Establishment of A Mouse Model of Renal Fibrosis via Unilateral Ischemia-Reperfusion Injury And Delayed Contralateral Nephrectomy

Summary

Here, a protocol is presented to establish a mouse model of renal fibrosis using unilateral ischemia-reperfusion injury followed by delayed contralateral nephrectomy. This strategy enables longitudinal studies of the AKI-to-CKD transition and precise assessment of residual kidney function at the endpoint.

Abstract

Renal ischemia-reperfusion injury (IRI) contributes to acute kidney injury (AKI) and is a major driver of progression to chronic kidney disease (CKD). A reproducible animal model is necessary to facilitate the evaluation of maladaptive repair and fibrosis mechanisms. Herein is reported a standard mouse model of renal fibrosis established by unilateral ischemia-reperfusion injury (uIRI) followed by contralateral nephrectomy (CNx). This strategy ensures long-term animal survival during disease progression while enabling the precise assessment of residual renal function of the injured kidney at the time of harvest. Male C57BL/6 mice were subjected to warm ischemia for 28 min at 37 °C, and a CNx was performed 24 h prior to each study endpoint. Mice were sacrificed for histological and immunohistochemical evaluations at the following time points: 2 days, 5 days, 1 week, 2 weeks, 3 weeks, and 4 weeks post-IRI. On postoperative days 2 and 5, the kidneys primarily exhibited tubular necrosis, particularly at the corticomedullary junction, without evidence of interstitial fibrosis. Starting one week after surgery, these pathological features were replaced by inflammatory cell infiltration and early tubular atrophy; interstitial fibrosis also emerged and reached its peak severity by the second week. During the third and fourth weeks after surgery, the kidneys showed widespread tubular atrophy consistent with disuse atrophy. Notably, interstitial fibrosis was reduced compared to the second week, indicating progression to the quiescent, hypocellular scarring phase. This model effectively reproduces the clinical scenarios of the AKI-to-CKD transition and provides a reliable platform for exploring fibrotic mechanisms and screening therapeutic interventions.

Introduction

Renal ischemia-reperfusion injury (IRI) is one of the critical factors leading to acute kidney injury (AKI) in the clinical setting¹. Common etiologies include renal transplantation (involving cold and warm ischemia followed by reperfusion of the donor kidney), cardiovascular or renal surgeries requiring temporary vascular occlusion, and systemic hypoperfusion states caused by severe trauma, major hemorrhage, or sepsis².

The pathophysiology of IRI involves a complex biphasic process. Initially, during the ischemic phase, the interruption of oxygen and nutrient supply leads to metabolic derangement, ATP depletion, and cytoskeletal disruption in renal tubular epithelial cells (TECs)³. Subsequently, the restoration of blood flow during reperfusion, although necessary for salvaging the ischemic tissue, triggers a more complex "secondary insult."⁴ This phase of injury is driven by a cascade of events, including oxidative stress from the burst of reactive oxygen species (ROS), endothelial dysfunction, massive infiltration of inflammatory cells (such as neutrophils and macrophages), and activation of the complement system³. These complex cascades collectively exacerbate TECs' apoptosis and necrosis, constituting the primary pathological presentation of AKI.

Notably, IRI is not limited to acute, reversible damage. Substantial evidence indicates that incomplete or maladaptive repair following renal IRI is a core mechanism driving the transition to long-term chronic pathology. Under physiological conditions, the kidney initiates a repair program post-AKI, which effectively clears damaged cells and promotes the proliferation and differentiation of surviving TECs to restore the structure and function of the damaged tubules. However, following a severe or repetitive IRI insult, this repair process can become dysregulated, ultimately leading to tubular atrophy, renal interstitial fibrosis, and accelerating the development and progression of chronic kidney disease (CKD)^5,6.

Therefore, elucidating the mechanisms of the AKI-to-CKD transition and developing therapeutic interventions are urgent needs. Rodent models, particularly mouse models, have become essential and indispensable in vivo. tools for studying post-IRI fibrosis due to their well-defined genetic backgrounds and similarity to human disease progression. However, significant heterogeneity exists in current IRI methodologies (e.g., duration of ischemia, temperature control, unilateral vs. bilateral injury), which leads to poor reproducibility and limited inter-study comparability⁷.

To address these challenges, the present study details a standardized protocol for a modified unilateral ischemia-reperfusion injury (uIRI) model. Also, this protocol incorporates a contralateral nephrectomy (CNx) performed 24 h prior to the study endpoint. This strategy overcomes the high mortality associated with bilateral ischemia while enabling the precise assessment of residual renal function and fibrotic progression in the injured kidney. This model accurately recapitulates the dynamic AKI-to-CKD transition, providing a robust in vivo. platform for mechanistic exploration and therapeutic screening.

Protocol

All animal experiments were conducted in accordance with the Regulations for the Administration of Affairs Concerning Experimental Animals. The experimental protocol was approved by the Institutional Animal Care and Use Committee (IACUC) of Guangzhou Jennio Biotech Co., Ltd. (Approval No. JENNIO-IACUC-2025-A053). All procedures were performed in strict accordance with the Guide for the Care and Use of Laboratory Animals and institutional guidelines, fully adhering to the 3R principles. The timeline of the study, including the points of sacrifice and sample collection, is summarized in Figure 1.

1. Animal and instrument preparation

1. Use male C57BL/6 mice (8 weeks old, weighing 20–25 g) housed in specific pathogen-free (SPF) facilities for the study.
2. Divide the mice randomly into the uIRI group and the Sham group. For each of the six designated time points (2 days, 5 days, 1 week, 2 weeks, 3 weeks, and 4 weeks), allocate n = 4 mice per group.
   NOTE: Mice were maintained on a 12-h light/12-h dark cycle with free access to food and water. They were acclimatized to the laboratory environment for at least one week prior to experimentation.
3. Sterilize all surgical instruments via .high-pressure steam autoclaving prior to use.
4. Maintain core body temperature strictly at 37.0 °C ± 0.5 °C using a homeothermic monitoring system equipped with a rectal probe for continuous temperature monitoring. Insert the lubricated probe into the rectum immediately after induction of anesthesia and keep it in place throughout the procedure.
5. Anesthetize mice using a small-animal gas anesthesia system with isoflurane (5% for induction, 1.5% for maintenance, with an oxygen flow rate of 1.0 L/min via. a calibrated vaporizer).
   NOTE: Confirm deep anesthesia by the absence of a pedal withdrawal reflex. All surgical procedures must be performed only after the mouse has reached a state of deep anesthesia.
6. Shave the fur on the left dorsal flank area.

2. Surgical procedure

1. uIRI model (Day 0)
   1. Position the mouse in right lateral decubitus to expose the left flank.
   2. Make a 1 cm to 1.5 cm longitudinal skin incision on the left back, followed by a muscle incision to enter the retroperitoneal space.
   3. Gently push out the left kidney using a sterile cotton swab moistened with saline.
      NOTE: The kidney should only be moved by manipulating the peri-renal fat pad or using sterile cotton swabs. Forceps should never touch the kidney surface.
   4. Carefully dissect the renal pedicle using fine-tip forceps to isolate the renal artery and vein from the surrounding connective tissue.
   5. Clamp the left renal pedicle using a non-traumatic vascular micro-clip to induce ischemia.
      NOTE: The renal pedicle dissection must be performed cleanly and completely. The procedure should explicitly exclude the ureter from the clamp application. Inadvertent ureteral clamping would induce a hybrid IRI/Unilateral Ureteral Obstruction (UUO) injury, potentially introducing a permanent obstructive component.
   6. Start a timer immediately upon clamping. The duration of ischemia is set strictly to 28 min.
   7. Verify ischemia visually: the kidney color should change from bright red to dark purple within seconds.
   8. Cover the kidney with sterile gauze soaked in warm saline.
   9. After 28 min, remove the clamp to initiate reperfusion.
   10. Confirm reperfusion visually: the kidney color should return to pink/red within a few minutes.
   11. Gently return the kidney to the abdominal cavity.
   12. Close the muscle layer with 5-0 absorbable sutures and the skin incision with 5-0 nylon sutures.
2. Sham operation (Day 0)
   1. Perform the same anesthesia, incision, and kidney exteriorization steps as described in step 2.1.
   2. Dissect the renal pedicle but do not clamp the vessels.
   3. Keep the kidney exteriorized for the same duration (28 min) before returning it to the abdominal cavity and closing the incision.
3. CNx surgery
   NOTE: Perform CNx 24 h prior to the scheduled sample collection endpoint for each specific group to assess the residual function of the injured kidney.
   For the 2-day group: Perform CNx on Day 1 post-IRI.
   For the 5-day group: Perform CNx on Day 4 post-IRI.
   For the 1-week group: Perform CNx on Day 6 post-IRI.
   For the 2-week group: Perform CNx on Day 13 post-IRI.
   For the 3-week group: Perform CNx on Day 20 post-IRI.
   For the 4-week group: Perform CNx on Day 27 post-IRI.
   1. Anesthetize the mouse as described in step 1.5. Then, shave and disinfect the right dorsal flank.
   2. Make a small flank incision on the right side to expose the right kidney.
   3. Exteriorize the right kidney and gently dissect the renal pedicle.
   4. Ligate the renal pedicle (artery, vein, and ureter) tightly using a 4-0 non-absorbable silk suture.
   5. Excise the right kidney distal to the ligature.
      NOTE: Ensure there is no active bleeding from the stump.
   6. Return the pedicle stump into the abdominal cavity and close the incision as described in step 2.1.12.
   7. Allow the mouse to recover on a heating pad.

3. Postoperative care and monitoring

1. Keep the mice on the heating pad until they fully recover consciousness and mobility.
2. Return the mice to clean cages with easily accessible food and water.
3. Monitor the health status of the mice daily.
   1. Check for mortality daily.
   2. Monitor for changes in grooming, signs of distress or pain, and posture.
   3. Inspect incisions for signs of infection, inflammation, or dehiscence.

4. Postoperative assessments

1. Sample collection
   NOTE: Mice were sacrificed at designated time points: 2 days, 5 days, 1 week, 2 weeks, 3 weeks, and 4 weeks post-IRI to evaluate the progression of injury and fibrosis.
   1. Anesthetize the mice with 3% isoflurane.
   2. Collect blood samples for serum biochemical analysis (if required).
   3. Euthanize the mice by cervical dislocation.
   4. Harvest the left (injured) kidney rapidly.
   5. Section the kidney tissue: fix one portion in commercial 4% paraformaldehyde for histological analysis, and snap-freeze the remaining tissue in liquid nitrogen and store at -80 °C for molecular analysis.
2. Tissue processing and sectioning
   1. Dehydrate, clear, and embed the fixed tissues in paraffin wax, strictly following the tissue processing protocol described by Feldman and Wolfe⁸.
   2. Section the paraffin blocks at a thickness of 4 µm using a rotary microtome.
   3. Mount the tissue sections onto glass slides and bake them at 60 °C for 2 h to ensure tissue adhesion.
3. Hematoxylin and Eosin (H&E) staining
   1. Deparaffinize the tissue slides in xylene and rehydrate them through a descending graded ethanol series to distilled water.
   2. Perform H&E staining to evaluate morphological damage. Execute the staining and washing steps exactly as detailed in the standardized protocol by Feldman and Wolfe⁸.
   3. Dehydrate, clear, and coverslip the stained slides using a resinous mounting medium.
4. Masson’s Trichrome staining
   1. Deparaffinize and rehydrate the tissue sections to distilled water.
   2. Apply the mordant, nuclear, cytoplasmic, and collagen dyes sequentially to assess collagen deposition and renal fibrosis. Follow the exact reagent incubation times and wash steps outlined by Islam and Kumar⁹.
   3. Rinse, dehydrate, clear, and coverslip the slides for microscopic evaluation.
5. Immunohistochemistry (IHC)
   1. Deparaffinize and rehydrate the tissue sections.
   2. Perform heat-induced epitope retrieval using a 10 mM sodium citrate buffer containing 0.05% Tween-20 (pH 6.0) as outlined by Magaki et al.¹⁰.
   3. Block endogenous peroxidase activity by incubating the sections in 3% hydrogen peroxide.
   4. Block non-specific binding sites using 5% bovine serum albumin in PBS for 30 min at room temperature.
   5. Incubate the sections with the anti-alpha-smooth muscle actin (α-SMA) primary antibody (1:400 dilution) overnight at 4 °C.
   6. Wash the slides and incubate them with the secondary antibody for 1 h at room temperature.
   7. Visualize the target antigen using DAB and counterstain with hematoxylin, adhering to the procedural steps provided by Magaki et al.¹⁰.
   8. Dehydrate, clear, and coverslip the slides for final imaging.

5. Statistical analysis

1. Present all quantitative data as mean ± standard deviation (SD).
   NOTE: Statistical analyses and graph generation were performed using GraphPad Prism software version 10.0. Differences among multiple groups compared to a single control group (Sham) were analyzed using an Ordinary One-Way Analysis of Variance (ANOVA) followed by Dunnett’s multiple comparisons test. A P-value of < 0.05 was considered statistically significant (*P < 0.05, **P < 0.01, ***P < 0.001, ****P. < 0.0001). ns indicates no significant difference.

Results

Throughout the entire experimental period, the overall survival rate was 100% for both the Sham group and the uIRI group (n = 4 per time point). No mortality was observed during either the initial uIRI surgery or the subsequent delayed CNx procedure. This result confirms the high safety profile and reliability of this two-stage protocol compared to bilateral ischemia models, which typically suffer from significant uremia-induced mortality.

H&E staining, Masson’s trichrome staining, and IHC...

Discussion

The present study describes a standardized protocol for establishing a mouse model of renal interstitial fibrosis using uIRI, followed by CNx performed 24 h prior to the study endpoint. Dynamic histopathology screening revealed distinct pathological phases: acute tubular necrosis characterized by peak incidence within days 2–5, followed by active fibrogenesis and myofibroblast activation peaking at 1–2 weeks, and notably, by weeks 3–4, the tissue transitioned to a chronic phase characterized by stable c...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Anti-α-SMA Antibody	Proteintech	14395-1-AP	Primary antibody used in IHC to detect α-SMA expression and assess myofibroblast activation
Blood Urea Nitrogen (BUN) Assay Kit	Nanjing Jiancheng Biotechnology Research Institute Co., Ltd.	C013-2-1	Used to quantify serum BUN levels for assessing residual renal function
Creatinine (CRE) Assay Kit	Nanjing Jiancheng Biotechnology Research Institute Co., Ltd.	C011-2-1	Used to quantify serum creatinine levels for assessing residual renal function
Gas Anesthesia System	Nanjing Calvin Biotechnology Co., Ltd.	KW-MZJ-4	Equipped with isoflurane for general anesthesia
GraphPad Prism Software	GraphPad Software, Inc.	N/A	Used for statistical analysis and graphical representation of data (https://www.graphpad.com)
Homeothermic Monitoring System	Conduct Science	CS-HS-0001	Equipped with a rectal probe for closed-loop, continuous core body temperature monitoring and regulation
ImageJ Software	National Institutes of Health	N/A	Open-source software used for morphometric quantification of fibrotic areas and IHC signal
Isoflurane	RWD Life Science Co.,Ltd	R510-22	Inhalant anesthetic agent used during surgical procedures
Masson’s Trichrome Staining Kit	Beyotime	C0189M	To quantify collagen deposition
Non-traumatic Vascular Micro-clip	ROBOZ SURGICAL INSTRUMENT CO.	RS-5481T	Used to clamp renal pedicle for 28 min
Paraformaldehyde (4%)	Beyotime Biotech Inc	P0099-100ml	Used for rapid fixation of harvested kidney tissue samples
Sodium Citrate Buffer (10 mM, pH 6.0)	Thermo Fisher Scientific Inc.	005000	Used for heat-induced epitope retrieval prior to immunohistochemical staining
Stereotaxic Surgical Microscope	Beijing Zhongtian Guangzheng Technology Co., Ltd.	TS-39NK	Used for performing IRI surgery