A subscription to JoVE is required to view this content. Sign in or start your free trial.

In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

This study presents a highly reproducible large animal model of renal ischemia-reperfusion injury in swine using temporary percutaneous bilateral balloon-catheter occlusion of the renal arteries for 60 min and reperfusion for 24 h.

Abstract

Acute kidney injury (AKI) is associated with higher risk for morbidity and mortality post-operatively. Ischemia-reperfusion injury (IRI) is the most common cause of AKI. To mimic this clinical scenario, this study presents a highly reproducible large animal model of renal IRI in swine using temporary percutaneous bilateral balloon-catheter occlusion of the renal arteries. The renal arteries are occluded for 60 min by introducing the balloon-catheters through the femoral and carotid artery and advancing them into the proximal portion of the arteries. Iodinated contrast is injected in the aorta to assess any opacification of the kidney vessels and confirm the success of the artery occlusion. This is furtherly confirmed by the flattening of the pulse waveform at the tip of the balloon catheters. The balloons are deflated and removed after 60 min of bilateral renal artery occlusion, and the animals are allowed to recover for 24 h. At the end of the study, plasma creatinine and blood urea nitrogen significantly increase, while eGFR and urine output significantly decrease. The need for iodinated contrast is minimal and does not affect renal function. Bilateral renal artery occlusion better mimics the clinical scenario of perioperative renal hypoperfusion, and the percutaneous approach minimizes the impact of the inflammatory response and the risk of infection seen with an open approach, such as a laparotomy. The ability to create and reproduce this clinically relevant swine model eases the clinical translation to humans.

Introduction

Acute kidney injury (AKI) is a commonly diagnosed condition among surgical patients associated with significant morbidity and mortality1,2. Available data show that AKI can affect even half of all hospitalized patients worldwide and leads to 50% mortality rate in patients in the intensive care unit1,3. Despite its high prevalence, current AKI therapy remains limited to preventive strategies, such as fluid management and dialysis. Therefore, there is an ongoing interest in exploring alternative therapies for AKI4,5,6.

AKI is typically classified into pre-renal, intrinsic, and post-renal based on its etiology4,5,6. The majority of surgical patients with AKI are associated with pre-renal causes due to hypovolemia, resulting in ischemia-reperfusion injury (IRI) of the kidneys2. Clinically, urine output decreases, and creatinine levels increase due to decreased renal function. The kidney is a high-metabolic-rate organ and susceptible to ischemia. A highly reproducible large animal model of renal IRI is necessary to obtain a better insight into the pathophysiology of AKI and its potential therapeutic approaches5.

To mimic the clinical scenario of kidney hypoperfusion peri-operatively, a model of bilateral renal artery occlusion is deemed suitable. Previously described models entailing unilateral renal artery occlusion with or without resection of the contralateral kidney do not provide sufficient clinical applicability7,8. Although these models are sufficient for causing AKI, they do not resemble real-life clinical scenarios neither in terms of type nor duration of injury.

The aim of this paper is to present a porcine model of percutaneous bilateral temporary occlusion of the renal arteries by balloon-catheter occlusion under angiography. Bilateral renal artery occlusion mimics the clinical scenario of renal hypoperfusion, followed by the subsequent removal of the balloon for reperfusion9,10. The technical steps are described, including catheterization, catheter guidance, angiography, and hemodynamic monitoring. This method not only allows for a highly controlled and replicable occlusion of the renal arteries, but the percutaneous approach minimizes the impact of the inflammatory response by limiting the amount of insult to the body compared to an open approach.

Protocol

All in vivo studies were conducted in accordance with the National Institutes of Health's guidelines on animal care and use and were approved by the Boston Children's Hospital's Animal Care and Use Committee (Protocol 18-06-3715). All animals received humane care in compliance with the Guide for the Care and Use of Laboratory Animals. Figure 1 shows the timeline including anesthesia, surgical preparation, and timepoints for primary outcome measurements of this study.

1. Induction, anesthesia and intubation

  1. To prevent unnecessary stress and discomfort, sedate the swine by intramuscular injection of a mixture of tiletamine/zolazepam 4-6 mg/kg and xylazine 1.1-2.2 mg/kg as well as isoflurane 3% using a face mask.
  2. Cannulate the ear vein to obtain venous access with a 20 G IV cannula after disinfecting the area with 95% ethanol. Start a maintenance infusion (0.9% NaCl at 5 mL/kg/h).
  3. Intubate the pig with an endotracheal tube (size 7 for pigs weighing 40-50 kg) once anesthetic adequacy is confirmed. Perform balloon-ventilation with a frequency of 12 breaths/min and transport the pig to the operating theater.
  4. Place the animal on the operating table in the supine position. Immediately start mechanical positive pressure ventilation with FiO2 0.50, 10 mL/kg tidal volume, and a frequency of 12 breaths/min under continuous capnography.
  5. Place a pulse oximeter on the ear or the bottom lip for continuous monitoring.
  6. Maintain normothermia (37 °C) using an air-heated pad.
  7. To maintain general anesthesia, keep isoflurane administration at 0.5-4% through the endotracheal tube. Throughout the procedure, continuously monitor ECG, arterial blood pressure, temperature, and capnography to measure the depth of anesthesia.
  8. Insert a Foley catheter to check the fluid status of the animal and monitor the urine output by collecting urine in a drainage bag.
    NOTE: Female swine are preferred over males due to the anatomical features of their urethra which allows easier catheterization.

2. Surgical preparation and vascular access

  1. Drape the animal in a sterile fashion.
  2. Disinfect the right lateral area of the neck by applying betadine and then 95% ethanol for 3 times.
  3. Perform a cut-down for the catheterization of the right carotid artery and the right jugular vein. Retract the sternocleidomastoid muscle laterally and dissect it down to the right carotid artery and the right jugular vein.
  4. Insert a 5F angiography sheath in both the artery and the vein. Secure it with a silk 2-0 suture.
  5. Insert a 5F angiography sheath using the Seldinger technique into the left femoral artery.
    1. To perform the Seldinger technique puncture the femoral artery using a hollow needle. Insert a soft tip guidewire through the lumen and advance it into the femoral artery.
    2. Hold the guidewire secure with the hand while removing the needle. Pass the angiography sheath over the guidewire into the femoral artery and withdraw the guidewire. Use ultrasound guidance, if necessary.

3. Induction of renal ischemia-reperfusion injury

  1. Administer 200 IU/kg sodium heparin intravenously to achieve systemic anticoagulation (target activated clotting time (ACT) > 300 s).
  2. Perform an angiography by injecting an iodinated contrast agent under fluoroscopy to identify the renal arteries.
    NOTE: To reduce the risk for contrast-induced nephrotoxicity, dilute the iodinated contrast agent in a 1:1 solution with normal saline. Tabulate the dosage for all animals to ensure equivalent dosing.
  3. Identify the renal arteries, manually advance the guidewire in the guiding catheter.
  4. Position the 5F JL4 guiding catheter in the left renal artery through the right carotid artery (Figure 1A).
  5. Position the second 5F JL4 guiding catheter in the right renal artery through the left femoral artery (Figure 1A).
  6. Use the guidewires to direct a 5F percutaneous transluminal angioplasty (PTA) dilatation catheter in each renal artery.
    NOTE: It is preferable to position the balloon at the proximal renal artery so that no branches or collaterals of the renal artery are left patent after balloon inflation.
  7. Position each balloon catheter in place and connect a pressure line to each catheter.
  8. Check the presence of arterial pulse waveforms in the pressure monitor to ensure the correct positioning of the catheter.
  9. Inflate each balloon and aim for a pressure of approximately 2.5 atm inside the balloon (Figure 1B).
  10. To confirm the cessation of blood flow to the kidneys observe the flattening of the pulse waveform at the tip of the balloon catheter.
  11. Inject iodinated contrast medium (1:1 dilution) and check for any opacification of the renal vessels.
    NOTE: It is also possible to fill the balloon with an iodinated contrast agent for visualization of the inflated balloon. However, this method is not as sensitive as pulse waveform flattening to confirm occlusion of the renal arteries.
  12. After 60 min of occlusion, carefully deflate and remove the balloon catheters from the renal arteries.
  13. Perform an angiography (using 1:1 diluted contrast medium) to confirm renal artery patency and the establishment of renal reperfusion (Figure 1C).
  14. Remove the 5F angiography sheath from the left femoral artery.
  15. Apply firm pressure at the site of catheterization for 30 min.
  16. Reverse the effect of heparin by the administration of protamine (3 mg/kg) until ACT normalizes.
  17. To sample urine during the post-operative period, secure a tube to the Foley catheter with a silk 2-0 suture using an interrupted stitch on the skin.
  18. Leave the angiography sheaths in the right carotid artery and the right jugular vein in place and secure them with a silk 2-0 suture using an interrupted stitch to allow for blood sampling throughout the study.
  19. Close the neck incision with a silk 2-0 suture using a continuous stitch in 2 layers.
  20. Administer bupivacaine (3 mg/kg) at the incision site to minimize pain.
  21. Continue to hydrate the animal with 0.9% NaCl at 5 mL/kg/h for a total of 2 h following the end of ischemia.
  22. Place a fentanyl patch (25-50 µg/h) on the back of the animal to minimize post-operative pain.
  23. Administer an intramuscular injection of buprenorphine (0.005-0.1 mg/kg) to minimize post-operative pain.
  24. Monitor the animal and maintain it on mechanical ventilation until awaking.

4. Animal recovery

  1. Following awaking, accommodate the animal in a temperature-controlled room.
  2. Continue to turn the animal from one lateral side to the other until it regains full consciousness and ability to ambulate.
  3. Provide water and food ad libitum.

5. Functional assessment

  1. Collect the blood and the urine samples according to the desired protocol.
    NOTE: In this study, the following time points were designated: baseline (1 h following initiation of the hydration protocol and before occlusion of the renal arteries), end of ischemia, and reperfusion (2 h, 6 h, 24 h).
  2. Collect the arterial and the venous blood samples. Store them in lithium heparin or EDTA coated vacutainers for subsequent analysis.
    NOTE: Draw blood directly from the catheters in the carotid artery and jugular vein.
  3. Collect the urine samples from the Foley catheter and store them in 15 mL tubes for analysis.
    NOTE: Collect the urine from the drainage bag connected to the Foley catheter.
  4. To determine urine output, empty the drainage bag and collect urine for 1 h.
    NOTE: For the 6 h timepoint at which a drainage bag is not connected to the Foley catheter, close the tube connected to the Foley catheter for 30 min and then collect the urine with a 60 mL syringe to determine urine output.

6. Euthanasia

  1. Following the end of the reperfusion period, perform anesthesia and monitor as described above.
  2. Continue hydration with 0.9% NaCl at 5 mL/kg/h.
  3. Use the arterial and the venous catheters for blood sampling and the Foley catheter to determine urine output. Collect the final blood and urine samples and calculate the urine output.
  4. Perform a 15-cm midline laparotomy incision using a size 10 blade from the xiphoid down to the mid pelvis.
  5. Use a straight lateral retractor to retract the abdominal skin.
  6. Dissect the lateral peritoneal attachments of the abdominal wall to expose the right and left retroperitoneum.
  7. Identify and bluntly dissect both renal arteries and veins.
  8. Ligate both renal arteries and veins with a 2-0 silk suture and perform bilateral nephrectomies to collect whole tissue specimens for histological and metabolic analysis.
  9. Euthanize the animal with the preferred method of euthanasia (e.g., exsanguination, pentobarbital)

Results

Functional analysis
The representative results of this study arise from 6 animals and the data shown are mean ± standard error of the mean.Renal function is assessed by determining the urine output, estimated glomerular filtration rate (eGFR), plasma creatine, and blood urea nitrogen (BUN). The biomarkers of renal function are assessed using a portable chemistry analyzer. eGFR is calculated according to the following formula: eGFR =1.879 × BW1.092/PCr0.6 (...

Discussion

AKI is a common clinical disorder affecting up to 50% of hospitalized adult patients worldwide6,12. A clinically relevant animal model is needed to further investigate the pathophysiology of the disease and potential therapeutic targets. Although there are several murine models replicating AKI, these do not completely mimic their respective clinical scenarios and the anatomy of the human kidney. This study proposes a clinically relevant swine model to allow for t...

Disclosures

The authors declare no competing financial interests.

Acknowledgements

We would like to thank Dr. Arthur Nedder for his help and guidance. This work was supported by the Richard A. and Susan F. Smith President's Innovation Award, Michael B. Klein and Family, The Sidman Family Foundation, The Michael B. Rukin Charitable Foundation, The Kenneth C. Griffin Charitable Research Fund, and The Boston Investment Council.

Materials

NameCompanyCatalog NumberComments
0.9% sodium chloride injection, usp, 100 ml viaflex plastic containerBaxter2B1302For animal hydration
Agent contrast 100.0ml injection media btl ioversal 74%CARDINAL HEALTH133311For visualizing the vasculature
Bard Bardia Closed System Urinary Drainage BagBARD Inc802001For urine collection
BD Vacutainer K2 EDTABD367841For blood sample storage
BD Vacutainer Lithium HeparinBD366667For blood sample storage
BetadineHenry Schein6906950For skin disinfection
Bookwalker retractorCodmanFor skin retraction
Bupivacaine 0.25%HospiraAdminister at incision site for analgesia
Buprenorphine SRZoo Pharm10mg/ml bottle, Dose: 0.2mg/kg SC
Cath angio 5.0 Fr x100.0 cm 0.038 in JR4MERIT MEDICAL SYSTEM INC7523-21For identification of the renal arteries
Cuffed endotracheal tubeEmdamedTo establish a secure airway for the duration of the operation
EKG Medtronics- Physiocontrol LifePak 20 Oxygen saturation monitorGE Healthcare Madison WIFor oxygen saturation monitoring
Encore 26 inflatorBOSTON SCIENTIFIC710113For inflating the balloon catheters
Ethanol 95% (Ethyl alcohol)Henry ScheinFor skin disinfection
Fentanyl patchMylanDose: 25-50mcg/hr, TD
Gold silicone coated FoleyTELEFLEX MEDICAL INC180730160For urine collection
Heparin sodiumLEO Pharma A/SDose: 200 IU/kg IV
i33 ultrasound machinePhillipsUse ultrasonographic guidance for femoral catherization if necessary
Inqwire diagnostic guide wire - 0.035" (0.89 mm) - 260 cm (102") - 1.5 mm j-tipMERIT MEDICAL SYSTEM INC6609-33For guiding the balloon catheters to the renal arteries
Intravenous catheter, size 20 gaugeSanta Cruz BiotechnologyInc SC-360097For fluid administration
IsofluranePatterson Veterinary Supply, Inc.21283620Dose: 3%, INH
Metzenbaum blunt curved 14.5 cm - 5(3/4)"Rudolf MedicalRU-1311-14MFor tissue dissection and cutting
Neonatal disposable transducer kit with 30ml/hr flush device and double 4-way stopcocks for continuous monitoringArgon Medical041588505AFor pressure measurement
Powerflex pro PTA dilatation catheter 6 x 20 mm - shaft length (135cm)CARDINAL HEALTH4400602XFor occlusion of the renal arteries
Pressure monitoring lines mll/mll - 12" clear, mll/mllSmiths MedicalB1571/MX571For pressure measurement
Procedure packMolnlycke Health Care97027809Surgical drape, gauze pads, syringes, beaker etc
ProtamineHenry Schein1044148For heparin reversal
Scalpel blade - size #10Cardinal Health (Allegiance)32295-010For the skin incisions
Stopcock iv 4 way lrg bore rotg male ll adptr strlPeoplesoft1550For connecting tubings
Straight lateral retractorCodmanFor skin retraction
Suture perma hnd 18in 2-0 braid silk blkCARDINAL HEALTH 1A185HFor suturing incision site and securing catheters
Syringe contrast injection 10ml fixed male luer redMERIT MEDICAL SYSTEM INCMSS111-RTo administer the contrast agent
Syringe medical 60ml ll plst strl ltx free dispCARDINAL HEALTH 1BF309653For urine collection and flushing of the angiocath
Tilzolan (tiletamine/zolazepam)Patterson Veterinary Supply, Inc.07-893-1467Dose: 4-6 mg/kg, IM
XylazinePutney, INCDose: 1.1-2.2 mg/kg, IM

References

  1. 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).
  2. 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).
  3. 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).
  4. Bhargava, P., Schnellmann, R. G. Mitochondrial energetics in the kidney. Nature Reviews Nephrology. 13 (10), 629-646 (2017).
  5. 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).
  6. 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).
  7. 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).
  8. 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).
  9. 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).
  10. 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).
  11. 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).
  12. Rewa, O., Bagshaw, S. M. Acute kidney injury-epidemiology, outcomes and economics. Nature Reviews Nephrology. 10 (4), 193-207 (2014).
  13. 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).
  14. 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).

Reprints and Permissions

Request permission to reuse the text or figures of this JoVE article

Request Permission

Explore More Articles

Acute Kidney InjuryRenal IschemiaRenal Artery OcclusionLarge Animal ModelIschemia Reperfusion InjuryTherapeutic ModalitiesCatheterizationAngiography SheathSeldinger TechniqueSodium HeparinFluoroscopyRenal ArteriesPercutaneous Transluminal Angioplasty PTABalloon Catheter

This article has been published

Video Coming Soon

JoVE Logo

Privacy

Terms of Use

Policies

Research

Education

ABOUT JoVE

Copyright © 2025 MyJoVE Corporation. All rights reserved