A Modified Two Kidney One Clip Mouse Model of Renin Regulation in Renal Artery Stenosis

Summary

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.

Abstract

Renal artery stenosis is a common condition in patients with coronary or peripheral vascular disease where the renin angiotensin aldosterone system (RAAS) is overactivated. In this context, there is a narrowing of the renal arteries that stimulate an increase in the expression and release of renin, the rate-limiting protease in RAAS. The resulting rise in renin expression is a known driver of renovascular hypertension, frequently associated with kidney injury and end organ damage. Thus, there is a great interest in developing novel treatments for this condition. The molecular and cellular mechanism of renin control in renal artery stenosis is not fully understood and warrants further investigation. To induce renal artery stenosis in mice, a modified 2 kidney 1 clip (2K1C) Goldblatt mouse model was developed. The right kidney was stenosed in wild type mice and sham operated mice were used as control. After renal artery stenosis, we determined renin expression and kidney injury. Kidneys were harvested, and fresh cortices were used to determine protein and mRNA expression of renin. This animal model is reproducible and can be used to study pathophysiological responses, molecular and cellular pathways involved in renovascular hypertension and kidney injury.

Introduction

Renal artery stenosis (RAStenosis) is an intractable problem affecting about 6% of people over 65 and in up to 40% of people with coronary or peripheral vascular disease^1,2. Current treatments for the disease are limited; therefore, there is a critical need to develop new therapies to treat renovascular hypertension or resistant hypertension induced by RAStenosis. Renin angiotensin aldosterone system (RAAS) is the key pathway involved in the pathogenesis of RAStenosis induced hypertension or renovascular hypertension^3,4. Known therapies targeting RAAS, such as ACE inhibitors or angiotensin receptor blockers, alleviate hypertension, but need close examining for kidney failure and hyperkalemia^5,6,7. Renin catalyzes the rate-limiting step in RAAS; it converts angiotensinogen to angiotensin I. In atherosclerosis, plaque formation causes the narrowing of renal artery that drives renin secretion, resulting in renovascular hypertension and kidney damage⁸. A number of studies have reported increased levels of oxidative stress during renovascular hypertension in humans, which were corroborated with the two kidney one clip (2K1C) mice model as well as other hypertensive animal models^{2,9,10,11,12,13,14,15,16}. The molecular mechanism of renin expression control during RAStenosis induced renovascular hypertension is not well understood and warrants further investigation.

Experimental animal models that reliably and reproducibly recapitulate RAStenosis are important in elucidating the cellular and molecular mechanisms of renin expression control for the development of novel therapies. The 2K1C mouse model is a well-established experimental model to study the pathogenesis of renovascular hypertension^17,18,19,20. This model is generated by the constriction of the renal artery using a clip^17,20,21, therefore producing renal artery occlusion that results in an increase in renin expression and hypertension^17,19,20,21. However, there are no technical reports available, which describe a step by step procedure to generate renal artery stenosis in animal models.

Conventional U-shaped silver clips, polyurethane tubes and other clips have been used to constrict the renal artery to induce renal artery stenosis. Some studies have shown that the design and material of the clip are critical to obtaining reliable and reproducible data with the 2K1C animal model. According to Lorenz et al., the use of conventional U-designed silver clips induces a low success rate of hypertension (40-60%)²¹. Due to the clip design, the renal artery is press laterally, triggering a few constrictions and greater probability to be dislodged from the renal artery. Silver malleability and ductility may allow changes in clip widths; therefore, causing different hypertension levels among mice. Silver dioxides on the clip can cause perivascular inflammation, intimal proliferation, and tissue granulation, altering the renal artery diameter²². Due to the variability in the levels of hypertension obtained with the conventional U-design silver clip, Warner et al. and Lorenz et al. have successfully used a rounder-design polyurethane tubing to initiate renal artery stenosis in mice, generating a more reliable and consistent induction of the two kidney one clip animal model^20,21.

In this report, we describe a surgical protocol to generate experimental RAStenosis in mice, using the polyurethane tubing to constrict the renal artery. The polyurethane round-design cuff is a more reproducible, reliable and low-cost clip to generate stenosis in mouse. The goal of this experimental model is to study and define the molecular and cellular mechanism of renin expression control during renal artery stenosis. We confirmed the success of RAStenosis mice model by measuring renin expression and kidney injury marker neutrophil gelatinase-associated lipocalin (N-GAL).

Protocol

Mice were housed and cared at the Vanderbilt University Medical Center (VUMC) Division of Animal Care following the National Institutes of Health (NIH) guidelines and the Guide for the Care and Use of Laboratory Animals, US Department of Health and Human Services. All animal procedures were approved by the VUMC Institutional Animal Care and Use Committee prior to starting the experiments.

1. Animal preparation and dissection

1. Turn on the germinator and water pump of the heating pad about 30 min before starting surgery.
2. Cut 0.5 mm length polyurethane tubing with a sharp scalpel. Remove 0.2 mm of the circumference by making a cut lengthwise to make a cuff.
   NOTE: This is a critical part of the renal artery stenosis procedure that requires extreme precision, attention to details and patience. Try to cut several pieces of polyurethane tubing at a time. Perform all of this procedure under microscope.
3. Before proceeding any further, put on surgical sterile gloves and a surgical mask.
4. Use C57BL/6 wild-type (WT) mice of 6-8 weeks. Use an equal number of male and female mice to avoid any sex bias.
5. Record the weight of the mice before performing surgery. The ideal weight to perform renal artery stenosis with polyurethane tube is 18-22 g. Handle mice gently and do not agitate while injecting the anesthesia. Administer pre-operative analgesia (Ketofgen, 5 mg/kg body wieght).
   NOTE: While transferring the mice from their housing room to the surgery room, carry them with great gentleness and care to avoid agitation. Carrying mice cages in hands instead of a trolley is highly recommended.
6. Anesthetize the mice with a mixture of ketamine (100 mg/kg BW) and xylazine (10 mg/kg BW) via intraperitoneal (I.P.) injections.
7. Place the mouse back inside the cage until fully anesthetized. It takes about 4-5 minutes before the mouse is fully unconscious. Pinch the tail or toe with forceps to check if the mouse is fully unconscious and ready for the surgery.
8. Lay the mouse on its back on a paper towel. Remove the hair of the lateral abdomen using an electric hair clipper following the opposite direction of hair growth. After shaving the surgical site, clean the area with a surgical scrub containing iodine (or chlorhexidine) followed by a rinse with 70% ethanol (or sterile saline).  Repeat 3 times.
   NOTE: The hair removal procedure must be performed at some distance or preferably at a different bench than the surgery procedure bench to avoid any hair interference and hair contamination during the surgery procedure.
9. Cover the heating pad with a sterile sheet. Bring the mouse to the surgical bench and place on to the sterile sheet, facing the mouse’s right lateral side towards the microscope. Maintain a constant pad temperature of 37 °C with circulating water.
10. Open the sterilized bag containing all the surgical equipment. Using a dissecting microscope and sterile sharps scissors, make a small flank incision (close to 13^th thoracic rib, the last rib in mouse) and about 0.5 cm away from vertebrae. Proceed along the lumbar vertebrae and make a 1 inch incision.
11. Pull back the skin and muscle to expose the kidney.
12. Clean and remove the surrounding fat using sterile cotton swabs to isolate the renal artery. Isolate the renal nerve from the renal artery using curved forceps.
13. When performing the sham surgery, apply sutures to close the skin and apply antibiotic to the closed wound, then proceed to Post-operative care. If not, proceed with the following section to stenose the artery.
    NOTE: Every experiment should have sham animals as controls of the surgical procedure. Sham animals consist of mice that have gone through the surgical procedure of exposing the renal artery without placing a cuff on it.

2. Right renal artery stenosis

1. Place two nylon sutures under the right renal artery, make loose knots, and then place the cuff around the main renal artery approximately equidistant between the kidney and aorta bifurcation
2. Close the cuff using the nylon sutures. Make four knots for each suture to avoid the probability of losing the sutures after the surgery.
3. Close the incision in the muscle by applying a simple continuous suture.
4. Make simple interrupted sutures to close the skin.
5. Administer IACUC approved analgesics.
   NOTE: Autoclave surgical tools before every use. If more than one surgery is being performed at once, wipe all used tools with a sterile alcohol gauge and place them in hot germinator for 15-30 s after every surgery. Change sterile gloves also for each mouse.

3. Post-operative care

1. Return mice to their cage and leave the cage half on, half off, a circulating water heating pad for 2 - 3 hours. Add gel diet recovery food inside the cage.
2. Administer painkiller (ketoprofen) intraperitoneally (dose: 5 mg/kg BW) the next day.
3. Weigh the mice for the next two days; if some mice lose more than 20% of their weight consult with the veterinarian and decide if the animal needs to be euthanized following the appropriate IACUC authorized procedure.
4. Monitor the mice daily to assess for redness, swelling, pain or infection.
5. Seek veterinary consultation for postoperative complications in accordance with the institutional IACUC policies..

4. Tissue harvest

1. Harvest tissue 3 weeks after surgical procedure. Record the weight of each mouse.
2. Euthanize the mouse with an IACUC approved procedure.
3. Place the mouse on a sterile platform in supine position to dissect.
4. Secure and extend the limbs to limit movement.
5. Thoroughly spray the mice with 70% ethanol.
6. Make a midline incision to open the abdomen and chest area using sharp scissors.
7. Pull back the skin and peritoneal wall.
8. Carefully expose the heart and puncture the right ventricle and exsanguinate the mouse.
9. Remove both kidneys using forceps. Kidneys are located on the back of the mice.
   NOTE: Do not mix both kidneys. Be aware of stenosed, sham, and contralateral kidneys.
10. Remove the kidney capsule, clean them from any fat and record the weight of each kidney separately.
11. Cut a longitudinal section of both kidneys and fixed in 4% PFA overnight at 4 °C to be later processed for paraffin embedding, to perform in situ hybridization (ISH) and immunohistochemistry (IHC). Follow ISH and IHC protocols as reported^23,24.
12. Isolate the cortex of the remaining kidney and flash freeze in liquid nitrogen to perform western blot. Store samples at -80 °C until analysis.
13. Quantify renin, and N-GAL expressions with Western blot as described in literature^23,24.

5. Statistics

1. Use one-way or two-way ANOVA for experiments with three or more conditions followed by Bonferroni post-hoc tests for comparisons between individual groups. Consider a p-value equal or less than 0.05 significant. Use software (e.g., GraphPad Prism 8.2) to perform all statistical analysis.

Results

Renal artery constriction increases renin expression in the stenosed kidney while repressing expression in the contralateral kidney. The two kidney one clip (2K1C) or Goldblatt model of stenosis induces increased renin expression and kidney injury. This is recognized as the best representative model of unilateral renal artery stenosis in humans.

Expression of renin and prorenin (precursor of renin) were measured using immunoblotting. The data show that renin and prorenin expression increased i...

Discussion

Renal artery stenosis is an important cause of secondary or resistant hypertension, and kidney injury^1,29. The two kidney one clip (2K1C) Goldblatt model has been employed to study RAStenosis induced renovascular hypertension^1,17,18,19. A number of previous studies using various animals models have shown that stenosis in the renal arter...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Diet Gel	Clear H2O	Diet-Gel 76A	Surgery recovery diet
EMC Heated Hard pad	Hallowell	000A2788B	Heating pads were used to keep mice warm
Ethilon Nylon Suture	Ethicon	662G	4-0 (1.5 metric), This suture was used to close the peritoneum, and skin
Ethilon Nylon Suture	Ethicon	2815 G	8-0 (0.4 metric), This suture was used to close cuff to tie and constrict the artery
Germinator 500	Braintree Scientific Inc.	GER 5287	Sterilize surgical tools between surgeries
Ketoprofen	Zoetis	Ketofen	Painkiller
Polyurethane	Braintree Scientific Inc.	MRE-025	This tube was used to initiate stenosis
Povidone-iodine antiseptic swabsticks	Medline	MDS093901	It was applied after hair removal and surgery on the skin
Reflex 7 Clip Applier	Roboz Surgical Instrument Co	204-1000	This clip applier was used to apply clip in case one or more sutures went off
Sterile towel drapes	Dynarex	4410	It was used as a bedsheet for mice during surgery
Triple antibiotic ointment	Medi-First	22312
Water pump	Stryker	T/pump Professionals	Used to warm and circulate water in the heating hard pad to keep mice warm during and post-surgery