Bilateral Renal Ischemia-Reperfusion Model for Acute Kidney Injury in Mice

Summary

The ischemia-reperfusion injury (IRI) model can be used at different stages of acute kidney injury (AKI) development, especially during AKI to chronic kidney disease (CKD) progression. Here, we describe the procedure for IRI model development in mice via a trans-abdominal approach, clamping renal pedicles via a vascular clip to induce ischemic injury.

Abstract

Acute kidney injury (AKI) is defined as a rapid decline in renal function, in which persistent kidney dysfunction gradually progresses to chronic kidney disease (CKD) due to the irreversible loss of nephrons and their maladaptive repair. In recent years, the incidence of AKI has been increasing concerning diverse etiologies, including volume depletion, sepsis, nephrotoxicity, muscle injury, and major trauma, in which ischemia-reperfusion injury (IRI) accounts for most episodes. Development of the IRI model in mice is induced by surgical clamping of the renal pedicles, which provides powerful and controllable tools for preclinical models of AKI. Importantly, the IRI model is deployed at different stages of the AKI development, especially in the processes of AKI to CKD. Despite the IRI model being widely practiced in many laboratories, a series of variables still influence the results of this model. Here, we describe the procedure of IRI model development to provide a repeatable and reliable method for researchers to explore the underlying pathogenesis in the development of AKI and the progression of AKI to CKD.

Introduction

Acute kidney injury (AKI) is a severe clinical syndrome with significant morbidity and mortality, defined as an increase in serum creatinine of ≥ 0.3 mg/dL (26.5 µM/L) within 48 h or an increase in serum creatinine to ≥ 1.5 times baseline within 7 days, or urine volume < 0.5 mL/kg/h for 6 h^1,2,3. Despite decades of research, effective therapy for AKI is lacking to alleviate kidney damage or accelerate kidney recovery, and a considerable proportion of AKI patients progress to chronic kidney disease (CKD)^4,5,6. Complex molecules and pathways are involved in AKI and its progression in part, so preclinical models provide powerful tools to unravel these complexities for the development of efficient therapeutic modalities.

Clinically, ischemia-reperfusion injury (IRI) injury is the major cause of AKI in various conditions, including cardiac and hepatic surgeries, circulatory shock, volume depletion, sepsis, renal vascular occlusion or obstruction, kidney transplantation, and so on⁷. The IRI-AKI mouse model has been in use since the 1960s; this model was developed by surgical clamping of the renal pedicles with non-traumatic clamps in mice leading to ischemia and followed by reperfusion of renal blood flow by removing the clamps. The IRI-AKI model is typically characterized by renal tubular cell death and progressive kidney tissue damage. The IRI is one of the most common models used for the pathogenesis and therapeutic intervention in AKI for several reasons: (1) The simplicity and safety of the surgical procedure improve the survival rate and success rate of the IRI-AKI model⁸; (2) Since ischemia is a major etiology in human AKI, IRI-AKI model is better used for assessing clinical AKI event⁹; (3) The IRI model could present kidney injury and histopathology changes in different stages of AKI, which is also applicable to studying the progression from AKI to CKD¹⁰. Depending on the experimental design, IRI-induced AKI models include bilateral IRI, unilateral IRI with intact contralateral kidney, and unilateral IRI with simultaneous contralateral nephrectomy. Notably, the bilateral IRI model is considered more relevant to human pathological conditions of AKI because both kidneys have been affected by blood supply¹¹. The IRI model is applicable to simulate the effects of reduced renal blood flow after kidney transplantation, cardiac bypass, renal vascular, or nephron-sparing surgery, as well as in the setting of hypotension⁹. Here, we describe the procedure for a bilateral IRI model to provide a consistent and reliable method for researchers to explore the underlying pathogenesis in ischemia-induced AKI.

Protocol

Male C57BL/6J mice at 8 weeks of age and weighing 25 g were used to establish the AKI model by bilateral ischemia-reperfusion. As per previous studies, we maintain the body temperature at around 36.5 °C-37 °C, and the kidney ischemia duration is 30 min in the IRI surgery^12,13. A total of 6 mice were needed for each group, and sham-operated mice served as controls. The animal experiments in this study have been approved by the Institutional Animal Care and Use Committee (IACUC) of Zhejiang University to protect the welfare of animals. All animal research procedures were performed following the ethical guidelines and principles of Zhejiang University.

1. Pre-operative preparation

1. High-pressure sterilize all surgical instruments.
2. Prepare the anesthetic solution by adding 2 mL of ketamine and 0.4 mL of xylazine in 7.6 mL of sterile saline. Anesthetize the mouse with a mixture of ketamine (80 mg/kg) and xylazine (16 mg/kg) by intraperitoneal injection. Assess anesthetic depth using the toe pinch reflex.
3. Place the anesthetized mouse on the homeothermic blanket to ensure that its airway remains unobstructed. Maintain the body temperature in the range of 36.5-37 °C.
4. Cover the mouse's eyes with 1% tetracycline hydrochloride eye ointment to prevent dryness while under anesthesia.
5. Shave the hair of the abdomen with a hair clipper and clean the skin of the surgical area with a povidone-iodine solution for 3x.

2. Surgery

1. Make an abdominal median incision approximately 1-1.5 cm with surgical scissors via the skin and muscle layer and open the abdominal cavity with a spreader.
2. Expose the kidney by moving the retroperitoneal fat and pushing the intestines and other organs to the offside with a cotton swab. The kidney is located in the retroperitoneal space, around 0.5 cm lateral to the spine, and below the 13^th rib.
3. Dissect the renal pedicle with fine pointed forceps to separate and remove the fascia and adipose tissue and expose the left renal pedicles.
4. Clamp the renal pedicles with a vascular clip using holding forceps and ensure that the vascular damage is as minor as possible. Avoid clamping redundant renal sinus fat, which may lead to incomplete renal ischemia.
5. Set the kidney ischemia duration, starting with clamping for 30 min. The characteristic of successful ischemia is that the kidney gradually changes from red to deep purple within a few minutes.
6. Move the kidney into the retroperitoneal space. Repeat the procedure at the contralateral side to expose and clamp the right renal pedicles.
7. Record the ischemia time on each side separately to ensure that both kidneys receive the exact duration of ischemia. Reopen the incision and release the vascular clip at the end of the ischemia duration.
8. Replace the kidney into the retroperitoneal space, and then suture the muscle and skin layer by layer with the Vicryl 4-0 suture.
   NOTE: The surgical procedures should be performed under sterile conditions. Clean the surgical table and instruments with 75% ethanol during operation when needed.

3. Post-operative care

1. Administer 0.5-1 mL of warm and sterile saline by intraperitoneal injection to compensate for the fluid loss.
2. Maintain sternal recumbency with attention until the mouse has regained sufficient consciousness. Put the mouse on the homeothermic blanket until it gains full consciousness, then return to its cage. Do not return the mouse to the company of other animals until fully recovered.
3. Give buprenorphine 0.05-0.10 mg/kg every 12 h for the first 3 days to alleviate post-surgical pain. Monitor the surgical mice every day.

4. Model assessment

1. Hematoxylin-Eosin (HE) staining
   1. Euthanize animals using pentobarbital sodium by intraperitoneal injection on days 1, 3, 7, or 14 after IRI.
   2. Fix fresh kidney tissues with 4% paraformaldehyde (PFA) overnight, and store in 75% ethanol at 4 °C.
   3. After dehydration and embedding, cut the obtained samples into 8 µm thickness for staining.
   4. Dewax the tissue sections in xylene, and then rehydrate with decreasing concentrations of ethanol.
   5. Stain the tissue sections with hematoxylin and eosin.
   6. Dehydrate the tissue sections by increasing concentrations of ethanol and xylene.
2. Kidney function assay
   1. Collect blood samples using the eyeball blood collection method after anesthesia.
   2. Centrifuge blood samples at 12000 x g for 10 min to separate serum.
   3. Determine serum creatinine and blood urea nitrogen (BUN) by the automatic dry-chemistry analyzer to monitor renal function.
3. Real-time PCR (RT-PCR)
   1. Extract total RNA from kidney tissues using a quick RNA extraction kit, and then synthesize cDNA with a reverse transcriptase mix kit. Perform RT-PCR with SYBR Green Premix Kit and run on RT-PCR instrument. The primer sequences used here were reported before¹⁴: KIM1 forward, 5'-GCTGCTACTGCTCCTTGTGA-3'; reverse 5'-GGAAGGCAACCACGCTTAGA-3'; NGAL forward, 5'-GGCCAGTTCACTCTGGGAAA-3; reverse 5'- TGGCGAACTGGTTGTAGTCC-3'; GAPDH forward, 5'-GGTGAAGGTCGGTGTGAACG-3'; reverse 5'-CTCGCTCCTGGAAGATGGTG-3'.

Results

State of the kidney during surgery

The characteristic of successful ischemia is that the kidney gradually changes from red to deep purple within 1-2 min, and successful reperfusion is characterized by the kidney gradually changing from deep purple to red within 1-2 min.

Histology of the kidney after surgery

HE and Periodic Acid Schiff (PAS) staining are the direct ways to verify acute kidney injury. In th...

Discussion

In this paper, we have provided a detailed procedure on the renal IRI model, subsequently highlighting that it is a robust model for the progression of AKI and AKI to CKD. In addition, we demonstrate the impact of the two main criteria of kidney injury, including kidney histology and function.

Several key points in surgical procedures need to be emphasized for a repeatable and reliable model. For abdominal surgery, a midline incision is recommended to expose the kidney to minimize trauma assoc...

Materials

Name	Company	Catalog Number	Comments
Animal hair clipper	FEIYUBIO	19-7002
1-ml syringes	Longreen	SR60061
Ethanol	Macklin	E885996
Gauze	FEIYUBIO	19-5022
Homeothermic monitor system	Warmmate	40 x 50
Needle holder	DKBT	CZQ-00160
Spreader	RWD	R22029-03
Sterile saline	Biosharp	BL158A
Tissue scissors	DKBT	DC-YKJ1002
Tissue tweezers	DKBT	DK079904
Vascular clip	Fine Science Tools	18055-02
Vicryl suture	Shanghai Jinhuan	4 -0