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

Summary

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).

Abstract

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.

Introduction

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 nephrectomy^2,3,4 and by the reductions of RBF that occur in kidney failure^5,6,7. 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 ultrasound^9,11,12, MRI^4,13, and PET-CT^14,15 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 previously^{16,17,18,19,20,21}. 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.

Protocol

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

1. 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.
2. Place the rats in the cage to become familiar with the environment, food, and water system at least the week prior to surgery. Fast the rats from the day before the surgery because a high stomach content may interfere with the placement of the flow probe into the left renal artery and can cause tracheal aspiration.
3. Connect a 5 cm of polyurethane tubing (inner diameter 0.30 mm and outer diameter 0.64 mm) to the end of the 90 cm of polyurethane tubing (inner diameter 0.64 mm and outer diameter 1.02 mm) with PVC cement to make a femoral arterial catheter (see Table of Materials).
   1. Sterilize the catheters with an Ethylene oxide sterilizer, the flow probe with 2.5% glutaraldehyde, and the surgical instruments in a steam autoclave. Wipe surgical tables, microscopy, and lights with 1% sodium hypochlorite.

2. Surgery

1. Place the RBF probe following the steps below.
   1. Anesthetize the rats with 2.0%-2.5% Isoflurane to the degree that the rats do not respond to the pain stimulus. Place it on the surgical table set at 37 °C and inject 0.09 mg/kg of buprenorphine SR and 15 mg/kg of cefazolin (see Table of Materials) before surgery.
   2. Shave the entire abdomen with an electric clipper and a region on the nape of the neck around the 7^th cervical vertebrae where the catheter and flow prove wires will exit.
   3. After shaving, wipe the area with 70% ethanol, 10% povidone-iodine, and again with 70% ethanol.
   4. Place the rat in the prone position. Make a 1 cm cut using a scalpel on the nape of the neck and the left flank. Then, perform a blunt dissection with hemostatic forceps and clear a subcutaneous space from the flank incision to the back of the neck.
   5. Pass the flow probe through this subcutaneous tunnel from the neck to the flank incision with hemostatic forceps.
   6. Place the rat in the supine position. Make a 4-5 cm midline abdominal incision.
   7. Dissect the area around the renal artery with curved tweezers to expose a space sufficient to place the flow probe (see Table of Materials). Then bluntly pierce the left quadratus lumborum muscle with the hemostatic forceps and pull the head of the flow probe into the abdominal cavity.
   8. Hook the tip of the flow probe to the left renal artery and connect it to the flow meter (see Table of Materials). Add some gel around the probe tip, and the value of the flow rate will appear on the flow meter.
      NOTE: Although it depends on the size of the rat, a flow of about 3-5 mL/min will be observed in a 230 g rat.
   9. Glue the polyester fiber mesh attached to the probe with tissue adhesive to the abdominal wall and hold until dry and bonded (~1-2 min). Once the flow is in place, disconnect the flow probe from the flow meter and cover the abdomen with saline-soaked gauze and move on to the step of inserting the catheter.
2. Insert the femoral catheter following the steps below.
   NOTE: The method for inserting a fluid-filled catheter is the same as regular telemetry installations. Although telemetry is preferred, the arterial catheter enables pressure monitoring and period blood sampling from the conscious rat.
   1. First, fill the catheter with saline and clamp it with vascular forceps before making a 1 cm skin incision using a scalpel on the left thigh to dissect and expose the femoral artery. While blocking the flow at the proximal side of the femoral artery with a thread, insert the catheter.
   2. Flush with a small amount of saline, plug with appropriate size stainless wire, and tie the catheter with a thread to fix it.
   3. Once the ligature is tied around the catheter, create a subcutaneous tunnel by using a stainless-steel trocar from the thigh to the back of the neck to bring the catheter to the neck region. Secure it with 3-0 silk sutures placed in the trapezius muscle.
3. Suture the probe.
   1. Turn the rat to the prone position and stitch the circular loop of the flow probe subcutaneously at the flank. Suture the incision at the flank and the neck with 4-0 surgical suture (see Table of Materials).
   2. Attach a skin button to the flow probe and suture it with 3-0 silk at the back of the neck.
   3. Connect the flow probe to the flow meter again, turn the rat back to the dorsal position to check RBF, and make final adjustments of the flow probe to optimize its position on the renal artery.
   4. Finally, suture the muscle with 3-0 silk and the skin with 4-0 surgical suture.

3. Recovery of the animal

1. After careful observation, until the rats are fully recovered from the anesthesia, return the rats to a movement response caging system, connect the flow probe to the blood flow meter, and allow a recovery period of about a week to stabilize the probe and flow measurement.
   NOTE: Recording doesn't have to be done during this period.
2. Infuse 3% heparinized saline continuously throughout the study from the arterial catheter at the rate of 100 µL/h to prevent the clotting.
3. When the flow stabilizes after 5-6 days, set the flowmeter calibration to measure blood flow at 0-20 mL/min and begin the continuous recording of RBF.

Results

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...

Discussion

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...

Materials

List of materials used in this article

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