肾内注射<em>大肠杆菌</em>在肾性肾炎大鼠模型中
Summary
This manuscript describes a rat surgical model of pyelonephritis using direct intra-renal infection by Escherichia coli into the renal pelvis. The experimental procedure can be utilized to study the pathogenesis of pyelonephritis as well as the associated inflammation and fibrosis.
Abstract
Pyelonephritis is a bacterial infection of the kidney and is most commonly caused by Escherichia coli. Recurrent infections can cause significant renal inflammation and fibrosis ultimately resulting in declining kidney function. Before improved clinical management and prevention of pyelonephritis can be instituted, a reliable animal model must be established in order to study the mechanisms of progression, recurrence, and therapeutic efficacy. The transurethral infection model closely mimics human pyelonephritis but exhibits considerable variation due to its reliance on urethral reflux to transport the bacteria to the kidney. Herein, a detailed surgical protocol for performing bacterial injections into the rat renal pelvis is provided and confirmed by non-invasive Magnetic Resonance Imaging (MRI). Using this protocol, animals receive direct exposure to a desired concentration of E. coli bacteria and can fully recover from the surgical procedure with adequate post-operative care. This facilitates subsequent longitudinal MRI assessments of the experimental animal models for comparison with saline (sham) controls. Using this direct delivery approach, the severity of infection is controllable and applicable for mechanistic studies of progression as well as development of novel treatment strategies.
Introduction
啮齿动物模型已被用于研究许多人类疾病表现，包括肾盂肾炎和尿路感染（UTI）。 UTI是一个全球性的健康问题，可以影响所有年龄段的儿童，男性和女性。 1，2，3尿路感染的初始表现形式包括膀胱炎，如果感染沿输尿管上升，肾脏感染（肾盂肾炎）可以遵循。与此同时，全球糖尿病患病率接近四亿人。 4,5重要的是，尿路感染发病率可能高达谁是病人肥胖或有2型糖尿病，导致复发性尿路感染感染（入替），败血症，从肾盂肾炎肾纤维化和膀胱功能障碍的风险增加4倍以上升高。 6，7，8鼠类模型在研究UTI方面很重要，因为目前的抗生素疗法仅在UTI患者的一个子集中产生持续的预防性应答。为了改善临床UTI护理，关键步骤是了解rUTI及其从急性感染到炎症到纤维化的病理生理过程的机制以及2型糖尿病的影响。
改进动物模型的目标是开发可以更准确地评估疾病进展和治疗干预措施的技术。已经采用几种不同的方法在大鼠和/或小鼠中诱导肾盂肾炎，以研究肾损伤的病理生理学，抗生素治疗的效果以及UTI的自然过程的其他方面。建立逆行性UTI的常见方法是经尿道导尿。 10，11，12，13 </ sup>该方法通过尿道将细菌引入麻醉动物的膀胱。虽然这种技术密切模拟人类肾盂肾炎，但由于多种因素，肾盂肾炎感染的实际发生率和大小可能是非常可变的，包括在接种期间或之后紧随其后的输尿管反流自发或排尿不足。 11其结果是，在诱导上行肾盂肾炎的感染可以限制这种模型来研究肾脏感染以及治疗策略的效用的实验变化。
该报告描述了一种手术肾盂肾炎大鼠模型，其中将大肠杆菌直接注射入大鼠肾脏。尽管这种大鼠模型是侵入性的，但是可以有效地控制输送到肾脏的大肠杆菌的数量，从而能够强壮的肾脏感染和炎症。 14在这个过程中，我们还描述如何通过体内磁共振成像（MRI）纵向监测这些诱导的肾脏感染。
Protocol
Representative Results
Discussion
啮齿动物（即小鼠和大鼠）升高的急性肾盂肾炎可以通过经尿道导尿术产生。 16，17，18经尿道这种感染的方法是有利的，因为它是非侵入性的和模拟上行感染的人类病理生理学。 17，18，19，20然而，这种方法也可以遭受不?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
The authors would like to acknowledge the support of NIH/NIDDK K12 DK100014 (Lan Lu), the Case Comprehensive Cancer Center (NIH/NCI P30 CA43703), and the Clinical and Translation Science Collaborative of Cleveland (NIH/NCATS UL1 TR000439).
Materials
| Absorbing Pad | Fisher | 14-127-47 | |
| Sterile Cotton Gauze Pad | Fisher | 22-415-469 | |
| Latex Surgical Gloves | Henry Schein Animal Health | 21540 | |
| Curved Mayo Scissors | Fisher | S17341 | |
| Straight Blunt Foreceps | Fisher | 08-895 | |
| Scalpel Handle | Fisher | 08-913-5 | |
| Sterile Scalpel Blades | Fisher | 53220 | |
| 1mL Luer-Lok Syringe | BD Biosciences | 309628 | For bacterial injections |
| 20mL Luer-Lok Syringe | BD Biosciences | 301031 | For saline wash |
| Hemostat | Seneca Medical | 240267 | |
| 23 G 3/4 in. Needle | BD Biosciences | 305143 | |
| 30 G 1 in. Needle | BD Biosciences | 305128 | |
| U-100 Insulin Syringe | Exel International | 25846 | For medication injections |
| Isoflurane | Henry Schein Animal Health | 050033 | |
| Xylazine | Henry Schein Animal Health | 33197 | Inject IP |
| Ketamine | Patterson Vetrinary | 07-881-9413 | Inject IP |
| Yohimbine (Atipamezole) | Patterson Vetrinary | 07-867-7097 | Inject IP after surgery |
| Bupivacaine (Marcaine) | Patterson Vetrinary | 07-890-4584 | Inject SQ at site of incision |
| Carprofen (Rimadyl) | Patterson Vetrinary | 07-844-7425 | Should be kept at 4 ᵒC |
| 4-0 Chromic Gut Suture | Ethicon Inc. | U203H | |
| 4-0 Braided Vicryl Suture | Ethicon Inc. | J304H | |
| 1mL SubQ Syringe | BD Biosciences | 309597 | |
| E. coli UTI89 or CFT073 | ATCC | 700928 | |
| Surgicel Absorbable Hemostat | Ethicon Inc. | ETH1951CS | |
| Biospec 9.4T MRI | Bruker | 94/20 USR |
References
- Saliba, W., Barnett-Griness, O., Rennert, G. The association between obesity and urinary tract infection. Eur J Intern Med. 24 (2), 27-31 (2012).
- Semins, M., Shore, A., Makary, M., Weiner, J., Matlaga, B. The impact of obesity on urinary tract infection risk. Urology. 79 (2), 266-269 (2011).
- Zilberberg, M., Shorr, A. Secular trends in gram-negative resistance among urinary tract infection hospitalizations in the United States, 2000-2009. Infect Control Hosp Epidemiol. 34 (9), 940-946 (2013).
- Whiting, D., Guariguata, L., Weil, C., Shaw, J. IDF diabetes atlas: global estimates of the prevalence of diabetes for 2011 and 2030. Diabetes Res Clin Pract. 94 (3), 311-321 (2011).
- Wild, S., Roglic, G., Green, A., Sicree, R., King, H. Global prevalence of diabetes: estimates for the year 2000 and projections for 2030. Diabetes Care. 27 (5), 1047-1053 (2004).
- Ma, D., Gulani, V., Seiberlich, N., Liu, K., Sunshine, J., Duerk, J., et al. Magnetic resonance fingerprinting. Nature. 495 (7440), 187-192 (2013).
- Lu, L., Sedor, J., Gulani, V., Schelling, J., O’Brien, A., Flask, C. A., et al. Use of diffusion tensor MRI to identify early changes in diabetic nephropathy. Am J Nephrol. 34 (5), 476-482 (2011).
- Rosen, D., Hooton, T., Stamm, W., Humphrey, P., Hultgren, S. Detection of intracellular bacterial communities in human urinary tract infection. PLoS Med. 4 (12), e329 (2007).
- Torine, L. A. Urinary tract infection: diabetic women’s strategies for prevention. Br J Nurs. 20 (13), 791-792 (2011).
- Rosen, D., Hung, C., Kline, K., Hultgren, S. Streptozocin-induced diabetic mouse model of urinary tract infection. Infect Immun. 76 (9), 4290-4298 (2008).
- Larsson, P., Kaijser, B., Mattsby-Baltzer, I., Olling, S. An experimental model for ascending acute pyelonephritis caused by Escherichia coli or proteus in rats. J Clin Pathol. 33 (4), 408-412 (1980).
- Gupta, R., Ganguly, N., Ahuja, V., Joshi, K., Sharma, S. An ascending non-obstructive model for chronic pyelonephritis in BALB/c mice. J. Med. Microbiol. 43 (1), 33-36 (1995).
- Fernandes, P., Shipkowitz, N., Bower, R. Murine models for studying the pathogenesis and treatment of pyelonephritis. Adv. Exp. Med. Biol. 224, 35-51 (1987).
- Kaye, D. The effect of water diuresis on spread of bacteria through the urinary tract. J. Infect. Dis. 124 (3), 297-305 (1971).
- Fierer, J., Tainer, L., Braude, A. Bacteremia in the pathogenesis of retrograde E. coli pyelonephritis in the rat. Am. J. Pathol. 64 (2), 443-456 (1971).
- Nickel, J., Olson, M., Costerton, J. Rat model of experimental bacterial prostatitis. Infection. 19 (3), S126-S130 (1991).
- Hagberg, L., Engberg, I., Freter, R., Olling, S., Eden, C. Ascending, unobstructed urinary tract infection in mice caused by pyelonephritogenic Escherichia coli of human origin. Am Soc Microbiol. 40 (1), 273-283 (1983).
- Kurosaka, Y., Ishida, Y., Yamamura, E., Takase, H., Otani, T., Kumon, H. A non-surgical rat model of foreign body-associated urinary tract infection with Pseudomonas aeruginosa. Microbiol. Immunol. 45 (1), 9-15 (2001).
- Anderson, B., Jackson, G. Pyelitis, an important factor in the pathogenesis of retrograde pyelonephritis. J Exp Med. 114 (3), 375-384 (1961).
- Anderson, J. Vesico-ureteric reflux. J R Soc Med. 55 (6), 419-426 (1962).
