Name: intravascular delivery of biologics to the rat kidney
Uploaded: 2016-09-01T15:00:00
Description: Watch this Scientific Journal Video about Intravascular Delivery of Biologics to the Rat Kidney at JoVE.com

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
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	<li>Autoclave all surgical instruments before surgery. If multiple surgeries on different rats are ...

<ol>
	<li>Dejani, H., Eisen, T. D., Finkelstein, F. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Revascularization+of+renal+artery+stenosis+in+patients+with+renal+insufficiency.">Revascularization of renal artery stenosis in patients with renal insufficiency.</a> Am. J. Kidney Dis. 36 (4), 752-758 (2000).</li><li>Kang, D. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impaired+angiogenesis+in+the+remnant+kidney+model:+I.+Potential+role+of+vascular+endothelial+growth+factor+and+thrombospondin-1.">Impaired angiogenesis in the remnant kidney model: I. Potential role of vascular endothelial growth factor and thrombospondin-1.</a> J. Am. Soc. Nephrol. 12 (7), 1434-1447 (2001).</li><li>Kang, D. H., Hughes, J., Mazzali, M., Schreiner, G. F., Johnson, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impaired+angiogenesis+in+the+remnant+kidney+model:+II.+Vascular+endothelial+growth+factor+administration+reduces+renal+fibrosis+and+stabilizes+renal+function.">Impaired angiogenesis in the remnant kidney model: II. Vascular endothelial growth factor administration reduces renal fibrosis and stabilizes renal function.</a> J. Am. Soc. Nephrol. 12 (7), 1448-1457 (2001).</li><li>Kang, D. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+the+microvascular+endothelium+in+progressive+renal+disease.">Role of the microvascular endothelium in progressive renal disease.</a> J. Am. Soc. Nephrol. 13 (3), 806-816 (2002).</li><li>Chade, A. R., Kelsen, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reversal+of+renal+dysfunction+by+targeted+administration+of+VEGF+into+the+stenotic+kidney:+a+novel+potential+therapeutic+approach.">Reversal of renal dysfunction by targeted administration of VEGF into the stenotic kidney: a novel potential therapeutic approach.</a> Am. J. Physiol.- Renal Physiol. 302 (10), F1342-F1350 (2012).</li><li>Eirin, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+Glomerular+Filtration+Rate+After+Renal+Revascularization+Correlate+With+Microvascular+Hemodynamics+and+Inflammation+in+Swine+Renal+Artery+Stenosis.">Changes in Glomerular Filtration Rate After Renal Revascularization Correlate With Microvascular Hemodynamics and Inflammation in Swine Renal Artery Stenosis.</a> Circ.-Cardiovasc. Interv. 5 (5), 720-728 (2012).</li><li>Chade, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+Renal+Injury+in+Early+Atherosclerosis+and+Renovascular+Disease.">Distinct Renal Injury in Early Atherosclerosis and Renovascular Disease.</a> Circulation. 106 (9), 1165-1171 (2002).</li><li>Seddon, M., Saw, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atherosclerotic+renal+artery+stenosis:+review+of+pathophysiology,+clinical+trial+evidence,+and+management+strategies.">Atherosclerotic renal artery stenosis: review of pathophysiology, clinical trial evidence, and management strategies.</a> Can. J. Cardiol. 27 (4), 468-480 (2011).</li><li>Lao, D., Parasher, P. S., Cho, K. C., Yeghiazarians, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atherosclerotic+renal+artery+stenosis--diagnosis+and+treatment.">Atherosclerotic renal artery stenosis--diagnosis and treatment.</a> Mayo Clin Proc. 86 (7), 649-657 (2011).</li><li>Sharfuddin, A. A., Molitoris, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathophysiology+of+ischemic+acute+kidney+injury.">Pathophysiology of ischemic acute kidney injury.</a> Nat. Rev. Nephrol. 7, 189-200 (2011).</li><li>Noiri, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidative+and+nitrosative+stress+in+acute+renal+ischemia.">Oxidative and nitrosative stress in acute renal ischemia.</a> Am. J. Physiol.- Renal Physiol. 281 (5), F948-F957 (2001).</li><li>Koesters, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tubular+Overexpression+of+Transforming+Growth+Factor-&#206;1+Induces+Autophagy+and+Fibrosis+but+Not+Mesenchymal+Transition+of+Renal+Epithelial+Cells.">Tubular Overexpression of Transforming Growth Factor-&#206;1 Induces Autophagy and Fibrosis but Not Mesenchymal Transition of Renal Epithelial Cells.</a> Am. J. Pathol. 177 (2), 632-643 (2010).</li><li>Shanley, P. F., Rosen, M. D., Brezis, M., Silva, P., Epstein, F. H., Rosen, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Topography+of+focal+proximal+tubular+necrosis+after+ischemia+with+reflow+in+the+rat+kidney.">Topography of focal proximal tubular necrosis after ischemia with reflow in the rat kidney.</a> Am. J. 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The increasing incidence of chronic kidney disease raises the need for novel therapeutic approaches that can promote functional kidney recovery7,8. 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...

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-12-4. 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 function3-5. 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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			<td height="21" style="height:21px;width:257px;">Surgical Microscope</td>
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			<td style="width:376px;">Buprenorphine narcotic analgesic formulated in a polymer that slows absorption extending duration of action (72 hours duration of activity).&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; Liquid is viscous, warming to room temperature aids in drawing into syringe.&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; Recommended dosage: 1-1.2 mg/kg SC. DO NOT DILUTE.</td>
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			<td>Sterile ocular lubricant</td>
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			<td style="width:176px;">Baxter Healthcare Corp.</td>
			<td style="width:119px;">NDC0338-0117-02</td>
			<td>For body fluids replacement</td>
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			<td style="width:119px;">8884541300</td>
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			<td height="21" style="height:21px;width:257px;">4-0 silk suture (without needle)&#160;</td>
			<td style="width:176px;">Cardinal Heatlhcare</td>
			<td style="width:119px;">A183H</td>
			<td></td>
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			<td height="42" style="height:42px;width:257px;">Vessel Clip, Straight, 0.75 x 4mm Jaw</td>
			<td style="width:176px;">World Precision Instruments&#160;</td>
			<td style="width:119px;">501779-G</td>
			<td></td>
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			<td height="42" style="height:42px;width:257px;">I.V. Catheter, Straight Hub, Radiopaque, 24g x 3/4&#34;, FEP Polymer</td>
			<td style="width:176px;">Jelco</td>
			<td align="right" style="width:119px;">4053</td>
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			<td height="21" style="height:21px;width:257px;">Phosphate Buffered Saline</td>
			<td style="width:176px;">Life Technologies</td>
			<td align="right" style="width:119px;">10010023</td>
			<td></td>
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			<td height="42" style="height:42px;width:257px;">SURGIFOAM Absorbable Gelatin Sponge</td>
			<td style="width:176px;">Cardinal Healthcare</td>
			<td align="right" style="width:119px;">179082</td>
			<td></td>
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			<td height="42" style="height:42px;width:257px;">4-0 VICRYL PLUS (ANTIBACTERIAL) VIOLET 27&#34; RB-1 TAPER</td>
			<td style="width:176px;">Ethicon</td>
			<td style="width:119px;">VCP304H</td>
			<td>For muscle layer suturing</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">4-0 VICRYL PLUS (ANTIBACTERIAL) UNDYED 18&#34; PC-3 CUTTING</td>
			<td style="width:176px;">Ethicon</td>
			<td style="width:119px;">VCP845G</td>
			<td>For skin layer suturing</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:257px;">Triple antibiotic ointment</td>
			<td style="width:176px;">Actavis</td>
			<td style="width:119px;">NDC0472-0179-56</td>
			<td>For topical use on the site of the incision</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">Recombinant Rat VEGF 164 Protein</td>
			<td style="width:176px;">R&#38;D Sytems</td>
			<td style="width:119px;">564-RV</td>
			<td></td>
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			<td height="21" style="height:21px;width:257px;">Rabbit monoclonal VEGFA</td>
			<td style="width:176px;">Abcam</td>
			<td style="width:119px;">ab46154</td>
			<td></td>
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			<td height="21" style="height:21px;width:257px;">Rabbit monoclonal FLK1</td>
			<td style="width:176px;">Cell Signaling</td>
			<td align="right" style="width:119px;">9698</td>
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			<td height="21" style="height:21px;width:257px;">Rabbit monoclonal AKT</td>
			<td style="width:176px;">Cell Signaling</td>
			<td align="right" style="width:119px;">4691</td>
			<td></td>
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			<td height="42" style="height:42px;width:257px;">Rabbit monoclonal phosphoAKT (Ser 473)</td>
			<td style="width:176px;">Cell Signaling</td>
			<td align="right" style="width:119px;">4060</td>
			<td></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:257px;">Rabbit monoclonal p44/42 MAPK (ERK1/2)</td>
			<td style="width:176px;">Cell Signaling</td>
			<td align="right" style="width:119px;">4695</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">Rabbit monoclonal phospho p44/42 MAPK (Thr202 and Tyr 204)</td>
			<td style="width:176px;">Cell Signaling</td>
			<td align="right" style="width:119px;">4370</td>
			<td></td>
		</tr>
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intravascular delivery of biologics to the rat kidney

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.

We injected two different doses of recombinant rat VEGF (rrVEGF, 0.17 &#956;g/kg and 5 &#956;g/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&#38;E staining. While Sirius red staining did not show any increase in extracellular matrix deposition in response to t...

Watch this Scientific Journal Video about Intravascular Delivery of Biologics to the Rat Kidney at JoVE.com

Intravascular Delivery of Biologics to the Rat Kidney

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.

The renal microvascular compartment plays an important role in the progression of kidney disease and hypertension, leading to the development of End Stage ...

The overall goal of this procedure is to deliver biologics directly into the renal artery in order to induce a localized therapeutic effect in the renal microvascular compartment in rats. The main advantage of this technique over existing methods such as systemic administration of anti-inflammatory or antifibrotic drugs is that the intrarenal delivery technique minimizes any potential systemic side effects. The implications of this technique extend to a therapy of a variety of renal pathologies, because the renal microvascular function is involved in a wide spectrum of kidney diseases.Playing a key role in the progression of kidney damage. To prepare for surgery, begin by autoclaving all surgical instruments. After anesthetizing a rat, according to the text protocol, transfer the animal to a controlled heating pad, to preserve the animal's body temperature at 37 degrees Celsius.Use 1%to 2%isoflourane in one liter per minute of oxygen to maintain anesthesia. Administer an analgesic subcutaneously, and apply eye ointment to prevent dryness. To compensate for loss of body fluids, administer 10 milliliters per kilogram of 0.9%normal saline subcutaneously.Then, shave the abdominal area, and use povidone iodine and 70%ethanol pads to clean the skin. To begin surgery, ensure that the depth of sedation is adequate by monitoring physical reflexes such as a withdrawal from a toe pinch. Use a number 10 surgical scalpel blade to perform a laparotomy through a small midline incision.With cotton swabs, pull the intestine and colon to the right side of the abdomen, and cover them with sterile gauze, soaked in 0.9%normal saline to keep the organs moist. Gently retract upward the spleen, liver, stomach, and pancreas to expose the aorta and the left kidney artery. Next, under a surgical microscope, use blunt dissecting curved forceps with a repeated open close motion along the length of the vessels and sterile cotton swabs, to 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.The use of cotton swabs helps avoid damage to nerves and infected blood vessel, in particular to thin-walled veins. Then, place a 4-0 silk suture underneath the aorta, and using microvascular clips, clamp the aorta above and below the renal artery bifurcation. Now, using a 24 gauge intravenous catheter, puncture the aorta at the level of the left kidney artery bifurcation, and carefully advance the catheter into the renal artery.Because of the small size of the renal artery, the catheter might puncture through the vessel. It is important to work under a surgical microscope and to insert the needle as parallel to the artery as possible. Then, connect a syringe filled with the drug solution or saline to the catheter, and profuse the kidney.Immediately after profusion, use a microvascular clip to clamp the left renal vein and the left ureter before removing 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 with a cotton swap, gently apply pressure. At the same time, release the clamp from the aorta below the left renal artery bifurcation.After five minutes, release the clamp from the renal vein and ureter. Carefully release the clamp from the aorta above the left renal artery bifurcation, and allow the kidney to reperfuse. The total renal ischemia should last no longer than seven minutes.After ensuring that no active bleeding occurs, and closely observing the area for 10 more minutes, use 4-0 absorbable sutures and a continuous pattern to prevent infection, and close the abdominal incision in two layers. Apply topical antibiotic ointment over the incision area to prevent infections. Then, transfer the rat into the observation cage on a warm pad until complete recovery.After completion of all of the studies, euthanize the animals according to the text protocol. Two different doses of recombinant rat VEGF, or PBS as control, were injected into rats, and then animals were euthanized eight hours post surgery to examine the activation of the VEGF pathway. As seen here, by H&E staining, the surgical procedure did not effect the morphology of the perfused kidney, as compared to the control organ.In addition, serious red staining did not show an increase in extracellular matrix deposition in response to the ischemic time and the infusion of rrVEGF, as compared to control tissue. Finally, by Western blot analysis, a small but significant increase in the expression of proteins involved in the VEGF pathways was observed, including VEGF, FLK-1, Phospho-AKT and AKT, and Phospho-ERK and ERK. Once mastered, this technique can be done in about 45 minutes if it is performed properly.While attempting this procedure, it's important to follow institutional and national guidelines for the animal care and use, in particular, to alleviate the pain and discomfort by the administration of analgesics. Following this procedure, other methods, such as Western blotting, or histological staining of the kidney can be performed to determine the efficacy of different therapies as well as activation of specific pathways.

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