Intravascular Delivery of Biologics to the Rat Kidney

Summary

The administration of drugs for recovery of kidney function requires control of the localization and distribution of the therapeutic compound. Here, we describe in detail a simple technique for intrarenal delivery of drugs in rats. This procedure may be easily performed with no mortality and high reproducibility.

Abstract

The renal microvascular compartment plays an important role in the progression of kidney disease and hypertension, leading to the development of End Stage Renal Disease with high risk of death for cardiovascular events. Moreover, recent clinical studies have shown that renovascular structure and function may have a great impact on functional renal recovery after surgery. Here, we describe a protocol for the delivery of drugs into the renal artery of rats. This procedure offers significant advantages over the frequently used systemic administration as it may allow a more localized therapeutic effect. In addition, the use of rodents in pharmacodynamic analysis of preclinical studies may be cost effective, paving the way for the design of translational experiments in larger animal models. Using this technique, infusion of rat recombinant Vascular Endothelial Growth Factor (VEGF) protein in rats has induced activation of VEGF signaling as shown by increased expression of FLK1, pAKT/AKT, pERK/ERK. In summary, we established a protocol for the intrarenal delivery of drugs in rats, which is simple and highly reproducible.

Introduction

The renal microvasculature is involved in a wide spectrum of kidney diseases. Depending on the pathophysiology of disease, the endothelial cells may present structural or functional impairment, which may play a pivotal role in propagating kidney damage by creating an ischemic microenvironment. This renal microvascular dysfunction may catalyze the onset of a progressive deterioration of renal function over time, leading to chronic kidney disease (CKD), end-stage renal disease, hypertension and cardiorenal syndrome. In fact, untreated hypertension may have implications in renal arterioles, causing nephrosclerosis or glomerulosclerosis with significant reduction in vascular volume fraction, increase in vascular resistance and development of tubulointerstitial fibrosis¹.

Loss of renal microvasculature may be due to altered vascular homeostasis induced by local angiogenic/anti-angiogenic factors imbalance. This correlates with attenuated Vascular Endothelial Growth Factor (VEGF) signaling as well as elevated thrombospondin-1^2-4. Thus, using different animal models (mice, rats and pigs), the therapeutic effect of exogenous administration of VEGF has been recently investigated in some forms of renal disease, showing reduced interstitial fibrosis and stabilized renal and cardiac function^3-5. This effect is likely due to actions of VEGF on endothelial cells of the microvascular bed and inflammatory monocyte phenotype switching⁶.

For some preclinical studies, the use of rodents, the most commonly used laboratory animals, provides a good animal model for high throughput studies due to relatively low costs and ease of handling. Moreover, the use of genetically-altered rats as models of human diseases, such as hypertension, has become more and more frequent in the scientific community. Therefore, the aim of this protocol is to describe a useful intrarenal VEGF delivery technique in rats that is easy to perform and highly reproducible. Moreover, the same method can be used to selectively deliver other drugs.

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Protocol

The experiments were performed on female Sprague-Dawley rats, weighing 250-300 g. All animal procedures complied with the standards stated in the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, National Academy of Sciences, Bethesda, MD, USA) and were approved by the Mayo Clinic College of Medicine Institutional Animal Care and Use Committee (IACUC).

1. Preparation

1. Autoclave all surgical instruments before surgery. If multiple surgeries on different rats are planned in the same day, rinse instruments after each animal procedure and then sterilize using a hot bead sterilizer.
2. Anesthetize the rat with 4% isoflurane in 1 L/min O₂.
3. Transfer the rat to a controlled heating pad to preserve the body temperature at 37 °C. Maintain anesthesia with 1-2% isoflurane in 1 L/min O₂.
4. Administer the analgesic drug (Buprenorphine Sustained Release 0.6 mg/kg) subcutaneously.
5. Apply ointment to the eyes to prevent drying during the procedure.
6. In order to compensate for loss of body fluids due to laparotomy, it is important to administer 10 ml/kg of 0.9% normal saline subcutaneously preoperatively.
7. Shave the abdominal area and clean the skin with povidone-iodine and 70% ethanol pads.

2. Surgical Procedure

1. Ensure that the depth of sedation is adequate by monitoring physical reflexes, such as withdrawal from toe pinch, palpebral reflex, jaw tone, and respiration rate/pattern.
2. Perform a laparotomy through a small midline incision (2-2.5 cm in length) using a surgical scalpel blade No. 10.
3. Pull the intestine and colon to the right side of the abdomen by using cotton swabs and cover them with sterile gauze soaked in to 0.9% normal saline to maintain the organs moist.
4. Gently retract upward the spleen, liver, stomach and pancreas to expose the aorta and the left kidney artery.
5. With the help of a surgical microscope, carefully separate the abdominal aorta above and below the left kidney and the left renal artery from the veins, the fat and the surrounding connective tissue with blunt dissecting curved forceps and sterile cotton swabs.
   1. Use the forceps with a repeated open-close motion (blunt dissection) along the length of the vessels to remove the connective tissue and the cotton swabs with a lateral rolling motion to remove the fat.
      NOTE: The dissection of the peri-aortic region is a very delicate step as nerves and lymphatic vessels might be damaged. Make sure to keep the arteries moist with saline during the dissection procedure.
6. Place a 4-0 silk suture underneath the aorta.
7. Using microvascular clips, clamp the aorta above (just below the superior mesenteric artery) and below the renal artery bifurcation.
8. Puncture the aorta at the level of the left kidney artery bifurcation with a 24 G intravenous catheter and advance the catheter into the renal artery.
   NOTE: this is a critical step as puncture through the renal artery may occur.
9. Connect a syringe filled with the drug solution or saline (up to 500 μl) to the catheter and perfuse the kidney.
10. Immediately after perfusion, clamp the left renal vein and the left ureter with a microvascular clip and remove the catheter. Then, place a piece of absorbable hemostat gelatin sponge, with a small drop of tissue adhesive, over the punctured area of the aorta and gently apply pressure with a cotton swab.
11. At the same time, release the clamp from the abdominal aorta, below the left renal artery bifurcation. After 5 min, release the clamp from renal vein and ureter.
12. Carefully release the clamp from the aorta, above the left renal artery bifurcation, and allow for kidney reperfusion. The total renal ischemia should last no longer than 7 min.
13. Ensure that no active bleeding occurs and closely observe the area for 10 more min.
14. Close the abdominal incision in two layers (muscle and skin), using 4-0 absorbable sutures and a continuous pattern to prevent infection. In addition to the continuous pattern suture technique, another option would be to use a simple, interrupted technique, especially for body wall closure to prevent dehiscence.
15. Apply topical antibiotic ointment over the incision area to prevent infections.
16. Transfer the rat into a bedding-free observation cage on a warm pad until complete recovery with a temperature range set at 35-37 °C. Loose bedding should be covered (e.g. with a drape or paper towel) or removed from the cage until animals are fully recovered to prevent suffocation or aspiration of bedding.
17. After surgery, observe the animals continuously until breathing spontaneously, then hourly for a few hours. Re-dose the analgesic Buprenorphine SR 72 hr later if signs of discomfort are observed, such as lethargy, hunched and scruffy, grimace, not resuming normal activities.
18. After completion of all studies, euthanize animals with the inhalation of an overdose of CO₂ and harvest the renal tissues for ex vivo analyses such as histology and Western Blotting⁵.

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Results

We injected two different doses of recombinant rat VEGF (rrVEGF, 0.17 μg/kg and 5 μg/kg) or PBS. The animals were euthanized 8 hr post-surgery to examine the activation of the VEGF pathway. The surgical procedure did not affect the morphology of the perfused kidney (Figure 1A) when compared to control (Figure 1B), as shown by H&E staining. While Sirius red staining did not show any increase in extracellular matrix deposition in response to t...

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Discussion

The increasing incidence of chronic kidney disease raises the need for novel therapeutic approaches that can promote functional kidney recovery^7,8. Traditional therapies include the systemic administration of anti-inflammatory, anti-fibrotic drugs⁹. However, these strategies are frequently characterized by unwanted side effects due to off-target distribution of the injected drug. Therefore, in this manuscript, we describe a simple procedure for delivering drugs into the renal artery of rats. This pr...

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Materials

Name	Company	Catalog Number	Comments
Surgical Microscope	Leica	M125
Isoflurane 100 ml	Cardinal Healthcare	PI23238	Anesthetic
Buprenorphine HCL SR LAB 1 mg/ml, 5 ml	ZooPharm Pharmacy		Buprenorphine narcotic analgesic formulated in a polymer that slows absorption extending duration of action (72 hr duration of activity).                                                        Liquid is viscous, warming to RT aids in drawing into syringe.                                                           Recommended dosage: 1 - 1.2 mg/kg SC. DO NOT DILUTE.
Puralube Vet Ophthalmic Ointment	Dechra	NDC17033-211-38	Sterile ocular lubricant
Lactated Ringer's Injection, USP, 250 ml VIAFLEX Plastic Container	Baxter Healthcare Corp.	NDC0338-0117-02	For body fluids replacement
Sol Povidone-Iodine  Swabstick, 3'	Cardinal Heatlhcare	23405-010B
Sterile cotton tipped applicators	Kendall	8884541300
4-0 silk suture (without needle)	Cardinal Heatlhcare	A183H
Vessel Clip, Straight, 0.75 mm x 4 mm Jaw	World Precision Instruments	501779-G
I.V. Catheter, Straight Hub, Radiopaque, 24 g x 3/4", FEP Polymer	Jelco	4053
Phosphate Buffered Saline	Life Technologies	10010023
SURGIFOAM Absorbable Gelatin Sponge	Cardinal Healthcare	179082
4-0 VICRYL PLUS (ANTIBACTERIAL) VIOLET 27" RB-1 TAPER	Ethicon	VCP304H	For muscle layer suturing
4-0 VICRYL PLUS (ANTIBACTERIAL) UNDYED 18" PC-3 CUTTING	Ethicon	VCP845G	For skin layer suturing
Triple antibiotic ointment	Actavis	NDC0472-0179-56	For topical use on the site of the incision
Recombinant Rat VEGF 164 Protein	R&D Sytems	564-RV
Rabbit monoclonal VEGFA	Abcam	ab46154
Rabbit monoclonal FLK1	Cell Signaling	9698
Rabbit monoclonal AKT	Cell Signaling	4691
Rabbit monoclonal phosphoAKT (Ser 473)	Cell Signaling	4060
Rabbit monoclonal p44/42 MAPK (ERK1/2)	Cell Signaling	4695
Rabbit monoclonal phospho p44/42 MAPK (Thr202 and Tyr 204)	Cell Signaling	4370