Name: highly sensitive measurement of glomerular permeability in mice with fluorescein isothiocyanate-polysucrose 70
Uploaded: 2019-08-09T13:00:00
Description: Watch this Scientific Journal Video about Highly Sensitive Measurement of Glomerular Permeability in Mice with Fluorescein Isothiocyanate-polysucrose 70 at JoVE.com

The authors thank Christina Schwandt, Blanka Duvnjak, and Nicola Kuhr for their exceptional technical assistance and Dr. Dennis Sohn for his help with the fluorescence scan.This research was supported by a grant of the Deutsche Forschungsgemeinschaft (DFG) SFB 612 TP B18 to L.C.R. and L.S. The funder had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

The investigations were conducted according to the guidelines outlined in the Guide for Care and Use of Laboratory Animals (US National Institutes of Health Publication No. 85-23, revised 1996). All animal experiments were performed in accordance with the relevant institutional approvals (state government Landesamt f&#252;r Natur, Umwelt und Verbraucherschutz [LANUV] reference number 84-02.04.2012.A397).
1. Preparation of instruments, solutions, and equipment
<ol>
	<li>Reconstitute FITC-poly...

<ol>
	<li>Chronic Kidney Disease Prognosis Consortium, , 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=Association+of+estimated+glomerular+filtration+rate+and+albuminuria+with+all-cause+and+cardiovascular+mortality+in+general+population+cohorts:+a+collaborative+meta-analysis.">Association of estimated glomerular filtration rate and albuminuria with all-cause and cardiovascular mortality in general population cohorts: a collaborative meta-analysis.</a> Lancet. 375 (9731), 2073-2081 (2010).</li><li>Mori, K. P., 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=Increase+of+Total+Nephron+Albumin+Filtration+and+Reabsorption+in+Diabetic+Nephropathy.">Increase of Total Nephron Albumin Filtration and Reabsorption in Diabetic Nephropathy.</a> Journal of the American Society of Nephrology. 28 (1), 278-289 (2017).</li><li>Amsellem, 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=Cubilin+is+essential+for+albumin+reabsorption+in+the+renal+proximal+tubule.">Cubilin is essential for albumin reabsorption in the renal proximal tubule.</a> Journal of the American Society of Nephrology. 21 (11), 1859-1867 (2010).</li><li>Axelsson, J., Rippe, A., Oberg, C. M., Rippe, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid,+dynamic+changes+in+glomerular+permeability+to+macromolecules+during+systemic+angiotensin+II+(ANG+II)+infusion+in+rats.">Rapid, dynamic changes in glomerular permeability to macromolecules during systemic angiotensin II (ANG II) infusion in rats.</a> American Journal of Physiology-Renal Physiology. 303 (6), F790-F799 (2012).</li><li>Grande, 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=Unaltered+size+selectivity+of+the+glomerular+filtration+barrier+in+caveolin-1+knockout+mice.">Unaltered size selectivity of the glomerular filtration barrier in caveolin-1 knockout mice.</a> American Journal of Physiology-Renal Physiology. 297 (2), F257-F262 (2009).</li><li>Jeansson, M., Haraldsson, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glomerular+size+and+charge+selectivity+in+the+mouse+after+exposure+to+glucosaminoglycan-degrading+enzymes.">Glomerular size and charge selectivity in the mouse after exposure to glucosaminoglycan-degrading enzymes.</a> Journal of the American Society of Nephrology. 14 (7), 1756-1765 (2003).</li><li>Reis, L. 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The presented method enables the investigator to test glomerular permeability in mice in a very sensitive manner using a tracer. With this method, short-term increases in glomerular permeability can be diagnosed using only small amounts of urine. The most critical steps for successfully mastering this technique are 1) developing manual expertise in mouse surgery, especially in the cannulation of a central vein, 2) placing the urinary catheter without harming the mucosa, and 3) manual expertise in handling 384-well plates...

Functional or structural defects within the glomerular filtration barrier increase glomerular permeability to albumin, resulting in the detection of albumin in the urine (albuminuria). Albuminuria predicts cardiovascular outcome and is an important marker for glomerular injury1. Even low levels of albuminuria, lying within the normal range, are associated with an increased cardiovascular risk1.
Under physiological conditions, albumin is filtered through the glomerulus and is almost completely reabsorbed in the tubular system2,3. In mice, the detection of albumin in the urine is usually performed by an albumin enzyme-linked immunosorbent assay (ELISA) from 24 h of urine collection. If urine from a 24 h urine collection or spot urine is used, small differences in albumin concentrations may be missed due to assay sensitivity problems. Most researchers, therefore, use animal models in which albuminuria is induced by robust renal injury due to toxins, drugs, and renal surgery.
Therefore, the finding of a sensitive method to detect small and transient changes in glomerular permeability is very important to the field. Rippe et al. have presented a rat model to test glomerular permeability by applying a fluorescently labeled tracer, namely FITC-polysucrose 70 (i.e., FITC-Ficoll 70), at the size of albumin4. The tracer application allows the testing of short-term changes in glomerular permeability (within minutes) and is very sensitive4. Two studies have used the tracer method in mice5,6. Despite its benefits, this method, unfortunately, has disadvantages: it is technically very complex, radioactive, and invasive. Further analysis of the urine is only accomplished by using gel filtration or size-exclusion HPLC5,6.
Within this paper, we present an alternative, sensitive, nonradioactive, and fast method to measure glomerular permeability in mice using fluorescently labeled FITC-polysucrose 70. By introducing a transurethral catheter, urine collection is less invasive than bladder puncture, urethrotomy, and suprapubic catheter application, and allows urine collection at least every 30 min. Urine analysis is performed from small amounts (5&#181;L) using a fluorescent plate reader. Tracer concentrations in the urine are normalized to creatinine concentrations in the urine using an enzymatic creatinine assay.
Therefore, this novel method offers a sensitive tool to study early glomerular injury with increased glomerular permeability.

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			<td height="21" style="height:21px;width:257px;">Motic SMZ168 BL</td>
			<td style="width:77px;">Motic</td>
			<td style="width:101px;">SMZ168BL</td>
			<td style="width:235px;">microscope for mouse surgery</td>
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			<td height="84" style="height:84px;width:257px;">KL1500LCD</td>
			<td style="width:77px;">Pulch and Lorenz microscopy</td>
			<td style="width:101px;">150500</td>
			<td>light for mouse surgery</td>
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			<td height="42" style="height:42px;width:257px;">Microfederschere</td>
			<td style="width:77px;">Braun, Aesculap</td>
			<td style="width:101px;">FD100R</td>
			<td>fine scissors</td>
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			<td height="42" style="height:42px;width:257px;">Durotip Feine Scheren</td>
			<td style="width:77px;">Braun, Aesculap</td>
			<td style="width:101px;">BC210R</td>
			<td>for neck cut</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:257px;">Anatomische Pinzette</td>
			<td style="width:77px;">Braun, Aesculap</td>
			<td style="width:101px;">BD215R</td>
			<td>for surgery&#160;</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:257px;">Pr&#228;parierklemme</td>
			<td style="width:77px;">Aesculap</td>
			<td style="width:101px;">BJ008R</td>
			<td>for surgery&#160;</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:257px;">Seraflex</td>
			<td style="width:77px;">Serag Wiessner</td>
			<td style="width:101px;">IC108000</td>
			<td>silk thread</td>
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			<td height="21" style="height:21px;width:257px;">Ketamine 10%</td>
			<td style="width:77px;">Medistar</td>
			<td style="width:101px;"></td>
			<td>anesthesia</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:257px;">Rompun (Xylazin) 2%</td>
			<td style="width:77px;">Bayer</td>
			<td style="width:101px;"></td>
			<td>anesthesia</td>
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			<td height="42" style="height:42px;width:257px;">Fine Bore Polythene Tubing ID 0.28mm OD 0.61mm</td>
			<td style="width:77px;">Portex</td>
			<td style="width:101px;">800/100/100</td>
			<td style="width:235px;">Catheter</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:257px;">Fine Bore Polythene Tubing ID 0.58mm OD 0.96mm</td>
			<td style="width:77px;">Portex</td>
			<td style="width:101px;">800/100/200</td>
			<td style="width:235px;">Catheter</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:257px;">Harvard apparatus 11 Plus</td>
			<td style="width:77px;">Harvard Apparatus</td>
			<td style="width:101px;">70-2209</td>
			<td>syringe pump</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:257px;"></td>
			<td style="width:77px;"></td>
			<td style="width:101px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">BD Insyte Autoguard</td>
			<td style="width:77px;">BD</td>
			<td style="width:101px;">381823</td>
			<td>&#160;urinary catheter</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:257px;">Multimode Detector DTX 880</td>
			<td style="width:77px;">Beckman Coulter</td>
			<td style="width:101px;"></td>
			<td>plate reader</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:257px;">384 well microtiterplate</td>
			<td style="width:77px;">Nunc</td>
			<td style="width:101px;">262260</td>
			<td style="width:235px;">384 well platte</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:257px;">Creatinine Assay Kit</td>
			<td style="width:77px;">Sigma-Aldrich</td>
			<td style="width:101px;">MAK080</td>
			<td>to measure creatinine concentration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">96 well plate</td>
			<td style="width:77px;">Nunc</td>
			<td style="width:101px;">260836</td>
			<td>for creatinine assay&#160;</td>
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		<tr height="63">
			<td height="63" style="height:63px;width:257px;">FITC-labeled polysuccrose 70</td>
			<td style="width:77px;">TBD Consultancy</td>
			<td style="width:101px;">FP70</td>
			<td>FITC-ficoll</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">Angiotensin II</td>
			<td style="width:77px;">Sigma-Aldrich</td>
			<td style="width:101px;">A9525</td>
			<td>used to test glomerular permeability</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BP-98A</td>
			<td style="width:77px;">Softron</td>
			<td style="width:101px;"></td>
			<td>for blood pressure measurement</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">OTS 40.3040</td>
			<td style="width:77px;">Medite</td>
			<td style="width:101px;">01-4005-00</td>
			<td>heating plate for mouse surgery</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:257px;">Instillagel 6mL</td>
			<td style="width:77px;">Farco-Pharma GmbH</td>
			<td style="width:101px;"></td>
			<td>for urinary catheter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">Exacta</td>
			<td style="width:77px;">Aesculap</td>
			<td style="width:101px;">GT415</td>
			<td>shaver</td>
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highly sensitive measurement of glomerular permeability in mice with fluorescein isothiocyanate-polysucrose 70

The loss of albumin in urine (albuminuria) predicts cardiovascular outcome. Under physiological conditions, small amounts of albumin are filtered by the glomerulus and reabsorbed in the tubular system up until the absorption limit is reached. Early increases in pathological albumin filtration may, thus, be missed by analyzing albuminuria. Therefore, the use of tracers to test glomerular permselectivity appears advantageous. Fluorescently labeled tracer fluorescein isothiocyanate (FITC)-polysucrose (i.e., FITC-Ficoll), can be used to study glomerular permselectivity. FITC-polysucrose molecules are freely filtered by the glomerulus but not reabsorbed in the tubular system. In mice and rats, FITC-polysucrose has been investigated in models of glomerular permeability by using technically complex procedures (i.e., radioactive measurements, high-performance liquid chromatography [HPLC], gel filtration). We have modified and facilitated a FITC-polysucrose tracer-based protocol to test early and small increases in glomerular permeability to FITC-polysucrose 70 (size of albumin) in mice. This method allows repetitive urine analyses with small urine volumes (5 &#181;L). This protocol contains information on how the tracer FITC-polysucrose 70 is applied intravenously and urine is collected via a simple urinary catheter. Urine is analyzed via a fluorescence plate reader and normalized to a urine concentration marker (creatinine), thereby avoiding technically complex procedures.

As depicted in Figure 2, the method to test glomerular permeability in mice is built up in three phases. The first phase is called the preparation phase, in which a urinary catheter and a central venous catheter are placed. The second phase is called the equilibration phase, starting with an intravenous bolus injection of FITC-polysucrose 70 and followed by the continuous infusion of FITC-polysucrose 70 for 60 min. The last phase is called the experimental ph...

Watch this Scientific Journal Video about Highly Sensitive Measurement of Glomerular Permeability in Mice with Fluorescein Isothiocyanate-polysucrose 70 at JoVE.com

Highly Sensitive Measurement of Glomerular Permeability in Mice with Fluorescein Isothiocyanate-polysucrose 70

Here, we present a protocol to test glomerular permeability in mice using a highly sensitive, nonradioactive tracer. This method allows repetitive urine analyses with small urine volumes.

The loss of albumin in urine (albuminuria) predicts cardiovascular outcome. Under physiological conditions, small amounts of albumin are filtered by the ...

This method can help answer key questions in the nephrology field about the role of glomerular permeability in kidney disease. The main advantage of this technique is that small and transient increases of glomerular permeability are detected by using a fluorescent lead labeled tracer. After anesthesia, place a urinary catheter in the female mouse.Position the mouse in dorsal recumbency. Tighten the lower abdomen and find the urethral ostium. Obtain the plastic part of the catheter of a 22 G angiocatheter and lubricate it with xylocaine gel.Use the left index finger to place careful tension on the lower abdomen and introduce the catheter three millimeters into the urethral ostium while paralleling the distal urethral axis. Turn the top of the angiocatheter 180 degrees by keeping the tip within the urethral ostium and maintaining the axis of the urethra. Then, introduce the catheter seven millimeter further into the mouse so that it is placed within the bladder.Place a 1.5 milliliter brown tube over the top of the angiocatheter to collect the urine. Apply one milliliter of 0.9%sodium chloride subcutaneously to enhance urine production. Shave the neck of the mouse and place it in recumbency with the head toward the surgeon.Hyper extend the head of the mouse with a tape. Disinfect the neck with 70%isopropanol. Using a tweezer and a pair of scissors make a five millimeter skin incision below the jawline.Cut the skin approximately one centimeter in the direction of the sternum until the middle of the sternum is reached. Use a pair of scissors. Carefully dissect the skin to the right side of the neck.Make a rectangular incision of the skin to the right to expose the soft tissue of the neck. Be careful not to damage the jugular vein. Use a pair of scissors and a tweezer to fix the skin flaps with two clamps.Using the tip of the fine tweezer, carefully expose the jugular vein by blunt preparation. If necessary, remove tissue with fine scissors. Avoid injury to vein branches.Place and close a ligature with a silk thread at the distal part of the visible jugular vein which is toward the head of the mouse. Fix the silk thread with a tape to put tension on the ligature. This ensures slight tension on the jugular vein.Prepare a ligature around the proximal part of the jugular vein. After that, fill the catheter with the equilibration and fusion solution. Fix the catheter with a tape so that the catheter is aligning the jugular vein.Control for bubbles to avoid air embolism. Lift the jugular vein with fine tweezers at the site of insertion. Align the tubing parallel to the jugular vein and puncture the jugular vein aiming for the lumen.Insert for approximately two to four millimeters paralleling the axis of the jugular vein. Avoid any brisk movements. Close the ligature to fix the catheter.Control for tightness of the ligature by carefully viewing through the microscope. Put a damp swab over the site of surgery. Place the tail cuff at the bottom of the mouse tail while the mouse lays in dorsal recumbency.Start the measurements and repeat them ten times per time point. If blood pressure measurements do not seem correct and false data are produced, adjust the position of the tail cuff. Before the equilibration phase, change the urinary collection tube and place it on ice.Fold a swab and put it around the small vessel catheter that is introduced into the jugular vein. Place a clamp over the swab to abolish retrograde blood flow or air embolism. Connect a 27 G needle with a one milliliter syringe filled with the FITC bolus to the end of the small vessel catheter.Open the clamp and apply the 100 microliter FITC bolus. Close the clamp again and connect the larger catheter with the smaller catheter. Reopen the clamp and start the syringe pump with a flow rate of 0.008 milliliter per minute.Continue the infusion for 60 minutes. In this phase, the effective drugs on glomerular perm selectivity is investigated. Change the urinary tube form time point zero minute to another 1.5 milliliter brown tube.Put a swab around the small vessel catheter and disconnect the large catheter from the small vessel catheter. Fill the syringe with the experimental phase solution. Place it into the syringe pump and let the infusion pump run so that the large catheter is filled with the experimental phase solution.Reconnect the catheters while avoiding air embolism and reopen the clamp. Start the syringe pump at the flow rate of 0.008 milliliter per minute. Continue running the syringe pump for 60 minutes and collect the urine within the urinary tube thereafter.Place the urine tube on ice. In this study, fluorescence scan of native mouse urine shows a peak at 395 nanometers. FITC polysucrose 70 at a concentration of 40 micrograms per milliliter in mouse urine displays a fluorescence maximum at 525 nanometers.Mouse urine does not disturb the fluorescence measurement of FITC polysucrose 70. At the admission of 525 nanometers increasing concentrations of FITC polysucrose 70 shows an increased fluorescence intensity. This method is also capable of detecting differences in glomerular permeability.The white columns represent FITC polysucrose 70 levels before the start of angiotensin II stimulation in the experimental phase. After dosages for 60 minutes, the glomerular permeability increased compared with control group. angiotensin II wash out decreased glomerular permeability.The FITC polysucrose 70 levels decreased after an additional 60 minutes of angiotensin II stimulation or 60 minutes of angiotensin II washout. The increase in glomerular permeability due to angiotensin II could be blocked by an angiotensin II receptor blocker. Blood pressure monitored by the tail cuff method did not show significant differences between the control and angiotensin II treated groups.While attempting this procedure it's important to avoid air embolism when changing infusion solutions.

Research

JoVE Journal

Medicine

Identification and Isolation of Slow-Dividing Cells in Human Glioblastoma Using Carboxy Fluorescein Succinimidyl Ester (CFSE)

Identification and Isolation of Slow-Dividing Cells in Human Glioblastoma Using Carboxy Fluorescein Succinimidyl Ester CFSE

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Quantifying Glomerular Permeability of Fluorescent Macromolecules Using 2-Photon Microscopy in Munich Wistar Rats

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An in vivo Assay to Test Blood Vessel Permeability

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Assessment of Kidney Function in Mouse Models of Glomerular Disease

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