球注射阿霉素对小鼠肾病综合征的诱导作用及缓释抑肽酶预防容积保留率的研究
1Department of Internal Medicine, Division of Endocrinology, Diabetology, Vascular Disease, Nephrology and Clinical Chemistry,University Hospital Tübingen, 2Institute of Diabetes Research and Metabolic Diseases (IDM) of the Helmholtz Center Munich,University Tübingen, 3German Center for Diabetes Research (DZD),University Tübingen
Summary
本文介绍了阿霉素快速球注射 129S1/SvImJ 小鼠实验性肾病综合征的诱导作用。我们还治疗肾病小鼠的缓释丸含有抑肽酶, 以抑制尿丝氨酸蛋白酶活性和防止钠保留。
Abstract
肾病综合征是蛋白尿肾病最极端的表现, 其特点是重蛋白尿, hypoalbuminemia, 水肿, 由于钠保留和高脂血症。为了研究这一综合症的病理生理学, 在注射了阿霉素等有毒物质造成足细胞损伤的基础上开发了啮齿动物模型。在小鼠中, 只有很少的菌株容易受到这种模型的影响。在 wildtype 129S1/SvImJ 小鼠中, 阿霉素通过快速静脉注射到球窦, 可诱发实验性肾病综合征, 包括钠保留和水肿在内的所有人类疾病症状。蛋白尿后, 小鼠的尿丝氨酸蛋白酶活性增加, 导致上皮钠通道 (ENaC) 的活化和钠的保留。缓释抑肽酶对尿丝氨酸蛋白酶的药理抑制作用废除 ENaC 活化, 防止钠的滞留。该模型是研究 proteasuria 的病理生理学的理想方法, i. e., 活性丝氨酸蛋白酶的排泄, 导致 ENaC 活化由其γ亚基的水解。这可以看作是蛋白尿肾脏疾病中 ENaC 活化和钠保留的主要机制。
Introduction
肾病综合征以重度蛋白尿、hypoalbuminemia、水肿、高脂血症为特征, 可视为蛋白尿肾病最极端的表现。在啮齿类动物中, 实验性肾病综合征可由单一注射 anthracyclines 或嘌呤霉素引起足细胞损伤, 类似于人的最小变化疾病和局灶节段性肾小球硬化 (FSGS) 1。在它的第一个描述以后在1955年由胡里奥·弗兰克et al。 2, 大鼠嘌呤霉素核苷肾病 (PAN) 已成为研究肾病综合征病理生理学的标准模型, 在许多研究中,3, 4, 5, 6.在小鼠中, 相应的模型可以由蒽阿霉素7诱导。然而, 有一个强大的应变依赖性, 是由至少两个遗传基因位点8决定的。此外, 肾病综合征的蛋白尿反应和病程有差异 9, 10.使用 129S1/SvImJ 小鼠和快速静脉注射阿霉素通过球窦, 蛋白尿反应达到的价值足以诱发肾病综合征的典型特点, 特别是体积保留特点腹水和几乎无钠尿7。实验性肾病综合征的钠保留被认为是在远端小管的上皮钠通道 (ENaC) 活化的结果, aberrantly 过滤丝氨酸蛋白酶, 如纤溶酶导致其γ亚单位的蛋白质降解4 ,11,12。最近, 这一概念被证明在肾病小鼠, 这是防止蛋白水解 ENaC 活化和钠保留的治疗与丝氨酸蛋白酶抑制剂抑肽酶, 同样有效的 ENaC 阻滞剂阿米洛利13。为了确保持续交付到远端小管, 抑肽酶是通过皮下植入缓释丸。未来的研究需要确定的丝氨酸蛋白酶, 负责蛋白水解 ENaC 活化的肾病综合征, 这被认为是平行的人的情况。为此, 阿霉素诱发的肾病综合征是一个有价值的模型, 可用于野生型小鼠或扩展到基因工程小鼠。其优点包括药物成本低、管理复杂性低、重现性好14。
本文通过快速静脉注射阿霉素对球窦的诱导和抑制尿丝氨酸蛋白酶的缓释微丸的植入, 证明了实验性肾病综合征用显色法测定的活动。
Protocol
所有的方法都是按照国家卫生研究院的指导, 对实验动物的护理和使用以及德国的动物福利法进行的, 并得到地方当局 (Regierungspräsidium Tübingen) 的批准。 1. 阿霉素注射液球窦诱导实验性肾病综合征 通过标记活塞的停止位置, 准备一个装有30G 套管的0.5 毫升注射器。 计算男性阿霉素的注射量 (7.25 µL/克体重 (体重) 等于14.5 µg/克阿霉素) 和雌性小鼠 (6.9 µL/克体重等?…
Representative Results
在异氟醚麻醉诱导后, 阿霉素通过左球窦快速注射。整个体积的7.25 µL/克体重注射没有任何阻力和渗出证明正确的静脉插管位置。老鼠从麻醉迅速地恢复了, 并且没有损伤在那天和尔后。特别是, 左眼没有损伤的迹象。在阿霉素注射液后, 由于阿霉素的一般毒性作用和此后恢复 (图 1a), 食物和液体摄入量在头3天下降。在注射后的头3天内, 由于不振 (<stron…
Discussion
在这里, 我们证明阿霉素注射液通过球窦注射诱发实验性肾病综合征的 129S1/SvImJ 小鼠蛋白尿, 钠保留, hypoalbumenia 和高脂血症。但是, 在使用此模型时, 必须考虑到两个关键问题。首先, 模型的诱导是严格的剂量依赖性和偏差的阿霉素剂量为0.3 µg/克影响的响应小鼠15。当注射低剂量, 如14µg/克或以下, 阿霉素只导致非肾病蛋白尿, 不与显性钠保留, 腹水和高脂血症。相反, 注射更高剂?…
Declarações
The authors have nothing to disclose.
Acknowledgements
这项研究得到了德国研究基金会 (DFG, AR 1092/2-1) 的资助。
Materials
| supplies | |||
| BD Micro FINE + U-40 (0.30 mm x 8 mm) | BD Deutschland GmbH, Heidelberg, Germany | PZN: 07468060 | syring |
| ETHILON*II (5/0, 16 mm, 3/8c, 45 cm) | Johnson&Johnson Medical GmbH, Ethicon Deutschland, Norderstedt, Germany) | EH7823H | suture |
| Name | Company | Catalog Number | Comments |
| reagents | |||
| aprotinin (6000 KIU/mg) | LOXO, Heidelberg, Germany | CAS 9087-70-1 | |
| Bepanthen, Augen- und Nasensalbe | Bayer Vital GmbH, Leverkusen, Germany | PZN: 1578681 | ointment |
| Chromogenix S-2251 | HAEMOCHROM DIAGNOSTICA GmbH, Essen, Germany | 82 0332 39 | chromogenic substrate |
| doxorubicin (2.0 µg/µL) | Cell Pharm, Bad Vilbel, Germany | CAS 25316-40-9 | doxorubicin |
| isofluran CP (1 mL/mL) | CP-Pharma, Burgdorf, Germany | CAS 26675-46-7 | isofluran |
| pellets with matrix-driven sustained release (custom-made) | Innovative Research of America, Sarasota, FL | X-999 | pellet |
| Name | Company | Catalog Number | Comments |
| equipment | |||
| ELx808 Absorbance Mikroplate Reader | BioTek, Bad Friedrichshall, Germany | ELx808 | microplate reader |
| MICROSTERIL – 436 | B.M.S., Trescore Balneario, Italy | GAL/436 | sterilizer |
| Hybridization oven/shaker | GE Healthcare UK Limited, Amersham LIFE SCIENCE, Little Chalfont, UK | RPN 2511 | heat chamber |
| Thermo MAT Pro 10 W, 15×25 cm, Lucky Reptile | Import Export Peter Hoch GmbH, Waldkirch, Germany | 61201 | mouse warming device |
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Citar este artigo
Bohnert, B. N., Artunc, F. Induction of Nephrotic Syndrome in Mice by Retrobulbar Injection of Doxorubicin and Prevention of Volume Retention by Sustained Release Aprotinin. J. Vis. Exp. (135), e57642, doi:10.3791/57642 (2018).