高灵敏度测量与氟素等西洋盐-聚糖小鼠的球状渗透性 70
Summary
在这里,我们提出了一个协议,用高度灵敏的非放射性示踪剂测试小鼠的球状渗透性。此方法允许重复尿分析与小尿量。
Abstract
尿液中白蛋白的流失(白蛋白尿)预测心血管结果。在生理条件下,少量的白蛋白被球蛋白过滤,并在管状系统中重新吸收,直到达到吸收极限。病理白蛋白过滤的早期增加可能错过通过分析白蛋白尿。因此,使用示踪剂来测试球状的渗透率似乎具有优势。荧光标记示踪剂氟辛等子素(FITC)-多糖(即FITC-纤维素),可用于研究球状渗透率。FITC-多糖分子被球蛋白自由过滤,但不在管状系统中重新吸收。在小鼠和大鼠中,使用技术复杂的程序(即放射性测量、高性能液相色谱[HPLC]、凝胶过滤)在球状渗透性模型中对FITC-Polyucrose进行了研究。我们修改并促进了基于FITC-多糖示踪剂的协议,以测试小鼠中FITC-Polyucrose 70(白蛋白大小)的球状渗透性的早期和微小增加。此方法允许重复尿分析与小尿体积 (5 μL)。该协议包含有关追踪器 FITC-Polyucrose 70 如何静脉注射和通过简单的尿导管收集尿液的信息。尿通过荧光板读取器进行分析,并归化为尿浓度标记(肌氨酸),从而避免技术上复杂的程序。
Introduction
球状过滤屏障内的功能或结构缺陷增加了白蛋白的球状渗透性,导致尿中白蛋白的检测(白蛋白)。白蛋白尿病预测心血管结果,是肾小球损伤的重要标志1。即使白蛋白尿水平较低,处于正常范围内,也与心血管风险增加1相关。
在生理条件下,白蛋白通过球蛋白过滤,在管状系统2,3中几乎完全重新吸收。在小鼠中,尿液中白蛋白的检测通常通过从24小时尿液收集中与白蛋白酶相关的免疫吸附剂测定(ELISA)进行。如果使用 24 小时尿收集或点尿的尿液,则由于测定敏感性问题,白蛋白浓度的微小差异可能会丢失。因此,大多数研究人员使用动物模型,其中白蛋白尿是由毒素、药物和肾脏手术引起的强健的肾损伤引起的。
因此,发现一种灵敏的方法来检测球状渗透性的微小和瞬态变化,对该领域非常重要。Rippe等人提出了一个大鼠模型,通过应用荧光标记的示踪剂,即FITC-聚糖70(即FITC-Ficoll 70),在白蛋白4的大小测试球状渗透性。示踪剂应用允许测试球状渗透性的短期变化(在几分钟内),并且非常敏感4。两项研究在小鼠5,6中使用了示踪法。尽管这种方法有其优点,但不幸的是,它的缺点:它在技术上非常复杂,放射性和侵入性。尿的进一步分析只能通过使用凝胶过滤或大小排除HPLC5,6完成。
在本文中,我们提出了一种替代的、灵敏的、非放射性的、快速的方法,用荧光标记的FITC-Polyucrose 70测量小鼠的球状渗透性。通过引入输尿管导管,尿液收集比膀胱穿刺、尿道和超尿管应用的侵入性小,并且允许至少每30分钟收集一次尿液。荧光板读取器。使用酶肌氨酸测定法,将尿液中的示踪剂浓度归化为尿液中的肌氨酸浓度。
因此,这种新方法为研究早期肾小球损伤和增加球状渗透性提供了一个敏感的工具。
Protocol
调查是根据《实验室动物护理和使用指南》(美国国家卫生研究院出版物,1996年修订)中概述的准则进行的。所有动物实验均按照相关机构批准进行(州政府兰德萨姆特·法尔·纳图尔,乌姆韦尔特·德·维尔布劳赫舒茨[LANUV] 参考号84-02.04.2012.A397)。 1. 仪器、解决方案和设备的准备 用0.9%无菌氯化钠(NaCl)将FITC-聚糖70重组为10mg/mL的最终浓度(即100毫克的纳L)。 Dialyze FITC-聚?…
Representative Results
如图2所示,测试小鼠球状渗透性的方法分三个阶段建立。第一阶段称为准备阶段,其中放置尿导管和中央静脉导管。第二阶段称为平衡阶段,从静脉注射FITC-Polyucrose 70开始,然后持续输注FITC-Polyucrose 7060分钟。最后一个阶段称为实验阶段。在此阶段,继续注入FITC-多糖70,并可以测试药物或其他物质影响球状渗透性。尿在每个阶段结束时收集。 <p class="jove_c…
Discussion
提出的方法使研究者能够使用示踪剂以非常敏感的方式测试小鼠的球状渗透性。使用这种方法,只需少量尿液即可诊断肾小球渗透性的短期增加。成功掌握此技术最关键的步骤是:1) 发展小鼠手术的手动专业知识,特别是在中央静脉的罐中,2) 在不损害粘管的情况下放置导管,以及 3) 手动处理专业知识384 孔板,样品体积小。
放置中央静脉导管时,导管不得穿透静脉静脉。为了避免渗?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
作者感谢克里斯蒂娜·施万特、布兰卡·杜夫尼亚克和尼古拉·库尔的出色技术援助,并感谢丹尼斯·索恩博士在荧光扫描方面给予的帮助。这项研究得到了德国福森斯格明舍夫特(DFG)SFB 612 TP B18的资助,并授予了洛杉矶和洛杉矶。在研究设计、数据收集和分析、决定出版或编写手稿方面,受资者没有作用。
Materials
| Motic SMZ168 BL | Motic | SMZ168BL | microscope for mouse surgery |
| KL1500LCD | Pulch and Lorenz microscopy | 150500 | light for mouse surgery |
| Microfederschere | Braun, Aesculap | FD100R | fine scissors |
| Durotip Feine Scheren | Braun, Aesculap | BC210R | for neck cut |
| Anatomische Pinzette | Braun, Aesculap | BD215R | for surgery |
| Präparierklemme | Aesculap | BJ008R | for surgery |
| Seraflex | Serag Wiessner | IC108000 | silk thread |
| Ketamine 10% | Medistar | anesthesia | |
| Rompun (Xylazin) 2% | Bayer | anesthesia | |
| Fine Bore Polythene Tubing ID 0.28mm OD 0.61mm | Portex | 800/100/100 | Catheter |
| Fine Bore Polythene Tubing ID 0.58mm OD 0.96mm | Portex | 800/100/200 | Catheter |
| Harvard apparatus 11 Plus | Harvard Apparatus | 70-2209 | syringe pump |
| BD Insyte Autoguard | BD | 381823 | urinary catheter |
| Multimode Detector DTX 880 | Beckman Coulter | plate reader | |
| 384 well microtiterplate | Nunc | 262260 | 384 well platte |
| Creatinine Assay Kit | Sigma-Aldrich | MAK080 | to measure creatinine concentration |
| 96 well plate | Nunc | 260836 | for creatinine assay |
| FITC-labeled polysuccrose 70 | TBD Consultancy | FP70 | FITC-ficoll |
| Angiotensin II | Sigma-Aldrich | A9525 | used to test glomerular permeability |
| BP-98A | Softron | for blood pressure measurement | |
| OTS 40.3040 | Medite | 01-4005-00 | heating plate for mouse surgery |
| Instillagel 6mL | Farco-Pharma GmbH | for urinary catheter | |
| Exacta | Aesculap | GT415 | shaver |
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Citazione di questo articolo
Königshausen, E., Potthoff, S. A., Woznowski, M., Stegbauer, J., Rump, L. C., Sellin, L. Highly Sensitive Measurement of Glomerular Permeability in Mice with Fluorescein Isothiocyanate-polysucrose 70. J. Vis. Exp. (150), e59064, doi:10.3791/59064 (2019).