Name: long-term continuous measurement of renal blood flow in conscious rats
Uploaded: 2022-02-08T15:00:00
Description: Watch this Scientific Journal Video about Long-Term Continuous Measurement of Renal Blood Flow in Conscious Rats at JoVE.com

This study was supported by grants for scientific research (P01 HL116264, RO1 HL137748). The authors would like to thank Theresa Kurth for her advice and help in maintaining the experimental environment as the lab manager.

The protocol is approved by the Medical College of Wisconsin Institutional Animal Care and Use. Dahl salt-sensitive rats (males and females), ~8 weeks of age, 200-350 g, were used for the experiments.
1. Animal preparation
<ol>
	<li>Install a movement response caging system for the rat, a perivascular flow module, syringe pump, recording device, and software (see Table of Materials) in the animal room.</li>
	<li>Place the rats in the cage to become familiar ...

<ol>
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W. <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+pressure+in+angiotensin+II-induced+renal+injury.">Role of pressure in angiotensin II-induced renal injury.</a> Hypertension. 43 (4), 752-759 (2004).</li><li>Mori, T., 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=High+perfusion+pressure+accelerates+renal+injury+in+salt-sensitive+hypertension.">High perfusion pressure accelerates renal injury in salt-sensitive hypertension.</a> Journal of the American Society of Nephrology. 19 (8), 1472-1482 (2008).</li><li>Polichnowski, A. J., Cowley, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pressure-induced+renal+injury+in+angiotensin+II+versus+norepinephrine-induced+hypertensive+rats.">Pressure-induced renal injury in angiotensin II versus norepinephrine-induced hypertensive rats.</a> Hypertension. 54 (6), 1269-1277 (2009).</li><li>Polichnowski, A. J., Jin, C., Yang, C., Cowley, A. W. <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+renal+perfusion+pressure+versus+angiotensin+II+renal+oxidative+stress+in+angiotensin+II-induced+hypertensive+rats.">Role of renal perfusion pressure versus angiotensin II renal oxidative stress in angiotensin II-induced hypertensive rats.</a> Hypertension. 55 (6), 1425-1430 (2010).</li><li>Evans, L. C., 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=Increased+perfusion+pressure+drives+renal+T-cell+infiltration+in+the+dahl+salt-sensitive+rat.">Increased perfusion pressure drives renal T-cell infiltration in the dahl salt-sensitive rat.</a> Hypertension. 70 (3), 543-551 (2017).</li><li>Shimada, S., 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=Renal+perfusion+pressure+determines+infiltration+of+leukocytes+in+the+kidney+of+rats+with+angiotensin+II-induced+hypertension.">Renal perfusion pressure determines infiltration of leukocytes in the kidney of rats with angiotensin II-induced hypertension.</a> Hypertension. 76 (3), 849-858 (2020).</li><li>Cousins, M. J., Mazze, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anaesthesia,+surgery+and+renal+function:+Immediate+and+delayed+effects.">Anaesthesia, surgery and renal function: Immediate and delayed effects.</a> Anaesthesia and Intensive Care. 1 (5), 355-373 (1973).</li><li>Cousins, M. J., Skowronski, G., Plummer, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anaesthesia+and+the+kidney.">Anaesthesia and the kidney.</a> Anaesthesia and Intensive Care. 11 (4), 292-320 (1983).</li><li>Schiffer, T. A., Christensen, M., Gustafsson, H., Palm, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+inactin+on+kidney+mitochondrial+function+and+production+of+reactive+oxygen+species.">The effect of inactin on kidney mitochondrial function and production of reactive oxygen species.</a> PLOS ONE. 13 (11), 0207728 (2018).</li><li>Evans, R. G., 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=Chronic+renal+blood+flow+measurement+in+dogs+by+transit-time+ultrasound+flowmetry.">Chronic renal blood flow measurement in dogs by transit-time ultrasound flowmetry.</a> Journal of Pharmacological and Toxicological Methods. 38 (1), 33-39 (1997).</li><li>Bell, T. D., DiBona, G. F., Biemiller, R., Brands, M. 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The present protocol describes a technique that utilizes commercially available instrumentation to record RBF and arterial pressure continuously over many weeks. In addition, urine can be collected using the device described in step 1.1. It can also be used to evaluate metabolites in the urine and, when an arterial catheter is implanted, blood sampling for analysis.
Traditionally, RBF measurements have been obtained acutely in surgically prepared anesthetized animals or estimated by PAH cleara...

The kidneys are only 0.5% of the bodyweight but rich in blood flow, receiving 20%-25% of the total cardiac output1. The regulation of renal blood flow (RBF) is central to kidney function, body fluid, and electrolyte homeostasis. The importance of blood flow regulation to the kidney is nicely illustrated by the substantial increase of RBF in the remaining kidney after unilateral nephrectomy2,3,4 and by the reductions of RBF that occur in kidney failure5,6,7. Whether such changes in RBF occur in response to alterations in kidney function or a decrease in function due to reduction of RBF has been challenging to ascertain in anesthetized surgically prepared animals or human subjects. Temporal studies are required in which the events can be determined before and following a defined change and observed in the same animal during the progression of events. In the animal and human studies, RBF has been estimated indirectly by the clearance of para-amino hippuric acid (PAH)8,9,10 and in more recent time by imaging techniques such as ultrasound9,11,12, MRI4,13, and PET-CT14,15 which give helpful snapshot images of each kidney and which can follow the progression of the disease. It is challenging to evaluate RBF in small animals by ultrasound or MRI scans without anesthesia. It has been impossible to continuously measure RBF under conscious conditions in the same rat over prolonged periods.
The present protocol, therefore, developed techniques that enable simultaneous continuous 24 h/day measurements of RBF, which has been combined with continuous blood pressure measurement methods for freely moving rats as described previously16,17,18,19,20,21. This technology allows for the temporal evaluation of RBF in various models of rats to study cause-effect relationships in various renal disorders in the future.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1RB probe</td>
			<td>Transonic</td>
			<td>1RB</td>
			<td>ultrasonic flow probe</td>
		</tr>
		<tr>
			<td>Betadine</td>
			<td>Avrio Health</td>
			<td></td>
			<td>povidone-iodine</td>
		</tr>
		<tr>
			<td>Buprenorphine SR-LAB</td>
			<td>ZooPharm</td>
			<td></td>
			<td>Buprenorphine</td>
		</tr>
		<tr>
			<td>Cefazolin</td>
			<td>APOTEX</td>
			<td>NDC 60505</td>
			<td>Cefazolin</td>
		</tr>
		<tr>
			<td>Crile Hemostats</td>
			<td>Fine Surgical Instruments</td>
			<td>13004-14</td>
			<td>Hemostats for blunt dissection</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Piramal</td>
			<td>NDC 66794</td>
			<td>Isoflurane</td>
		</tr>
		<tr>
			<td>Medium Clear PVC cement</td>
			<td>Oatey</td>
			<td></td>
			<td>PVC cement</td>
		</tr>
		<tr>
			<td>Mersilene polyester fiber mesh</td>
			<td>Ethicon</td>
			<td></td>
			<td>polyester fiber mesh</td>
		</tr>
		<tr>
			<td>MetriCide28</td>
			<td>Metrex</td>
			<td>SKU 10-2805</td>
			<td>2.5% glutaraldehyde</td>
		</tr>
		<tr>
			<td>Micro-Renathane 0.025 x 0.012</td>
			<td>Braintree Scientific</td>
			<td>MRE 025</td>
			<td>use for catheter</td>
		</tr>
		<tr>
			<td>MINI HYPE-WIPE</td>
			<td>Current Technologies</td>
			<td>#9803</td>
			<td>1% sodium hypochlorite</td>
		</tr>
		<tr>
			<td>Oatey Medium Clear PVC Cement</td>
			<td>Oatey</td>
			<td>#31018</td>
			<td>PVC cement</td>
		</tr>
		<tr>
			<td>PHD2000 syringe pump</td>
			<td>Harvard apparatus</td>
			<td>71-2000</td>
			<td>syringe pump</td>
		</tr>
		<tr>
			<td>Ponemah software</td>
			<td>DSI</td>
			<td></td>
			<td>recording software</td>
		</tr>
		<tr>
			<td>Precision 3630 Tower</td>
			<td>Dell</td>
			<td></td>
			<td>Computer for recording</td>
		</tr>
		<tr>
			<td>Raturn Stand-Alone System</td>
			<td>BASi</td>
			<td>MD-1407</td>
			<td>a movement response caging system</td>
		</tr>
		<tr>
			<td>RenaPulse High Fidelity Pressure Tubing 0.040 x 0.025</td>
			<td>Braintree Scientific</td>
			<td>RPT 040</td>
			<td>use for catheter</td>
		</tr>
		<tr>
			<td>Silicone cuff</td>
			<td>Transonic</td>
			<td>AAPC102</td>
			<td>skin button</td>
		</tr>
		<tr>
			<td>Surgical lubricant sterile bacteriostatic</td>
			<td>Fougera</td>
			<td>0168-0205-36</td>
			<td>gell for flow probe</td>
		</tr>
		<tr>
			<td>Tergazyme</td>
			<td>Alconox</td>
			<td></td>
			<td>protease contained anionic detergent</td>
		</tr>
		<tr>
			<td>TS420 Perivascular Flow Module</td>
			<td>Transonic</td>
			<td>TS420</td>
			<td>perivascular flow module</td>
		</tr>
		<tr>
			<td>Vetbond</td>
			<td>3M</td>
			<td>1469SB</td>
			<td>tissue adhesive</td>
		</tr>
		<tr>
			<td>WinDaq software</td>
			<td>DATAQ</td>
			<td></td>
			<td>recording software</td>
		</tr>
	</tbody>
</table>

long-term continuous measurement of renal blood flow in conscious rats

The kidneys play a crucial role in maintaining the homeostasis of body fluids. The regulation of renal blood flow (RBF) is essential to the vital functions of filtration and metabolism in kidney function. Many acute studies have been carried out in anesthetized animals to measure RBF under various conditions to determine mechanisms responsible for the regulation of kidney perfusion. However, for technical reasons, it has not been possible to measure RBF continuously (24 h/day) in unrestrained unanesthetized rats over prolonged periods. These methods allow the continuous determination of RBF over many weeks while also simultaneously recording blood pressure (BP) with implanted catheters (fluid-filled or by telemetry). RBF monitoring is carried out with rats placed in a circular servo-controlled rat cage that enables the unrestrained movement of the rat throughout the study. At the same time, the tangling of cables from the flow probe and arterial catheters is prevented. Rats are first instrumented with an ultrasonic flow probe placement on the left renal artery and an arterial catheter implanted in the right femoral artery. These are routed subcutaneously to the nape of the neck, and connected to the flowmeter and pressure transducer, respectively, to measure RBF and BP. Following surgical implantation, rats are immediately placed in the cage to recover for at least one week and stabilize the ultrasonic probe recordings. Urine collection is also feasible in this system. The surgical and post-surgical procedures for continuous monitoring are demonstrated in this protocol.

The mean arterial pressure data (Figure 1A) and blood flow data (Figure 1B) from a representative male Dahl salt-sensitive rat are shown. The Dahl salt-sensitive rats are maintained in a colony and bred at the Medical College of Wisconsin. The surgery was done at the age of 8 weeks, and the bodyweight was 249 g at the time of surgery. Rats were fed with a 0.4% NaCl diet, and the diet was changed to a 4% NaCl diet at the age of 10 weeks. Measurements were continu...

Watch this Scientific Journal Video about Long-Term Continuous Measurement of Renal Blood Flow in Conscious Rats at JoVE.com

Long-Term Continuous Measurement of Renal Blood Flow in Conscious Rats

The present protocol describes a long-term continuous measurement of renal blood flow in conscious rats and simultaneously recording blood pressure with implanted catheters (fluid-filled or by telemetry).

The kidneys play a crucial role in maintaining the homeostasis of body fluids. The regulation of renal blood flow (RBF) is essential to the vital ...

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