临时双性肾动脉闭塞的急性肾损伤大型动物模型
Summary
本研究提出了一种高度可重复的大型动物模型，即猪肾缺血-再灌注损伤，使用暂时皮质双边气球导管将肾脏动脉闭合60分钟，再灌注24小时。
Abstract
急性肾损伤 （AKI） 与术后发病率和死亡率的较高风险相关。缺血-再灌注损伤（IRI）是AKI最常见的原因。为了模拟这种临床场景，本研究使用肾动脉的临时皮质双边导管闭合，在猪中提出了一个高度可重复的大型动物模型肾IRI。肾脏动脉通过通过股骨动脉和胡萝卜动脉引入气球导管并将其推进动脉的近部，被遮挡60分钟。在大动脉中注入异位对比，以评估肾血管的任何不透明化，并确认动脉闭塞的成功。气球导管尖端脉冲波形的扁平进一步证实了这一点。气球在60分钟的双性肾动脉闭塞后被放气并取出，动物们可以恢复24小时。在研究结束时，血浆肌氨酸和血尿素氮显著增加，而eGFR和尿液输出显著减少。对异性对比的需求微乎其微，不影响肾功能。双边肾动脉闭合更好地模拟腹腔内低灌注的临床情况，皮下方法可最大限度地减少炎症反应的影响和通过开放方法（如腹腔切除术）看到的感染风险。能够创建和复制这种临床相关的猪模型，可以简化临床翻译给人类。
Introduction
急性肾损伤 （AKI） 是手术患者中常见的诊断疾病，与重大发病率和死亡率1，2相关。现有数据显示，AKI甚至可影响全球半数住院病人，导致重症监护病房1、3名病人死亡率达50%。尽管其高流行率，目前的AKI治疗仍然仅限于预防策略，如液体管理和透析。因此，人们一直有兴趣探索AKI4，5，6的替代疗法。
AKI通常根据其病因4、5、6分为肾前、内在和肾后。大多数手术患者与肾前原因由于低伏血症，导致缺血-再灌注损伤（IRI）的肾脏2。临床上，尿液输出减少，肌酸水平增加，由于肾功能下降。肾脏是一种高代谢率器官，易患缺血。肾IRI的高度可重复的大型动物模型是必要的，以更好地了解AKI的病理生理学及其潜在的治疗方法5。
为了模拟肾输液的临床场景，双性肾动脉闭合模型被认为是合适的。先前描述的模型涉及单边肾动脉闭合与或没有切除的逆向肾脏不提供足够的临床适用性7，8。虽然这些模型足以引起AKI，但它们与现实生活中的临床场景不同，无论是在伤害类型还是持续时间方面。
本文的目的是提出一个猪模型，通过气管闭合下血管造影术，对肾脏动脉进行皮外双边暂时闭合。双边肾动脉闭合模拟肾输液的临床情景，随后取出气球进行再灌注9、10。介绍了技术步骤，包括导管、导管制导、血管造影和血液学监测。这种方法不仅允许高度控制和可复制的肾脏动脉闭塞，而且皮下的方法通过限制对身体的侮辱量来尽量减少炎症反应的影响，而不是开放的方法。
Protocol
所有活体研究均根据国家卫生研究院关于动物护理和使用的准则进行，并经波士顿儿童医院动物护理和使用委员会（第18-06-3715号议定书）批准。所有动物都按照《实验室动物护理和使用指南》得到人道护理。 图1 显示了这项研究的主要结果测量的时间表，包括麻醉、手术准备和时间点。 1. 诱导、麻醉和管教 为了防止不必要的压力和不适，通过?…
Representative Results
泛函分析本研究的代表性结果来自6种动物，所显示的数据均值为平均值±标准误差。肾功能通过确定尿液输出、估计球状过滤率（eGFR）、血浆肌酸和血尿素氮（BUN）来评估。肾功能的生物标志物使用便携式化学分析仪进行评估。eGFR 根据以下公式计算： eGFR =1.879 × BW1.092/PCr0.6 （BW： 体重（千克;PCr： 等离子肌氨酸在毫克/dL）10?…
Discussion
AKI是一种常见的临床疾病，影响全球6，12名住院成人患者中高达50%。需要临床相关的动物模型来进一步研究该病的病理生理学和潜在的治疗目标。虽然有几个木耳模型复制AKI，这些并没有完全模仿各自的临床场景和人体肾脏的解剖。这项研究提出了一个临床相关的猪模型，允许翻译给人类13。
在这里，协议描述了?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们要感谢阿瑟·内德博士的帮助和指导。这项工作得到了理查德·阿和苏珊·史密斯总统创新奖、迈克尔·克莱因和家庭、西德曼家庭基金会、迈克尔·鲁金慈善基金会、肯尼思·格里芬慈善研究基金和波士顿投资委员会的支持。
Materials
| 0.9% sodium chloride injection, usp, 100 ml viaflex plastic container | Baxter | 2B1302 | For animal hydration |
| Agent contrast 100.0ml injection media btl ioversal 74% | CARDINAL HEALTH | 133311 | For visualizing the vasculature |
| Bard Bardia Closed System Urinary Drainage Bag | BARD Inc | 802001 | For urine collection |
| BD Vacutainer K2 EDTA | BD | 367841 | For blood sample storage |
| BD Vacutainer Lithium Heparin | BD | 366667 | For blood sample storage |
| Betadine | Henry Schein | 6906950 | For skin disinfection |
| Bookwalker retractor | Codman | For skin retraction | |
| Bupivacaine 0.25% | Hospira | Administer at incision site for analgesia | |
| Buprenorphine SR | Zoo Pharm | 10mg/ml bottle, Dose: 0.2mg/kg SC | |
| Cath angio 5.0 Fr x100.0 cm 0.038 in JR4 | MERIT MEDICAL SYSTEM INC | 7523-21 | For identification of the renal arteries |
| Cuffed endotracheal tube | Emdamed | To establish a secure airway for the duration of the operation | |
| EKG Medtronics- Physiocontrol LifePak 20 Oxygen saturation monitor | GE Healthcare Madison WI | For oxygen saturation monitoring | |
| Encore 26 inflator | BOSTON SCIENTIFIC | 710113 | For inflating the balloon catheters |
| Ethanol 95% (Ethyl alcohol) | Henry Schein | For skin disinfection | |
| Fentanyl patch | Mylan | Dose: 25-50mcg/hr, TD | |
| Gold silicone coated Foley | TELEFLEX MEDICAL INC | 180730160 | For urine collection |
| Heparin sodium | LEO Pharma A/S | Dose: 200 IU/kg IV | |
| i33 ultrasound machine | Phillips | Use ultrasonographic guidance for femoral catherization if necessary | |
| Inqwire diagnostic guide wire – 0.035" (0.89 mm) – 260 cm (102") – 1.5 mm j-tip | MERIT MEDICAL SYSTEM INC | 6609-33 | For guiding the balloon catheters to the renal arteries |
| Intravenous catheter, size 20 gauge | Santa Cruz Biotechnology | Inc SC-360097 | For fluid administration |
| Isoflurane | Patterson Veterinary Supply, Inc. | 21283620 | Dose: 3%, INH |
| Metzenbaum blunt curved 14.5 cm – 5(3/4)" | Rudolf Medical | RU-1311-14M | For tissue dissection and cutting |
| Neonatal disposable transducer kit with 30ml/hr flush device and double 4-way stopcocks for continuous monitoring | Argon Medical | 041588505A | For pressure measurement |
| Powerflex pro PTA dilatation catheter 6 x 20 mm – shaft length (135cm) | CARDINAL HEALTH | 4400602X | For occlusion of the renal arteries |
| Pressure monitoring lines mll/mll – 12" clear, mll/mll | Smiths Medical | B1571/MX571 | For pressure measurement |
| Procedure pack | Molnlycke Health Care | 97027809 | Surgical drape, gauze pads, syringes, beaker etc |
| Protamine | Henry Schein | 1044148 | For heparin reversal |
| Scalpel blade – size #10 | Cardinal Health (Allegiance) | 32295-010 | For the skin incisions |
| Stopcock iv 4 way lrg bore rotg male ll adptr strl | Peoplesoft | 1550 | For connecting tubings |
| Straight lateral retractor | Codman | For skin retraction | |
| Suture perma hnd 18in 2-0 braid silk blk | CARDINAL HEALTH 1 | A185H | For suturing incision site and securing catheters |
| Syringe contrast injection 10ml fixed male luer red | MERIT MEDICAL SYSTEM INC | MSS111-R | To administer the contrast agent |
| Syringe medical 60ml ll plst strl ltx free disp | CARDINAL HEALTH 1 | BF309653 | For urine collection and flushing of the angiocath |
| Tilzolan (tiletamine/zolazepam) | Patterson Veterinary Supply, Inc. | 07-893-1467 | Dose: 4-6 mg/kg, IM |
| Xylazine | Putney, INC | Dose: 1.1-2.2 mg/kg, IM |
References
- Ali Pour, P., Kenney, M. C., Kheradvar, A. Bioenergetics consequences of mitochondrial transplantation in cardiomyocytes. Journal of the American Heart Association. 9 (7), 014501 (2020).
- Giraud, S., Favreau, F., Chatauret, N., Thuillier, R., Maiga, S., Hauet, T. Contribution of large pig for renal ischemia-reperfusion and transplantation studies: The Preclinical Model. Journal of Biomedicine and Biotechnology. 2011, 14 (2011).
- Amdisen, C., et al. Testing Danegaptide effects on kidney function after ischemia/reperfusion injury in a new porcine two week model. PLoS ONE. 11 (10), 1-13 (2016).
- Bhargava, P., Schnellmann, R. G. Mitochondrial energetics in the kidney. Nature Reviews Nephrology. 13 (10), 629-646 (2017).
- Bonventre, J. V., Weinberg, J. M. Recent advances in the pathophysiology of ischemic acute renal failure. Journal of the American Society of Nephrology. 14 (8), 2199-2210 (2003).
- Case, J., Khan, S., Khalid, R., Khan, A. Epidemiology of Acute Kidney Injury in the Intensive Care Unit. Critical Care Research and Practice. 2013, 9 (2013).
- Jabbari, H., et al. Mitochondrial transplantation ameliorates ischemia/reperfusion-induced kidney injury in rat. Biochimica et Biophysica Acta (BBA) – Molecular Basis of Disease. 1866 (8), 165809 (2020).
- Malagrino, P. A., et al. Catheter-based induction of renal ischemia/reperfusion in swine: Description of an experimental model. Physiological Reports. 2 (9), 1-13 (2014).
- Freeman, R. V., et al. Nephropathy requiring dialysis after percutaneous coronary intervention and the critical role of an adjusted contrast dose. American Journal of Cardiology. 90 (10), 1068-1073 (2002).
- Gasthuys, E., et al. Postnatal maturation of the glomerular filtration rate in conventional growing piglets as potential juvenile animal model for preclinical pharmaceutical research. Frontiers in Pharmacology. 8 (431), 1-7 (2017).
- Doulamis, I. P., et al. Mitochondrial transplantation by intra-arterial injection for acute kidney injury. American Journal of Physiology – Renal Physiology. 319 (3), 403-413 (2020).
- Rewa, O., Bagshaw, S. M. Acute kidney injury-epidemiology, outcomes and economics. Nature Reviews Nephrology. 10 (4), 193-207 (2014).
- Grossini, E., et al. Levosimendan Protection against Kidney Ischemia/Reperfusion Injuries in Anesthetized Pigs. Journal of Pharmacology and Experimental Therapeutics. 342 (2), 376-388 (2012).
- Laskey, W. K., et al. Volume-to-creatinine clearance ratio. A pharmacokinetically based risk factor for prediction of early creatinine increase after percutaneous coronary intervention. Journal of the American College of Cardiology. 50 (7), 584-590 (2007).
Cite This Article
Doulamis, I. P., Guariento, A., Saeed, M. Y., Nomoto, R. S., Duignan, T., del Nido, P. J., McCully, J. D. A Large Animal Model for Acute Kidney Injury by Temporary Bilateral Renal Artery Occlusion. J. Vis. Exp. (168), e62230, doi:10.3791/62230 (2021).