A Modified Two Kidney One Clip Mouse Model of Renin Regulation in Renal Artery Stenosis
October 26th, 2020
A modified 2 kidney 1 clip (2K1C) Goldblatt mouse model was developed using polyurethane tubing to initiate renal artery stenosis, inducing an increase in renin expression and kidney injury. Here, we describe a detailed procedure of preparing and placing the cuff onto the renal artery to generate a reproducible and consistent 2K1C mouse model.
The 2K1C mouse model is a reliable and reproducible method to induce renal artery stenosis. This protocol is valuable in elucidating molecular mechanisms involved in renin expression control during renal vascular hypertension. This animal model mimics unilateral renal artery stenosis in humans.
The reliability and reproducibility make it a good model to discern new therapeutic targets for treating renal vascular hypertension. This technique requires a basic knowledge of performing surgery in mice. There are two critical parts:tubing preparation, and placement of tubing in the renal artery.
Both are described in detail. Constriction of the right renal artery using precisely-cut tubing is key. This video of the procedure will show learners how to cut the tubing, then access and constrict the artery.
Begin by cutting the tubing for the stenosis procedure. Cut a 0.5-millimeter-length piece of polyurethane tubing with a sharp scalpel. Then make a cuff by removing 0.2-millimeters of the circumference with a lengthwise cut.
Record the weight of the mice, which should be between 18 and 22 grams, to perform renal artery stenosis with the polyurethane tube. The weight of the mice is critical to perform the surgery. After anesthetizing and administering the painkiller to the mouse, place it back inside the cage until it is fully unconscious.
Pinch the toe with forceps to check if the mouse is fully anesthetized and ready for the surgery. Lay the mouse on its back on a paper towel away from the surgery area and remove the hair of the lateral abdomen with an electric hair clipper following the opposite direction of hair growth. Clean the shaved area with a sterile alcohol gauze pad and apply topical povidone-iodine solution.
Open the sterilized bag containing all the surgical equipment. Working under the dissecting microscope, use sterile sharp scissors to make a small flank incision about 0.5 centimeters away from the vertebrae. Proceed along the lumbar vertebrae and make a one-inch incision.
Pull back the skin and muscle to expose the kidney. Then, clean and remove the surrounding fat with cotton swabs to isolate the renal artery. Isolate the renal nerve from the renal artery using the curved forceps.
Clean the oozed fluid near the renal artery with a cotton swab. Place two nylon sutures under the right renal artery, make loose knots, and place the cuff around the main renal artery approximately equidistant between the kidney and aorta bifurcation. Close the cuff using the nylon sutures, making three knots for each suture to avoid the probability of losing the sutures after the surgery.
Close the incision in the muscle by applying a simple continuous suture and make simple interrupted sutures to close the skin. Return the mice to their cage and leave the half cage on a circulating water heating pad for two to three hours for animals to recover from the surgery. Provide the animals with gel diet recovery food.
On the next day, administer painkiller. Weigh the mice for the next two days and consult with the veterinarian if a mouse loses more than 20%of its weight. Inspect the mice daily to assess for redness, swelling, pain, or infection.
Record the weight of each mouse. After euthanizing the animal, place it on a sterile platform in supine position. Secure and extend the limbs to limit movement and thoroughly spray the mouse with 70%ethanol.
Use scissors to make a midline incision and open the abdomen and chest area. Pull back the skin and peritoneum wall, then carefully expose the heart and puncture the right ventricle. Remove both kidneys using forceps, keeping track of which kidney was stenosed.
Remove the kidney capsules, clean them from any fat, and record the weight of each kidney separately. Cut a longitudinal section of both kidneys and fix them in 4%PFA overnight at four degrees Celsius for in situ hybridization and immunohistochemistry. Isolate the cortex of the remaining kidney and flash freeze it in liquid nitrogen to perform a Western blot.
Store the samples at minus 80 degrees Celsius until further analysis. Renal artery constriction increases renin expression in the stenosed kidney, while repressing expression in the contralateral kidney. The two-kidney one-clip model of stenosis induced renin expression and increased kidney injury.
Expression of renin and prorenin was measured using immunoblotting. The data show that renin and prorenin expression increased in the stenosed kidney compared to contralateral and sham kidneys, suggesting that the cuff was constricting the renal artery and causing changes in renal perfusion. Immunohistochemistry was performed to visualize the localization of renin expression.
The images confirmed increased expression of renin in the clipped kidney. Moreover, juxtaglomerular cell recruitment along the afferent arterial was seen in the stenosed kidney. To investigate the effect on renin mRNA expression levels, in situ hybridization was performed.
The data suggests increased renin mRNA and JG cell recruitment in the stenosed kidney. Another characteristic of renal artery stenosis is the upregulation of kidney injury markers due to changes in kidney perfusion, superoxide production, and hypertension. Therefore, N-GAL was measured with immunoblotting, showing that the acute kidney injury marker was highly upregulated in the stenosed kidney.
When attempting this protocol, remember to place the nylon sutures around the renal artery before placing the cuff. It helps expedite the procedure.
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Overview
The 2K1C mouse model is a reliable method to induce renal artery stenosis, facilitating the study of renin expression and renal vascular hypertension. This protocol is crucial for understanding the molecular mechanisms involved in these conditions.
Key Study Components
Area of Science
- Neuroscience
- Renal Physiology
- Hypertension Research
Background
- The 2K1C model mimics unilateral renal artery stenosis in humans.
- It is used to explore therapeutic targets for renal vascular hypertension.
- Understanding renin expression is vital for hypertension research.
- The model's reliability and reproducibility are significant advantages.
Purpose of Study
- To elucidate molecular mechanisms of renin expression control.
- To provide a consistent model for studying renal vascular hypertension.
- To identify new therapeutic targets for treatment.
Methods Used
- Preparation of polyurethane tubing for renal artery cuff.
- Placement of the cuff to induce stenosis.
- Monitoring of renal function and renin expression.
- Utilization of surgical techniques in mice.
Main Results
- The model successfully induces renal artery stenosis.
- Increased renin expression was observed post-surgery.
- Consistent results across multiple trials confirm model reliability.
- Insights gained into potential therapeutic interventions.
Conclusions
- The 2K1C mouse model is effective for studying renal hypertension.
- It provides a platform for exploring new treatments.
- Further research can build on findings related to renin expression.
Frequently Asked Questions
Executive Industry Relevance
The modified two kidney one clip (2K1C) mouse model enables precise interrogation of renin regulation and kidney injury mechanisms in the context of renal artery stenosis. This model provides a reproducible platform for target validation and mechanistic de-risking in early cardiovascular and renal drug discovery. Its translational alignment with human renovascular hypertension supports predictive confidence for portfolio advancement decisions.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Enables functional validation of renin as a therapeutic target in renovascular hypertension.
- Supports mechanistic de-risking by clarifying molecular pathways of RAAS activation.
- Facilitates hypothesis-driven interrogation of kidney injury markers and their regulation.
- Provides a robust system for evaluating target engagement and pathway modulation.
Screening & Assay Development
- Delivers validated biological samples for downstream quantitative assays of renin and injury markers.
- Supports assay standardization through reproducible induction of renal artery stenosis.
- Enables reliable measurement of protein and mRNA expression for screening readiness.
- Prepares tissue for immunoblotting and in situ hybridization to assess molecular endpoints.
Translational & Preclinical Research
- Aligns with disease-relevant mechanisms observed in human renal artery stenosis.
- Enables continuity from discovery-stage findings to preclinical biomarker validation.
- Supports risk-adjusted advancement by modeling both hypertension and kidney injury phenotypes.
- Provides mechanistic insights for translational biomarker development.
Pipeline & Workflow Integration
This model integrates into the discovery-to-preclinical continuum by enabling hypothesis testing, target validation, and quantitative biomarker analysis in a disease-relevant system.
- Discovery Biology: Supports null hypothesis testing of renin regulation and injury marker expression under controlled stenosis conditions.
- Screening: Provides reproducible tissue samples for quantitative immunoblotting and mRNA analysis.
- Analytics: Enables statistical comparison of renin and injury marker levels across experimental groups.
- Translational Research: Models human-like pathophysiology for preclinical biomarker alignment.
- Enterprise Reuse: Offers a standardized, reusable in vivo platform for cardiovascular and renal target evaluation.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence in target validation and mechanistic understanding of RAAS-driven pathology.
- Operational Value: Delivers reproducible, scalable in vivo data for cross-functional R&D teams.
- Strategic Value: Improves go/no-go decision quality by reducing biological ambiguity early in the pipeline.
- Portfolio Impact: Enables risk-adjusted prioritization of renal and cardiovascular therapeutic programs.
Implementation Considerations
- Requires surgical expertise in small animal models and precise vascular manipulation.
- Demands access to analytical platforms for protein, mRNA, and histological analysis.
- Necessitates standardized protocols for cross-team reproducibility and data comparability.
- Adaptable to various genetic backgrounds and experimental conditions for broader applicability.
- Potential limitations include technical variability and the need for rigorous post-surgical monitoring.
