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Medicina

机械通气仔猪肾小球滤过率的透皮测量

Published: September 13, 2022
doi:
10.3791/64413
, , , , ,
1Department of Physiology,College of Medicine University of Tennessee Health Science Center

Summary

肾小球滤过率（GFR）是评估肾功能的理想标志物。然而，使用菊粉注射液和连续血液和尿液分析的标准测量方法是不切实际的。本文描述了一种经皮测量仔猪GFR的实用方法。

Abstract

肾小球滤过率（GFR）的透皮测量已被用于评估有意识动物的肾功能。这种技术在啮齿动物中已经建立起来，用于研究急性肾损伤和慢性肾脏疾病。然而，使用透皮系统测量GFR尚未在猪中得到验证，猪是一种与人类具有相似肾脏系统的物种。因此，我们研究了脓毒症对麻醉和机械通气新生猪透皮GFR的影响。多种微生物败血症由盲肠结扎和穿刺 （CLP） 诱发。由微型荧光传感器组成的透皮GFR测量系统连接到猪的剃光皮肤上，以确定荧光素 – 异硫氰酸酯（FITC）结合的sinistrin（一种静脉注射的GFR示踪剂）的清除率。我们的结果表明，在CLP后12 h，血清肌酐随着GFR的降低而增加。这项研究首次证明了透皮GFR方法在确定机械通气新生猪肾功能方面的实用性。

Introduction

肾功能的实际和定量评估是肾小球滤过率（GFR）测量，它根据清除原则1来说明肾脏过滤血液的程度。测量GFR的早期方法需要静脉注射外源性化合物，如菊粉或sinistrin，对血浆/尿液水平进行连续测量以检测其清除率2，3。这种方法比较麻烦，需要连续采集血浆和尿液样本4。另一种方法是测量内源性代谢终产物，如肌酐。然而，这很耗时，有时也不准确，因为它不仅被肾小球过滤，而且还被小管分泌5，6。此外，肌酐水平受性别、年龄、饮食和肌肉质量的影响7，8，9。

一种更精确、微创和广泛使用的 GFR 测量方法是使用透皮 GFR 监测仪，用于测量动物的实时 GFR4，10。Sinistrin是一种高度可溶性和自由过滤的外源性肾脏标志物，用荧光素-异硫氰酸酯（FITC）标记。这种偶联化合物通过静脉注射，无需采集血液和尿液样本即可实时评估肾功能11。透皮 GFR 测量已在啮齿动物 12、狗13 和猫14 中得到验证，但在猪中未得到验证。

猪种与人类具有多种解剖学和生理学特征，使其成为研究各种人类疾病的理想动物15。猪在转化生物医学研究中的使用越来越受欢迎，并且比啮齿动物模型更受欢迎，因为它模仿人类生理学和病理生理学16。新生猪对了解儿科患者特有的疾病机制感兴趣17.此外，最近猪到人体器官移植的进展促使人们迫切需要扩大临床前和临床试验的诊断工具18，19，20，21。本文首次为使用透皮装置测量新生猪GFR提供了指南。

Protocol

这些程序是根据实验动物护理和使用的国家标准编写的，并得到了田纳西大学健康科学中心（UTHSC）机构动物护理和使用委员会（IACUC）的批准。 注意：实验组的仔猪进行盲肠结扎和穿刺，而假手术组仅进行腹部开口而不进行盲肠结扎或穿刺。两组仔猪在手术后麻醉12小时，以使实验组有足够的时间发生败血症和急性肾损伤（AKI）。透皮GFR测量将在手术后8小时进行，总共12小?…

Representative Results

在本节中，我们首次介绍了在新生猪中使用透皮GFR的代表性数据。我们使用盲肠结扎和穿刺模型，该模型先前已被证明可以降低肾功能28。因此，我们假设在我们的CLP猪中，与AKI对应的GFR应该急剧下降，这应该在透皮GFR装置上检测为清除时间增加（t1/2），从而验证其在猪中的使用。纳入7头雄性仔猪，3头假仔猪和4头败血症仔猪。两组的体重相当（图3A</stro…

Discussion

本文描述了在机械通气、麻醉的新生猪模型中使用微型透皮 GFR 监测仪和 FITC-siniststrin 确定猪肾功能的实际步骤。以前的论文已经在啮齿动物11，12，14中建立了实验性透皮GFR方案，但在猪中不存在方案。

最近，人们一直在探索替代动物模型，以解决顽固性疾病并减轻人类肾脏疾病的负担。不幸的是，由于?…

Declarações

The authors have nothing to disclose.

Acknowledgements

这项研究得到了美国国立卫生研究院授予Adebiyi博士的R01 DK120595和R01 DK127625的支持。本文内容完全由作者负责，并不一定代表美国国立卫生研究院的官方观点。感谢MediBeacon GmbH现场总监Daniel Schock-Kusch博士的建议。

Materials

Alpha – Chloralose Sigma-Aldrich C0128-25G Used for maintanining anesthesia
Black braided silk  3-0 Surgical Specialties SP117 Silk tie for blood vessel traction and ligation
Centrifugation machine AccuSpin 8C Fischer Scientific 75-008-821 Used to extract plasma from whole blood sample
Endotracheal Tube 3.0 uncuffed Progressive Medical International 1109021995 Inserted through tracheostomy
FITC-Sinistrin 1.0 g MediBeacon Inc. FTCF S001 Store at room temp and protect from light
GEM Premier 3000 Blood gas analyzer Instrumentation Laboratory 5700 For bedside blood gas analysis
Heating Pad medium size 20 in x 29 in Adroit Medical Systems V029 Connects to heat therapy pump
HTP-Heat Therapy Pump Adroit Medical Systems HTP Allows you to set temperature as needed.
IDEXX Catalyst One IDEXX Laboratories 89-92525-00 Plasma creatinine analysis
Invasive blood pressure catheter 3.5Fr Millar SPR-524 Inserted in femoral artery
IV adminstration set with flow regulator True Care TCRTCBINF033G Used to connect IV fluid bag to vein catheter
Ketamine Covetrus 68317 Used for induction of Anesthesia
MediBeacon analysis software version 3.0 MediBeacon Inc. N/A Software program used for analysing data to obtain sinistrin clearance half life and curve
Millex-GV Syringe Filter Unit 0.22 µm Millipore Sigma SLGVR33RS Syringe filter for chloralose injection
Neonate/Infant Ventilator Sechrist Millennium 20409 Connected to air supply to provide ventilation through endotracheal tube
Phenobarbital Sodium + Phenytoin Sodium (Euthasol) Covetrus 72934 Used for euthanasia
Ringer Lactate 500 mL bag Baxter 2B2323Q Maintanence fluid infusion
Sterile Gloves Henry Schein 104-5920 Used by operator during surgery
Sterile Gown Halyard Health 95021 Used by operator during surgery
Steril Towel Medline 42131704 Used as drape to maintaine sterile field when operating
Suture 3-0 silk reverse cutting needle Ethicon NC1842168 Used for suturing abdominal wall layers
Transdermal Mini GFR Monitor MediBeacon Inc. TDM004 Battery and USB connector included in package
Transdermal monitor adhesive patch MediBeacon Inc. PTC-SM001 Doubl sided adhesive patch for GFR probe
Umbilical Tape 1/8 in x 20 yds Fisher Scientific NC9303017 To secure endotracheal tube
Venous Catheter size PE/5 Micro medical tubing BB31695 For femoral vein cannulation
Xylazine Covetrus 61035 Used for induction of anesthesia
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Referências

  1. Pasala, S., Carmody, J. B. How to use… serum creatinine, cystatin C and GFR. Archives of Disease in Childhood Education and Practice Edition. 102 (1), 37-43 (2017).
  2. Smith, H. W. . The Kidney: Structure and Function in Health and Disease. , (1951).
  3. Gutman, Y., Gottschalk, C. W., Lassiter, W. E. Micropuncture study of inulin absorption in the rat kidney. Science. 147 (3659), 753-754 (1965).
  4. Ellery, S. J., Cai, X., Walker, D. D., Dickinson, H., Kett, M. M. Transcutaneous measurement of glomerular filtration rate in small rodents: through the skin for the win. Nephrology. 20 (3), 117-123 (2015).
  5. Eisner, C., et al. Major contribution of tubular secretion to creatinine clearance in mice. Kidney International. 77 (6), 519-526 (2010).
  6. Wendt, M., Waldmann, K. H., Bickhardt, K. Comparative studies of the clearance of inulin and creatinine in swine. Zentralblatt fur Veterinarmedizin. Reihe A. 37 (10), 752-759 (1990).
  7. Schwartz, G. J., Brion, L. P., Spitzer, A. The use of plasma creatinine concentration for estimating glomerular filtration rate in infants, children, and adolescents. Pediatric Clinics of North America. 34 (3), 571-590 (1987).
  8. Boer, D. P., de Rijke, Y. B., Hop, W. C., Cransberg, K., Dorresteijn, E. M. Reference values for serum creatinine in children younger than 1 year of age. Pediatric Nephrology. 25 (10), 2107-2113 (2010).
  9. Guignard, J. P., Drukker, A. Why do newborn infants have a high plasma creatinine. Pediatrics. 103 (4), 49 (1999).
  10. Friedemann, J., Schock-Kusch, D., Shulhevich, Y. Transcutaneous measurement of glomerular filtration rate in conscious laboratory animals: state of the art and future perspectives. Reporters, Markers, Dyes, Nanoparticles, and Molecular Probes for Biomedical Applications IX. 10079, 63-71 (2017).
  11. Herrera Pérez, Z., Weinfurter, S., Gretz, N. Transcutaneous assessment of renal function in conscious rodents. Journal of Visualized Experiments. (109), e53767 (2016).
  12. Scarfe, L., et al. Transdermal measurement of glomerular filtration rate in mice. Journal of Visualized Experiments. (140), e58520 (2018).
  13. Mondritzki, T., et al. Transcutaneous glomerular filtration rate measurement in a canine animal model of chronic kidney disease. Journal of Pharmacological and Toxicological Methods. 90, 7-12 (2018).
  14. Steinbach, S., et al. A pilot study to assess the feasibility of transcutaneous glomerular filtration rate measurement using fluorescence-labelled sinistrin in dogs and cats. PLoS One. 9 (11), 111734 (2014).
  15. Almond, G. W. Research applications using pigs. The Veterinary Clinics of North America Food Animal Practice. 12 (3), 707-716 (1996).
  16. Bassols, A., et al. The pig as an animal model for human pathologies: A proteomics perspective. Proteomics Clinical Applications. 8 (9-10), 715-731 (2014).
  17. Ayuso, M., Irwin, R., Walsh, C., Van Cruchten, S., Van Ginneken, C. Low birth weight female piglets show altered intestinal development, gene expression, and epigenetic changes at key developmental loci. FASEB Journal. 35 (4), 21522 (2021).
  18. Pierson, R. N. Progress toward pig-to-human xenotransplantation. The New England Journal of Medicine. 386 (20), 1871-1873 (2022).
  19. Montgomery, R. A., et al. Results of two cases of pig-to-human kidney xenotransplantation. The New England Journal of Medicine. 386 (20), 1889-1898 (2022).
  20. Reardon, S. First pig kidneys transplanted into people: what scientists think. Nature. 605 (7911), 597-598 (2022).
  21. Lu, T., Yang, B., Wang, R., Qin, C. Xenotransplantation: current status in preclinical research. Frontiers in Immunology. 10, 3060 (2019).
  22. Pattison, R. J., English, P. R., MacPherson, O., Roden, J. A., Birnie, M. Hypothermia and its attempted control in newborn piglets. Proceedings of the British Society of Animal Production. 1990, 81 (1972).
  23. Tucker, B. S., Petrovski, K. R., Kirkwood, R. N. Neonatal piglet temperature changes: effect of intraperitoneal warm saline injection. Animals. 12 (10), 1312 (2022).
  24. Alcalá Rueda, I., et al. A live porcine model for surgical training in tracheostomy, neck dissection, and total laryngectomy. European Archives of Oto-Rhino-Laryngology. 278 (8), 3081-3090 (2021).
  25. Swindle, M. M., Smith, A. C. . Swine in the Laboratory: Surgery, Anesthesia, Imaging, and Experimental Techniques, Third Edition. , (2016).
  26. Steinbacher, R., von Ritgen, S., Moens, Y. P. Laryngeal perforation during a standard intubation procedure in a pig. Laboratory Animals. 46 (3), 261-263 (2012).
  27. Ettrup, K. S., et al. Basic surgical techniques in the Göttingen minipig: intubation, bladder catheterization, femoral vessel catheterization, and transcardial perfusion. Journal of Visualized Experiments. (52), e2652 (2011).
  28. Soni, H., Adebiyi, A. Early septic insult in neonatal pigs increases serum and urinary soluble Fas ligand and decreases kidney function without inducing significant renal apoptosis. Renal Failure. 39 (1), 83-91 (2017).
  29. Bütz, D. E., Morello, S. L., Sand, J., Holland, G. N., Cook, M. E. The expired breath carbon delta value is a marker for the onset of sepsis in a swine model. Journal of Analytical Atomic Spectrometry. 29 (4), 606-613 (2014).
  30. Turner, A. S., McIlwraith, C. W. . Techniques in Large Animal Surgery. , (1989).
  31. Steinbach, S., et al. A pilot study to assess the feasibility of transcutaneous glomerular filtration rate measurement using fluorescence-labelled sinistrin in dogs and cats. PLoS One. 9 (11), 111734 (2014).
  32. Mondritzki, T., et al. Transcutaneous glomerular filtration rate measurement in a canine animal model of chronic kidney disease. Journal of Pharmacological and Toxicological Methods. 90, 7-12 (2018).
  33. Schock-Kusch, D., et al. Transcutaneous measurement of glomerular filtration rate using FITC-sinistrin in rats. Nephrology Dialysis Transplantation. 24 (10), 2997-3001 (2009).
  34. Peters, A. M. Expressing glomerular filtration rate in terms of extracellular fluid volume. Nephrology Dialysis Transplantation. 7 (3), 205-210 (1992).
  35. Groth, S., Christensen, A. B., Nielsen, H. CdTe-detector registration of 99mTc-DTPA clearance. European Journal of Nuclear Medicine. 8 (6), 242-244 (1983).
  36. Guyton, A. C., Hall, J. E. The body fluid compartments: extracellular and intracellular fluids; interstitial fluid and edema. Textbook of Medical Physiology. 9, 306-308 (2000).
  37. Luis-Lima, S., et al. Iohexol plasma clearance simplified by dried blood spot testing. Nephrology, Dialysis, Transplantation. 33 (9), 1597-1603 (2018).
  38. Kobayashi, E., Hishikawa, S., Teratani, T., Lefor, A. T. The pig as a model for translational research: overview of porcine animal models at Jichi Medical University. Transplantation Research. 1 (1), 8 (2012).
  39. Swindle, M. M., et al. Swine as models in biomedical research and toxicology testing. Veterinary Pathology. 49 (2), 344-356 (2012).
  40. Ibrahim, Z., et al. Selected physiologic compatibilities and incompatibilities between human and porcine organ systems. Xenotransplantation. 13 (6), 488-499 (2006).
  41. Judge, E. P., et al. Anatomy and bronchoscopy of the porcine lung. A model for translational respiratory medicine. American Journal of Respiratory Cell and Molecular Biology. 51 (3), 334-343 (2014).
  42. Stevens, L. A., Levey, A. S. Measured GFR as a confirmatory test for estimated GFR. Journal of the American Society of Nephrology. 20 (11), 2305-2313 (2009).
  43. Bankir, L., Yang, B. New insights into urea and glucose handling by the kidney, and the urine concentrating mechanism. Kidney International. 81 (12), 1179-1198 (2012).
  44. Ruiz, S., et al. Sepsis modeling in mice: ligation length is a major severity factor in cecal ligation and puncture. Intensive Care Medicine Experimental. 4 (1), 22 (2016).
  45. Schock-Kusch, D., et al. Transcutaneous assessment of renal function in conscious rats with a device for measuring FITC-sinistrin disappearance curves. Kidney International. 79 (11), 1254-1258 (2011).
  46. Frennby, B., Sterner, G. Contrast media as markers of GFR. European Radiology. 12 (2), 475484 (2002).
  47. Burchardi, H., Kaczmarczyk, G. The effect of anaesthesia on renal function. European Journal of Anaesthesiology. 11 (3), 163-168 (1994).
  48. Fusellier, M., et al. Influence of three anesthetic protocols on glomerular filtration rate in dogs. American Journal of Veterinary Research. 68 (8), 807811 (2007).
  49. Arant, B. S. Functional immaturity of the newborn kidney-paradox or prostaglandin. Homeostasis, Nephrotoxicity, and Renal Anomalies in the Newborn. , 271-278 (1986).
  50. Gattineni, J., Baum, M. Developmental changes in renal tubular transport-an overview. Pediatric Nephrology. 30 (12), 2085-2098 (2015).
  51. Gu, X., Yang, B. Methods for assessment of the glomerular filtration rate in laboratory animals. Kidney Diseases. , 1-11 (2022).
  52. Mullins, T. P., Tan, W. S., Carter, D. A., Gallo, L. A. Validation of non-invasive transcutaneous measurement for glomerular filtration rate in lean and obese C57BL/6J mice. Nephrology. 25 (7), 575-581 (2020).

Tags

Transdermal GFR MeasurementKidney FunctionSwine ModelXenotransplantationPigletsCecal LigationTracheostomyVessel CannulationReal-time AssessmentNon-invasive

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Fanous, M. S., Afolabi, J. M., Michael, O. S., Falayi, O. O., Iwhiwhu, S. A., Adebiyi, A. Transdermal Measurement of Glomerular Filtration Rate in Mechanically Ventilated Piglets. J. Vis. Exp. (187), e64413, doi:10.3791/64413 (2022).
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