Unilateral Ureteral Obstruction Model for Investigating Kidney Interstitial Fibrosis

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.

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.

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.

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.

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 manner (Figure 1A).

Western blot
Fibronectin (FN), collagen I (Col-1), and α-smooth muscle actin (α-SMA) are commonly used markers to assess kidney fibrosis. Compared to the sham group, the expression levels of these fibrotic proteins were significantly elevated after UUO and positively correlated with the duration of obstruction (Figure 1B).

Real-time PCR
Compared to the sham group, the mRNA levels of fibrotic markers (FN, Col-1a1, α-SMA, and transforming growth factor-β1 (TGF-β1)), along with inflammatory cytokines such as monocyte chemoattractant protein-1 (MCP-1), tumor necrosis factor-α (TNF-α), and C-X-C motif chemokine ligand 1 (CXCL-1), were significantly increased in the UUO group (Figure 1C).

Influence of biological and surgical factors on UUO outcomes
The strain, age, and sex of mice significantly influence outcomes in UUO studies. A study established a reversible UUO mouse model consisting of six days of obstruction followed by seven days of reversal. While BALB/c mice exhibited nearly complete recovery of renal function to levels comparable to sham-operated controls, C57BL/6 mice experienced irreversible loss of renal function. For further details on renal function, refer to Puri et al.²⁰.

Sex differences also play a critical role in UUO models. In a comparison of male and female C57BL/6J mice after two weeks of UUO, males exhibited significantly higher levels of kidney interstitial fibrosis and increased collagen IV protein deposition in the tubular interstitium. Age is another key factor affecting interstitial damage following obstruction. Older mice (50 weeks) demonstrated more severe tubular dilation and atrophy compared to younger counterparts (16 weeks)²¹.

In addition to biological variations among mice, surgical parameters also influence UUO outcomes. Notably, TGF-β1 protein and mRNA expression were significantly suppressed in mice anesthetized with propofol compared to those receiving sevoflurane during the UUO procedure²². Furthermore, the duration of obstruction is directly correlated with renal function decline. Mice subjected to UUO for one to two days experienced complete recovery upon removal of the obstruction, whereas those obstructed for three days or longer developed time-dependent renal failure²⁰.

Figure 1: Kidney interstitial fibrosis induced by unilateral ureteral obstruction (UUO) on days 3, 7, and 14. (A) Periodic acid-Schiff (PAS), Masson's trichrome staining (MTS), and Sirius red staining of renal sections from sham and UUO kidneys. Scale bars = 100 µm. (B) Western blot analysis of fibronectin (FN), collagen I (Col-1), and α-smooth muscle actin (α-SMA) in sham and UUO kidneys. (C) Real-time PCR analysis of fibrosis-related markers and inflammatory cytokines in sham and UUO kidneys. Data are presented as mean ± standard error of the mean (SEM). Statistical analysis was performed using one-way ANOVA and Student's t-test. Statistical significance is indicated as follows: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Abbreviations: UUO, unilateral ureteral obstruction; PAS, periodic acid-Schiff; MTS, Masson's trichrome staining; FN, fibronectin; Col-1, collagen I; α-SMA, α-smooth muscle actin; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; TGF-β1, transforming growth factor-β1; MCP-1, monocyte chemoattractant protein-1; TNF-α, tumor necrosis factor-α; CXCL-1, C-X-C motif chemokine ligand 1.Please click here to view a larger version of this figure.

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 mice, BALB/c mice are less susceptible or even resistant to UUO²⁰. In studies involving male and female C57BL/6 mice, male mice exhibited greater Masson's trichrome staining and scoring, as well as increased collagen IV expression, following UUO²³. Additionally, the effects of sex differences on kidney fibrosis in UUO vary among transgenic and knockout mouse models²⁴. Age is another critical factor influencing UUO outcomes; older mice (50 weeks) demonstrate more severe tubulointerstitial atrophy compared to younger mice (16 weeks)²¹. Similar age-related effects on UUO outcomes have been observed in the Sprague-Dawley rat UUO model²⁵.

Extended anesthesia during surgery is associated with increased animal mortality²⁶. Rapid induction of surgical anesthesia was achieved using isoflurane, an inhaled anesthetic, which resulted in anesthesia onset within 5-10 min. This method allows the surgeon to more easily initiate, maintain, and terminate anesthesia compared to the intraperitoneal injection of sodium pentobarbital (100-200 mg/kg). In the UUO model, different anesthetics have varying effects on the severity of renal fibrosis. For example, propofol has been shown to exert protective effects against renal injury by downregulating inducible nitric oxide synthase expression in the UUO mouse model²²; therefore, its use should be avoided in UUO modeling.

Exposure of the kidney and ureter is essential for the success of the experiment. A key consideration is ensuring complete ligation of the ureter. A crucial aspect of this step involves carefully dissecting the adipose tissue surrounding the renal hilum. Selecting mice with lower body weight can be advantageous, as they typically have less adipose tissue around the kidney, facilitating easier exposure of the kidney and ureter while minimizing the risk of tearing surrounding tissue. This approach not only shortens the duration of the procedure but also reduces the amount of anesthesia required, thereby decreasing trauma and technical difficulty.

During kidney exposure, evaporation may cause blood vessels to adhere to the surgical tweezers, potentially leading to ureteral damage or even ureteral fistulas. Therefore, maintaining a moist operative field by continuously applying sterile normal saline is essential. To minimize clutter, it is advisable to cut the suture for ligation to a reasonable length (approximately 10 cm) before the procedure, allowing the operator to manage the ends more effectively¹⁴. Additionally, the second ligation should not be placed directly on top of the first, as this may prevent adequate blockage of the ureter.

Recovery of renal function in the UUO model is influenced by the duration of obstruction²⁷. The severity of fibrosis correlates directly with the duration of obstruction²⁸. In the classic UUO model described herein, appropriate time points for assessment are days 3, 7, and 14¹⁵. At day 3, early changes in cellular injury can be observed, including increased interstitial macrophage infiltration. By day 7, damage escalates, presenting a more differentiated pattern characterized by the initial signs of interstitial fibrosis and areas of tubular atrophy. Day 14 represents the optimal time point for evaluating the full phenotype, which includes significant hydronephrosis and loss of renal parenchyma. Studies in rats have demonstrated that permanent damage occurs after 72 h of obstruction, whereas 24 h of obstruction allows for complete recovery of the glomerular filtration rate within 14 days following reversal of the obstruction²⁷.

Post-surgical care is crucial for minimizing animal loss. Immediately after surgery, warm normal saline is administered via intraperitoneal injection to prevent dehydration in the mouse. Once the mouse is returned to its cage, providing easily accessible water and food is essential. The surgical procedure may impair the animal's mobility; therefore, if food and water are placed at elevated positions, mashed food should be kept on the floor of the cage for easier access. When monitoring the mice's post-operative condition, the dosage of analgesic medication should be appropriately increased for those exhibiting poor grooming, decreased feeding, or abnormal posture.

Several potential limitations of the UUO model should be noted. First, this model does not accurately track changes in renal function. Endogenous filtration markers, such as creatinine and blood urea nitrogen, are commonly used to assess renal function; however, their levels often remain stable because the non-obstructed kidney compensates for the function lost in the obstructed kidney. Second, the UUO model is unsuitable for studying cellular and tissue regeneration, as well as subsequent tissue remodeling following the release of the obstruction, due to the absence of tubular repair. Finally, the UUO mouse model has limitations in translating study findings from bench to bedside, as most clinical cases of UUO involve partial obstruction rather than complete obstruction.

In conclusion, the UUO model in mice is widely employed for research on kidney interstitial fibrosis. It has potential applications in studies aimed at identifying and characterizing markers of kidney fibrosis, examining pathogenic mechanisms, and exploring potential treatment approaches for CKD. Although variability in the mouse model can arise from numerous factors, the detailed procedure outlined in this study may assist researchers in developing a highly reproducible UUO model. Therefore, a proficient and skilled operator can produce a consistent UUO model by adhering to the described techniques and paying close attention to technical details.

The authors declare no conflict of interest.

This work was supported by National Science Foundation of China Grants (82370686/2024YFA1107704), Jiangsu Specially-Appointed Professor Grant, Nanjing Science and Technology Innovation Project, Jiangsu Province Hospital High-level Talent Cultivation Program (Phase I) (CZ0121002010037), Natural Science Foundation of Jiangsu Province (BK20240055), and Jiangsu Medical Innovation Team to JR; Jiangsu Province Hospital (the First Affiliated Hospital with Nanjing Medical University) Clinical Capacity Enhancement Project (JSPH-MA-2023-4), Priority Academic Program Development of Jiangsu Higher Education Institutions (China) and National Natural Science Foundation of China (81970639/82151320) to HM.