An Immature Murine Model of Reversible Unilateral Ureteral Obstruction

Summary

The present protocol describes a step-by-step, reproducible model of unilateral ureteral obstruction.

Abstract

Unilateral ureteral obstruction (UUO) is a common cause of chronic kidney disease (CKD), leading to the progression of renal interstitial fibrosis and ultimately resulting in irreversible kidney damage. The alleviation of UUO is crucial. Several animal models of reversible unilateral ureteral obstruction (RUUO) have been established in the literature, enabling the observation of structural changes and functional damage while also simulating physiological and pathophysiological changes following the relief of ureteral obstruction. In this study, a reversible obstruction model was established in the unilateral murine ureter using a silicone tube. Significant renal damage was observed prior to obstruction relief, with partial recovery noted afterward. Unlike UUO, this model prevents progressive hydronephrosis, leading to distinct pathological outcomes. This simple surgical procedure demonstrates a high success rate and holds promise as a classical model for investigating reversible obstructive nephropathy and potential treatments for renal interstitial fibrosis. Furthermore, it provides a practical platform for studying the mechanisms of recovery from obstructive nephropathy, renal cell regeneration, and tissue remodeling.

Introduction

Urethral obstruction significantly contributes to renal interstitial fibrosis and chronic kidney disease (CKD), potentially leading to irreversible structural damage and functional impairments in the kidney¹. While unilateral ureteral obstruction (UUO) is widely used to study kidney injury and CKD, it does not accurately replicate the spontaneous recovery mechanisms that occur after the removal of an obstruction. The UUO model involves ligating the left ureter with sutures, resulting in permanent obstruction, ureteral dilation, hydronephrosis, compression of the renal parenchyma, and cortical thinning. Histological examination typically reveals tubular dilation, tubular epithelial cell necrosis, and progressive interstitial inflammation and fibrosis². This model primarily investigates renal interstitial fibrosis and irreversible kidney function loss due to persistent obstruction.

However, many renal diseases encountered in clinical practice, such as obstruction caused by ureteral calculi or tumors, are reversible. The reversible unilateral ureteral obstruction (RUUO) model allows for the partial restoration of kidney structure and urinary tract function, ultimately resolving hydronephrosis. Recovery can be assessed through imaging techniques, histological examination, and biomarker analysis to quantify the reduction in kidney injury and fibrosis³. This model closely mimics the recovery phase of obstructive nephropathy in clinical settings and is more suitable than UUO for studying key processes such as inflammation, immune responses, cell regeneration, and tissue remodeling^4,5,6,7,8.

The RUUO model enables researchers to analyze renal repair and regeneration following injury relief, addressing the limitations of UUO in dynamic studies. By comparing different time points before and after obstruction, researchers can investigate molecular pathways involved in injury and repair, including inflammation, apoptosis, fibrosis, and regeneration. This approach enhances understanding of renal recovery mechanisms and identifies potential therapeutic targets^{2,3,4,5,8,9,10}. While renal fibrosis is often considered irreversible, clinical observations suggest that early relief of obstruction during initial fibrosis stages may halt or even reverse disease progression. The RUUO model provides a valuable experimental platform for investigating this phenomenon¹¹.

Moreover, the RUUO model facilitates the study of fibrosis reversal following obstruction relief, offering insights into recovery mechanisms and potential antifibrotic therapies^3,4. Consequently, this model is highly practical for translational research. The primary objective of this experimental model is to induce obstructive nephropathy through ureteral cannulation, followed by standardized relief at a predefined time point to ensure consistency. It is optimized for simplicity, reproducibility, and safety, making it an effective tool for experimental research.

Protocol

This animal study adhered to the guidelines of the Declaration of Helsinki and was approved by the Research Ethics Committee of the Children's Hospital of Chongqing Medical University. A total of 27 male Sprague Dawley (SD) rats were commercially obtained and housed in the Laboratory Animal Center of the Children's Hospital of Chongqing Medical University (SPF, license number: SYXK (Chongqing) 2007-0016). The rats were maintained under controlled temperature conditions with a 12-h light/dark cycle and had ad libitum access to food and water.

The protocol was conducted on male SD rats aged 6-8 weeks and is applicable to rats of all ages with bilateral ureters. In this study, fifteen 6-week-old male SD rats were randomly assigned to three groups: the native group (n = 5), the UUO group (n = 5), and the RUUO group (n = 5). Additionally, five 8-week-old SD rats (n = 5) were included as an additional control group. To establish the RUUO model, 12 rats were used, with 7 additional rats procured to account for potential risks such as intraoperative and postoperative mortality, surgical failures, incomplete obstruction, and unsuccessful reversal. This ensured a minimum of 5 rats per group for subsequent analyses.

All surgical procedures were conducted in strict accordance with institutional and national guidelines for laboratory animal care and use. Surgical staff adhered to personal protective equipment (PPE) protocols, including surgical masks, gloves, and gowns. Sterile surgical instruments were used for each procedure and were autoclaved before and after use to maintain sterility. Waste materials, including sharps and biological specimens, were disposed of in compliance with hazardous waste management protocols to mitigate contamination risks and ensure safety.

1. Animal and instrument preparation

1. Conduct all procedures using sterile (autoclaved) instruments and consumables. Cut the sterilized silicone tube (inner diameter: 1.5 mm, outer diameter: 2.5 mm) into approximately 1 cm segments. Make a longitudinal incision along one side of the tube wall for subsequent use.
2. Anesthetize the rats via intraperitoneal injection of pentobarbital (40 mg/kg) (following institutionally approved protocols). Confirm adequate anesthesia by checking for the absence of reflex responses, such as the pedal withdrawal reflex, upon toe pinch. Apply veterinary ophthalmic ointment to the eyes to prevent corneal drying during anesthesia.
3. Depilate the rat's abdomen from the xiphoid process to the pubic symphysis and extend bilaterally to the midline.
4. Position the rat in a supine position on a heated surgical pad and secure its limbs with rubber ropes.
5. Drape a sterile fenestrated sheet to maintain a sterile field. Prepare the skin with povidone-iodine solution. Make a midline skin incision along the abdomen, extending from the subxiphoid region to just below the umbilicus, to provide adequate exposure of the kidneys and upper ureters.
6. Incise the subcutaneous tissues and fascia along the midline using surgical scissors. Dissect the skin and underlying tissues meticulously layer by layer, and fully expose the retroperitoneal space using tissue forceps.

2. Obstructive surgery for reversible unilateral ureteral obstruction

1. Retract the bowel to the right side of the abdominal cavity using a sterile swab to facilitate direct visualization of the left ureter. Cover the ureter with saline-soaked gauze to prevent desiccation.
2. Dissect and mobilize the left ureter using microscopic forceps, freeing approximately 1.5 cm from the surrounding tissues.
3. Place a 1 cm long silicone tube (inner diameter: 1.5 mm, outer diameter: 2.5 mm) beneath the freed ureter. Use forceps to ensure complete encasement within the tube.
4. Ligate the silicone tube and the middle portion of the ureter using 3-0 silk thread to induce ureteral obstruction. Avoid excessive ligation force. Gradually pull the silicone tube along the ureter's longitudinal axis to ensure secure but non-slipping ligation.
5. Reposition the bowel within the peritoneal cavity carefully, ensuring proper alignment without tension or obstruction.
6. Suture the abdominal muscle and fascial layers using a 2-0 non-absorbable suture with a curved cutting needle in a continuous fashion to provide adequate tensile strength. Close the skin with a 4-0 non-absorbable suture, ensuring anatomical alignment and even tension to promote healing and minimize the risk of wound dehiscence.
7. Disinfect the incision site with povidone-iodine solution. Allow the rats to recover under controlled conditions at a constant temperature for 7 days.

3. Relief surgery of reversible unilateral ureteral obstruction

1. Prepare the necessary animals and instruments, ensuring a sterile setup for abdominal reopening and full exposure of the abdominal cavity.
2. Dissect the knot of the silicone tube carefully using a scalpel blade. Remove the tube and irrigate the abdominal cavity with normal saline to minimize adhesion and infection risk.
3. Reposition the intestine and suture the abdominal wall incision in layers using 4-0 non-absorbable sutures. Sterilize the incision site with povidone-iodine solution. Place the rat in a controlled-temperature environment for a 7-day postoperative recovery period.
4. On postoperative day 14, anesthetize the rats (following the procedure mentioned in step 1.2) and collect kidney samples by transversely sectioning the kidneys into two halves⁹.
5. Store one half in 4% paraformaldehyde for histopathological examination, and rapidly freeze the other half in liquid nitrogen for storage at −80 °C for subsequent molecular analysis. Collect blood samples for biochemical analyses.
6. Perform euthanasia via CO₂ asphyxiation followed by cervical dislocation following ethical guidelines.

4. Follow-up assessments

1. Track body weight post-RUUO to assess overall recovery. Compare weight changes with the control and UUO groups.
2. Measure kidney weight and kidney volume to evaluate renal recovery.
3. Monitor serum creatinine (Scr) levels as an indicator of renal function improvement⁹.
4. Inject methylene blue into the renal pelvis to confirm ureteral patency. Observe ureteral peristalsis and coloration to assess post-obstruction recovery.
5. Perform H&E staining to assess tubular integrity and renal structure after RUUO^8,10.
6. Conduct Masson's trichrome staining to evaluate renal interstitial fibrosis regression^8,10.
7. Compare renal damage scores^3,8 between the UUO and RUUO groups to quantify tissue recovery.

Results

The effects of UUO and its subsequent release (RUUO) on body weight, kidney weight, kidney volume, and serum creatinine (Scr) levels were evaluated, as summarized in Table 1. Data are presented as mean ± standard deviation (SD), with n = 5 per group.

At 6 weeks, the native group exhibited a mean body weight of 234 g ± 16 g, kidney weight of 0.9107 g ± 0.0475 g, and kidney volume of 0.8962 cm³ ± 0.0502 cm³. By 8 weeks, the control group showed sign...

Discussion

This model employs a silicone tube to encircle the ureter, providing structural support, followed by ligation with a silk thread to induce complete ureteral obstruction through compression. After seven days, the ligation and silicone tube are removed to facilitate kidney decompression and the restoration of urinary tract integrity and functionality.

Silicone tubing, manufactured from silicone elastomers, offers excellent flexibility, biocompatibility, chemical resistance, and thermal stability...

Materials

Name	Company	Catalog Number	Comments
Forceps	Shanghai Medical Devices Co.,Ltd	20220032
Gauze	Sichuan Kelun Co., Ltd	20172140152
Hematoxylin and Eosin Stain Kit	Solarbio	G1120
Insulin needles	KDL Medical Devices	20193140938
Masson’s Trichrome Stain Kit	Solarbio	G1340
Medical Cotton balls	Sichuan Kelun Co., Ltd	20170037
Medical Cotton sticks	Sichuan Kelun Co., Ltd	20172140026
Methylene blue	Tianjin Dengfeng Chemical Reagent Factory	14038-43-8
Microscopic forceps	Suqian Shifeng Medical Devices Co., Ltd	S50985
Needle holders	Suqian Shifeng Medical Devices Co., Ltd	S7005
Povidone-iodine Solution	Sichuan Kelun Co., Ltd	514001
Saline	Sichuan Kelun Co., Ltd	20220004
SD Rats	SPF(Beijing)Biotechnology Co.,Ltd	D025
Silicone tubing	Taizhou Chunshi New Materials Co., Ltd	CS356
Silk suture	Qiangsheng Medical Devices Co.,Ltd	SA84G
Surgical blade	Huanan Yunyue Medical Devices Co.,Ltd	CE0434
Surgical scissors	Shanghai Medical Devices Co.,Ltd	J21130
Syringe	Tongmai medical devices	20183140304
Tissue Forceps	Jiangxi Yuyuan Medical Equipment Co., Ltd	J36030