Unilateral Ureteral Obstruction Model for Investigating Kidney Interstitial Fibrosis

Summary

This protocol describes a method for inducing unilateral ureteral obstruction (UUO) in mice to study the progression of tissue fibrosis in obstructive nephropathy. It includes surgical procedures, post-operative care, and methods for fibrosis assessment.

Abstract

Kidney fibrosis is the final pathological outcome of progressive chronic kidney disease (CKD). The unilateral ureteral obstruction (UUO) model is widely used to elucidate the molecular and cellular mechanisms underlying kidney interstitial fibrosis and to identify potential therapeutic targets. This model is established in mice through surgical ligation of a unilateral ureter, a procedure that is relatively straightforward and easy to perform. However, the UUO mouse model is known to exhibit significant variability and inconsistency, influenced by factors such as mouse strain, age, sex, anesthesia type, duration of surgery, body temperature during the procedure, the operator's surgical skills, feeding conditions, and the overall health status of the mice. Variations in surgical techniques, suture placement, and the duration of obstruction contribute to the variability in outcomes. Additionally, inconsistent sampling of obstructed kidneys further increases variability in the assessment of kidney fibrosis. This study outlines the process of developing the UUO mouse model and evaluating interstitial fibrosis, discusses the technical challenges contributing to the model's unpredictability, and proposes potential solutions. These insights aim to establish a more standardized and universally applicable approach for investigating kidney fibrosis.

Introduction

Chronic kidney disease (CKD) affects over 10% of the global population, and its prevalence is increasing¹. Various urinary tract conditions, including congenital anatomical anomalies, nephrolithiasis, prostatic hyperplasia, and bladder tumors, can lead to ureteral obstruction². As a result, the unilateral ureteral obstruction (UUO) mouse model is a key tool for identifying new mechanisms of kidney interstitial fibrosis, understanding disease progression, and evaluating potential treatment strategies. It has been widely used to investigate the origin of myofibroblasts, (myo)fibroblast subclusters, tubular cell metabolism, and cell cycle arrest, partial epithelial-mesenchymal transition, and other related processes^3,4,5,6,7,8.

In addition to UUO-induced kidney interstitial fibrosis, other commonly used rodent models of kidney interstitial fibrosis include toxin-induced models, such as those using aristolochic acid, folic acid, and adenine, as well as surgically induced models like 5/6 nephrectomy and ischemia-reperfusion injury (IRI). The UUO model offers several advantages over alternative kidney fibrosis models. For example, toxin-induced kidney fibrosis requires a relatively long modeling period (approximately 1-2 months), and its toxic side effects on other organs can complicate the investigation of fibrosis mechanisms^9,10,11. Surgically induced models, such as 5/6 nephrectomy, can lead to significant kidney bleeding and infection, increasing the risk of post-operative mortality. Additionally, the extent of induced interstitial fibrosis is directly correlated with the volume of resected kidney tissue, making it challenging to consistently reproduce the same degree of fibrosis in each mouse¹².

The renal IRI model is a primary method for inducing acute kidney injury to CKD and has significant clinical relevance. The severity of fibrosis can be modulated by adjusting ischemic time and body temperature; however, compared to the UUO model, it is more surgically complex, and the induction of interstitial fibrosis requires a longer duration¹³. Compared to these models, the UUO model has several advantages, including a short modeling duration, minimal variability, repeatability, and a relatively simple surgical procedure. The UUO mouse model, which does not involve toxins, is created by ligating one ureter, leading to obstructive nephropathy within two weeks. This results in hydronephrosis, tubular dilatation, and interstitial fibrosis, closely resembling the pathological process observed in humans¹⁴. The severity of fibrosis -- mild, moderate, or severe -- can be controlled by adjusting the experiment's duration.

Although the UUO mouse model is simpler to perform than other insult-induced models for investigating CKD, several factors can significantly affect its stability. These factors include mouse strain, age, sex, type of anesthesia, surgery duration, body temperature during surgery, the surgical skills of the operator, and the feeding conditions and health status of the mice^15,16.

Minimizing surgical stress and infection while performing the procedure in a steady and organized manner under anesthesia is essential for creating a reproducible UUO mouse model. Additionally, research on the mechanisms and potential therapeutic targets of CKD can be compromised by inexperienced operators, leading to increased mouse loss and greater model heterogeneity. To address these challenges, key technical aspects of the surgical process -- before, during, and after the procedure -- are outlined, highlighting critical issues that require attention. Furthermore, the evaluation methodology for the UUO mouse model is detailed to provide researchers with a consistent and reliable approach.

Protocol

All animal procedures are conducted in accordance with agency guidelines and approved by the Institutional Animal Ethics Committee of Nanjing Medical University. To eliminate sex and strain differences and ensure comparability of results, only male CD1 mice aged 8-10 weeks and weighing 22-25 g are used. The details of the reagents and equipment used in this study are listed in the Table of Materials.

1. Animal and instrument preparation

1. Autoclave all surgical instruments before surgery.
2. Weigh and anesthetize the mice (following institutionally approved protocols). Properly connect the inhalation anesthesia device and place the mice in the induction chamber with 2% isoflurane mixed with oxygen at 1-2 L/min. Then, place the mouse nose into the nose cone with a constant supply of anesthesia. Confirm the appropriate level of sedation by assessing the lack of response to a toe pinch.
   NOTE: Regularly check the respiration rate and assess the level of anesthesia using the pedal reflex of the mice. Adjust the isoflurane as appropriate.
3. Place the mouse on an electronic heat pad in a supine position with the head and neck extended to keep the airway open. Secure the paws with low-adhesive tape.
4. Use a thermometer to monitor the ambient temperature and maintain it at 37-38 °C. Insert a rectal probe and keep the core body temperature of the mice around 36.5-37 °C during the procedure.
5. Apply ophthalmic ointment to the eyes to prevent corneal xerosis injury.
6. Shave the hair over the surgical area with hair removal cream and disinfect it with betadine solution three times.

2. Surgical procedure

NOTE: Once the body temperature stabilizes at the set point and the toe pinch reflex is absent, initiate the following surgical procedures.

1. Use a surgical blade to make a vertical surgical incision of approximately 1- 1.5 centimeters from the bladder to the lower-left edge of the ribs to expose the abdominal cavity.
2. Move the intestine to the right side of the abdominal cavity using a cotton swab moistened with sterile normal saline to expose the left kidney, located below the back of the spleen and adjacent to the spine.
3. Use curved iris forceps to clear the fat and connective tissue surrounding the left ureter. After gentle isolation, ligate the left ureter close to the kidney's lower pole using a 3/0 silk braided suture. Perform a second ligation of the left ureter between the first ligation and the bladder.
   NOTE: To minimize variability among the mice, ensure that the ligation position remains consistent across all subjects. Use the left-hand forceps to place the suture into the tips of the right-hand forceps and slide the suture back under the ureter¹⁷. Avoid including excess fat and connective tissue in the ligation, as this may lead to incomplete kidney obstruction. Achieve this by gently stroking the ureter with dry cotton swabs.
4. Carefully reposition the intestines into the peritoneal cavity. Suture the muscle and skin layers separately using a 3/0 silk braided suture.
   NOTE: Suturing the skin and muscle layers together may cause suture wound rupture and abdominal viscera damage.
5. When operating on multiple mice, clean the surgical instruments with running water and 75% ethanol before proceeding to the next mouse.

3. Post-surgical care and monitoring

1. Inject 0.5 mL of warm normal saline intraperitoneally to prevent dehydration.
2. Place the mice back into a clean cage until they regain full consciousness.
3. Administer buprenorphine (50 µg/kg) by subcutaneous injection for 3 days. Monitor the mice daily for signs of lack of grooming, decreased eating, and abnormal posture.

4. Post-operative assessments

1. Histology
   1. Euthanize the mice with an overdose of isoflurane (following institutionally approved protocols). Harvest the obstructed and contralateral non-obstructed kidneys¹⁸ after euthanasia.
   2. Use the largest cross-sectional area of the kidney section for histological examination.
   3. Fix kidney tissues in 4% paraformaldehyde and cut the obtained samples into 4 µm sections.
   4. Stain kidney sections with periodic acid-Schiff (PAS), Masson's trichrome staining (MTS), or Sirius red staining to detect renal tubular injury and kidney fibrosis.
   5. Select ten random fields of the kidney sections under a light microscope at a magnification of 200×.
   6. Calculate the proportion of the collagen-positive blue area using ImageJ software.
2. Western blot
   1. Extract approximately 20 mg of kidney tissue from the same pole of UUO kidneys using radioimmunoprecipitation assay (RIPA) buffer¹⁸.
   2. Quantify the protein concentration using a bicinchoninic acid assay¹⁹.
   3. Load equal amounts of protein samples onto 4%-10% Bis-Tris gels for electrophoresis and transfer them to polyvinylidene difluoride (PVDF) membranes.
   4. Block the PVDF membranes with 5% milk in Tris-Buffered Saline Tween (TBST) and incubate them with primary antibodies overnight at 4 °C.
   5. Wash the PVDF membranes with TBST and incubate them with secondary antibodies at room temperature for 1 h.
   6. Detect protein levels using an enhanced chemiluminescence (ECL) method and analyze them using ImageJ software, with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as the loading control.
3. Renal Function
   1. Anesthetize the mice with inhaled isoflurane (see step 1.2).
   2. Collect blood samples retro-orbitally after anesthesia.
   3. Centrifuge blood samples at 3,000 x g for 15 min at room temperature.
   4. Measure serum creatinine and blood urea nitrogen (BUN) using an automatic dry chemical analyzer to monitor renal function.

Results

Histology
Periodic acid-Schiff (PAS) staining revealed tubular dilation, loss of brush borders, cast formation, and tubular epithelial swelling. Masson's trichrome and Sirius red staining showed interstitial fibrosis following UUO, in contrast to the normal compact tubules with discernible lumens observed in the sham group. The degree of renal interstitial fibrosis, indicated by blue areas in Masson's trichrome staining and red areas in Sirius red staining, increased in a time-dependent man...

Discussion

A comprehensive procedure for establishing the UUO model, a widely used approach for investigating kidney interstitial fibrosis, is provided. Additionally, the identification and assessment of the model, including evaluations of renal function and histological alterations, are demonstrated. The variables contributing to the model's heterogeneity and modifiable technical factors are discussed.

Susceptibility to UUO varies significantly based on age, sex, and mouse strain. Compared to C57BL/6 mi...

Materials

Name	Company	Catalog Number	Comments
1 mL Syringe	Mingankang	/
3/0 silk braided suture	Jinhuan Medical	F301
75% Ethanol	Lircon	6303060031
Anesthesia Air Pump	RWD Life Science	R510-29
Anesthesia Induction Chambers	RWD Life Science	V102-V
Animal hair clipper	Jinke	/
Betadine solution	Lircon	6303030036
Buprenorphine (analgesic)	RWD Life Science	/
Curved iris forceps	jinke	/
Electronic heat pad	Reptizoo	AHM23
fine straight forceps	Jinke	/
Gas Filter Canister	RWD Life Science	R510-31-6
Gauze Pads	Winner Medical	601-026576
Iris Scissors	Jinke	/
Isoflurane (anesthetic)	RWD Life Science	R510-22-10
Multi-output Animal Anesthesia Machine	RWD Life Science	R550IE
Needle holder	jinke	/
Ophthalmic ointment	Dechra NDC	17033-211-38
Sterile Cotton swab	Winner Medical	601-015213
Sterile saline	Shimen	H20066533