Name: assessment of kidney function in mouse models of glomerular disease
Uploaded: 2018-06-30T14:00:00.000+00:00
Duration: 9 min 16 s
Description: Watch this Scientific Journal Video about Assessment of Kidney Function in Mouse Models of Glomerular Disease at JoVE.com

这项工作得到了英国心脏基金会, 理查德明亮的 VEGF 研究信托和湄公河的支持。

<ol>
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作者没有什么可透露的。

本议定书描述了肾小球疾病的小鼠模型中应进行的全肾脏工作, 从而能够从一只老鼠身上获得关于肾脏和肾小球功能的大量信息。每种方法的关键步骤都允许对肾小球功能进行详细的功能、结构和机械分析, 包括评估肾脏的整体通透性 (uACR 和血浆肌酐测量), 通透性个体肾小球 (肾小球 LpA/V), 检查结构改变 (PAS, 三色蓝色和 EM), 蛋白质定位 (如果), 和肾小球基因表达 (rt-pcr 和西方印迹)。这些方法是对肾脏疾病模型小鼠肾小球功能的全面评估的关键。
在评估 GFB 的渗透性时, 许多研究选择使用常规 uACR 或24小时白蛋白排泄率作为有效的措施17,18。虽然这些技术允许评估 GFB 渗透性作为整体, 它不允许单独肾小球通透性评估和变异在肾小球之间。以前的研究已经发现, 对肾小球 Lpa/V 的测量我是一个更敏感的措施, 改变 GFB 渗透率5,9。事实上, 在本文所展示的代表性结果中, 在14周后诱导 vegf-a 高, vegf-a 高 X Neph VEGF-165小鼠有明显低于 VEGF-a 型小鼠 uACR;然而, 这一结果没有反映在肾小球 Lpa/V测量, 其中 VEGF-165b 没有显著地防止增加 GFB 通透性 (图 1和图 2)5。这表明使用多种化验方法来评估肾脏通透性和个体肾小球的渗透性的重要性。此外, 肾小球 Lponcometric 的实验表明, 同一肾脏的单个肾小球通透性有很大差异, 特别是在疾病模型5,10,19. 测量肾小球 LpA/V 的一个局限性是它只能在实验终点进行;因此, 需要常规的 uACR 测量来表示实验终点。
除了评估功能表型, 目前的方法也鼓励评估的结构和超微表型。这可以通过选择的污点, 如 PAS, 三色, 和银污渍;每个评估肾小球形态学的不同方面。在急性肾小球疾病模型中, 通常是老鼠模型中的例子, 除非你是一个训练有素的肾脏病理学家, 否则很难用这些污渍检测出任何主要的结构异常。因此, 建议对 GFB 的超微结构进行评估, 使其能定量测量肾小球、内皮吕克茶大小、数量和足细胞特征等参数。这种测量需要最少的训练来进行, 并使调查人员能够确定受疾病模型影响的细胞类型/结构。在代表性结果显示的例子中, VEGF-一只高鼠被发现是一种轻度肾小球疾病模型, 因此, 在 PAS 染色中没有出现重大的结构异常。然而, 足细胞特异性 VEGF-A 在检查肾小球超结构5时, 诱导了足和内皮细胞的变化。不幸的是, 目前的方法所描述的 EM 肾脏的制备并不能检测到内皮糖萼, 这对 GFB19的通透性也有显著影响。为了准确测量糖萼的深度, 肾脏应灌注固定与2.5% 戊二醛与1% 阿利新蓝为内皮糖萼标签, 如 Oltean et19所述。
一旦评估功能和结构表型, 不同基因和通路的表达/激活模式就可以在肾小球中具体评估。先前的超结构评估可以提供有关细胞类型/肾小球结构的一些信息, 表明是否应该检查足细胞或内皮特异基因/通路。例如, 在 VEGF-a 高鼠的代表性结果中, 观察到内皮吕克茶数的减少 (图 3D);因此, 研究了一种已知参与 VEGF 的内皮标记的肾小球蛋白表达--通路;VEGFR-2 (图 4B)5。除了蛋白在肾小球中的表达, 它们的定位也可以用 IF 来可视化。在张et al20的研究中, GLUT1 的足细胞表达在足中被证实, 如果将增加的 GLUT1 与 podocin 进行协调。
与文献中提出的用于评估肾小球功能的其他方法相比, 本文概述的方法用于评估肾小球疾病模型小鼠肾脏功能, 使肾小球表型能充分评估从多个方面。利用该方法, 研究人员能够确定模型的肾脏表型, 并对表型的形成机理进行评估。在研究这些模型中潜在的治疗途径时, 需要了解有关疾病机制的重要信息。这种方法可以很容易地应用于今后对肾小球功能的研究, 无论是在评估疾病表型和潜在疗法。
总之, 这种通用的和适应性的协议描述了肾小球疾病小鼠模型的全肾脏工作, 使大量有关肾脏和肾小球功能的信息可以从一只老鼠身上获得。这些方法可以对肾小球疾病的所有小鼠模型进行详细的功能、结构和机械分析。

使用小鼠模型模仿人类肾脏疾病越来越普遍。这种小鼠模型包括自发性高血压大鼠 (SHR)1、链脲佐菌素 (STZ) 诱发的糖尿病大鼠和小鼠2, 以及 db/db ii 型糖尿病老鼠3, 基因工程模型, 如原发性足细胞特异性局灶节段性肾小球硬化 (FSGS) 模型4, 足细胞特定血管内皮生长因子 a (vegf a) 敲出 (vegf a) 模型5, Alport 综合征模型6, 并获得模型如5/6 肾切除术7和单侧输尿管梗阻 (UUO)8。为了评估这些模型中肾小球功能的不同方面, 有几种技术可供选择。本方法的目的是为了充分评估肾小球功能, 在小鼠肾脏疾病模型中进行综合性的研究。
使用这种方法的基本原理是它能使肾小球表型从多个方面得到充分的评价。这包括评估肾小球通透性, 无论是蛋白质和水, 肾小球结构异常, 和变化的基因和蛋白质的表达/剪接对正常肾小球功能至关重要。利用该方法, 研究人员能够确定模型的肾脏表型, 并对表型的形成机理进行评估。这是关于疾病机制的重要信息, 在研究这些模型中的潜在治疗途径时, 这是必需的。
在文献中, 这是一个常见的发生, 提出了一个小鼠肾小球疾病模型, 其中表型是由增加水平的白蛋白在尿液中。然而, 有证据表明, 单一的方法来确定肾小球功能并不总是有效的;测量尿白蛋白排泄率或尿白蛋白肌酐比值 (uACR) 只提供有关总肾功能, 而不是单个肾小球的信息。以往的研究表明, 不同肾小球的通透性可以从同一个肾脏的5,9,10不等。另外, 评估单个肾小球的通透性是评估肾小球功能的一种较为敏感的方法;测量单个肾小球通透性的方法 (LpuACR) 对肾小球功能变化的敏感性比测量9。这种方法对抗蛋白尿的小鼠模型有益, 如 c57BL/6 背景11。本方法的优点是, 它检查了总肾通透性的白蛋白以及个别肾小球通透性的水。
对肾小球结构异常的检查通常是由一电池的污点, 如周期性酸希夫 (PAS), 三色, 和银污渍评估。这使经过训练的肾脏病理学家可以通过评分方法来评估肾脏疾病的水平。虽然所有的好方法, 肾小球宏观结构的变化并不总是观察到急性肾损伤模型12。该方法建议, 除了实施上述肾脏组织学技术外, 还应通过电子显微镜 (EM) 对肾小球超微结构进行评估。染色的肾小球在常规光镜下看起来相对正常;然而, 通过对 EM 的评估, 分析了肾小球基底膜宽度、足细胞足过程避嫌、内皮 fenestrations 和亚足细胞空间覆盖率的小变化。因此, 对肾小球超结构和微结构进行评估, 确定肾小球功能障碍的机制至关重要。
除了评估肾小球结构异常外, 还应检查 mRNA 和蛋白表达和剪接的变化, 以及蛋白质活化 (如磷酸化), 以进一步阐明肾小球疾病的机制。当观察肾小球疾病, 或例如, 当高/超表达一个基因专门在肾小球细胞, 如在足细胞特定 VEGF-一个高鼠5, 重要的是, 蛋白质和 mRNA 的变化只检查在肾小球细胞, 而不是整个肾脏。本协议描述了一种从小鼠肾皮质分离肾小球的方法, 然后分离出蛋白质/RNA。这允许对疾病模型肾小球中蛋白质/mRNA 失调的具体分析。
本议定书描述了肾小球疾病的小鼠模型中应进行的全肾脏工作, 从而能够从一只老鼠身上获得关于肾脏和肾小球功能的大量信息。这些方法可以对肾小球疾病的所有小鼠模型进行详细的功能、结构和机械分析。

<table><tbody><tr><td>Metabolic Cages</td><td>Harvard Apparatus</td><td>52-6731</td><td></td></tr><tr><td>Tris buffered saline (10x)</td><td>Sigma-Aldrich</td><td>T5912-1L</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma-Aldrich</td><td>A2058</td><td></td></tr><tr><td>Mouse Albumin ELISA Quantitation Set</td><td>Bethyl Laboratories</td><td>E90-134</td><td></td></tr><tr><td>TMB ELISA Substrate solution</td><td>ThermoFisher Scientific</td><td>34028</td><td></td></tr><tr><td>Sulphuric acid</td><td>Sigma-Aldrich</td><td>339741</td><td></td></tr><tr><td>SPECTRstar nano</td><td>BMG Labtech</td><td>SPECTRstar nano or equivalent</td><td></td></tr><tr><td>RNAlater stabilisation solution</td><td>ThermoFisher Scientific</td><td>AM7020</td><td></td></tr><tr><td>4-15% precast protein gel</td><td>BIORAD</td><td>4568084</td><td></td></tr><tr><td>4x Laaemmli Sample buffer</td><td>BIORAD</td><td>161-0747</td><td></td></tr><tr><td>Mini Trans-Blot cell</td><td>BIORAD</td><td>1703930</td><td></td></tr><tr><td>10x Tris running buffer</td><td>BIORAD</td><td>1610732</td><td></td></tr><tr><td>Coomassie Brilliant Blue Dye</td><td>ThermoFisher Scientific</td><td>20278</td><td></td></tr><tr><td>Creatinine Companion</td><td>Exocell</td><td>1012 Strip Plate</td><td></td></tr><tr><td>Glutaraldehyde Solution</td><td>Sigma-Aldrich</td><td>G5882</td><td></td></tr><tr><td>Sodium Cacodylate</td><td>Sigma-Aldrich</td><td>C0250</td><td></td></tr><tr><td>10x Phosphate Buffered Saline</td><td>ThermoFisher Scientific</td><td>AM9625</td><td></td></tr><tr><td>Sodium Chloride</td><td>Sigma-Aldrich</td><td>S7653</td><td></td></tr><tr><td>Sodium Acetate</td><td>Sigma-Aldrich</td><td>S2889</td><td></td></tr><tr><td>Sodium Phosphate</td><td>Sigma-Aldrich</td><td>342483</td><td></td></tr><tr><td>Sodium Bicarbonate</td><td>Sigma-Aldrich</td><td>S5761</td><td></td></tr><tr><td>Magnesium Sulfate</td><td>Sigma-Aldrich</td><td>M2643</td><td></td></tr><tr><td>Calcium Chloride</td><td>Sigma-Aldrich</td><td>C5670</td><td></td></tr><tr><td>D(+)Glucose</td><td>Sigma-Aldrich</td><td>G8270</td><td></td></tr><tr><td>EDTA Blood Collection tubes</td><td>BD</td><td>367835</td><td></td></tr><tr><td>23-25G Needle</td><td>BD</td><td>PMC0735</td><td></td></tr><tr><td>EDTA</td><td>Sigma-Aldrich</td><td>E9884</td><td></td></tr><tr><td>10 ml Glass Vial</td><td>Thomas Scientific</td><td>0914X10</td><td></td></tr><tr><td>Falcon 10 ml Polypropylene Tubes</td><td>ThermoFisher Scientific</td><td>10110101</td><td></td></tr><tr><td>0.5 ml Tubes</td><td>ThermoFisher Scientific</td><td>10681894</td><td></td></tr><tr><td>Disposable Tissue Molds</td><td>ThermoFisher Scientific</td><td>22-363-553</td><td></td></tr><tr><td>Mouse Surgical Disection Kit</td><td>ThermoFisher Scientific</td><td>13-005-204</td><td></td></tr><tr><td>Optimal Cutting Medium</td><td>ThermoFisher Scientific</td><td>23-730-571</td><td></td></tr><tr><td>4% Paraformaldehyde</td><td>ThermoFisher Scientific</td><td>AAJ19943K2</td><td></td></tr><tr><td>Glass Capillary Tubes</td><td>Harvard Apparatus</td><td>EC1 64-0770</td><td></td></tr><tr><td>Glomerular Permeability Rig</td><td>Built at the Univeristy of Bristol - not comercially available</td><td>Citation of LpA rig: Salmon et al. 2006; J. Physiol</td><td></td></tr><tr><td>Stainless Steel Sieves</td><td>Cole-Parmer</td><td>WZ-59984</td><td></td></tr><tr><td>Periodic Acid-Schiff (PAS) Staining System</td><td>Sigma-Aldrich</td><td>395B-1KT</td><td></td></tr><tr><td>Hematoxylin</td><td>Sigma-Aldrich</td><td>H3136</td><td></td></tr><tr><td>Xylene</td><td>MerckMillipore</td><td>108298</td><td></td></tr><tr><td>Poly-Prep Slides</td><td>Sigma-Aldrich</td><td>P0425-72EA</td><td></td></tr><tr><td>Mounting Medium</td><td>ThermoFisher Scientific</td><td>8030</td><td></td></tr><tr><td>Osmium tetroxide solution</td><td>Sigma-Aldrich</td><td>75632</td><td></td></tr><tr><td>Aradite Resin</td><td>Agar Scientific</td><td>CY212</td><td></td></tr><tr><td>Uranyl Acetate</td><td>Agar Scientific</td><td>AGR1260A</td><td></td></tr><tr><td>Lead Citrate</td><td>Agar Scientific</td><td>AGR1210</td><td></td></tr><tr><td>Cryostat</td><td>ThermoFisher Scientific</td><td>e.g. 957000H</td><td></td></tr><tr><td>Hydrophobic Pen</td><td>Abcam</td><td>ab2601</td><td></td></tr><tr><td>Nephrin (1243-1256) Antibody</td><td>Acris</td><td>BP5030</td><td></td></tr><tr><td>Anti-Podocin</td><td>Sigma-Aldrich</td><td>P0372-200UL</td><td></td></tr><tr><td>Anti-CD31</td><td>BD Biosciences</td><td>550274</td><td></td></tr><tr><td>Alexa Fluor Secondary Antibody</td><td>ThermoFisher Scientific</td><td>A32732</td><td></td></tr><tr><td>Vectashield Mounting Medium with DAPI</td><td>Vector Labs</td><td>H-1200</td><td></td></tr><tr><td>NP40 Cell Lysis Buffer</td><td>ThermoFisher Scientific</td><td>FNN0021</td><td></td></tr><tr><td>Halt Protease and Phosphatase Inhibitor Cocktail</td><td>ThermoFisher Scientific</td><td>78437X4</td><td></td></tr><tr><td>10x Transfer Buffer</td><td>BIORAD</td><td>1610734</td><td></td></tr><tr><td>PVDF Membrane</td><td>ThermoFisher Scientific</td><td>LC2002</td><td></td></tr><tr><td>HRP-Conjugated Secondary Antibodies</td><td>Abcam</td><td>ab6721</td><td></td></tr><tr><td>ECF Substrate for Western Blotting</td><td>Fisher</td><td>10713387</td><td></td></tr><tr><td>TRIzol</td><td>ThermoFisher Scientific</td><td>15596018</td><td></td></tr><tr><td>Dnase I</td><td>New England Biolabs</td><td>M0303S</td><td></td></tr><tr><td>M-MLV Reverse Transcriptase</td><td>New England Biolabs</td><td>M053S</td><td></td></tr><tr><td>Oligo dT</td><td>ThermoFisher Scientific</td><td>18418012</td><td></td></tr><tr><td>Random Primers</td><td>ThermoFisher Scientific</td><td>48190011</td><td></td></tr><tr><td>dNTP</td><td>ThermoFisher Scientific</td><td>18427088</td><td></td></tr><tr><td>Ribonuclease Inhibitor</td><td>ThermoFisher Scientific</td><td>10777019</td><td></td></tr><tr><td>DEPC Water</td><td>ThermoFisher Scientific</td><td>AM9915G</td><td></td></tr><tr><td>Fluorescent Light Miscroscope</td><td>Leica Microsystems</td><td></td><td></td></tr><tr><td>Image J Analysis Software</td><td>Image J</td><td></td><td></td></tr><tr><td>PCR Thermocycler</td><td>ThermoFisher Scientific</td><td></td><td></td></tr><tr><td>TEM Microscope</td><td>Britannica</td><td></td><td></td></tr></tbody></table>

assessment of kidney function in mouse models of glomerular disease

使用小鼠模型模仿人类肾脏疾病越来越普遍。我们的研究重点是评估肾小球功能的糖尿病肾病和足细胞特异性 VEGF-一个敲出小鼠;因此, 本协议描述了我们实验室中用于评估肾小球疾病小鼠模型的全肾脏工作, 使一只老鼠可以获得大量关于肾脏和肾小球功能的信息。与文献中提出的评估肾小球功能的其他方法相比, 本文概述的方法可以使肾小球表型从多个方面得到充分评价。利用该方法, 研究人员可以确定模型的肾脏表型, 并对表型的形成机制进行评估。在研究这些模型中潜在的治疗途径时, 需要了解有关疾病机制的重要信息。这些方法可以通过测量尿白蛋白肌酐比值和单个肾小球水渗透性, 以及结构和超结构检查, 对肾小球滤过屏障进行详细的功能评估。采用周期性酸席夫碱染色和电镜。此外, 分析基因失调在 mRNA 和蛋白质水平上能够对肾小球功能进行机械分析。本协议概述了可应用于所有小鼠肾小球疾病模型的通用但适应性强的方法。

用野生型代谢笼收集尿液, 诱导足细胞特异 vegf-一个敲出 (vegf a 高) 和 vegf-a 高 X Neph vegf165b 小鼠 (vegf-高表达人 vegf 的高鼠-165b 异构体在足中本构方式)。在0、4、10、14周的尿白蛋白肌酐比值测定后, 在血管内皮生长因子-a 高、vegf-a 的作用下, 与 littermate 控制相比, 高鼠逐渐形成蛋白尿10周。绝对值可以在图 2A中看到, 并且规范化到图 2B中每个鼠标值的基线。然而, 在 vegf 中没有观察到的蛋白尿--X Neph-vegf-165b 小鼠 (图 2), 表明 vegf-A165b 在蛋白尿5的模型中是保护性的。
肾小球 LpAV我是测量的单个肾小球筛出的体重, vegf a 高, 和 vegf-一个 X Neph VEGF-165b 肾。图 3A显示了一个肾小球被捕获的例子, 以及当与 8% BSA perifused 时观察到的收缩情况。这种收缩, 然后用于确定肾小球 LpA/V我为每个肾小球 (图 3B)。vegf-a 型小鼠肾小球 Lpa/V 在 14周后 vegf-高诱导与控制肾小球相比显著增加。虽然 vegf--一个 X Neph-vegf-165b 小鼠的低, 但肾小球 Lpa /V 的增加没有预防 vegf 的过度表达-165b 14 周5。
内皮生长因子诱导后14周肾皮质部分的 PAS 染色未发现 vegf 的肾小球结构异常-a 高或 vegf-X Neph vegf-165小鼠 (图 4A)。然而, 通过 EM 对肾小球超微结构的分析, VEGF-A 型小鼠发展了肾小球的宽度增加, 内皮吕克茶减少, SPS 覆盖率下降, 平均足细胞狭缝宽度增加 (图 4C-4F).平均足细胞脚加工宽度和缝数保持不变 (图 3B 和 3G)。vegf 的过度表达-165b 在血管内皮生长因子-一个高鼠阻止了改变的肾小球和狭缝宽度 (图 4C 和 4F)。然而, VEGF-A165b 对改变的吕克茶数和 SPS 覆盖率没有影响 (图 4D 和 4E)5。
对从筛出的肾小球中提取的 RNA 进行 rt-pcr PCR 显示, 人 vegf--165b mRNA 仅存在于 vegf 中--高 X Neph-vegf-165b 小鼠 (图 5A)。当从被筛的肾小球中提取蛋白质并通过西方印迹对蛋白质水平进行评估时, VEGFR-2 的肾小球蛋白表达减少, vegf-一个高的小鼠, 这是预防的血管内皮生长因子的过度表达-165b (图 5B 和 5C)5。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57764/57764fig1.jpg"> 图1。肾小球通透性 (LpA) 平台的图解建立.(a)肾小球被夹在(B)微内的持卡人使用吸力, 它被夹在支架上以稳定。(C)矩形 microslide。(D) 4X 用摄像机拍摄的物镜。(E) 1% BSA 溶液升温至37摄氏度。(F) 8% BSA 溶液升温至37摄氏度。(G)带有两条 perifusate 的遥控水龙头, 允许快速 perifusate 交换。(H) perifusate 对 microslide 的路线, 然后将肾小球沐浴。<a href="https://www.jove.com/files/ftp_upload/57764/57764fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57764/57764fig2.jpg"> 图2。尿白蛋白肌酐比值。(a)在0、4、10和14周后的 uACR 值, 在血管内皮生长因子的诱导下, Neph、vegf a 和 vegf--一个 X--vegf--165b 小鼠。(B)与每个鼠标的基线值 (0 周) 规范化的相同 uACR 值。uACR 明显增加 vegf-一个小鼠在周10和14与 littermate 控制, 这是预防的 vegf-X Neph vegf-165b 小鼠 (* p < 0.05;双向方差分析法, 对对比较的校正;n = 4-12 小鼠每时间点;误差条: 平均值的标准误差 [SEM])。这个数字已经从史蒂文斯等5修改。<a href="https://www.jove.com/files/ftp_upload/57764/57764fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57764/57764fig3.jpg"> 图3。肾小球水渗透性的测量。(A)肾小球通过吸力被微;perifusate 从 1% bsa (Ai) 转换为 8% bsa (全部), 并观察肾小球萎缩。(B)在 8% BSA 开关之前和之后进行的测量用于确定肾小球 LpA/V.(C) vegf--一个高鼠肾小球 Lp一/Vi . 在14周后诱导 vegf-a 比对照。这在 vegf-X Neph-vegf-165b 小鼠 (* p < 0.05) 中没有明显的预防作用;单程方差分析, Bonferroni 校正为比较之间对;n = 4-9 小鼠, 15-30 肾小球;误差条: SEM)。<a href="https://www.jove.com/files/ftp_upload/57764/57764fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57764/57764fig4.jpg"> 图4。肾小球结构分析。(A)肾皮质 PAS 染色并没有表明从体重、vegf a 和 vegf--X Neph-vegf-165b 小鼠 (Ai iii) 的肾小球中的任何结构异常。EM 揭示 VEGF 的超结构异常--高肾小球 (禽流感)。(B)各组之间的平均 FPW 没有变化。(C)血管内皮生长因子-一个高肾小球, 由 vegf (165b) 所阻止, 在 vegf 中增加. (D)血管内皮生长因子 (吕克茶) 的数量减少, vegf-a-165b. (E) SPS 覆盖率血管内皮生长因子 (高肾小球) 减少, vegf 也保持不变-165b. (F) vegf 的平均狭缝宽度增加-a 高肾小球, 由 vegf 预防-165b. (G)狭缝数未改变三组之间 (p < 0.05;单程方差分析, Bonferroni 校正为比较之间对;n = 3 只老鼠, 9 个肾小球;误差条: SEM)。这个数字已经从史蒂文斯等5修改。<a href="https://www.jove.com/files/ftp_upload/57764/57764fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57764/57764fig5.jpg"> 图5标记物的 mRNA 和蛋白表达。(a) rt-pcr 表明, 人 vegf-165b mRNA 表达仅在筛肾血管内皮生长因子-高 X Neph-vegf-165小鼠中可见。(B)西方印迹表明, VEGFR-2 蛋白表达下降调节 vegf-高肾小球, 这是预防 vegf-高 X Neph-vegf-165B 肾小球 (* p < 0.05;单程方差分析, Bonferroni 校正为比较之间对;n = 3-6 小鼠;误差条: SEM)。这个数字已经从史蒂文斯等5修改。<a href="https://www.jove.com/files/ftp_upload/57764/57764fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Assessment of Kidney Function in Mouse Models of Glomerular Disease at JoVE.com

Assessment of Kidney Function in Mouse Models of Glomerular Disease

本议定书描述了肾小球疾病小鼠模型中应进行的全肾脏工作。这些方法可以对肾小球疾病的所有小鼠模型进行详细的功能、结构和机械分析。

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 ...

assessment-of-kidney-function-in-mouse-models-of-glomerular-disease

Research

JoVE Journal

Medicine

17.5K 次观看.  University of Exeter. 本议定书描述了肾小球疾病小鼠模型中应进行的全肾脏工作。这些方法可以对肾小球疾病的所有小鼠模型进行详细的功能、结构和机械分析。

视频: Assessment of Kidney Function in Mouse Models of Glomerular Disease

Ischemia-reperfusion Model of Acute Kidney Injury and Post Injury Fibrosis in Mice

Ischemia-reperfusion induced acute kidney injury (IR-AKI) is widely used as a model of AKI in mice, but results are often quite variable with high, often unreported mortality rates that may confound analyses. Bilateral renal pedicle clamping is commonly used to induce IR-AKI, but differences between effective clamp pressures and/or renal responses to ischemia between kidneys often lead to more variable results. In addition, shorter clamp times are known to induce more variable tubular injury, and while mice undergoing bilateral injury with longer clamp times develop more consistent tubular injury, they often die within the first 3 days after injury due to severe renal insufficiency. To improve post-injury survival and obtain more consistent and predictable results, we have developed two models of unilateral ischemia-reperfusion injury followed by contralateral nephrectomy. Both surgeries are performed using a dorsal approach, reducing surgical stress resulting from ventral laparotomy, commonly used for mouse IR-AKI surgeries. For induction of moderate injury BALB/c mice undergo unilateral clamping of the renal pedicle for 26 min and also undergo simultaneous contralateral nephrectomy. Using this approach, 50-60% of mice develop moderate AKI 24 hr after injury but 90-100% of mice survive. To induce more severe AKI, BALB/c mice undergo renal pedicle clamping for 30 min followed by contralateral nephrectomy 8 days after injury. This allows functional assessment of renal recovery after injury with 90-100% survival. Early post-injury tubular damage as well as post injury fibrosis are highly consistent using this model.

We describe models of moderate and severe ischemia-reperfusion-induced kidney injury in which the mice undergo unilateral renal pedicle clamping followed by simultaneous or delayed contralateral nephrectomy, respectively. These models consistently give rise to renal dysfunction and post-injury fibrosis, but injury severity and survival are dependent on mouse background, age and surgical equipment.

急性肾损伤和伤后纤维化小鼠缺血再灌注模型

Ischemia-reperfusion induced acute kidney injury (IR-AKI) is widely used as a model of AKI in mice, but results are often quite variable with high, often ...

科研

医学

Renal Ischaemia Reperfusion Injury: A Mouse Model of Injury and Regeneration

Renal ischaemia reperfusion injury (IRI) is a common cause of acute kidney injury (AKI) in patients and occlusion of renal blood flow is unavoidable during renal transplantation. Experimental models that accurately and reproducibly recapitulate renal IRI are crucial in dissecting the pathophysiology of AKI and the development of novel therapeutic agents. Presented here is a mouse model of renal IRI that results in reproducible AKI. This is achieved by a midline laparotomy approach for the surgery with one incision allowing both a right nephrectomy that provides control tissue and clamping of the left renal pedicle to induce ischaemia of the left kidney. By careful monitoring of the clamp position and body temperature during the period of ischaemia this model achieves reproducible functional and structural injury. Mice sacrificed 24 hr&#160;following surgery demonstrate loss of renal function with elevation of the serum or plasma creatinine level as well as structural kidney damage with acute tubular necrosis evident. Renal function improves and the acute tissue injury resolves during the course of 7 days following renal IRI such that this model may be used to study renal regeneration. This model of renal IRI has been utilized to study the molecular and cellular pathophysiology of AKI as well as analysis of the subsequent renal regeneration.

The mouse model of renal ischaemia reperfusion injury described here comprises of a right nephrectomy that provides control tissue and clamping of the left renal pedicle to induce ischaemia that results in acute kidney injury. This model uses a midline laparotomy approach with all steps performed via one incision.

肾缺血再灌注损伤:损伤的小鼠模型和再生

Renal ischaemia reperfusion injury (IRI) is a common cause of acute kidney injury (AKI) in patients and occlusion of renal blood flow is unavoidable ...

Mouse Kidney Transplantation: Models of Allograft Rejection

Rejection of the transplanted kidney in humans is still a major cause of morbidity and mortality. The mouse model of renal transplantation closely replicates both the technical and pathological processes that occur in human renal transplantation. Although mouse models of allogeneic rejection in organs other than the kidney exist, and are more technically feasible, there is evidence that different organs elicit disparate rejection modes and dynamics, for instance the time course of rejection in cardiac and renal allograft differs significantly in certain strain combinations. This model is an attractive tool for many reasons despite its technical challenges. As inbred mouse strain haplotypes are well characterized it is possible to choose donor and recipient combinations to model acute allograft rejection by transplanting across MHC class I and II loci. Conversely by transplanting between strains with similar haplotypes a chronic process can be elicited were the allograft kidney develops interstitial fibrosis and tubular atrophy. We have modified the surgical technique to reduce operating time and improve ease of surgery, however a learning curve still needs to be overcome in order to faithfully replicate the model. This study will provide key points in the surgical procedure and aid the process of establishing this technique.

Here, we present a protocol to study the immunology of rejection. The surgical model presented reports a short operating time and a concise technique. Depending on the donor-recipient strain combination, the transplanted kidney may develop acute cellular rejection or chronic allograft damage, defined by interstitial fibrosis and tubular atrophy.

鼠肾移植:移植排斥反应模型

Rejection of the transplanted kidney in humans is still a major cause of morbidity and mortality. The mouse model of renal transplantation closely ...

Transcutaneous Assessment of Renal Function in Conscious Rodents

Glomerular filtration rate (GFR) is the gold standard to assess overall kidney function. However, traditional methods to evaluate GFR are cumbersome and time-consuming. In addition, serial blood or urine samples are required, with the associated stress for the experimental animals. A recent technique significantly reduces the investment in time and resources, minimizing the invasiveness and the animal stress, but being equally valid as the traditional approaches. The method measures transcutaneously renal function. Using an optical device and the exogenous renal marker fluorescein isothiocyanate (FITC)-sinistrin, this technique is capable of measuring the elimination kinetics of the marker through the skin. With neither blood nor urine samples nor the associated laboratory assays needed, the results of the transcutaneous measurement are almost instantaneously available. The method has been already validated in different species and successfully applied in several models of renal pathology. Moreover, due to its minimally invasive characteristics, it is suitable for sequential measurements within the same animal. Here is provided a detailed protocol to carry out the transcutaneous assessment of renal function in rodents.

Determination of glomerular filtration rate (GFR) is the gold standard to assess overall kidney function. However, traditional procedures to measure this parameter are cumbersome and require a large investment of time. Here we describe a faster and minimally invasive method to determinate GFR transcutaneously.

在鼠害自觉肾功能的评估皮

Glomerular filtration rate (GFR) is the gold standard to assess overall kidney function. However, traditional methods to evaluate GFR are cumbersome and ...

A High-throughput Method for Measurement of Glomerular Filtration Rate in Conscious Mice

The measurement of glomerular filtration rate (GFR) is the gold standard in kidney function assessment. Currently, investigators determine GFR by measuring the level of the endogenous biomarker creatinine or exogenously applied radioactive labeled inulin (3H or 14C). Creatinine has the substantial drawback that proximal tubular secretion accounts for ~50% of total renal creatinine excretion and therefore creatinine is not a reliable GFR marker. Depending on the experiment performed, inulin clearance can be determined by an intravenous single bolus injection or continuous infusion (intravenous or osmotic minipump). Both approaches require the collection of plasma or plasma and urine, respectively. Other drawbacks of radioactive labeled inulin include usage of isotopes, time consuming surgical preparation of the animals, and the requirement of a terminal experiment. Here we describe a method which uses a single bolus injection of fluorescein isothiocyanate-(FITC) labeled inulin and the measurement of its fluorescence in 1-2 &mu;l of diluted plasma. By applying a two-compartment model, with 8 blood collections per mouse, it is possible to measure GFR in up to 24 mice per day using a special work-flow protocol. This method only requires brief isoflurane anesthesia with all the blood samples being collected in a non-restrained and awake mouse. Another advantage is that it is possible to follow mice over a period of several months and treatments (i.e. doing paired experiments with dietary changes or drug applications). We hope that this technique of measuring GFR is useful to other investigators studying mouse kidney function and will replace less accurate methods of estimating kidney function, such as plasma creatinine and blood urea nitrogen.

Measurement of glomerular filtration rate (GFR) is the gold standard for kidney function assessment. Here we describe a high-throughput method which allows the determination of GFR in conscious mice by using a single bolus injection, determination of fluorescein isothiocyanate (FITC)-inulin in plasma and calculation of GFR by a two-phase exponential decay model.

高通量清醒小鼠肾小球滤过率的测定方法

The measurement of  glomerular filtration rate (GFR) is the gold standard in kidney function  assessment. Currently, investigators determine GFR by ...

生物学

Transdermal Measurement of Glomerular Filtration Rate in Mice

Transdermal analysis of glomerular filtration rate (GFR) is an established technique that is used to assess renal function in mouse and rat models of acute kidney injury and chronic kidney disease. The measurement system consists of a miniaturized fluorescence detector that is directly attached to the skin on the back of conscious, freely moving animals, and measures the excretion kinetics of the exogenous GFR tracer, fluorescein-isothiocyanate (FITC) conjugated sinistrin (an inulin analog). This system has been described in detail in rats. However, because of their smaller size, measurement of transcutaneous GFR in mice presents additional technical challenges. In this paper we therefore provide the first detailed practical guide to the use of transdermal GFR monitors in mice based on the combined experience of three different investigators who have been performing this assay in mice over a number of years.

Here we describe a protocol to measure glomerular filtration rate (GFR) in conscious, freely moving mice using a transdermal GFR monitor.

小鼠肾小球滤过率的透皮测定

Transdermal analysis of glomerular filtration rate (GFR) is an established technique that is used to assess renal function in mouse and rat models of ...

Using 2-Photon Microscopy to Quantify the Effects of Chronic Unilateral Ureteral Obstruction on Glomerular Processes

Applying novel microscopy methods to suitable animal disease models to explore the dynamic physiology of the kidney remains a challenge. Rats with surface glomeruli provide a unique opportunity to investigate physiological and pathophysiological processes using intravital 2-photon microscopy. Quantification of glomerular capillary blood flow and vasoconstriction and dilatation in response to drugs, permeability, and inflammation are just some of the processes that can be studied. In addition, transgenic rats, i.e., podocytes labeled with fluorescent dyes and other molecular biomarker approaches, provide increased resolution to directly monitor and quantify protein-protein interactions and the effects of specific molecular alterations.
In mice, which lack surface glomeruli after four weeks of age, unilateral ureteral obstruction (UUO) for several weeks has been used to induce surface glomeruli. As this induction model does not allow for baseline studies, we quantified the effects of UUO on glomerular processes in the UUO model in Munich Wistar Fr&#246;mter (MWF) rats, which have surface glomeruli under physiologic conditions. The UUO model for five weeks or more induced significant alterations to gross renal morphology, the peritubular and glomerular microvasculature, as well as the structure and function of tubular epithelia. Glomerular and peritubular red blood cell (RBC) flow decreased significantly (p &#60; 0.01), probably due to the significant increase in the adherence of white blood cells (WBCs) within glomerular and peritubular capillaries. The glomerular sieving coefficient of albumin increased from 0.015 &#177; 0.002 in untreated MWFs to 0.045 &#177; 0.05 in 5-week-old UUO MWF rats. Twelve weeks of UUO resulted in further increases in surface glomerular density and glomerular sieving coefficient (GSC) for albumin. Fluorescent albumin filtered across the glomeruli was not reabsorbed by the proximal tubules. These data suggest that using UUO to induce surface glomeruli limits the ability to study and interpret normal glomerular processes and disease alterations.

Here, we present a protocol using 2-photon microscopy in Munich Wistar Fromter rats with surface glomeruli to quantifythe effects of prolonged ureteral obstruction on glomerular dynamics and function.

使用2光子显微镜量化慢性单侧输尿管梗阻对肾小球过程的影响

Applying novel microscopy methods to suitable animal disease models to explore the dynamic physiology of the kidney remains a challenge. Rats with surface ...

A Murine Model of Hemodialysis Access-Related Hand Dysfunction

Chronic kidney disease is a major public health problem, and the prevalence of end-stage renal disease (ESRD) requiring chronic renal replacement therapies such as hemodialysis continues to increase. Autogenous arteriovenous fistula (AVF) placement remains a primary vascular access option for ESRD patients. Unfortunately, approximately half of the hemodialysis patients experience dialysis access-related hand dysfunction (ARHD), ranging from subtle paresthesia to digital gangrene. Notably, the underlying biologic drivers responsible for ARHD are poorly understood, and no adequate animal model exists to elucidate the mechanisms and/or develop novel therapeutics for the prevention/treatment of ARHD. Herein, we describe a new mouse model in which an AVF is created between the left common iliac artery and vein, thereby facilitating the assessment of limb pathophysiology. The microsurgery includes vessel isolation, longitudinal venotomy, creation of arteriovenous anastomosis, and venous reconstruction. Sham surgeries include all the critical steps except for AVF creation. Iliac AVF placement results in clinically relevant alterations in central hemodynamics, peripheral ischemia, and impairments in hindlimb neuromotor performance. This novel preclinical AVF model provides a useful platform that recapitulates common neuromotor perturbations reported by hemodialysis patients, allowing researchers to investigate the mechanisms of ARHD pathophysiology and test potential therapeutics.

This protocol details the surgical steps of murine common iliac arteriovenous fistula creation. We developed this model to study hemodialysis access-related limb pathophysiology.

血液透析通路相关手功能障碍的小鼠模型

Chronic kidney disease is a major public health problem, and the prevalence of end-stage renal disease (ESRD) requiring chronic renal replacement ...

Glomerular Outgrowth as an Ex Vivo Assay to Analyze Pathways Involved in Parietal Epithelial Cell Activation

Parietal epithelial cell (PEC) activation is one of the key factors involved in the development and progression of glomerulosclerosis. Inhibition of pathways involved in parietal epithelial cell activation could therefore be a tool to attenuate the progression of glomerular diseases. This article describes a method to culture and analyze parietal epithelial cell outgrowth of encapsulated glomeruli isolated from mouse kidney. After dissecting isolated mouse kidneys, the tissue is minced, and glomeruli are isolated by sieving. Encapsulated glomeruli are collected, and single glomeruli are cultured for 6 days to obtain glomerular outgrowth of parietal epithelial cells. During this period, parietal epithelial cell proliferation and migration can be analyzed by determining the cell number or the surface area of outgrowing cells. This assay can therefore be used as a tool to study the effects of an altered gene expression in transgenic- or knockout-mice or the effects of culture conditions on parietal epithelial cell growth characteristics and signaling. Using this method, important pathways involved in the process of parietal epithelial cell activation and consequently in glomerulosclerosis can be studied.

This article describes a method for culturing and analyzing glomerular parietal epithelial cell outgrowths of encapsulated glomeruli isolated from mouse kidney. This method can be used to study pathways involved in parietal epithelial cell proliferation and migration.

球状外生长作为前维沃分析分析涉及的路径的表皮上皮细胞激活

Parietal epithelial cell (PEC) activation is one of the key factors involved in the development and progression of glomerulosclerosis. Inhibition of ...

透明化小鼠肾脏中肾小球的荧光标记用于显微镜检查

An Efficient and Fast Method for Labeling and Analyzing Mouse Glomeruli

The glomeruli are fundamental units in the kidney; hence, studying the glomeruli is pivotal for understanding renal function and pathology. Biological imaging provides intuitive information; thus, it is of great significance to label and observe the glomeruli. However, the glomeruli observation methods currently in use require complicated operations, and the results may lose label details or three-dimensional (3D) information. The clear, unobstructed brain imaging cocktails and computational analysis (CUBIC) tissue clearing technology has been widely used in renal research, allowing for more accurate detection and deeper detection depth. We found that mouse glomeruli can be rapidly and effectively labeled by tail vein injection of medium molecular weight FITC-Dextran followed by the CUBIC clearing method. The cleared mouse kidney could be scanned by a light-sheet microscope (or a confocal microscope when sliced) to obtain three-dimensional image stacks of all the glomeruli in the entire kidney. Processed with appropriate software, the glomeruli signals could be easily digitized and further analyzed to measure the number, volume, and frequency of the glomeruli.

作者聚焦：通过标记透明化组织中的肾小球来辅助肾脏生物学研究 

This study presents an easy-to-use, complete, and simple set of methods to label and analyze glomeruli from CUBIC-cleared mouse kidneys. Data such as glomerulus number and volume can be obtained easily and reliably using fluorescein isothiocyanate (FITC)-Dextran, light sheet fluorescence microscopy (LSFM), or common confocal microscopy and software such as Imaris.

一种高效、快速的小鼠肾小球标记和分析方法

The glomeruli are fundamental units in the kidney; hence, studying the glomeruli is pivotal for understanding renal function and pathology. Biological ...