Name: analyses of proteinuria, renal infiltration of leukocytes, and renal deposition of proteins in lupus-prone mrl/lpr mice
Uploaded: 2022-06-08T13:00:00
Description: Watch this Scientific Journal Video about Analyses of Proteinuria, Renal Infiltration of Leukocytes, and Renal Deposition of Proteins in Lupus-prone MRL/lpr Mice at JoVE.com

我们感谢流式细胞术核心设施、组织病理学实验室、弗吉尼亚理工学院 Fralin 成像中心和州立大学的技术支持。这项工作得到了各种NIH和内部赠款的支持。

本议定书由弗吉尼亚理工大学的机构动物护理和使用委员会（IACUC）批准。由于狼疮病在女性中的发病率较高，因此仅使用雌性MRL / lpr小鼠。样本采集从4周龄开始，15周结束。小鼠是从商业来源获得的（参见 材料表），并按照机构指南在特定的无病原体环境中繁殖和维护。
1. 蛋白尿试验
<ol><li>按照以下步骤收集尿液。<ol><li>通过喷洒70%乙醇对罩进行...

<ol>
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LN是SLE患者死亡的主要原因，加重该病的因素尚不清楚。该方案的应用是使用多种方法表征肾功能，包括蛋白尿的测量，分离的肾白细胞的FACS分析以及冷冻肾切片的免疫荧光染色。
收集尿液时要考虑的一个要点是，必须与一天中的时间和尿液收集的位置一致。小鼠是夜间活动的动物，因此在小鼠开始活跃之前的下午晚些时候收集最准确的样本。选择这个时间的另一个原因是MR...

肾脏的主要功能是通过尿液消除有毒物质，同时保持水和盐的稳态1。该功能在系统性红斑狼疮（SLE）患者中受到威胁，导致所谓的狼疮性肾炎（LN）。LN是免疫系统攻击肾脏的结果，导致持续的肾脏炎症，因此失去了清除血液中废物和调节盐和液体浓度的能力。这最终将导致肾功能衰竭，这可能是致命的。在肾炎过程中，循环的B细胞，T细胞和单核细胞被招募到肾脏，分泌趋化因子，细胞因子和免疫复合物形成自身抗体。这最终导致内皮细胞损伤，膜损伤，肾小管萎缩和纤维化2。
容易出现狼疮的小鼠是一种经典的小鼠模型，表现出类似于人类SLE3的狼疮样临床体征。该模型有助于了解SLE患者死亡的主要原因之一，狼疮性肾炎（LN）4。在人和小鼠SLE中，LN的特征在于由肾沉积的免疫复合物引发的逐渐炎症，随后是补体激活，炎症细胞募集和肾功能丧失5。免疫复合物沉积是内在肾细胞诱导趋化因子和细胞因子产生的第一步，其通过招募免疫细胞6来扩展炎症反应。目前的方案提出了几种跟踪肾脏疾病进展的技术，分析细胞浸润和免疫复合物沉积。
每周收集的尿液可以检测和可视化狼疮发作之前、期间和之后的蛋白尿病程。蛋白尿作为生物标志物可以决定LN的生物学进展。该技术的其他优点是它是非侵入性的，具有成本效益的，并且易于实施7。当肾脏完美工作时，蛋白尿水平始终很低;然而，在MRL / lpr小鼠中，在8-9周龄后，观察到蛋白尿水平的逐渐增加，最终高到足以引起肾功能衰竭8。市面上有多种试剂条和比色试剂来监测问题。然而，Bradford测定在确定蛋白尿的发作和狼疮性肾炎的病程方面是廉价且非常准确的。该测定速度快，试剂不受样品中可能存在的溶剂、缓冲液、还原剂和金属螯合剂的影响9，10，11。
要考虑的一个重要方面是肾脏中的细胞浸润。这些浸润通过触发可溶性因子（如细胞因子）的分泌来促进发病机制，从而加重炎症12。为了更好地了解浸润中存在哪些细胞群，一种有用的方法是分离白细胞13。这里，以检测B细胞肾浸润为例。该过程从脱氧核糖核酸酶（DNase）和胶原酶的消化过程开始，然后是密度梯度分离，以去除碎片，红细胞和致密的粒细胞。分离B细胞（CD19 +）和浆细胞（CD138 +）的原因是狼疮肾可以浓缩这些细胞14。有人认为，肾脏中小聚集体中存在B细胞可以表明克隆性扩增，从而产生免疫球蛋白（Ig）。众所周知，浆细胞也存在于这些聚集体中15。一旦分离出白细胞，荧光活化细胞分选（FACS）可用于在用不同的荧光偶联抗体染色时分析感兴趣的细胞。
免疫荧光是免疫组化（IHC）检测方法之一，允许在4μm厚的肾脏组织样品中对蛋白质进行荧光可视化。其他IHC检测方法取决于分析物的性质、结合化学和其他因素16。免疫荧光是一种快速鉴定方法，可将抗原暴露于用特定荧光染料（或荧光染料）标记的对应抗体中。当激发时，它产生荧光显微镜可以检测到的光。该技术可用于观察补体C3和IgG2a17的沉积。过量的补体级联激活可能与不受控制的免疫反应和功能丧失有关18.肾脏中抗双链DNA（抗dsDNA）自身抗体的免疫沉积是一个主要问题19，其中具有IgG2a同种型的抗体与LN20相关。具体而言，抗dsDNA抗体对核材料表现出更多的致病性和亲和力，形成免疫复合物21。当存在IgG2a时，包括C3在内的补体级联反应被激活以清除免疫复合物22。C3和IgG2a标记可以单独量化或叠加以建立其相关性。
值得注意的是，血清肌酐测量是另一种可靠的技术，可以与显微镜下血尿和肾活检一起使用来诊断LN23。然而，蛋白尿的存在是肾小球损伤的强烈指标。从这个意义上说，监测狼疮期间的蛋白尿水平可以检测疾病发作并补充其他诊断狼疮的方法。此外，沉积在肾小球中的免疫复合物可以诱导炎症反应，激活补体系统，并招募更多的炎症细胞。该协议的另一个值得注意的点是B细胞浸润在肾脏中。这与浸润的T细胞一起放大了引发器官损伤的局部免疫反应。重要的是，LN的分类不仅基于显微镜下可见的肾小球形态变化，还基于免疫荧光观察到的免疫沉积物。因此，在该协议中，在实验室环境中提供了用于分析肾功能的准确性和成本效益的方法。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10x Tris-Buffered Saline (TBS)</td>
			<td>Thermo Fisher Scientific</td>
			<td>J60764.K2</td>
			<td></td>
		</tr>
		<tr>
			<td>2-mercaptoethanol</td>
			<td>Thermo Fisher Scientific</td>
			<td>21985-023</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Human/Mouse C3</td>
			<td>Cedarlane</td>
			<td>CL7632F</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Mouse CD138 BV711</td>
			<td>Biolegend</td>
			<td>142519</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Mouse CD45 AF700</td>
			<td>Biolegend</td>
			<td>103127</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A9418-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase D</td>
			<td>Sigma-Aldrich</td>
			<td>11088882001</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal Microscope LSM 880</td>
			<td>Zeiss</td>
			<td>LSM 880</td>
			<td></td>
		</tr>
		<tr>
			<td>Coplin jar</td>
			<td>Fisher Scientific</td>
			<td>50-212-281</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryomold</td>
			<td>Fisher Scientific</td>
			<td>NC9511236</td>
			<td></td>
		</tr>
		<tr>
			<td>Density gradient medium</td>
			<td>GE Healthcare</td>
			<td>17-1440-02</td>
			<td>Percoll</td>
		</tr>
		<tr>
			<td>DEPC-Treated water</td>
			<td>Thermo Fisher Scientific</td>
			<td>AM9906</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase I</td>
			<td>Sigma-Aldrich</td>
			<td>D4527</td>
			<td></td>
		</tr>
		<tr>
			<td>dsDNA-EC</td>
			<td>InvivoGen</td>
			<td>tlrl-ecdna</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic Acid</td>
			<td>Fisher Scientific</td>
			<td>S311-500</td>
			<td>EDTA</td>
		</tr>
		<tr>
			<td>EVOS M5000 Microscope imaging system</td>
			<td>Thermo Fisher Scientific</td>
			<td>AMF5000</td>
			<td></td>
		</tr>
		<tr>
			<td>FACS Fusion Cell sorter</td>
			<td>BD Biosciences</td>
			<td>FACS Fusion</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum - Premium, Heat Inactivated</td>
			<td>R&#38;D systems</td>
			<td>S11150H</td>
			<td></td>
		</tr>
		<tr>
			<td>Fisherbrand 96-Well Polystyrene Plates</td>
			<td>Fisher Scientific</td>
			<td>12-565-501</td>
			<td></td>
		</tr>
		<tr>
			<td>Graphpad prism</td>
			<td>GraphPad</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Hank&#8217;s Balanced Salt Solution</td>
			<td>Thermo Fisher Scientific</td>
			<td>14175-079</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Thermo Fisher Scientific</td>
			<td>15630-080</td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ software</td>
			<td>National Institutes of Health</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Lactobacillus reuteri Kandler et al.</td>
			<td>ATCC</td>
			<td>23272</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM non-essential amino acids</td>
			<td>Thermo Fisher Scientific</td>
			<td>11140-050</td>
			<td></td>
		</tr>
		<tr>
			<td>MRL/MpJ-Fas lpr /J Mice (MRL/lpr)</td>
			<td>Jackson Lab</td>
			<td>485</td>
			<td></td>
		</tr>
		<tr>
			<td>Nail enamel</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Any conventional store</td>
		</tr>
		<tr>
			<td>O.C.T compound</td>
			<td>Tisse-Tek</td>
			<td>4583</td>
			<td></td>
		</tr>
		<tr>
			<td>PAP pen</td>
			<td>Sigma-Aldrich</td>
			<td>Z377821</td>
			<td></td>
		</tr>
		<tr>
			<td>Peel-A-Way Disposable Embedding Molds</td>
			<td>Polysciences</td>
			<td>R-30</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Thermo Fisher Scientific</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline (PBS)</td>
			<td>Thermo Fisher Scientific</td>
			<td>70011069</td>
			<td></td>
		</tr>
		<tr>
			<td>Pierce 20x TBS Buffer</td>
			<td>Thermo Fisher Scientific</td>
			<td>28358</td>
			<td></td>
		</tr>
		<tr>
			<td>Pierce Coomassie Plus (Bradford) Assay kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>23236</td>
			<td>Albumin standard included</td>
		</tr>
		<tr>
			<td>ProLon Gold Antifade Mountant</td>
			<td>ThermoFisher</td>
			<td>P36934</td>
			<td></td>
		</tr>
		<tr>
			<td>Purified Rat Anti-Mouse CD16/CD32</td>
			<td>BD Biosciences</td>
			<td>553141</td>
			<td>FcR block</td>
		</tr>
		<tr>
			<td>RPMI 1640</td>
			<td>Thermo Fisher Scientific</td>
			<td>11875-093</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>Thermo Fisher Scientific</td>
			<td>11360-070</td>
			<td></td>
		</tr>
		<tr>
			<td>SpectraMax M5</td>
			<td>Molecular Devices</td>
			<td>N/A</td>
			<td>SoftMax Pro 6.1 software</td>
		</tr>
		<tr>
			<td>Sterile cell Strainers 100 &#181;M</td>
			<td>Fisher Scientific</td>
			<td>22363549</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Fisher Scientific</td>
			<td>BP337-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Vancomycin Hydrochloride</td>
			<td>Goldbio</td>
			<td>V-200-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Zombie Aqua</td>
			<td>Biolegend</td>
			<td>423102</td>
			<td>fluorescent dye for flow cytometry analysis</td>
		</tr>
	</tbody>
</table>

analyses of proteinuria, renal infiltration of leukocytes, and renal deposition of proteins in lupus-prone mrl/lpr mice

系统性红斑狼疮（SLE）是一种自身免疫性疾病，没有已知的治愈方法，其特征是许多器官（包括肾脏）的持续炎症。在这种情况下，肾脏失去了清除血液中的废物并调节盐和液体浓度的能力，最终导致肾功能衰竭。女性，特别是育龄妇女，被诊断出的频率是男性的九倍。肾脏疾病是系统性红斑狼疮患者死亡的主要原因。本方案描述了一种快速简单的方法来测量收集的尿液中排泄的蛋白质水平，跟踪狼疮随时间的进展。此外，根据大小和密度选择，提供了一种分离肾脏单核细胞的方法，以研究白细胞的肾脏浸润。此外，已经开发了一种免疫组织化学方法来表征肾小球中的蛋白质沉积和肾小管间质空间中的白细胞浸润。总之，这些方法可以帮助研究与易患狼疮的MRL / lpr小鼠的肾脏相关的慢性炎症的进展。

该方案使用多种方法来评估狼疮性肾炎的MRL / lpr小鼠。首先，描述了一种程序来研究由于肾功能障碍而引起的蛋白尿水平随时间增加。如图 1所示，雌性小鼠以口服强饲法200μL磷酸盐缓冲盐水（1x PBS）作为对照组，益生菌 罗伊氏乳杆菌 作为治疗组，浓度为109 cfu / mL，每周两次。治疗从3周龄开始，到15周龄结束。用 罗伊氏乳杆菌 治疗的小鼠组比对照组?...

Watch this Scientific Journal Video about Analyses of Proteinuria, Renal Infiltration of Leukocytes, and Renal Deposition of Proteins in Lupus-prone MRL/lpr Mice at JoVE.com

Analyses of Proteinuria, Renal Infiltration of Leukocytes, and Renal Deposition of Proteins in Lupus-prone MRL/lpr Mice

本方案描述了一种跟踪小鼠狼疮进展的方法。提出了另外两种程序来表征基于细胞浸润和肾脏中蛋白质沉积的狼疮性肾炎。

分析易患狼疮的MRL/lpr小鼠的蛋白尿、白细胞肾浸润和蛋白质的肾脏沉积

Systemic lupus erythematosus (SLE) is an autoimmune disorder with no known cure and is characterized by persistent inflammation in many organs, including ...

这些方法可以帮助研究狼疮患者肾脏相关慢性炎症的进展。我们提供可靠且具有成本效益的替代方案，可用于监测狼疮的病程。通过喷洒 70% 乙醇开始对罩子进行消毒，并在表面上放置无菌塑料片。然后准备吸头，1.5毫升管和移液管以收集约20微升尿液。取笼子并喷洒 70% 乙醇，然后将其放入引擎盖内。然后，用一只手握住鼠标，用另一只手从上到下轻轻按摩腹侧区域。使用1.5毫升管或移液管，直接收集尿液。或者，如果样品滴从塑料片上收集它。然后将鼠标放回笼子里。将解剖的肾脏切成颗粒大小，在含有胶原蛋白酶和 DNase 1 的 5 毫升消化缓冲液中在含有 HEPES 的 RPMI 1640 培养基中消化一小时，在 37 摄氏度下连续轻轻摇动。加入含有10毫摩尔EDTA的10毫升冰冷PBS，在冰上孵育10分钟。然后涡旋悬浮液两次。之后，通过100微米过滤器过滤细胞悬液，并用10毫升HBSS满水洗涤。之后，在室温下以350倍G离心10分钟。准备30%、37%和70%储备等渗Percoll溶液，并在70%溶液上添加一层37%Percoll溶液。将细胞重新悬浮在五毫升30%储备等渗Percoll中。使用巴斯德移液器缓慢加载顶部 37% 的 5 毫升和底部 70% 的 5 毫升，使库存等渗 Percoll 梯度。然后，在室温下以1000倍G离心9号和下零号离心30分钟。之后，从37%至70%界面收集白细胞，并将它们重新悬浮在5毫升C 10中。再次，在 350 倍 G 下在 4 摄氏度下离心 10 分钟。随后将细胞沉淀重新悬浮在三毫升传真缓冲液中，并在 350 倍 G 下在 4 摄氏度下离心五分钟。然后弃去上清液，留下约50微升的总体积。每管加入 50 微升 FC 受体阻滞剂。按照这种混合物，在黑暗中在冰上孵育10分钟。之后，加入两毫升传真缓冲液进行洗涤。然后，在 350 倍 G 下在 4 摄氏度下离心五分钟，丢弃上清液，留下约 50 微升的总体积。接下来，加入20微升适当的荧光染料，让它在冰上静置15至30分钟。每管加入 50 微升抗体混合物，含有 CD 138 亮紫七 11 和 CD 45 Alexa Fluor 700，并在黑暗中的冰上孵育 15 至 30 分钟。然后，加入三毫升传真缓冲液。在 350 倍 G 下在 4 摄氏度下离心五分钟。通过真空除去上清液，留下约50微升的总体积。之后，将细胞沉淀重新悬浮在200微升PBS中。为了收集样品，将半个肾嵌入具有最佳切割温度化合物的小塑料冷冻机中。然后将干冰加入聚苯乙烯泡沫盒中，并小心地将冷冻机放在干冰上。接下来，取出冷冻模具，用铝箔包裹并储存在零下 80 摄氏度直至进一步使用。然后用冷丙酮将干燥的四微米部分固定 10 分钟。固定载玻片后，风干0.5至1小时，并在零下20摄氏度短时间或零下80摄氏度长时间存放。要染色样品，请将载玻片重新固定在冷丙酮中 5 到 10 分钟，并让切片加热至室温。然后，使用轻拍笔，在该部分周围画一个疏水圆圈并使其干燥。然后将载玻片放入谷轮罐中的TBS中20分钟，然后将罐子放在摇床上轻轻摇晃。随后，倒出TBS，并用TBS 5%BSA和0.1%TW 20填充罐子。之后，通过在摇床上轻轻摇晃将罐子孵育 20 分钟。然后，取出载玻片并擦拭载玻片的底部。向切片中加入100微升偶联抗体C3适合C和IgG2 APE，并在加湿室中室温孵育一小时。在摇床上用PBS 5%BSA和0.1%TW 20清洗切片3次10分钟，然后摇干载玻片。用防褪色试剂安装切片，然后用盖玻片安装。然后将载玻片存放在四摄氏度，直到在显微镜下观察。对于使用图像J软件进行图像分析，请勾勒出所需区域。之后，通过单击“分析”设置标准参数，然后设置测量和测量区域以获得结果。接下来，将从度量窗口获取的数据传输到电子表格。完成后，单击“分析”以测量区域并将数据再次传输到上一个电子表格。随着时间的推移检测MRL LPR小鼠尿液中的蛋白质水平，其中用磷酸盐缓冲盐水作为对照组和罗伊氏乳杆菌作为治疗组进行处理。用罗伊氏乳杆菌治疗的小鼠组比对照组更积极地进行蛋白尿进展。对分离的肾白细胞进行传真分析以分析浆细胞。此外，用万古霉素或万古霉素以及大肠杆菌双链DNA处理小鼠。尽管预计大肠杆菌双链DNA会触发肾脏浸润，但浆细胞的百分比没有发现显着差异。补体C3和IgG2a通过女性MRL IPR肾脏切片的免疫荧光染色进行检测。比较了动物设施中内部小鼠面包和从供应商处购买的小鼠面包对肾脏沉积C3和IgG2a的有效环境因素。免疫组织化学分析未显示两组之间有任何差异。添加每层百分比库存ISO 20 Percoll溶液可能具有挑战性，如果它们混合，则必须重新开始。该程序有助于流式细胞仪和玻璃体实验，因此您无法研究不同的肾细胞类型群体。这个过程使我们能够研究肾脏中浸润的B细胞，文献中没有很多信息。

Research

JoVE Journal

Immunology and Infection

Isolation of Brain-infiltrating Leukocytes

脑浸润白细胞的分离

We describe a method for preparing brain infiltrating leukocytes (BILs) from mice. We demonstrate how to infect mice with Theiler's murine ...

科研

免疫与感染

Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice

Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice

脑幽静的白细胞的分离与分析<em&gt;伯氏疟原虫</emANKA感染的小鼠

We describe a method for isolation and  characterization of adherent inflammatory cells from brain blood vessels of P. berghei ANKA-infected mice. ...

Cecal Ligation and Puncture-induced Sepsis as a Model To Study Autophagy in Mice

盲肠结扎穿孔致脓毒症作为模型小鼠中自噬研究

Experimental sepsis can be induced in mice using the cecal ligation and puncture (CLP) method, which causes polymicrobial sepsis. Here, a protocol is ...

Analyzing the Effects of Stromal Cells on the Recruitment of Leukocytes from Flow

对白细胞从流程的分析招聘基质细胞的影响

Stromal cells regulate the recruitment of circulating leukocytes during inflammation through cross-talk with neighboring endothelial cells. Here we ...

Isolation of Leukocytes from the Human Maternal-fetal Interface

从人类母胎界面分离白细胞

Pregnancy is characterized by the infiltration of leukocytes in the reproductive tissues and at the maternal-fetal interface (decidua basalis and decidua ...

Selective Harvesting of Marginating-pulmonary Leukocytes

Marginating肺白细胞的选择性收获

Marginating-pulmonary (MP) leukocytes are leukocytes that adhere to the inner endothelium of the lung capillaries. MP-leukocytes were shown to exhibit ...

Selective Harvesting of Marginating-hepatic Leukocytes

Marginating肝白细胞的选择性收获

Marginating-hepatic (MH) leukocytes (leukocytes adhering to the sinusoids of the liver), were shown to exhibit unique composition and characteristics ...

Phenotypic Characterization of Macrophages from Rat Kidney by Flow Cytometry

通过流式细胞仪大鼠肾脏巨噬细胞的表型特征

There is increasing evidence suggesting the important role of inflammation and, subsequently, macrophages in the development and progression of renal ...

Bronchoalveolar Lavage of Murine Lungs to Analyze Inflammatory Cell Infiltration

支气管肺泡灌洗小鼠肺分析炎症细胞浸润

Bronchoalveolar Lavage (BAL) is an experimental procedure that is used to examine the cellular and acellular content of the lung lumen ex vivo to gain ...

Renal Subcapsular Transplantation of 2'-Deoxyguanosine-Treated Murine Embryonic Thymus in Nude Mice

2'-脱氧核苷在裸体小鼠中治疗鼠胚胎胸腺的肾下囊移植

The thymus is an important central immune organ, which plays an essential role in the development and differentiation of T cells. Thymus transplantation ...