我们感谢Edanz Group（www.edanzediting.com）的Sarah Williams博士编辑了这份手稿的草稿。

动物实验经吉智医科大学动物伦理委员会批准，并符合《吉智医科大学实验动物指南》中的实验动物使用和护理指南。
1. 从HIGA小鼠中获取肾脏样本
注意:HIGA小鼠在8,9,10,11岁25周后显示出稳定的IgA肾病表型。应选择Balb/c小鼠作为对照组8、9、10、11。获得25周龄雌性HIGA小鼠（n = 10）和25周龄雌性Balb / c小鼠（n = 10）。有必要提前确定实验所需的小鼠数量。对于10只HIGA小鼠和10只Balb / c小鼠的样本量，此步骤需要约7-8小时。
<ol><li>准备这些物品:HIGA小鼠（25周龄，雌性），Balb / c小鼠（25周龄，雌性），吸入麻醉器，异氟醚，软木板，针，磷酸盐缓冲盐水（PBS），注射器，针头，手术剪刀和镊子。</li>
	<li>使用4%-5%的异氟醚和吸入麻醉装置麻醉HIGA小鼠，并使用销钉将其安装在软木板上的背侧位置。将异氟醚的浓度调节至2%-3%作为诱导麻醉后的维持剂量。 注意:麻醉深度保持在完全消除与侵入性手术相关的疼痛的水平。特别是，在切除胸部等组织时，异氟醚浓度应调节至4-5%。脱毛和眼膏不是必需的。如果可以快速执行该过程，则无需将鼠标安装在加热垫上。</li>
	<li>用手术剪刀和镊子在小鼠腹壁上做一个3-4厘米的中线切口，并仔细识别肾脏的两侧。</li>
	<li>用手术剪刀和镊子切开肋骨和横膈膜，露出心脏。切开右心房后，将PBS注射到左心室，直到肾脏颜色变为淡黄色（此过程意味着小鼠的整个身体都注入了PBS。</li>
	<li>切开肾动脉、肾静脉和输尿管，切除肾脏。将肾脏分成30毫克片，用于下一步。 注意:IgA肾病的病理主要涉及肾皮质（肾脏外部）中的肾小球。虽然很难在肉眼下完全区分皮层和髓质，但希望主要收集肾脏的外部。这些样品可在–80°C下储存数月。</li>
</ol>2. 纯化肾脏样本中的总RNA
注意:在此步骤中，市售miRNA分离试剂盒用于提取总RNA。此外，还使用生物聚合物粉碎离心柱。有关更多详细信息，请参阅 材料表 。miRNA 分离试剂盒包含基于硅胶膜的离心柱、基于苯酚/胍的裂解试剂、胍/乙醇洗涤缓冲液（洗涤缓冲液 1）、乙醇洗涤缓冲液（洗涤缓冲液 2）和无核酸酶的水。对于10只HIGA小鼠和10只对照小鼠的样本量，此步骤需要约3小时。
<ol><li>准备以下物品:1.5 或 2.0 mL 收集管、100% 乙醇、氯仿、硅均质器、微量移液器、移液器吸头、离心机、生物聚合物粉碎离心柱、硅胶膜离心柱、苯酚/胍基裂解试剂、胍/乙醇洗涤缓冲液（洗涤缓冲液 1）、乙醇洗涤缓冲液（洗涤缓冲液 2）和无核酸酶水。</li>
	<li>在室温下，使用硅匀浆器和 700 μL 苯酚/胍基裂解试剂匀浆 30 mg 肾脏样品。 注意:样品中的miRNA在暴露于基于苯酚/胍的裂解试剂之前是脆弱的，因此应立即执行这些步骤。</li>
	<li>将裂解物转移到生物聚合物粉碎离心柱中，并在室温下以15,000× g 离心2分钟。</li>
	<li>向滤液中加入 140 μL 氯仿并剧烈混合 15 秒。放置2-3分钟，然后在4°C下以12,000 ×g 离心15分钟。</li>
	<li>轻轻地将透明上清液不接触中间层转移到新的收集管中，并加入测定体积（见下文）的100%乙醇。涡旋5秒。 注意:需要100%乙醇，为获得的上清液体积的1.5倍。</li>
	<li>将样品（上限 700 μL）转移到基于硅胶膜的离心柱中，并在室温下以 8,000 x g 离心样品 15 秒。离心后弃去滤液。</li>
	<li>向基于硅胶膜的离心柱中加入 700 μL 洗涤缓冲液 1，并在室温下以 8,000 x g 离心柱 15 秒。离心后弃去滤液。</li>
	<li>将 500 μL 洗涤缓冲液 2 加入基于硅胶膜的离心柱中，并在室温下以 8,000 x g 离心 15 秒。离心后弃去滤液。</li>
	<li>将 500 μL 洗涤缓冲液 2 加入基于硅胶膜的离心柱中，并在室温下以 8,000 x g 离心 15 秒。离心后弃去滤液。</li>
	<li>再次离心基于硅胶膜的离心柱，而不添加任何东西以除去任何额外的乙醇，在室温下以15,000× g 离心1分钟。离心后扔掉滤液。</li>
	<li>将连接到离心柱的管更换为新的收集管。</li>
	<li>向基于硅胶膜的离心柱中加入 30 μL 无核酸酶水，并在室温下以 8,000 x g 离心 1 分钟。 注意:所得的 30 μL 溶液含有高浓度的总 RNA。该样品可在–80°C下储存数月。</li>
</ol>3. 从总RNA合成cDNA
注意:在此步骤中，使用市售的逆转录试剂盒。有关更多详细信息，请参阅 材料表 。该试剂盒包含核酸混合物、逆转录酶混合物和缓冲液。此过程必须在冰上进行，以防止反应进展。对于10只HIGA小鼠和10只对照小鼠的样本量，此步骤需要约3小时。
<ol><li>准备以下物品:1.5 mL 收集管、八孔试管、微量移液器、移液器吸头、分光光度计、无核酸酶水、冰、核酸混合物、逆转录酶混合物、缓冲液和热循环仪。</li>
	<li>使用分光光度计测量总RNA的浓度。然后，计算所需体积的无核酸酶水，使 12 μL 含有 1 μg 总 RNA。</li>
	<li>制备含有以下内容的预混液:2.0 μL 核酸混合物、2.0 μL 逆转录酶混合物和 4.0 μL 缓冲液，每个样品总共 8.0 μL。</li>
	<li>向八孔试管的每个孔中加入 8 μL 预混液。</li>
	<li>稀释以3.2计算的无核酸酶水体积中的总RNA。然后，向每个八孔试管孔中加入 12 μL 总 RNA 溶液（含有 1 μg 总 RNA）。</li>
	<li>使用热循环仪将样品在37°C的8孔试管中孵育60分钟。然后，使用热循环仪在95°C孵育5分钟。 注意:孵育后的溶液含有高浓度的cDNA。</li>
	<li>将该溶液转移到 1.5 mL 管中，加入 200 μL 无核酸酶水。 注意:所得的 200 μL 溶液可用作 qRT-PCR 模板 cDNA。该样品可在–80°C下储存约1年。</li>
</ol>4. miRNA 的 qRT-PCR
注意:在此步骤中，使用市售PCR试剂盒。有关更多详细信息，请参阅 材料表 。该试剂盒包含PCR混合物、通用引物和无核酸酶水。样品应一式两份制备，并在每种情况下考虑结果的准确性。miRNA的表达水平通过ΔΔCT方法定量。对于10只HIGA小鼠和10只对照小鼠的样本量，此步骤需要约4小时。
<ol><li>准备以下物品:1.5 mL 收集管、96 孔反应板、用于 96 孔反应板的粘合膜、微量移液器、移液器吸头、PCR 混合物、通用引物、无核酸酶水、miRNA 特异性引物和实时荧光定量 PCR 仪器。</li>
	<li>制备含有以下内容的预混液:每孔 12.5 μL PCR 混合物、2.5 μL 通用引物、1.25 μL 5 μM miRNA 特异性引物和 6.25 μL 无核酸酶水。 注意:在该测定中，使用了对miRNA[RNA，U6小核2（RNU6-2），miR-155-5p，miR-146a-5p和miR-21-5p]特异性引物。RNU6-2用作内源性对照14。</li>
	<li>向 96 孔反应板的每个孔中加入 22.5 μL 预混液。</li>
	<li>将步骤 3.7 中制备的 2.5 μL cDNA 加入 96 孔板的各个孔中。</li>
	<li>用粘附膜覆盖96孔反应板，然后以1,000× g 离心30秒。</li>
	<li>运行实时荧光定量 PCR 仪器。对PCR仪器进行编程如下:在95°C下初始活化15分钟，然后在94°C下变性40个循环15秒，在55°C下退火30秒，并在70°C下延伸30秒。</li>
	<li>使用 ΔΔCT 方法定量基因表达。相对表达水平确定为 2–ΔΔCT。 注意:应考虑从结果中排除异常的PCR扩增曲线。</li>
</ol>

<ol>
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提交人声明他们没有利益冲突。

我们能够使用这种新方法测量IgA肾病小鼠模型（HIGA小鼠）肾脏中miRNA的表达水平。IgA肾病是一种无法解释的疾病，需要进一步研究以阐明其病因和治疗靶点1,3。然而，获取人类肾脏样本是高度侵入性的。这种新技术的优点在于它允许使用小动物研究IgA肾病，并且应该有助于未来对IgA肾病和miRNA的研究。在未来的研究中，该方法可以应用于治疗IgA肾病的新型miRNA的研究。HIGA小鼠中miRNA的人工调节可能会影响IgA肾病的表型。在这种情况下，该方法可能有助于验证miRNA的变化。该方法不用于分析血清中的miRNA，但可以通过修改协议将其用于此目的。
这种方法的理由是根据以前的报告。我们使用生物聚合物粉碎离心柱来破坏样品中的生物聚合物，这在以前已被证明是一种有效的技术12。过去的报告还证明了使用逆转录从总RNA合成cDNA和使用制备的cDNA作为模板进行PCR的准确性13。作为该方法的关键步骤，PCR结果的评估很重要。有必要从结果中排除异常的扩增曲线。此外，如果未获得稳定的结果，应考虑内源性对照的变化14。
这种方法可能遇到的一个主要问题是miRNA的异常低表达。miRNA是高度可降解的化合物，因此应快速执行该方法。此外，需要消除核糖核酸酶（RNase）在实验环境中的影响。研究人员应始终注意皮肤和头发中RNase的存在17。实验时必须戴上一次性手套、口罩和发帽。此外，必须清洁微量移液器、移液器吸头和收集管等实验室设备，以确保不含RNase17。必要时应使用高压灭菌器使RNA酶失活17。否则，研究人员应使用无RNase认证的项目17。所有样品都应在推荐的条件下妥善储存，以降低RNase污染的风险。
我们的方法有三个重要的局限性。首先，我们没有证明它是否可以应用于其他动物和其他器官。这是相关的，因为已知miRNA在IgA肾病2,6,7的血细胞中起重要作用，但我们没有研究我们的方法是否可以用于血细胞。其次，我们实验的样本量可能很小。样本量应根据研究设计、统计分析、所需时间和所需成本来确定。因此，每个研究人员必须在开始此实验之前确定适当的样本量。第三，HIGA小鼠IgA肾病的严重程度和表型可能因个体而异9。此外，性别和年龄的偏见尚未得到充分调查。如有必要，建议添加免疫组织化学检查以确认IgA肾病的严重程度和表型。此外，建议根据需要使用PCR以外的方法，包括微阵列、北方印迹和核糖核酸酶保护测定。
总之，我们开发了一种可靠的方法来测量IgA小鼠模型（HIGA小鼠）肾脏中miRNA的表达水平。

免疫球蛋白A（IgA）肾病是一种原发性肾小球肾炎，其特征是IgA在肾小球系膜区域1,2中的异常沉积。它是最常见的原发性肾小球肾炎，在20%-40%的患者中导致终末期肾衰竭2。病因尚不清楚，但与持续性黏膜感染有关1,3。皮质类固醇、免疫抑制剂和肾素-血管紧张素系统抑制剂已被提出作为治疗方法1,3，但尚未完全确立3。因此，需要进一步研究明确治疗IgA肾病的病因和治疗方法。
microRNA（miRNA）是小的非编码RNA，在调节基因表达中起重要作用4,5。据报道，miRNA参与各种疾病的发病机制，有些已被确定为疾病生物标志物和治疗剂4,5。近年来，miRNA与IgA肾病发病机制之间的关联也有报道2,6,7。例如，miR-148b被证明与IgA肾病2,6,7患者的IgA结构异常有关，而miR-148b和let-7b被记录为检测IgA肾病的新型生物标志物7。尽管了解miRNA对IgA肾病的影响可能有助于进一步阐明病因和治疗2，但尚未建立使用小动物模型分析IgA肾病中miRNA的标准方法2。
我们在此开发了一种简单可靠的方法来测量IgA肾病小鼠模型（HIGA小鼠）肾脏中的miRNA表达水平。HIGA小鼠是一种特征性的ddY菌株，显示血清IgA水平特别高，肾小球8,9,10,11中IgA的异常沉积。因此，HIGA小鼠可用作IgA肾病小鼠模型8,9,10,11。我们的方法包括四个主要步骤:首先，通过手术从HIGA小鼠获得肾脏样本;其次，使用基于硅胶膜的离心柱均质化样品并纯化总RNA;第三，使用逆转录从总RNA合成互补DNA（cDNA）;第四，通过定量逆转录聚合酶链反应（qRT-PCR）检测miRNA的表达水平。这种方法的基本原理和结果的可靠性基于以前的报告12,13。我们表明，这是一种准确测量IgA肾病小鼠模型中miRNA表达水平的有用技术，并且可用于促进未来对IgA肾病中miRNA的研究。

<table><tbody><tr><td>BALB/cCrSlc (25-week-old, female)</td><td>Japan SLC, Inc.</td><td>none</td><td>Mouse for control</td></tr><tr><td>HIGA/NscSlc (25-week-old, female)</td><td>Japan SLC, Inc.</td><td>none</td><td>IgA nephropathy mouse model</td></tr><tr><td>MicroAmp Optical 96 well reaction plate for qRT-PCR</td><td>Thermo Fisher Scientific</td><td>4316813</td><td>96-well reaction plate</td></tr><tr><td>MicroAmp Optical Adhesive Film</td><td>Thermo Fisher Scientific</td><td>4311971</td><td>adhesive film for 96-well reaction plate</td></tr><tr><td>miScript II RT kit</td><td>Qiagen</td><td>218161</td><td>Experimental kit for synthesis of cDNA</td></tr><tr><td>miRNeasy Mini kit</td><td>Qiagen</td><td>217004</td><td>Experimental kit fot extraction of total RNA</td></tr><tr><td>miScript Primer Assay (RNU6-2)</td><td>Qiagen</td><td>MS00033740</td><td>miRNA-specific primer</td></tr><tr><td>miScript Primer Assay (miR-155-5p)</td><td>Qiagen</td><td>MS00001701</td><td>miRNA-specific primer</td></tr><tr><td>miScript Primer Assay (miR-146a-5p)</td><td>Qiagen</td><td>MS00001638</td><td>miRNA-specific primer</td></tr><tr><td>miScript Primer Assay (miR-21-5p)</td><td>Qiagen</td><td>MS00009079</td><td>miRNA-specific primer</td></tr><tr><td>miScript SYBR Green PCR kit</td><td>Qiagen</td><td>218073</td><td>Experimental kit for real-time PCR</td></tr><tr><td>QIA shredder</td><td>Qiagen</td><td>79654</td><td>biopolymer-shredding spin column</td></tr><tr><td>QuantStudio 12K Flex Flex Real-Time PCR system</td><td>Thermo Fisher Scientific</td><td>4472380</td><td>real-time PCR instrument</td></tr><tr><td>QuantStudio 12K Flex Software version 1.2.1.</td><td>Thermo Fisher Scientific</td><td>4472380</td><td>real-time PCR instrument software</td></tr><tr><td>takara biomasher standard</td><td>Takara Bio</td><td>9790B</td><td>silicon homogenizer</td></tr></tbody></table>

detection of microrna expression in the kidneys of immunoglobulin a nephropathic mice

免疫球蛋白A（IgA）肾病是一种原发性肾小球肾炎，其特征是IgA沉积异常，导致终末期肾衰竭。近年来，在IgA肾病的发病机制中报道了microRNA（miRNA）的参与。然而，没有使用小动物模型分析IgA肾病中miRNA的既定方法。因此，我们开发了一种可靠的方法来分析IgA小鼠模型（HIGA小鼠）肾脏中的miRNA。该协议的目标是检测与对照小鼠肾脏中的水平相比，HIGA小鼠肾脏中miRNA的表达水平改变。简而言之，该方法包括四个步骤:1）从HIGA小鼠获得肾脏样本;2）从肾脏样本中纯化总RNA;3）从总RNA合成互补DNA;4）miRNA的定量逆转录聚合酶链反应（qRT-PCR）。使用这种方法，我们成功地检测了HIGA小鼠肾脏中几种miRNA（miR-155-5p，miR-146a-5p和miR-21-5p）的表达水平。这种新方法可以应用于其他分析IgA肾病中miRNA的研究。

我们研究了miRNA在HIGA小鼠肾脏中的表达水平（n = 10）。该结果完全基于所描述的协议获得。选择Balb / c小鼠的肾脏作为对照（n = 10）。在两组中，均选择25周龄。供应商仅提供雌性HIGA小鼠。三种miRNA（miR-155-5p、miR-146a-5p和miR-21-5p）的表达水平;图1）被检测到，之前报道与 IgA 肾病有关15,16。采用t检验比较两组相对miRNA表达水平，P<0.05有统计学意义。与对照组相比，HIGA组miR-155-5p的表达水平高3.3倍（P < 0.001），而HIGA组的miR-21-5p表达水平高1.58倍（P = 0.007）。两组miR-146a-5p表达差异无统计学意义。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61535/61535fig01.jpg"> 图1:miRNA在HIGA小鼠肾脏中的表达水平。 qRT-PCR测定了miR-155-5p，miR-146a-5p和miR-21-5p在HIGA小鼠（n = 10）和Balb / c小鼠（n = 10）中的相对表达水平。相对表达水平显示为平均值和标准误差。（A）HIGA组miR-155-5p表达高3.3倍（P <0.001）。（B）两组miR-146a-5p表达无显著差异。（C）HIGA组miR-21-5p表达高1.58倍（P=0.007）。定量实时逆转录聚合酶链反应（qRT-PCR）;微核糖核酸（miRNA）;不显著（NS）;*，第 <0.05 页。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61535/61535fig01large.jpg" target="_blank">请点击此处查看此图的大图。</a>

Watch this Scientific Journal Video about Detection of MicroRNA Expression in the Kidneys of Immunoglobulin A Nephropathic Mice at JoVE.com

Detection of MicroRNA Expression in the Kidneys of Immunoglobulin A Nephropathic Mice

microRNA参与IgA肾病的发病机制。我们已经开发出一种可靠的方法来检测IgA肾病小鼠模型（HIGA小鼠）肾脏中的microRNA表达水平。这种新方法将有助于检查miRNA是否参与IgA肾病。

检测免疫球蛋白A肾病小鼠肾脏中的microRNA表达

Immunoglobulin A (IgA) nephropathy is a type of primary glomerulonephritis characterized by the abnormal deposition of IgA, leading to the end-stage renal ...

detection-microrna-expression-kidneys-immunoglobulin-nephropathic

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

1.8K 次观看.  Jichi Medical University. microRNA参与IgA肾病的发病机制。我们已经开发出一种可靠的方法来检测IgA肾病小鼠模型（HIGA小鼠）肾脏中的microRNA表达水平。这种新方法将有助于检查miRNA是否参与IgA肾病。

视频: Detection of MicroRNA Expression in the Kidneys of Immunoglobulin A Nephropathic Mice

Detection of microRNA Expression in Peritoneal Membrane of Rats Using Quantitative Real-time PCR

MicroRNAs (miRNAs) are small noncoding RNAs that regulate messenger RNA expression post-transcriptionally. The miRNA expression profile has been investigated in various organs and tissues in rat. However, standard methods for the purification of miRNAs and detection of their expression in rat peritoneal membrane have not been well established. We have developed an effective and reliable method to purify and quantify miRNAs using quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR) in rat peritoneal membrane. This protocol consists of four steps: 1) purification of peritoneal membrane sample; 2) purification of total RNA including miRNA from peritoneal membrane sample; 3) reverse transcription of miRNA to produce cDNA; and 4) qRT-PCR to detect miRNA expression. Using this protocol, we successfully determined that the expression of six miRNAs (miRNA-142-3p, miRNA-21-5p, miRNA-221-3p, miRNA-223-3p, miRNA-327, and miRNA-34a-5p) increased significantly in the peritoneal membrane of a rat peritoneal fibrosis model compared with those in control groups. This protocol can be used to study the profile of miRNA expression in the peritoneal membrane of rats in many pathological conditions.

Here we present a protocol for the detection of microRNA expression in rat peritoneal membrane using quantitative real-time reverse-transcription polymerase chain reaction. This method is suitable for studying the microRNA expression profile in rat peritoneal membrane in several pathological conditions.

使用定量实时PCR检测大鼠腹膜中的微小RNA表达

MicroRNAs (miRNAs) are small noncoding RNAs that regulate messenger RNA expression post-transcriptionally. The miRNA expression profile has been ...

科研

遗传学

Isolation of Glomeruli and In Vivo Labeling of Glomerular Cell Surface Proteins

Proteinuria results from the disruption of the glomerular filter that is composed of the fenestrated endothelium, glomerular basement membrane, and podocytes with their slit diaphragms. The delicate structure of the glomerular filter, especially the slit diaphragm, relies on the interplay of diverse cell surface proteins. Studying these cell surface proteins has so far been limited to in vitro studies or histologic analysis. Here, we present a murine in vivo biotinylation labeling method, which enables the study of glomerular cell surface proteins under physiologic and pathophysiologic conditions. This protocol contains information on how to perfuse mouse kidneys, isolate glomeruli, and perform endogenous immunoprecipitation of a protein of interest. Semi-quantitation of glomerular cell surface abundance is readily available with this novel method, and all proteins accessible to biotin perfusion and immunoprecipitation can be studied. In addition, isolation of glomeruli with or without biotinylation enables further analysis of glomerular RNA and protein as well as primary glomerular cell culture (i.e., primary podocyte cell culture).

Isolation of Glomeruli and In Vivo Labeling of Glomerular Cell Surface Proteins

Here we present a protocol for murine in vivo labeling of glomerular cell surface proteins with biotin. This protocol contains information on how to perfuse mouse kidneys, isolate glomeruli, and perform endogenous immunoprecipitation of the protein of interest.

肾小球的分离及肾小球细胞表面蛋白的活体标记

Proteinuria results from the disruption of the glomerular filter that is composed of the fenestrated endothelium, glomerular basement membrane, and ...

生物化学

Quantitative Real-Time PCR Evaluation of microRNA Expressions in Mouse Kidney with Unilateral Ureteral Obstruction

MicroRNAs (miRNAs) are single stranded, non-coding RNA molecules that typically regulate gene expression at the post-transcriptional level by binding to partially complementary target sites in the 3&#39; untranslated region (UTR) of messenger RNA (mRNA), which reduces the mRNA&#39;s translation and stability. The miRNA expression profiles in various organs and tissues of mice have been investigated, but standard methods for the purification and quantification of miRNA in mouse kidney have not been available. We have established an effective and reliable method for extracting and evaluating miRNA expression in mouse kidney with renal interstitial fibrosis by quantitative reverse-transcription polymerase chain reaction (qRT-PCR). The protocol required five steps: (1) creation of sham and unilateral ureteral obstruction (UUO) mice; (2) extraction of kidney samples from the UUO mice; (3) extraction of total RNA, which includes miRNA, from the kidney samples; (4) complementary DNA (cDNA) synthesis with reverse transcription from miRNA; and (5) qRT-PCR using the cDNA. Using this protocol, we successfully confirmed that compared to the controls, the expression of miRNA-3070-3p was significantly increased and those of miRNA-7218-5p and miRNA-7219-5p were significantly decreased in the kidneys of a mouse model of renal interstitial fibrosis. This protocol can be used to determine the miRNA expression in the kidneys of mice with UUO.

We describe a method for evaluating the microRNA expression in the kidneys of mice with unilateral ureteral obstruction (UUO) by quantitative reverse-transcription polymerase chain reaction. This protocol is suitable for studying kidney microRNA expression profiles in mice with UUO and in the context of other pathological conditions.

小鼠肾中微RNA表达与单体尿阻的定量实时PCR评价

MicroRNAs (miRNAs) are single stranded, non-coding RNA molecules that typically regulate gene expression at the post-transcriptional level by binding to ...

Quantitative Real-Time Polymerase Chain Reaction Evaluation of MicroRNA Expression in Kidney and Serum of Mice with Age-Dependent Renal Impairment

MicroRNAs (miRNAs) are small, noncoding RNAs consisting of 21-25 bases. They are not translated into proteins but rather work to impede the functioning of their target messenger RNAs (mRNAs) by destabilizing them and disrupting their translation. Although the miRNA expression profiles in various mouse organs and tissues have been investigated, there have been no standard methods for purifying and quantifying mouse kidney and serum miRNAs. We have established an effective and reliable method for extracting and evaluating the miRNA expression in the serum and kidney of mice with age-dependent renal impairment. 
The method uses quantitative reverse-transcription-polymerase chain reaction (qRT-PCR), and the protocol requires six steps: (1) preparing senescence-accelerated mouse resistance 1 (SAMR1) mice and senescence-accelerated mouse prone (SAMP1) mice; (2) extracting serum samples from these mice; (3) extracting a kidney sample from each mouse; (4) extracting total RNA (including miRNA) from kidney and serum samples from each mouse; (5) the synthesis of complementary DNA (cDNA) with reverse transcription from the miRNA; (6) conducting a qRT-PCR using the cDNA obtained. 
This protocol was used to confirm that, compared to the controls, the expression of miRNA-7218-5p and miRNA-7219-5p was significantly changed in the kidney and serum of a mouse model of age-dependent renal impairment. This protocol also clarified the relationship between the kidney and serum of the mouse model of age-dependent renal impairment. This protocol can be used to determine miRNA expression in the kidney and serum of mice with age-dependent renal impairment.

We present a method for evaluating microRNA expression in the kidney and serum of mice with age-dependent renal impairment by quantitative reverse-transcription polymerase chain reaction.

年龄依赖性肾损伤小鼠肾脏和血清中MicroRNA表达的定量实时聚合酶链反应评估

MicroRNAs (miRNAs) are small, noncoding RNAs consisting of 21-25 bases. They are not translated into proteins but rather work to impede the functioning of ...

Assessment of Kidney Function in Mouse Models of Glomerular Disease

The use of murine models to mimic human kidney disease is becoming increasingly common. Our research is focused on the assessment of glomerular function in diabetic nephropathy and podocyte-specific VEGF-A knock-out mice; therefore, this protocol describes the full kidney work-up used in our lab to assess these mouse models of glomerular disease, enabling a vast amount of information regarding kidney and glomerular function to be obtained from a single mouse. In comparison to alternative methods presented in the literature to assess glomerular function, the use of the method outlined in this paper enables the glomerular phenotype to be fully evaluated from multiple aspects. By using this method, the researcher can determine the kidney phenotype of the model and assess the mechanism as to why the phenotype develops. This vital information on the mechanism of disease is required when examining potential therapeutic avenues in these models. The methods allow for detailed functional assessment of the glomerular filtration barrier through measurement of the urinary albumin creatinine ratio and individual glomerular water permeability, as well as both structural and ultra-structural examination using the Periodic Acid Schiff stain and electron microscopy. Furthermore, analysis of the genes dysregulated at the mRNA and protein level enables mechanistic analysis of glomerular function. This protocol outlines the generic but adaptable methods that can be applied to all mouse models of glomerular disease.

This protocol describes a full kidney work-up that should be carried out in mouse models of glomerular disease. The methods allow for detailed functional, structural, and mechanistic analysis of glomerular function, which can be applied to all mouse models of glomerular disease.

小鼠肾小球疾病模型肾脏功能的评价

The use of murine models to mimic human kidney disease is becoming increasingly common. Our research is focused on the assessment of glomerular function ...

医学

MicroRNA In situ Hybridization for Formalin Fixed Kidney Tissues

In this article we describe a method for colorimetric detection of miRNA in the kidney through in situ hybridization with digoxigenin tagged microRNA probes. This protocol, originally developed by Kloosterman and colleagues for broad use with Exiqon miRNA probes1, has been modified to overcome challenges inherent in miRNA analysis in kidney tissues. These include issues such as structure identification and hard to remove residual probe and antibody. Use of relatively thin, 5 mm thick, tissue sections allowed for clear visualization of kidney structures, while a strong probe signal was retained in cells. Additionally, probe concentration and incubation conditions were optimized to facilitate visualization of microRNA expression with low background and nonspecific signal. Here, the optimized protocol is described, covering the initial tissue collection and preparation through the mounting of slides at the end of the procedure. The basic components of this protocol can be altered for application to other tissues and cell culture models.

MicroRNA In situ Hybridization for Formalin Fixed Kidney Tissues

This article describes an in situ hybridization protocol optimized for colormetric detection of microRNA expression in formalin fixed kidney sections.

微RNA<em&gt;原位</em&gt;杂交的福尔马林固定的肾组织

In this article we describe a method for colorimetric detection of miRNA in the kidney through in situ hybridization with digoxigenin tagged microRNA ...

生物学

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

Systemic lupus erythematosus (SLE) is an autoimmune disorder with no known cure and is characterized by persistent inflammation in many organs, including the kidneys. Under such circumstances, the kidney loses its ability to clean waste from the blood and regulate salt and fluid concentrations, eventually leading to renal failure. Women, particularly those of childbearing age, are diagnosed nine times more often than men. Kidney disease is the leading cause of mortality in SLE patients. The present protocol describes a quick and simple method to measure excreted protein levels in collected urine, tracking lupus progression over time. In addition, an approach to isolate kidney mononuclear cells is provided based on size and density selection to investigate renal infiltration of leukocytes. Furthermore, an immunohistochemical method has been developed to characterize protein deposition in the glomeruli and leukocyte infiltration in the tubulointerstitial space. Together, these methods can help investigate the progression of chronic inflammation associated with the kidneys of lupus-prone MRL/lpr mice.

The present protocol describes a method to track lupus progression in mice. Two additional procedures are presented to characterize lupus nephritis based on cell infiltration and protein deposition in the kidneys.

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 ...

免疫与感染

A Quantitative Detection Method for MicroRNAs in the Kidney of an Ischemic Kidney Injury Mouse Model

MicroRNAs (miRNAs) are involved in various disease states and are effective biomarkers for the early diagnosis of diseases and treatment in mice. However, standard protocols for the purification of miRNAs and detection of their expression in the kidneys of acute kidney injury (AKI) mice have not been well established. This study developed an effective and simple protocol to purify and quantify miRNAs in the kidneys of an AKI mouse model induced by renal ischemia-reperfusion using quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR). This protocol comprises five steps: 1) induction of AKI by renal ischemia-reperfusion, 2) harvesting of kidneys, 3) purification of total RNA, including miRNAs, from kidneys, 4) cDNA synthesis by reverse transcription of miRNA, and 5) qRT-PCR to detect miRNA expression. Using this protocol, the renal ischemia-reperfusion injury model can be generated with mild to severe forms of AKI. Additionally, if the procedure is followed properly, a consistent AKI model with minimal individual differences can be obtained. This qRT-PCR assay shows a very wide dynamic range and enables the discrimination of mature miRNAs, which can be accurately quantified with high specificity. This protocol can be used to study the miRNA expression profile in AKI kidneys.

This article presents a protocol for detecting microRNA expression in the kidneys of an acute kidney injury mouse model using quantitative real-time reverse-transcription polymerase chain reaction. This protocol emphasizes an ischemic kidney injury mouse model and the careful extraction of microRNA samples.

MicroRNAs (miRNAs) are involved in various disease states and are effective biomarkers for the early diagnosis of diseases and treatment in mice. However, ...

Delivery of Exogenous Artificially Synthesized miRNA Mimic to the Kidney Using Polyethylenimine Nanoparticles in Several Kidney Disease Mouse Models

microRNAs&#160;(miRNAs), small noncoding RNAs (21-25 bases) that are not translated into proteins, inhibit lots of target messenger RNAs (mRNAs) by destabilizing and inhibiting their translation in various kidney diseases. Therefore, alternation of miRNA expression by exogenous artificially synthesized miRNA mimics is a potentially useful treatment option for inhibiting the development of many kidney diseases. However, because serum RNAase immediately degrades systematically administered exogenous miRNA mimics in vivo, delivery of miRNA to the kidney remains a challenge. Therefore, vectors that can protect exogenous miRNA mimics from degradation by RNAase and significantly deliver them to the kidney are necessary. Many studies have used viral vectors to deliver exogenous miRNA mimics or inhibitors to the kidney. However, viral vectors may cause an interferon response and/or genetic instability. Therefore, the development of viral vectors is also a hurdle for the clinical use of exogenous miRNA mimics or inhibitors. To overcome these concerns regarding viral vectors, we developed a nonviral vector method to deliver miRNA mimics to the kidney using tail vein injection of polyethylenimine nanoparticles (PEI-NPs), which led to significant overexpression of target miRNAs in several mouse models of kidney disease.

Here, we deliver exogenous artificially synthesized miRNA mimics to the kidney via tail vein injection of a nonviral vector and polyethylenimine nanoparticles in several kidney disease mouse models. This led to significant overexpression of target miRNA in the kidney, resulting in inhibited progression of kidney disease in several mouse models.

在几种肾脏疾病小鼠模型中使用聚乙烯亚胺纳米颗粒将外源人工合成的miRNA模拟物递送到肾脏

microRNAs&#160;(miRNAs), small noncoding RNAs (21-25 bases) that are not translated into proteins, inhibit lots of target messenger RNAs (mRNAs) by ...

<meta charset="utf8"></head><body>在小鼠模型中用 Dioscin 治疗免疫球蛋白 A 肾病 （IgAN）

Dioscin Mediated IgA Nephropathy Alleviation by Inhibiting B Cell Activation In Vivo and Decreasing Galactose-Deficient IgA1 Production In Vitro

The increase of circulating galactose-deficient IgA1 (Gd-IgA1) is caused by excessive activation of IgA-positive secretory cells in the process of mucosal immune responses, which is a critical link in the pathogenesis of IgA nephropathy (IgAN). Peyer&#39;s patch, the prominent place where B lymphocytes are transformed into IgA-secreting plasma cells, is the primary source of IgA. In addition, the lower expression of core 1&#946;-1,3-galactosyltransferase (C1GalT1) and its molecular chaperone, C1GalT1-specific molecular chaperone (Cosmc), is related to abnormal glycosylation of IgA1 in IgAN patients. Our clinical experience shows that Dioscoreae Nipponicae Rhizoma&#39;s (DNR) herbal medicine can relieve proteinuria and hematuria and improve renal function in IgAN patients. Dioscin (DIO) is one of the main active ingredients of DNR, which has various pharmacological activities. This study explores DIO&#39;s possible mechanism in treating IgAN.The IgAN model mouse was established by mucosal immune induction. The mice were divided into the control, model, and DIO gavage groups. The glomerular IgA deposition in mice, renal pathological changes, and B cell markers CD20 and CXCR5 expression in Peyer&#39;s patch were detected by immunofluorescence and immunohistochemistry. After lipopolysaccharide (LPS) stimulation, DIO&#39;s effects on DAKIKI cells proliferation, IgA and Gd-IgA1 secretion, C1GalT1, and Cosmc expression were studied by cell counting kit-8 (CCK-8) assay, enzyme-linked immunosorbent assay (ELISA) test, quantitative real-time polymerase chain reaction (QRT-PCR), and western blotting (WB). In in vivo studies, IgA deposition accompanied by glomerular mesangial hyperplasia and increased expression of CD20 and CXCR5 in Peyer&#39;s patch in the IgAN model mouse was alleviated by DIO. In vitro studies showed 0.25 &#181;g/mL to 1.0 &#181;g/mL DIO inhibited LPS-induced DAKIKI cell proliferation, IgA and Gd-IgA1 secretion, and up-regulated the mRNA and protein expression of C1GalT1 and Cosmc. This study demonstrates that DIO may reduce Gd-IgA1 production by inhibiting excessive activation of IgA-secreting cells and up-regulating C1GALT1/Cosmc expression.

<meta charset="utf8"></head><body>作者聚焦：研究中草药活性薯片素治疗 IgA 肾病的潜力

This study provides experimental data for treating immunoglobulin A nephropathy (IgAN) with Dioscin (DIO), the active ingredient of Dioscoreae Nipponicae Rhizoma (DNR), and a paradigm for studying herbal medicine's effects and underlying mechanisms in vivo and in vitro.

Dioscin 通过抑制体内 B 细胞活化和减少体外半乳糖缺乏型 IgA1 的产生来缓解 IgA 肾病

The increase of circulating galactose-deficient IgA1 (Gd-IgA1) is caused by excessive activation of IgA-positive secretory cells in the process of mucosal ...