Establishing a Standardized Renal Artery Ligation Procedure for Hypertension in Syrian Golden Hamsters

Summary

This study established and optimized a partial renal artery ligation model in Syrian hamsters (2K1C), which stably induces RAS-related secondary hypertension in a background of hyperlipidemia, providing a highly reproducible platform for cardiovascular research.

Abstract

Hypertension is considered a major global health threat, which​ can lead to severe complications, including vascular remodeling, heart failure, myocardial infarction, and end-stage renal disease. Syrian golden hamsters, a model with naturally expressing cholesteryl ester transfer protein (CETP), are susceptible to diet-induced hyperlipidemia. To establish a reliable and reproducible hamster model for studying the relationship between renal artery stenosis (RAS), secondary hypertension, and cardiovascular disease, the two-kidney, one-clip (2K1C) Goldblatt model was employed. This study details a refined surgical procedure for inducing RAS in Syrian golden hamsters via partial renal artery ligation, emphasizing critical factors such as surgical isolation technique and ligature material selection, which significantly impact outcomes. Implementation of this standardized surgical protocol resulted in approximately 90% of treated hamsters exhibiting a consistent systolic blood pressure elevation of 25–30 mmHg. This optimized renal artery ligation procedure provides a robust method for generating a hypertensive hamster model with high consistency, enhanced reproducibility, and utility for cardiovascular disease research.

Introduction

Renal artery stenosis (RAS) is a leading cause of secondary hypertension and constitutes a significant risk factor for cardiovascular morbidity¹. A primary challenge in clinical research is the difficulty in delineating the causal pathway connecting dyslipidemia, hypertension, and end-organ damage, as comorbidities and concurrent medications frequently confound patient studies. This underscores the critical need for an animal model that accurately recapitulates this complex pathophysiology in a controlled, reproducible experimental setting.

The two-kidney, one-clip (2K1C) Goldblatt model is a well-established method for inducing renovascular hypertension in rodents. However, conventional species such as mice and rats present considerable limitations. Notably, these rodents lack cholesteryl ester transfer protein (CETP), a key enzyme in human high-density lipoprotein metabolism². As a result, they are resistant to diet-induced hypercholesterolemia and atherosclerosis, failing to mimic the metabolic profile commonly observed in patients with RAS. Furthermore, the hypertensive response to renal artery clipping in these models is often attenuated and highly variable; for instance, C57BL/6 mice exhibit a minimal blood pressure increase, while Sprague-Dawley rats show considerable inter-individual variability, which impedes reproducible mechanistic and therapeutic investigations^3,4.

In contrast, the Syrian golden hamster (Mesocricetus auratus) possesses unique physiological advantages for such research. Its natural expression of CETP enables the development of a human-like lipoprotein profile, including elevated LDL-C and slightly increased HDL-C, upon feeding a high-fat and high-cholesterol diet, leading to the spontaneous formation of atherosclerotic lesions in the aorta and coronary arteries⁵. This provides a highly relevant metabolic and vascular background for studying cardiovascular disease. Moreover, recent evidence indicates that hamsters mount a significantly more robust blood pressure response to renin-angiotensin-aldosterone system (RAAS) activation compared to mice, and chymase, making them particularly suitable for hypertension research⁶.

Despite these advantages, the utility of the hamster 2K1C model has been limited by the lack of a standardized and survivable surgical protocol. Existing techniques, which range from complete ligation with high mortality rates to operator-dependent partial ligation, yield inconsistent results and poor reproducibility⁷. Therefore, to fully exploit the hamster’s dual susceptibility to metabolic and hypertensive disease, we developed a refined and highly reproducible surgical approach for the 2K1C model. This optimized protocol ensures consistent induction of hypertension against a background of diet-induced dyslipidemia, thereby offering a robust and integrated experimental platform to investigate the synergistic effects of RAS, hyperlipidemia, and cardiovascular injury.

Protocol

The animal protocols were approved by the laboratory animal ethics committee of Jiangmen International Health Innovation Institute (N2023008) and Peking University (DLASBE0730). Male Syrian golden hamsters, 10–12 weeks of age, were used for the experiment. The details of the reagents and equipment used were listed in the Table of Materials.

1. Preoperative preparation

1. Feed the animals a normal chow diet and provide clear water under standard conditions (temperature 21–24 °C, humidity 40%–60%, 12-h light-dark cycle).
2. Sterilize all surgical instruments and materials before use. Disinfect the surgical platform with 75% ethanol. Preheat a heating pad to approximately 38 °C before surgery.
3. Place the hamster in an anesthesia induction chamber supplied with 3% isoflurane (following institutionally approved protocols).
4. Confirm deep anesthesia by the absence of a pedal withdrawal reflex. Maintain anesthesia via a nose cone with 1.5%–2% isoflurane.
5. Remove the hair from the mid-left lumbar region using electric clippers.
6. Disinfect the exposed skin with povidone-iodine.

2. Surgical procedure

1. Make an approximately 1.5–2 cm longitudinal incision in the skin along the midline of the left lumbar region using a scalpel. Bluntly dissect the underlying muscles to separate the latissimus dorsi from the iliocostalis layer. Create an approximately 1 cm access channel along the direction of the muscle fibers.
2. Gently retract the retroperitoneal fat using blunt forceps to expose the kidney. Exteriorize the left kidney and identify the renal pedicle. Separate the renal artery and vein using blunt forceps.
3. Place a 0.27 mm diameter needle adjacent to the renal artery. Loop a 6-0 polypropylene suture around both the artery and the needle. Tie a single overhand knot loosely.
   1. Carefully withdraw the needle, leaving a circumferential constriction that reduces the luminal diameter to 0.27 mm. Confirm successful stenosis by observing visible narrowing, but not complete cessation, of blood flow accompanied by a mild change in renal color.
4. Return the kidney to the renal fossa. Close the muscle layer and skin separately using 4-0 and 3-0 nonabsorbable polypropylene sutures, respectively. Disinfect the surgical area with povidone-iodine followed by ethanol.
5. Place the animal on a heating pad and monitor respiratory status until consciousness returns. Administer carprofen (5 mg/kg, subcutaneously) for up to 3 days after surgery. House the hamster individually for at least 1 week.

3. Blood pressure measurement

NOTE: Measure blood pressure non-invasively at 2 and 4 weeks post-operation using a Tail-cuff blood pressure monitoring system.

1. Anesthetize the hamsters in a chamber supplied with 3% isoflurane.
2. Place the animal in a prone position on an operating board and fit a mask to maintain anesthesia.
3. Attach a cuff to the forelimb and record blood pressure for at least 2 min for each animal.

4. Ultrasound examination

NOTE: Assess renal hemodynamics and structural adaptation 4 weeks after surgery using color Doppler and three-dimensional volume ultrasound systems.

1. Anesthetize the hamsters in a chamber supplied with 3% isoflurane.
2. Place the animal in a prone position on an operating board and fit a mask to maintain anesthesia.
3. Remove abdominal hair using a depilatory cream to ensure optimal acoustic contact. Apply coupling gel before placing the ultrasound probe.
4. Position the probe over the left kidney and obtain a clear longitudinal B-mode image.
5. Measure renal length and cortical thickness using the caliper function.
6. Activate color Doppler mode and visualize renal blood flow.
7. Record peak systolic velocity, end-diastolic velocity, and calculate the resistive index.
8. Perform three-dimensional scanning to reconstruct renal volume.
9. Save all images and measurement data.
10. Remove the probe and monitor the animal until recovery.

Results

In this study, these hamsters were randomly divided into two groups: 8 in the sham surgery group and 11 in the surgical group. All data are presented as means ± standard deviations unless otherwise stated. The approximate operative time was 20–25 min per animal. There were no intraoperative deaths, and the survival rate within 9 weeks of surgery was 100%. 2 weeks after surgery, noninvasive blood pressure was measured. The surgery, with or without ligation, had a lesser effect on appetite, and body weight in both groups s...

Discussion

The two-kidney, one-clip (2K1C) model is a classic animal model in renal vascular hypertension research. With historical roots dating back to the last century, it effectively simulates the pathogenesis of human renovascular hypertension. The underlying mechanism involves renal artery stenosis-induced ischemia, which activates the renin-angiotensin system (RAS), ultimately leading to systemic hypertension. This model not only closely mirrors the pathological progression of human hypertension but also offers significant ad...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.37 wire diameter suture	Johnson & Johnson MedTech, China	JH-18-5100
6-0 Polypropylene suture	Yangzhou Huanyu Medical Equipment Co., Ltd., China	N/A
Animal anesthesia machine	Midmark Corporation, USA	V2330299
Carprofen	Macklin, China	C830557-5g
Cholesterol Assay Kit	Zhongsheng Beikong, China	100000180
Color Doppler ultrasound system	Mindray Medical, China	Resona8
Glucose Assay Kit	Zhongsheng Beikong, China	100000240
High-density lipoprotein cholesterol assay kit	Zhongsheng Beikong, China	100020235
LDLR KO Syrian golden hamster	Peking University, China	N/A
Muromachi MK-2000ST Non-Invasive Blood Pressure Monitor for Mice & Rats	Muromachi Kikai Co., Ltd., Japan	140305MU
Pet heating pad	Meitai, China	DXT30*35-1X
Rat Renin ELISA kit	Covide, China	KWD-E7398R-B
Suture needle	Johnson & Johnson MedTech, China	JH-18-5037
Triglyceride Assay Kit	Zhongsheng Beikong, China	100000220
Vertical pressure steam sterilizer	Yamato Scientific, Japan	J3170372