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 derange....

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 ....

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