RF. Receiver operating characteristic (ROC) curve was used to determine the efficacy of D, D^*, f values of left renal cortex and medulla in the diagnosis and staging of RF.Results In the RF group, the D and f values of left renal cortex and medulla decreased gradually with RF. The D^* values of left renal cortex were significantly different between RF-4W and pre-RF, RF-1W, RF-2W subgroups (P all＜0.05). However, the D^* values of the left renal medulla did not differ significantly over time. The D^* and f values of the right renal cortex and medulla gradually increased with RF. However, the D values of the right kidney cortex and medulla did not differ significantly over time. In the control group, there was no significant difference in IVIM parameters between left and right renal cortex and medulla over time. The D and D^* values of the left renal cortex and medulla in the RF-4W subgroup were statistically different (P all <0.05). The f values of the left renal cortex and medulla in the pre-RF, RF-3W and RF-4W subgroups were statistically different (P all <0.05). The f values of the right renal cortex and medulla in the pre-RF and RF-4W subgroups were statistically different (P all <0.05). Spearman correlation test showed that the D and f values of left renal cortex and the f value of left renal medulla were moderately or strongly negatively correlated with the degree of RF (r=-0.595, -0.717, -0.412, P all <0.01). The D^* values of left renal cortex and the D、D^* values of the left renal medulla were not correlated with the degree of RF (P all >0.05). The f value was the best to identify pre-RF and RF-1W-RF-4W, pre-RF-RF-1W and RF-2W-RF-4W. The D value was the best to identify pre-RF-RF-2W and RF-3W-RF-4W, pre-RF-RF-3W and RF-4W.Conclusions MR-IVIM can reflect the occurrence and development of RF from two aspects of perfusion and diffusion respectively. It has a great value in evaluating and staging RF.

[关键词] 肾纤维化；肾动脉狭窄；体素内不相干运动；磁共振成像

[Keywords] renal fibrosis；renal artery stenosis；intravoxel incoherent motion；magnetic resonance imaging

作者信息

查婷婷 陈杰 俞胜男 刘国强 陈婧邢伟 ^*

苏州大学附属第三医院放射科，常州　213003

通信作者：邢伟，E-mail：suzhxingwei@suda.edu.cn

作者贡献声明：邢伟设计本研究的方案，对稿件重要的学术内容进行了修改；查婷婷起草和撰写稿件，获取、分析或解释本研究的数据，获得了常州市卫健委重大科技项目、常州市卫生健康青苗人才培养工程项目和苏州大学放射医学与辐射防护国家重点实验室项目资助；陈杰、俞胜男、刘国强、陈婧获取、分析或解释本研究的数据，对稿件的重要学术内容进行了修改；全体作者均同意发表最后的修改稿，同意对本研究的所有方面负责，确保本研究的准确性和诚信。

出版信息

基金项目： 常州市卫健委重大科技项目 ZD202211 常州市卫生健康青苗人才培养工程项目 CZQM2020037 苏州大学放射医学与辐射防护国家重点实验室资助项目 GZK12023046

收稿日期：2023-08-01

接受日期：2023-10-27

中图分类号：R445.2 R-332

文献标识码：A

DOI: 10.12015/issn.1674-8034.2023.11.021

本文引用格式：查婷婷, 陈杰, 俞胜男, 等. MR体素内不相干运动成像评价兔肾动脉狭窄肾纤维化的研究[J]. 磁共振成像, 2023, 14(11): 128-135. DOI:10.12015/issn.1674-8034.2023.11.021.

正文

0 前言

慢性肾病（chronic kidney disease, CKD）已经成为一个全球性的公共卫生问题，发病率逐年升高^[¹^]。大部分CKD将不可避免地因肾纤维化（renal fibrosis, RF）而发展为慢性肾衰竭。RF是CKD进展的最重要预测因子之一^[²^,³^]。RF形成过程分为三期，一是可逆期（炎症反应期），二是纤维化形成期，三是瘢痕期（不可逆期）。如在可逆阶段进行临床药物干预或解除致病因素，可以阻止或逆转RF的病理损伤。因此，早期检测和动态评估RF的进程，对逆转肾功能损伤、提高CKD患者生存质量至关重要^[⁴^,⁵^,⁶^]。

RF进程的监测一直依赖肾活检，但存在取样偏差、潜在的出血等并发症、难以重复施行，无法满足动态观察的需要，血清学指标亦不能反映单肾功能^[⁷^]。影像学检查能弥补上述检查方法的缺点。功能MRI已经成为肾脏病研究的热点方向，研究涉及肾脏宏观结构、血流动力学、氧合以及微观结构相关的MRI生物标志物等^[⁸^,⁹^,¹⁰^,¹¹^,¹²^]。

基于双指数拟合模型的体素内不相干运动（intravoxel incoherent motion, IVIM）成像分别评估组织微灌注和扩散信息，可以更准确地揭示水分子运动与组织结构的相互作用，提供关于扩散和灌注特性的完整信息^[¹³^,¹⁴^,¹⁵^,¹⁶^,¹⁷^]，已用于评估肾功能损伤和移植肾纤维化。在一项对97位CKD患者的研究中发现，IVIM参数值与肾皮质的毛细血管密度正相关，IVIM可以反映肾纤维化的毛细血管损失和低灌注改变^[¹⁸^]。BANE等^[¹⁹^]通过对27例肾移植患者移植肾的研究发现，IVIM的灌注分数（perfusion fraction, f）和真扩散系数（true diffusion coefficient, D）值与血管和肾小球损伤的Banff评分显著相关。也有部分研究^[²⁰^]采用单侧输尿管梗阻（unilateral ureteral obstruction, UUO）模型分析肾脏IVIM参数与RF的关系。但UUO模型会导致肾盂扩张积血积液，对肾髓质信号存在潜在影响，不利于髓质的评估。此外，对侧健肾的代偿作用研究较少。

本研究采用肾动脉狭窄法构建兔RF，通过IVIM参数监测肾脏皮髓质信号随纤维化进展的动态变化，同时分析了对侧肾脏的代偿作用，探讨MR-IVIM成像分析评价兔肾动脉狭窄RF的价值，以期为RF的早期诊断、精准分期和疗效评价提供无创可靠的影像学方法。

1 材料与方法

1.1 动物分组

本研究得到了苏州大学附属第三医院伦理委员会批准（批文号：2023 科第057 号）。采用左肾动脉狭窄法构建实验动物模型。取健康新西兰大白兔[江苏科标医学技术集团有限公司提供，实验动物使用许可证号：SYXK（苏）2021-0012] 78 只，3-4 月龄，体质量为2.0-2.5 kg，采用简单随机分组法将实验兔随机分为对照组（8 只）和肾动脉狭窄RF 组（70 只）。其中RF 组随机分为6个亚组，分别为建模前pre-RF亚组、建模后1、2、3、4 周亚组（RF-1W、RF-2W、RF-3W、RF-4W，每个亚组12 只），以及动态观察（RF-longitude, RF-L）亚组（10只）。实验设计流程图见图1。

点击查看大图

图1 实验设计分组流程图。RF：肾纤维化；pre-RF：RF建模前；RF-1W：RF建模后1周；RF-2W：RF建模后2周；RF-3W：RF建模后3周；RF-4W：RF建模后4周；RF-L：RF纵向观察亚组。
Fig. 1 The experimental design and assignment of rabbits. pre-RF: pre-renal fibrosis; RF-1W: 1 week after RF; RF-2W: 2 weeks after RF; RF-3W: 3 weeks after RF; RF-4W: 4 weeks after RF; RF-L: renal fibrosis-longitude.

1.2 模型制备

RF组实验兔3%氨基甲酸乙酯溶液（广州甄皓贸易有限公司，中国；1 mL/kg）肌内注射麻醉，并用小动物麻醉仪异氟烷吸入维持麻醉。麻醉后实验兔取右侧卧位，左肾区备皮，逐层切开左侧腹壁并暴露左肾，分离左肾动脉，用0.5 mm内径的硅胶套套扎左肾动脉，逐层关闭腹腔。对照组实验兔除不套扎左肾动脉，其余手术操作相同。

1.3 MRI检查

所用仪器为Philips Ingenia 3.0 T磁共振扫描仪（飞利浦，Ingenia，荷兰），采用16通道头颈线圈。pre-RF、RF-1W、RF-2W、RF-3W和RF-4W亚组分别于术前、术后1、2、3和4 W行MR轴位T2WI序列和IVIM序列扫描。所有实验兔麻醉后分别取左、右侧卧位，头先进置于MR检查床上扫描双侧肾脏。T2WI序列扫描参数：横断位，TR 2500 ms，TE 100 ms，视野（field of view, FOV）150 mm×150 mm，矩阵252×196，层厚4 mm，层间距0.4 mm，激励次数2。IVIM序列扫描参数：采用单次激发自旋回波平面成像（echo planar imaging, EPI）序列，TR 3000 ms，TE 75 ms，FOV 150 mm×150 mm，矩阵68×68，层厚3 mm，层间距1 mm，激励次数4，扩散敏感梯度方向数3，10个b值分别为0、10、20、50、100、200、400、600、800、1200 s/mm²。扫描范围从双肾上极至下极，轴位扫描定位线分别垂直于左右肾长轴，中心位于肾门水平。

1.4 图像后处理及分析

IVIM图像分析采用MITK Diffusion后处理软件（德国癌症研究中心，v2022.10,https://www.mitk.org/wiki/Downloads#MITK_Diffusion）完成，原始数据导入后自动生成参数D、伪扩散系数（pseudo-diffusion coefficient, D^*）和f图。由两位分别具有9年和5年泌尿系统影像诊断经验的副主任医师和主治医师采用双盲法独立完成图像分析。选择左右肾脏的肾门水平轴位图像进行分析，与组织病理学检测的层面保持一致。在肾脏IVIM图像的轴位正中层面，避开出血区、伪影、皮质和髓质的边缘，分别手工勾画肾皮质和髓质区。应用IVIM双指数模型进行拟合，获得D、D^*、f值。

1.5 肾脏组织病理学检查

所有实验兔末次MRI检查结束后立即处死，取出双肾。本次实验中实验兔双肾均未发现存在附肾动脉的情况。采用10%甲醛固定液浸泡，石蜡包埋，切片后行Masson染色。选择垂直于双肾长轴的肾门横断面切片进行病理学分析。在显微镜放大100倍视野下，在皮质和髓质区分别随机选取5个非重复视野，观察Masson染色的蓝染区域（代表纤维化），使用Image J软件计算纤维面积占总截面积的平均百分比。

1.6 统计学分析

采用SPSS 26.0（Statistical Product and Service Solutions, v26.0.0.0, IBM, USA）和GraphPad Prism 9.4（v9.4，GraphPad Software, USA）软件进行统计学分析。用Shapiro-Wilk分析连续变量是否为正态分布，符合正态分布的连续变量采用均值±标准差（x¯±s）表示，不符合正态分布的参数采用中位数（四分位距）表示。连续变量根据是否为正态分布分别采用独立样本t检验或者Mann-Whitney U检验。采用组内相关系数（intraclass correlation coefficient, ICC）评价2名阅片者测量各参数的一致性。不同时间点左右肾皮质和髓质D、D^*、f值差异的比较采用重复测量方差分析^[²¹^]。同一时间点组间双肾皮、髓质D、D^*、f值的差异比较采用独立样本t检验。采用Spearman相关性分析比较左肾皮、髓质D、D^*、f值与RF程度的相关性。采用受试者工作特征（receiver operating characteristic, ROC）曲线判断左肾皮、髓质D、D^*、f值对RF诊断和分期的效能。P＜0.05为差异有统计学意义。

2 结果

2名医师测量pre-RF、RF-1W、RF-2W、RF-3W及RF-4W各亚组的D值、f值、D^*值一致性好，ICC均＞0.75。

2.1 T2WI及IVIM图像特点

正常肾脏T2WI图像显示皮髓质分界清晰，髓质信号高于皮质。RF早期左肾皮髓质开始出现分界不清，髓质信号开始减低。随着RF的进展，左肾皮髓质分界欠清，皮髓质扩散受限，IVIM图像信号逐渐减低（图2）。

点击查看大图

图2 pre-RF、RF-1W-RF-4W亚组实验兔左肾T2WI和体素内不相干运动（IVIM）图像。2A-2E：T2WI图像；2F-2J：IVIM融合灰度图；2K-2O：真扩散系数（D）伪彩图（蓝色到红色过渡表示D值逐渐升高，单位μm²/ms）；2P-2T：伪扩散系数（D^*）伪彩图（蓝色到红色过渡表示D^*值逐渐升高，单位μm²/ms）；2U-2Y：灌注分数（f）伪彩图（蓝色到红色过渡表示f值逐渐升高，单位%）。pre-RF亚组显示左肾皮髓质分界清晰，RF-1W-RF-4W亚组显示肾皮髓质分界欠清，皮质、髓质信号逐渐减低。RF：肾纤维化；pre-RF：RF建模前；RF-1W：RF建模后1周；RF-2W：RF建模后2周；RF-3W：RF建模后3周；RF-4W：RF建模后4周。
Fig. 2 T2WI and intravoxel incoherent motion (IVIM) images of the left kidney of the pre-RF and RF-1W-RF-4W subgroup. 2A-2E: T2WI image; 2F-2J: IVIM fusion gray scale image; 2K-2O: D pseudo color image (the blue to red transition indicates a gradual increase in D values, unit μm²/ms); 2P-2T: D^* pseudo color image (the blue to red transition indicates a gradual increase in D^* values, unit μm²/ms); 2U-2Y: f pseudo color image (the blue to red transition indicates a gradual increase in f values, unit %). In the pre-RF subgroup, the boundary between the left renal cortex and medulla is clear, while in the RF-1W-RF-4W subgroup, the boundary between the left renal cortex and medulla is unclear. The cortical and medullary signal intensity gradually decreased. pre-RF: pre-renal fibrosis; RF-1W: 1 week after RF; RF-2W: 2 weeks after RF; RF-3W: 3 weeks after RF; RF-4W: 4 weeks after RF.

2.2 各组IVIM参数值随时间的动态比较

（1）RF组左肾皮质和髓质IVIM参数值随时间的动态变化结果：RF组左肾皮质和髓质的D值和f值均逐渐减低。皮质D值在RF-4W和pre-RF、RF-1W-RF-3W亚组，RF-3W和pre-RF、RF-1W-RF-2W亚组比较差异有统计学意义（P均＜0.05）；皮质f值在pre-RF和RF-1W-RF-4W亚组，RF-1W和RF-2W-RF-4W亚组比较差异有统计学意义（P均＜0.05）；皮质D^*值在RF-4W和pre-RF、RF-1W-RF-2W亚组比较差异有统计学意义（P＜0.05）。髓质D值在RF-4W和pre-RF、RF-1W-RF-2W亚组比较差异有统计学意义（P均＜0.05）；髓质f值在pre-RF和RF-2W-RF-4W亚组，RF-1W和RF-4W亚组比较差异均有统计学意义（P均＜0.05）；髓质D^*值在各亚组差异均无统计学意义（表1）。

（2）RF组右肾皮质和髓质IVIM参数值随时间的动态变化结果：RF组右肾皮质和髓质的D^*值和f值均逐渐升高。皮质D^*值在pre-RF和RF-2W-RF-4W亚组，RF-1W和RF-3W-RF-4W亚组，RF-2W和RF-4W亚组比较差异均有统计学意义（P均＜0.05）；皮质f值在RF-4W和pre-RF、RF-1W-RF-3W亚组，RF-3W和pre-RF、RF-1W亚组比较差异有统计学意义（P均＜0.05）；髓质D^*值在pre-RF和RF-1W-RF-4W亚组，RF-1W和RF-4亚组比较差异有统计学意义（P均＜0.05）；髓质f值在RF-4W和pre-RF、RF-1W-RF-3W亚组，RF-3W和pre-RF亚组比较差异有统计学意义（P均＜0.05）；右肾皮质和髓质的D值在各亚组差异无统计学意义（表2）。

（3）对照组左、右肾的皮质、髓质的IVIM参数值随时间比较差异均无统计学意义。

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表1 左肾皮髓质各时间点体素内不相干运动参数值动态比较
Tab. 1 Dynamic comparison of intravoxel incoherent motion parameter values of left kidney cortex and medulla at different time points

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表2 右肾皮髓质各时间点体素内不相干运动参数值动态比较
Tab. 2 Dynamic comparison of intravoxel incoherent motion parameters at different time points in right kidney cortex and medulla

2.3 各组肾脏同一时间点皮质、髓质的IVIM参数值比较

结果显示，左肾皮质与髓质在RF-4W亚组D值、D^*值差异均有统计学意义（P均＜0.05）；pre-RF、RF-3W和RF-4W亚组左肾皮质与髓质的f值比较差异均有统计学意义（P均＜0.05）；pre-RF、RF-4W亚组右肾皮质与髓质的f值比较差异均有统计学意义（P均＜0.05）。详见图3。

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图3 各组肾脏同一时间点皮质、髓质的体素内不相干运动（IVIM）成像参数值比较。3A、3B：左肾皮质与髓质在RF-4W亚组的真扩散系数（D）、伪扩散系数（D^*）值差异均有统计学意义（P均＜0.05）；3C：左肾皮质与髓质在pre-RF、RF-3W和RF-4W亚组的灌注分数（f）值差异均有统计学意义（P均＜0.05）；3D：右肾皮质与髓质在pre-RF、RF-4W亚组的f值差异有统计学意义（P均＜0.05）。RF：肾纤维化；pre-RF：RF建模前；RF-1W：RF建模后1周；RF-2W：RF建模后2周；RF-3W：RF建模后3周；RF-4W：RF建模后4周。
Fig. 3 Comparison of intravoxel incoherent motion (IVIM) parameters of renal cortex and medullary in each group at same time. 3A, 3B: The true diffusion coefficient (D) and pseudo-diffusion coefficient (D^*) values of the left renal cortex and medulla in the RF-4W subgroup are statistically different (P all＜0.05); 3C: The perfusion fraction (f) values of the left renal cortex and medulla in the pre-RF, RF-3W and RF-4W subgroups are statistically different (P all＜0.05); 3D: The f values of the right renal cortex and medulla in the pre-RF and RF-4W subgroups are statistically different (P all＜0.05). pre-RF: pre-renal fibrosis; RF-1W: 1 week after RF; RF-2W: 2 weeks after RF; RF-3W: 3 weeks after RF; RF-4W: 4 weeks after RF.

2.4 组织病理学结果

肉眼观：pre-RF亚组左肾皮髓质分界清晰，结构正常（图4A）；RF初期（1 W），左肾体积较右肾稍缩小，色泽未见明显改变，皮髓质开始分界欠清；随着RF进展（2 W-4 W），左肾体积呈进行性缩小，表面颜色苍白泛黄，肾实质逐渐萎缩，皮髓质分界不清，右肾代偿性增大（图4B-4E）。

镜下观（Masson染色）：pre-RF亚组左肾肾小球、肾小管分布、排列正常，肾内毛细血管丰富（图4F）；RF-1W亚组肾小球囊未见明显扩张，肾小管未见明显损伤，肾内毛细血管减少（图4G）；RF-2W亚组肾小球囊见轻度扩张，部分肾小管轻度扩张、内见管型形成，肾间质内胶原纤维数量增加，间质蓝染程度加深（图4H）；RF-3W、RF-4W亚组肾小球囊进一步扩张，肾小球毛细血管团萎缩，多发肾小管管腔塌陷、数量减少，肾间质内胶原纤维数量进一步增加，间质蓝染程度更深，无法辨认正常肾结构（图4I-4J）。

Image J软件胶原纤维半定量分析显示，pre-RF和RF-1W-RF-4W亚组的Masson染色镜下蓝染面积百分比依次为0.46%±0.06%、4.70%±0.86%、20.98%±2.29%、55.34%±2.73%、65.16%±1.57%。

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图4 pre-RF及RF-1-RF-4W各亚组双肾正中横断面标本肉眼观及各亚组左肾镜下Masson染色（×100）。4A、4F：pre-RF的标本肉眼观及镜下观；4B-4E、4G-4J：分别为RF-1W、RF-2W、RF-3W、RF-4W的标本肉眼观及镜下观。RF：肾纤维化；pre-RF：RF建模前；RF-1W：RF建模后1周；RF-2W：RF建模后2周；RF-3W：RF建模后3周；RF-4W：RF建模后4周。
Fig. 4 Macroscopic view of bilateral median renal cross-sectional specimens in each subgroup of pre-RF and RF-1W, RF-2W, RF-3W, RF-4W and Masson staining under the left nephroscope in each subgroup (×100). 4A, 4F: The specimens of pre-RF under the naked eye and microscope; 4B-4E, 4G-4J: The macroscopic and microscopic views of specimens of RF-1W, RF-2W, RF-3W, RF-4W, respectively. pre-RF: pre-renal fibrosis; RF-1W: 1 week after RF; RF-2W: 2 weeks after RF; RF-3W: 3 weeks after RF; RF-4W: 4 weeks after RF.

2.5 IVIM参数值与RF程度的相关性

Spearman相关性检验显示左肾皮质D、f值及髓质f值分别与纤维化程度呈中等或强负相关（r值分别为-0.595、-0.717、-0.412，P均＜0.01）。而左肾皮质D^*值、髓质D、D^*值分别与纤维化程度无明显相关性（r值分别为-0.259、-0.305、0.217，P值分别为0.107、0.056、0.179）（图5）。

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图5 体素内不相干运动（IVIM）参数值与肾纤维化程度的相关性分析。5A、5C、5F显示左肾皮质真扩散系数（D）、灌注分数（f）值、髓质f值与肾纤维化程度呈中等或强负相关（r值分别为-0.595、-0.717、-0.412，P均＜0.01）；5B、5D、5E显示左肾皮质伪扩散系数（D^*）值、髓质D和D^*值与纤维化程度无明显相关性（r值分别为-0.259、-0.305、0.217，P值分别为0.107、0.056、0.179）。
Fig. 5 Correlation between intravoxel incoherent motion (IVIM) parameters and the degree of renal fibrosis. 5A, 5C, 5F show that the true diffusion coefficient (D), perfusion fraction (f) values of left renal cortex and f values of left renal medulla are moderately or strongly negatively correlated with the degree of renal fibrosis (r=-0.595, -0.717, -0.412, P all＜0.01); 5B, 5D, 5E show that the pseudo-diffusion coefficient (D*) values of left renal cortex, and D, D* values of left medulla degree of fibrosis (r=-0.259, -0.305, 0.217, P=0.107, 0.056, 0.179).

2.6 IVIM参数对RF诊断和分期的效能

由IVIM参数值评估RF的ROC曲线得出，f值对鉴别pre-RF和RF-1W-RF-4W、pre-RF-RF-1W和RF-2W-RF-4W的效能最优，ROC曲线下面积分别为0.960、0.893；D值对鉴别pre-RF-RF-2W和RF-3W-RF-4W、pre-RF-RF-3W和RF-4W的效能最优，ROC曲线下面积分别为0.855、0.926（图6）。

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图6 左肾皮、髓质体素内不相干运动（IVIM）各参数对肾纤维化的分期诊断受试者工作特征（ROC）曲线。6A：左肾皮、髓质IVIM各参数鉴别肾纤维化pre-RF和RF-1W-RF-4W；6B：左肾皮、髓质IVIM各参数鉴别肾纤维化pre-RF-RF-1W和RF-2W-RF-4W；6C：左肾皮、髓质IVIM各参数鉴别肾纤维化pre-RF-RF-2W和RF-3W-RF-4W；6D：左肾皮、髓质IVIM各参数鉴别肾纤维化pre-RF-RF-3W和RF-4W。RF：肾纤维化；pre-RF：RF建模前；RF-1W：RF建模后1周；RF-2W：RF建模后2周；RF-3W：RF建模后3周；RF-4W：RF建模后4周。
Fig. 6 Receiver operating characteristic (ROC) curve analysis of intravoxel incoherent motion (IVIM) parameters in the left renal cortex and medulla for staging renal fibrosis. 6A: ROC curves of IVIM parameters in the left renal cortex and medulla for differentiating pre-RF from RF-1-RF-4W with renal fibrosis; 6B: ROC curves of IVIM parameters in the left renal cortex and medulla for differentiating pre-RF-RF-1W and RF-2W-RF-4W with renal fibrosis; 6C: ROC curves of IVIM parameters in the left renal cortex and medulla for differentiating pre-RF-RF-2W and RF-3W-RF-4W renal fibrosis; 6D: ROC curve of IVIM parameters of left renal cortex and medulla in differentiating pre-RF-RF-3W and RF-4W of renal fibrosis. pre-RF: pre-renal fibrosis (RF); RF-1W: 1 week after RF; RF-2W: 2 weeks after RF; RF-3W: 3 weeks after RF; RF-4W: 4 weeks after RF.

3 讨论

本研究采用肾动脉狭窄法^[²²^]成功构建实验兔单侧RF，探讨肾脏皮髓质的IVIM参数D、D^*和f随纤维化进展的动态变化，发现左肾皮质D、f值，髓质f值分别与纤维化程度呈中等或强负相关；f值是反映RF发生发展最敏感的标志物，D值对鉴别中晚期RF价值最大。MR-IVIM成像可以从灌注和扩散两个层面评价兔肾动脉狭窄RF微循环减少和间质胶原沉积的动态变化，为RF的长期监测和临床管理提供影像学依据。

3.1 RF组左肾皮质和髓质IVIM参数值随时间动态变化

本研究结果显示RF组左肾皮质和髓质的D值和f值随着RF的进展均逐渐减低。D值反映的是组织的真实扩散，而f是血管和管状液体体积与组织中总液体的比率^[²³^,²⁴^]。表明RF早期，炎症细胞浸润导致的细胞密度增加和细胞肿胀导致的细胞外空间缩小，降低了肾实质的D值^[²⁵^]。随着RF的进展，肾脏的间质成纤维细胞增加、胶原纤维沉积，阻碍了间质中水的扩散，使得D持续减低^[²⁶^,²⁷^,²⁸^]。RF发生时诱导肾素-血管紧张素系统的激活，从而促进血管收缩，f值减低，此后随着肾血管硬化，功能性肾小球数量减少^[²⁹^]，f值亦逐渐降低。D^*反映血管和小管的流体速度^[²³^,²⁴^]。本研究中左肾皮质和髓质的D^*值随时间无显著差异，这与之前的研究结果相似^[²⁵^,³⁰^]。但也有研究发现D^*值在RF晚期降低，其敏感度低于f值^[³¹^,³²^]，可能是不同实验对象和序列参数导致了差异。D^*值在RF评估中的价值有待进一步探究和讨论。

3.2 各时间点肾皮质与髓质IVIM参数值的比较

本研究中，RF-4W亚组左肾皮质与髓质的D值、D^*值差异均有统计学意义，RF-3W和RF-4W亚组的左肾皮质与髓质的f值差异均有统计学意义，皮质均低于髓质。先前的研究发现^[²⁵^,³²^]，采用输尿管梗阻模型构建的RF实验中，髓质由于是相对乏血供区，更易受水分子扩散和灌注的影响，髓质参数的改变要比皮层显著且早，这与我们的结果不同。可能是因我们采用了肾动脉狭窄的慢性缺血模型构建RF，使得皮质的血供减少比髓质显著，反而削弱了皮髓质的差异。RF后期，在灌注和扩散的双重作用下，皮质参数较髓质更低^[³³^,³⁴^]。

3.3 对侧肾脏的代偿作用

此外，本研究分析了对侧（右）肾脏的代偿作用。RF组右肾皮质和髓质的D^*值和f值均逐渐升高，而D值随时间无显著差异。RF-4W亚组右肾皮质的f值明显高于髓质。表明由于左肾功能的进行性丢失，右肾的灌注逐渐增加，流体速度加快，起到了代偿的作用。病理标本也显示右肾体积逐渐增大，肾小管细胞肥大。代偿肾f值的升高反映有效滤过率的升高，可能对肾脏内血液过滤和水再吸收的机制具有重要意义。而既往研究^[²⁵^]发现对侧肾IVIM参数在单侧UUO建模前后无显著变化，无明显代偿作用，可能因实验观察时间较短，仅为UUO建模后7天，无法观察到对侧健肾远期的变化。本研究也是在RF-3W、RF-4W组观察到对侧右肾f值的改变。对侧健肾的长期动态观察往往容易忽略，但却至关重要，长期的影像学监测有利于为临床综合管理及干预提供更多的依据。

3.4 IVIM参数对RF诊断和分期的效能

ROC曲线分析显示，f值对鉴别pre-RF和RF-1W-RF-4W、pre-RF-RF-1W和RF-2W-RF-4W的效能最优，提示血流灌注在RF的早期改变较为显著；D值对鉴别pre-RF-RF-2W和RF-3W-RF-4W、pre-RF-RF-3W和RF-4W的效能最优，说明D值在鉴别RF的中晚期中有较高价值。虽然本研究使用的是肾动脉狭窄的模型构建的RF，早期f值的降低可能与肾脏慢性缺血有关，但在一些输尿管梗阻的模型中亦发现了早期f值就减低的现象，且f值下降早于D值下降^[³⁵^,³⁶^]，说明RF启动后，渐进性毛细血管损失与肾脏瘢痕形成同时发生，毛细血管损失和低灌注既是纤维化肾脏的关键特征，也是进一步推动纤维化损伤的驱动因素^[³⁷^]。f值可能是IVIM三个参数中反映RF最敏感的标志物。f和D值可以作为RF早期诊断和准确分期的可靠指标。

3.5 局限性

本研究存在一定的局限性：第一，样本量较小，需要进一步扩大样本量增加实验数据的稳定性和准确性；第二，未将血生化等临床常用实验室指标纳入研究；第三，本实验采用的MR检查线圈非小动物专用线圈，图像的信噪比欠佳。

4 结论

综上所述，MR-IVIM可以分别从灌注和扩散两方面反映RF发生发展的过程，对评价RF具有可行性和较大的应用价值。

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