This work was supported by the Vanderbilt Center for Kidney Disease (VCKD), and was in part funded by the following grants: DOD PR161028 and R01DK112688 (Mark de Caestecker)
We acknowledge support to LS, PM and BW by the MRC, EPSRC and BBSRC-funded UK Regenerative Medicine Platform &#34;Safety and Efficacy, focussing on Imaging Technologies Hub&#34; (MR/K026739/1).

All animal experiments were performed in accordance with local guidelines in the UK and USA. Experiments conducted at the University of Liverpool were performed under a license granted under the UK Animals (Scientific Procedures) Act 1986 and were approved by the University of Liverpool ethics committee. All animal experiments conducted at Vanderbilt University Medical Center were approved by the Vanderbilt Institutional Animal Care and Use Committee.
1. Preparing the FITC-sinistrin
<ol>
	<li>Prepare 40 mg/mL FITC-sinistrin in phosphate buffered saline (PBS). 
	NOTE: Aliquots can be stored at -20 &#176;C for several months with no noticeable decrease in quality; however multiple freeze-thaw cycles should be avoided. FITC-sinistrin is light sensitive - keep the tube protected from light.</li>
	<li>Calculate the volume of FITC-sinistrin required for each mouse:
	<ol>
		<li>Weigh each mouse on each day of measurement.</li>
		<li>The recommended dose is 0.15 mg FITC-sinistrin per gram body weight.</li>
	</ol>
	</li>
</ol>
2. Mouse Preparation
<ol>
	<li>Prepare separate cages for the mice while undergoing GFR measurements. Provide absorbent paper towels and a few pellets of food.</li>
</ol>
3. Removing Hair from the Mouse (1-2 days before GFR Measurement)
<ol>
	<li>Anesthetize the mouse with 3% isoflurane, and once the mouse is asleep, maintain anesthesia with 1.5&#8211;2% isoflurane, depending on the breathing rate of the mouse. Place the mouse prone on a heat pad.</li>
	<li>Use an electric shaver, going against the direction of the fur, to remove most of the fur from one side of the mouse&#8217;s back. Shave from the top of the hind legs up to the neck, and across the ribs.</li>
	<li>Apply a thin layer of depilation cream to the shaved area using a cotton bud (Figure 1A). Move the cotton bud against the direction of the fur to ensure that the cream is applied as close to the skin as possible.</li>
	<li>Remove the cream after 1&#8211;3 min by washing it off with cotton swabs and warm water. Do not perform the measurement if the skin appears very red and irritated after measurement, and do not repeat depilation within 72 h to avoid damaging the skin.</li>
</ol>
4. Preparing the Transdermal GFR Monitor
<ol>
	<li>Use one of the two sizes of patches that are available. The first is 2.5 x 3 cm2 in size and can be used for measurements in mice directly. The other patches are 6 x 3 cm2 in size and are meant to be used in rats or larger animals but can be cut to a smaller size for use in mice.</li>
	<li>Peel the backing off one side of the patch and stick the GFR device on the adhesive side, positioning the LEDs exactly above the clear window.</li>
	<li>Cut the excess adhesive patch to fit the size of the battery and stick one side of the patch to the battery.</li>
</ol>
5. Attaching the Transdermal GFR Monitor
<ol>
	<li>Anaesthetize the mouse with isoflurane as described in step 3.1 and place the mouse prone on a heat pad. Anesthetize mice only for placement of the transdermal GFR monitor and injection of FITC-sinistrin; allow to recover from anesthesia for the measurement of FITC decay.</li>
	<li>Clean the pre-shaved skin with 70% ethanol. Place approximately 12 cm of hypoallergenic silk tape under the mouse (Figure 1B; the width of the tape should be reduced to 1.5&#8211;2 cm so that it is not too wide for the mouse).</li>
	<li>Position the tape so that only approximately 2 cm is on the mouse&#8217;s right side, and the rest is on the left. Fold over one edge of the right side of tape for easy placement and removal after the measurement. The left-right instructions for steps 5.3 and 5.6 are for device placement on the right side of the animal and can be swapped for device placement on the left side of the animal if required.</li>
	<li>Connect the battery to the device, remove the backing from the battery and securely place it on top of the device. The device is ready to use and data acquisition starts when the blue light emitting diodes (LEDs) start blinking.</li>
	<li>Remove the backing from the device and place on to the shaved skin. Position the device such that the window exposing the LEDs is over the ribs &#8211; do not have it too close to the spine or limbs (Figure 1C).</li>
	<li>Secure the device with the white tape. Secure the right side first (Figure 1D), wrapping it tightly around all edges of the device, then wrap the left side around the mouse and device (Figure 1E). Ideally, the left side of the tape only covers the device, and the right side ends under the mouse&#8217;s abdomen.</li>
	<li>Attach the tape by pressing it alongside the circumference of the mouse&#8217;s body. The tape needs to be attached firmly, but not tightly. If it is too loose then the device will move around too much and cause movement artefacts. However, it should not be so tight that it restricts breathing or movement or puts too much pressure on the skin.</li>
	<li>Leave the device untouched for 3 minutes before the FITC-sinistrin injection to allow a steady background reading to be taken. In this time, warm the tail with a heat pad or glove filled with warm water to prepare for tail vein injection (if using this route).</li>
</ol>
6. FITC-sinistrin Injection
<ol>
	<li>Prepare an insulin syringe with the calculated amount of FITC-sinistrin required for injection (this can be rounded to the nearest 10 &#956;L).</li>
	<li>Administer FITC-sinistrin by tail vein or retro-orbital injection. FITC-sinistrin should be administered in one smooth but rapid bolus to avoid multiple peaks on the clearance curve. It is better to administer only a partial dose than to have multiple attempts at administering the FITC-sinistrin.</li>
</ol>
7. Measuring the GFR
<ol>
	<li>Place the mouse in a cage on its own to recover from isoflurane anesthesia and for the duration of the measurement period.</li>
	<li>Observe the mouse in the cage for 1.5 h and then remove the device. Removing the device from the conscious mouse is fast, efficient, and generally well-tolerated by the mouse, but new users may prefer to anaesthetize the mouse for this step.
	<ol>
		<li>As one option, anaesthetize the mouse with isoflurane.</li>
		<li>As the other option, place the mouse on the wire rack on top of the cage, allowing the mouse to grasp the metal bars whilst the device is removed.</li>
	</ol>
	</li>
	<li>Pull off the white plaster tape from underneath the belly in one quick, smooth movement, and remove the device and black plaster from the skin. Be careful that the battery does not disconnect from the device yet.</li>
	<li>Return the mouse to its home cage.</li>
</ol>
8. Reading and Evaluating the Data
<ol>
	<li>Carefully disconnect the battery from the device</li>
	<li>Connect the device to the USB cable and then connect the cable to the computer</li>
	<li>Open the reading software (Sensor_ctrl_app.exe)</li>
	<li>In order, click &#8220;connect&#8221;, &#8220;read&#8221;, &#8220;re-name&#8221;, and &#8220;save&#8221;, then close the program</li>
	<li>Process and evaluate data in the analysis software as described in the respective manual</li>
</ol>

<ol>
	<li>Schreiber, 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=Transcutaneous+measurement+of+renal+function+in+conscious+mice.">Transcutaneous measurement of renal function in conscious mice.</a> American Journal of Physiology - Renal Physiology. 303 (5), F783-F788 (2012).</li><li>Friedemann, J., 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=Improved+kinetic+model+for+the+transcutaneous+measurement+of+glomerular+filtration+rate+in+experimental+animals.">Improved kinetic model for the transcutaneous measurement of glomerular filtration rate in experimental animals.</a> Kidney International. 90 (6), 1377-1385 (2016).</li><li>Schock-Kusch, D., 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=Reliability+of+transcutaneous+measurement+of+renal+function+in+various+strains+of+conscious+mice.">Reliability of transcutaneous measurement of renal function in various strains of conscious mice.</a> PLoS One. 8 (8), e71519 (2013).</li><li>Schock-Kusch, D., 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=Transcutaneous+assessment+of+renal+function+in+conscious+rats+with+a+device+for+measuring+FITC-sinistrin+disappearance+curves.">Transcutaneous assessment of renal function in conscious rats with a device for measuring FITC-sinistrin disappearance curves.</a> Kidney International. 79 (11), 1254-1258 (2011).</li><li>Herrera P&#233;rez, Z., Weinfurter, S., Gretz, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcutaneous+Assessment+of+Renal+Function+in+Conscious+Rodents.">Transcutaneous Assessment of Renal Function in Conscious Rodents.</a> Journal of Visualized Experiments. (109), 53767 (2016).</li><li>Cowley, A. W., 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=Progression+of+glomerular+filtration+rate+reduction+determined+in+conscious+Dahl+salt-sensitive+hypertensive+rats.">Progression of glomerular filtration rate reduction determined in conscious Dahl salt-sensitive hypertensive rats.</a> Hypertension. 62 (1), 85-90 (2013).</li><li>Scarfe, L., 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=Measures+of+kidney+function+by+minimally+invasive+techniques+correlate+with+histological+glomerular+damage+in+SCID+mice+with+adriamycin-induced+nephropathy.">Measures of kidney function by minimally invasive techniques correlate with histological glomerular damage in SCID mice with adriamycin-induced nephropathy.</a> Scientific Reports. 5, 13601 (2015).</li><li>Lazzeri, 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=Endocycle-related+tubular+cell+hypertrophy+and+progenitor+proliferation+recover+renal+function+after+acute+kidney+injury.">Endocycle-related tubular cell hypertrophy and progenitor proliferation recover renal function after acute kidney injury.</a> Nature Communications. 9 (1), 1344 (2018).</li><li>Street, J. M., 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=The+role+of+adenosine+1a+receptor+signaling+on+GFR+early+after+the+induction+of+sepsis.">The role of adenosine 1a receptor signaling on GFR early after the induction of sepsis.</a> American Journal of Physiology - Renal Physiology. 314 (5), F788-F797 (2018).</li><li>Skrypnyk, N. I., Harris, R. C., de Caestecker, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ischemia-reperfusion+model+of+acute+kidney+injury+and+post+injury+fibrosis+in+mice.">Ischemia-reperfusion model of acute kidney injury and post injury fibrosis in mice.</a> Journal of Visualized Experiments. (78), (2013).</li><li>Wang, W., 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=Endotoxemic+acute+renal+failure+is+attenuated+in+caspase-1-deficient+mice.">Endotoxemic acute renal failure is attenuated in caspase-1-deficient mice.</a> American Journal of Physiology - Renal Physiology. 288 (5), F997-F1004 (2005).</li><li>Eisner, 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=Major+contribution+of+tubular+secretion+to+creatinine+clearance+in+mice.">Major contribution of tubular secretion to creatinine clearance in mice.</a> Kidney International. 77 (6), 519-526 (2010).</li><li>Bankir, L., Yang, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+insights+into+urea+and+glucose+handling+by+the+kidney,+and+the+urine+concentrating+mechanism.">New insights into urea and glucose handling by the kidney, and the urine concentrating mechanism.</a> Kidney International. 81 (12), 1179-1198 (2012).</li><li>Qi, Z., 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=Serial+determination+of+glomerular+filtration+rate+in+conscious+mice+using+FITC-inulin+clearance.">Serial determination of glomerular filtration rate in conscious mice using FITC-inulin clearance.</a> American Journal of Physiology - Renal Physiology. 286 (3), F590-F596 (2004).</li><li>Peters, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+kinetic+basis+of+glomerular+filtration+rate+measurement+and+new+concepts+of+indexation+to+body+size.">The kinetic basis of glomerular filtration rate measurement and new concepts of indexation to body size.</a> European Journal of Nuclear Medicine and Molecular Imaging. 31 (1), 137-149 (2004).</li><li>Chapman, M. E., Hu, L., Plato, C. F., Kohan, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioimpedance+spectroscopy+for+the+estimation+of+body+fluid+volumes+in+mice.">Bioimpedance spectroscopy for the estimation of body fluid volumes in mice.</a> American Journal of Physiology - Renal Physiology. 299 (1), F280-F283 (2010).</li></ol>

D S-K, JF and YS are employees at MediBeacon GmbH the manufacturer and distributor of the transdermal GFR monitor. 
D S-K and JF are inventors on patents and patent applications for the presented technology.

This manuscript and the accompanying training video provide practical guidelines for the use of transdermal GFR monitors in mice. The most critical steps in the procedure are the correct attachment of the device on the animal&#39;s back, and securely wrapping the tape around the abdomen. The best position is either slightly left or right of the midline, over the ribcage. The patch and device need to be firmly attached to the skin, but they should not be so tight that they restrict breathing, movement, or affect skin blood circulation under the device, as this would lead to faulty/inaccurate measurements. In addition, since monitoring occurs in conscious mice after they have recovered from anesthesia, correct placement of the device on the part of the body with lowest interference from movement results in transdermal measurements with little movement artefacts. For this reason, it is important that the device is not placed too close to the upper limbs so that the mice can move their shoulders freely.
It is important to depilate the mice one to two days before GFR measurement, as depilation affects the measurement of FITC-sinistrin clearance, with preliminary data indicating that depilation immediately before measurement of transdermal GFR increases the apparent half-life of FITC-sinistrin. The mechanism for this is unknown. Therefore, in order to obtain reliable measurements over multiple time points and between experiments, it is advisable to depilate the mice in advance, to allow the skin to recover from this process before proceeding with GFR measurements. Depilation cream should not be applied to the same area of skin within 72 h of a prior application, to avoid chemical damage to the skin. In many cases, the fur re-growth takes several days or up to a week, and so re-application of depilation cream within 72 h can easily be avoided.&#160;
Because up to 50% of serum creatinine is excreted by tubular section in mice13, and because there is increased reabsorption of urea from renal tubules when mice are dehydrated14, serum creatinine and BUN are poor markers of renal function. However, because of their convenience, these assays continue to be used as the main measure of renal function in preclinical studies of AKI and CKD in mice. However, consistent with major contribution of tubular secretion to creatinine excretion in mice with normal or near normal renal function13, serum creatinine showed little correlation with FITC-sinistrin clearance at high clearance rates (low FITC-sinistrin half-life), indicating that creatinine is an insensitive measure of renal function in mice with mild kidney injury. In contrast, while BUN correlates well with FITC-sinistrin clearance in mice with mild renal impairment, there is poor correlation between BUN and FITC-sinistrin clearance in mice with more severe kidney injury (high FITC-sinistrin half-life). This is likely caused by effects of urea reabsorption associated with dehydration in sick animals with severe kidney injury.
A major advantage of the transdermal GFR measurement, compared to all other bolus clearance or constant infusion techniques for GFR measurement, is that it does not require carefully timed blood or urine collections. These can be particularly challenging in mice as they have low total blood volumes and urinary output as compared with rats. Moreover, mice need to be handled only for attachment of the device and injection, but not for multiple venipunctures, as required for classical bolus clearance experiments15.&#160;Furthermore, the duration of anesthesia is short, and as such it is possible to perform repeated measurements in individual mice over time. The frequency at which measurements can be performed primarily depends on the health status of the mice, the researcher&#8217;s aptitude for intravenous injections, and local institutional regulations on repeated anesthesia sessions. In healthy, uninjured mice, transdermal GFR measurements can be performed daily, with minimal or no adverse effects on the mouse. However, injured mice suffering from AKI or CKD are unlikely to tolerate repeated anesthesia sessions as well as the healthy mice, and thus the frequency of measurements should be reduced.&#160;
The main limitation of transdermal GFR measurement, as compared with bolus clearance methods to measure GFR in mice is that the excretion kinetics are only measured as change in relative fluorescence intensity over time, and not as absolute tracer concentrations. Because of this, it is only possible to measure the rate constant of the single exponential decay of the excretion kinetic, which is a very close estimate of GFR normalized on extracellular volume16. To express GFR in mL/min, the extracellular volume of the animal has to be estimated using a conversion factor that was established in prior studies in which simultaneous measurements of plasma concentrations of FITC-sinistrin were performed1. However, this conversion factor may not correctly estimate extracellular fluid volumes equally well in all mice, since fluid volume may be affected by a variety of extraneous factors including age, sex, hydration status (which may be affected by surgical interventions as well as kidney injury), and weight17. However, unlike the bolus dosing method to assess GFR in mice, transcutaneous GFR measurement is subject to less operator-dependent variability as it is not affected by dosing errors or by errors in timing of blood collections.
Another limitation of the transcutaneous GFR measurement technique is that baseline signal shifts may occur during the course of the measurement due to bleaching of skin fluorophores and the anesthesia required for device attachment and tracer injection. This limitation was addressed by Friedemann and colleagues by implementing a correction algorithm3. The implementation of this algorithm led to an improvement in precision of the transdermal technique comparable to a constant infusion technique of GFR assessment.
A frequently asked question is whether skin pigmentation in different mouse strains affects the transdermal FITC-sinistrin clearance. Skin pigmentation reduces the FITC-sinistrin signal intensity since dark pigments absorbs the blue excitation and the green emission signals from FITC-sinistrin measurements. However, the excretion rate of FITC-sinistrin is independent of the overall signal intensity. Furthermore, while the measured signal is lower, the background signal is also lower in pigmented mice. Because the background signal is a mixture of autofluorescence of skin fluorophores and reflection of the excitation light, we have found that the background-to-maximum signal ratio is comparable, or even improved, in pigmented animals. In addition, movement artifacts, which are caused by exposure of the surrounding skin to reflected light, are reduced in pigmented mice since the reflected light is also absorbed by pigmented skin.
In conclusion, the technique we have presented allows precise measurement of GFR in conscious, freely moving mice of all skin types. As the technique is independent of blood sampling, it can be used repeatedly on the same animal for longitudinal observations in CKD models, as well as for the measurement of rapid changes of GFR that occur after induction of AKI.

The use of transcutaneous GFR monitors in mice was first reported by Schreiber and colleagues in 2012 and was validated by comparing GFR measurements obtained using this technique, with results obtained by direct measurement of FITC-sinistrin bolus clearance from serial blood samples1. To date, there have been 35 peer-reviewed publications in which transcutaneous GFR monitors have been used in rats and mice (a regularly updated list of journal articles and conference abstracts in which the preclinical GFR monitor was used can be found at the MediBeacon website2). Transdermal GFR measurements in rats and mice has been described in a number of publications1,3,4,5, and a video tutorial demonstrating its use in rats has been published6. However, measurement in mice presents additional technical challenges. Here, we provide the first detailed practical guide to the use of transdermal GFR monitors in mice.
There are a variety of reasons why investigators are starting to favor the use of transdermal GFR monitors to assess renal function in rodent models. Transdermal measurement of FITC-sinistrin clearance has been shown to provide a more sensitive and accurate measure of renal function compared to the traditional parameters of renal function such as serum creatinine and blood urea nitrogen (BUN)7,8. By implementing an improved evaluation algorithm, Friedemann and colleagues demonstrated that the system reaches precision comparable to the gold standard, the constant infusion technique for GFR measurement3. Recent studies have also shown that sequential analysis using transcutaneous GFR monitors can be used to study early changes in renal function as well as functional recovery after induction of acute kidney injury (AKI) without interfering with the animals&#39; blood volume or hemodynamics, since the assay does not require sequential blood sampling9,10. The ability to measure GFR with high precision and sensitivity repeatedly in the same animal makes this technique attractive for a variety of different research disciplines. Transdermal GFR monitors have been used by pharmaceutical companies to assess the toxicity of novel compounds, as well as in universities for basic and translational research.

<table>
	<tbody>
		<tr>
			<td>Transdermal GFR monitor (comes with 1 device, 2 batteries and 1 charger)</td>
			<td>MediBeacon GmbH</td>
			<td>TDM-MH001</td>
			<td>Reading software: MPD Lab; Analysis software: MPD Studio</td>
		</tr>
		<tr>
			<td>Additional Batteries</td>
			<td>MediBeacon GmBH</td>
			<td>PWR-BT0001</td>
			<td></td>
		</tr>
		<tr>
			<td>Attachment patches</td>
			<td>MediBeacon GmbH</td>
			<td>small: PTC-SM001; large: PTC-LG001</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC-sinistrin</td>
			<td>MediBeacon GmbH</td>
			<td>FTC-FS001</td>
			<td></td>
		</tr>
		<tr>
			<td>Hypoallergenic silk tape</td>
			<td>e.g. Durapore (1538-2), or Kendall (7138C), or Leukosilk (01032-00)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Anaesthesia chamber, isoflurane, oxygen</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Heat pad</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Electric shaver</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Depilatory (hair removal) cream</td>
			<td>e.g. Veet or Nair</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cotton buds</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cotton swabs</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Timer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Scales</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>70% ethanol wipes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

transdermal measurement of glomerular filtration rate in mice

Transdermal analysis of glomerular filtration rate (GFR) is an established technique that is used to assess renal function in mouse and rat models of acute kidney injury and chronic kidney disease. The measurement system consists of a miniaturized fluorescence detector that is directly attached to the skin on the back of conscious, freely moving animals, and measures the excretion kinetics of the exogenous GFR tracer, fluorescein-isothiocyanate (FITC) conjugated sinistrin (an inulin analog). This system has been described in detail in rats. However, because of their smaller size, measurement of transcutaneous GFR in mice presents additional technical challenges. In this paper we therefore provide the first detailed practical guide to the use of transdermal GFR monitors in mice based on the combined experience of three different investigators who have been performing this assay in mice over a number of years.

In this section we present representative results of the use of the transdermal GFR monitor. The transdermal monitor has been used in a variety of mouse strains and models of AKI and CKD2.
Figure 2 shows representative FITC-sinistrin clearance curves in male BALB/c mice before and after ischemia reperfusion injury (IRI) with simultaneous contralateral nephrectomy. FITC-sinistrin is rapidly cleared from the circulation in healthy mice (Figure 2A), but clearance is dramatically delayed in mice with AKI (Figure 2B, C). In mice with very severe AKI, there may not be any change in FITC-sinistrin fluorescence during the 90-minute measurement period, indicating a complete absence of glomerular filtration (Figure 2C).
Transdermal GFR measurement is minimally invasive and can be used to monitor changes in kidney function in the same mice over multiple time points. Figure 3 depicts changes in GFR determined by sequential transdermal FITC-sinistrin clearance measurements at baseline, and 1, 2 and 4 days after inducing IRI (unilateral ischemia with simultaneous contralateral nephrectomy). Data shown includes FITC-sinistrin clearance half-life (Figure 3A), and GFR (Figure 3B) calculated from the measured FITC-sinistrin clearance half-life, as described by Schreiber et al1.
In Figure 4, chronic kidney disease (CKD) was induced in male BALB/c mice by performing prolonged unilateral IRI followed by delayed contralateral nephrectomy, as described11. GFR was assessed by transdermal FITC-sinistrin clearance on day 26 after the initial IRI. The increase in FITC-sinistrin half-life (Figure 4A), and therefore the decrease in GFR (Figure 4B), indicates impaired renal function in these mice. These data demonstrate that transcutaneous GFR measurement can be used to measure changes in renal function in mice with CKD.
Figure 5A shows that FITC-sinistrin half-life correlates closely with semi-quantitative histological assessment of tubular injury over the full range of GFR measurements in uninjured mice and in mice with different severities of IRI-induced AKI. In contrast, serum creatinine and blood urea nitrogen (BUN) showed a positive but weaker correlation with FITC-sinistrin clearance (Figure 5B, C), indicating that transcutaneous GFR measurements provide a more reliable measure of renal injury (tubular injury scores) following IRI-induced AKI than either serum creatinine or BUN.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58520/58520fig1.jpg" /> 
Figure 1: Attaching the transdermal GFR monitor. Photographs of hair removal (A), placement of the tape under the mouse (B), placement of the device on the mouse&#39;s skin (C), and securing the device by wrapping the tape around the mouse and device (D-E) <a href="https://www.jove.com/files/ftp_upload/58520/58520fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58520/58520fig2.jpg" /> 
Figure 2: Example FITC-sinistrin clearance curves in male BALB/c mice before and after ischemia reperfusion injury (IRI) with simultaneous contralateral nephrectomy. Clearance curves at baseline (A), and one day after IRI surgery (B) in the same mouse, indicating impaired renal function in this mouse. (C) Clearance curve from a more severely injured mouse one day after IRI surgery. There was no clearance of FITC-sinistrin during the measurement period, indicating renal failure. Black data points represent raw data, blue lines represent the 3-compartment fit, and green lines represent 95% confidence intervals. <a href="https://www.jove.com/files/ftp_upload/58520/58520fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58520/58520fig3.jpg" /> 
Figure 3: Male BALB/c mice, age 8-10 weeks underwent unilateral ischemia with simultaneous contralateral nephrectomy (n=5). GFR was assessed at baseline and on days 1, 2, and 4 after surgery, and compared with sham-operated control mice (n=5). FITC-sinistrin half-life in (A) was used, along with the body weight of the mice, to calculate GFR in (B). Data points represent individual animals, and error bars represent mean and standard error. <a href="https://www.jove.com/files/ftp_upload/58520/58520fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58520/58520fig4.jpg" /> 
Figure 4: Male BALB/c mice age 8-10 weeks underwent unilateral ischemia with delayed contralateral nephrectomy at day 8 (n=5). GFR was assessed by on day 26 and was compared to age-matched healthy control mice (n=5). FITC-sinistrin half-life in (A) was used, along with the body weight of the mice, to calculate GFR in (B). Data points represent individual animals, and error bars represent mean and standard deviation. Tubular injury was scored 0-50 based on the degree of necrosis and cast formation by a blinded observer (L.R.) on Periodic acid-Schiff-stained kidney sections. This method was adapted from Wang and colleagues12. <a href="https://www.jove.com/files/ftp_upload/58520/58520fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/58520/58520fig5.jpg" /> 
Figure 5: Correlation of three measures of kidney function/damage (histological evaluation of tubular injury (n=39), serum creatinine (n=30) and blood urea nitrogen (BUN) (n=30)) with FITC-sinistrin clearance (half-life). Male BALB/c mice underwent varying periods of unilateral renal pedicle clamping (25&#8211;45 min) or sham surgery, with simultaneous contralateral nephrectomy to induce different severity of AKI, and renal function parameters and histopathology were assessed at day 4 after IRI. The tubular injury score showed a strong positive correlation with FITC-sinistrin clearance (A; R2 = 0.88), whereas serum creatinine (B), and BUN (C) both showed positive but weaker correlation with FITC-sinistrin clearance (R2 = 0.64 and 0.52, respectively). <a href="https://www.jove.com/files/ftp_upload/58520/58520fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Transdermal Measurement of Glomerular Filtration Rate in Mice at JoVE.com

Transdermal Measurement of Glomerular Filtration Rate in Mice

Here we describe a protocol to measure glomerular filtration rate (GFR) in conscious, freely moving mice using a transdermal GFR monitor.

Transdermal analysis of glomerular filtration rate (GFR) is an established technique that is used to assess renal function in mouse and rat models of ...

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Pharmacokinetics: Drug Excretion and Clearance

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21.2K 조회수.  Vanderbilt University Medical Center. Here we describe a protocol to measure glomerular filtration rate (GFR) in conscious, freely moving mice using a transdermal GFR monitor.

비디오: Transdermal Measurement of Glomerular Filtration Rate in Mice

Implantation of Radiotelemetry Transmitters Yielding Data on ECG, Heart Rate, Core Body Temperature and Activity in Free-moving Laboratory Mice

The laboratory mouse is the animal species of choice for most biomedical research, in both the academic sphere and the pharmaceutical industry. Mice are a manageable size and relatively easy to house. These factors, together with the availability of a wealth of spontaneous and experimentally induced mutants, make laboratory mice ideally suited to a wide variety of research areas.
In cardiovascular, pharmacological and toxicological research, accurate measurement of parameters relating to the circulatory system of laboratory animals is often required. Determination of heart rate, heart rate variability, and duration of PQ and QT intervals are based on electrocardiogram (ECG) recordings. However, obtaining reliable ECG curves as well as physiological data such as core body temperature in mice can be difficult using conventional measurement techniques, which require connecting sensors and lead wires to a restrained, tethered, or even anaesthetized animal. Data obtained in this fashion must be interpreted with caution, as it is well known that restraining and anesthesia can have a major artifactual influence on physiological parameters1, 2.
Radiotelemetry enables data to be collected from conscious and untethered animals. Measurements can be conducted even in freely moving animals, and without requiring the investigator to be in the proximity of the animal. Thus, known sources of artifacts are avoided, and accurate and reliable measurements are assured. This methodology also reduces interanimal variability, thus reducing the number of animals used, rendering this technology the most humane method of monitoring physiological parameters in laboratory animals3, 4. Constant advancements in data acquisition technology and implant miniaturization mean that it is now possible to record physiological parameters and locomotor activity continuously and in realtime over longer periods such as hours, days or even weeks3, 5.
Here, we describe a surgical technique for implantation of a commercially available telemetry transmitter used for continuous measurements of core body temperature, locomotor activity and biopotential (i.e. onelead ECG), from which heart rate, heart rate variability, and PQ and QT intervals can be established in freeroaming, untethered mice. We also present pre-operative procedures and protocols for post-operative intensive care and pain treatment that improve recovery, well-being and survival rates in implanted mice5, 6.

A surgical technique for implantation of commercially available telemetry transmitters used for continuous measurement of biopotential (one-lead ECG), heart rate, core body temperature and locomotor activity in freely moving mice is shown. Recommendations and protocols for post-operative care and pain relief, improving recovery, well being and survival rate are also presented.

ECG, 심장 박동, 코어 바디 온도 및 자유 이동 실험실 마우스의 활동에 데이터를 항복 Radiotelemetry 송신기의 주입

The laboratory mouse is the animal species of choice for most biomedical research, in both the academic sphere and the pharmaceutical industry. Mice are a ...

연구

의학

The Measurement and Treatment of Suppression in Amblyopia

Amblyopia, a developmental disorder of the visual cortex, is one of the leading causes of visual dysfunction in the working age population. Current estimates put the prevalence of amblyopia at approximately 1-3%1-3, the majority of cases being monocular2. Amblyopia is most frequently caused by ocular misalignment (strabismus), blur induced by unequal refractive error (anisometropia), and in some cases by form deprivation.
Although amblyopia is initially caused by abnormal visual input in infancy, once established, the visual deficit often remains when normal visual input has been restored using surgery and/or refractive correction. This is because amblyopia is the result of abnormal visual cortex development rather than a problem with the amblyopic eye itself4,5 . Amblyopia is characterized by both monocular and binocular deficits6,7 which include impaired visual acuity and poor or absent stereopsis respectively. The visual dysfunction in amblyopia is often associated with a strong suppression of the inputs from the amblyopic eye under binocular viewing conditions8. Recent work has indicated that suppression may play a central role in both the monocular and binocular deficits associated with amblyopia9,10 .
Current clinical tests for suppression tend to verify the presence or absence of suppression rather than giving a quantitative measurement of the degree of suppression. Here we describe a technique for measuring amblyopic suppression with a compact, portable device11,12 . The device consists of a laptop computer connected to a pair of virtual reality goggles. The novelty of the technique lies in the way we present visual stimuli to measure suppression. Stimuli are shown to the amblyopic eye at high contrast while the contrast of the stimuli shown to the non-amblyopic eye are varied. Patients perform a simple signal/noise task that allows for a precise measurement of the strength of excitatory binocular interactions. The contrast offset at which neither eye has a performance advantage is a measure of the "balance point" and is a direct measure of suppression. This technique has been validated psychophysically both in control13,14 and patient6,9,11 populations.
In addition to measuring suppression this technique also forms the basis of a novel form of treatment to decrease suppression over time and improve binocular and often monocular function in adult patients with amblyopia12,15,16 . This new treatment approach can be deployed either on the goggle system described above or on a specially modified iPod touch device15.

Amblyopia is a developmental disorder of the visual cortex that is often accompanied by strong suppression of one eye. We present a new technique for measuring and treating interocular suppression in patients with amblyopia that can be deployed using virtual reality goggles or a portable iPod Touch device.

약시의 억제의 측정 및 치료

Amblyopia, a developmental disorder of the visual cortex, is one of the leading causes of visual dysfunction in the working age population. Current ...

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

Kidney diseases involving urinary loss of large essential macromolecules, such as serum albumin, have long been thought to be caused by alterations in the permeability barrier comprised of podocytes, vascular endothelial cells, and a basement membrane working in unison. Data from our laboratory using intravital 2-photon microscopy revealed a more permeable glomerular filtration barrier (GFB) than previously thought under physiologic conditions, with retrieval of filtered albumin occurring in an early subset of cells called proximal tubule cells (PTC)1,2,3.
Previous techniques used to study renal filtration and establishing the characteristic of the filtration barrier involved micropuncture of the lumen of these early tubular segments with sampling of the fluid content and analysis4. These studies determined albumin concentration in the luminal fluid to be virtually non-existent; corresponding closely to what is normally detected in the urine. However, characterization of dextran polymers with defined sizes by this technique revealed those of a size similar to serum albumin had higher levels in the tubular lumen and urine; suggesting increased permeability5.
Herein is a detailed outline of the technique used to directly visualize and quantify glomerular fluorescent albumin permeability in vivo. This method allows for detection of filtered albumin across the filtration barrier into Bowman's space (the initial chamber of urinary filtration); and also allows quantification of albumin reabsorption by proximal tubules and visualization of subsequent albumin transcytosis6. The absence of fluorescent albumin along later tubular segments en route to the bladder highlights the efficiency of the retrieval pathway in the earlier proximal tubule segments. Moreover, when this technique was applied to determine permeability of dextrans having a similar size to albumin virtually identical permeability values were reported2. These observations directly support the need to expand the focus of many proteinuric renal diseases to included alterations in proximal tubule cell reclamation.

A technique utilizing high resolution intavital 2-photon microscopy to directly visualize and quantify gloemrular filtration in surface glomeruli. This method allows for direct determination of permeability characteristics of macromolecules in both normal and diseased states.

뮌헨의 Wistar 쥐에서 2 광자 현미경을 사용하여 형광 고분자의 사구체 투과율을 정량화

Kidney  diseases involving urinary loss of large essential macromolecules, such as  serum albumin, have long been thought to be caused by alterations in ...

In utero Measurement of Heart Rate in Mouse by Noninvasive M-mode Echocardiography

Congenital heart disease (CHD) is the most frequent noninfectious cause of death at birth. The incidence of CHD ranges from 4 to 50/1,000 births (Disease and injury regional estimates, World Health Organization, 2004). Surgeries that often compromise the quality of life are required to correct heart defects, reminding us of the importance of finding the causes of CHD. Mutant mouse models and live imaging technology have become essential tools to study the etiology of this disease. Although advanced methods allow live imaging of abnormal hearts in embryos, the physiological and hemodynamic states of the latter are often compromised due to surgical and/or lengthy procedures. Noninvasive ultrasound imaging, however, can be used without surgically exposing the embryos, thereby maintaining their physiology. Herein, we use simple M-mode ultrasound to assess heart rates of embryos at E18.5 in utero. The detection of abnormal heart rates is indeed a good indicator of dysfunction of the heart and thus constitutes a first step in the identification of developmental defects that may lead to heart failure.

In utero Measurement of Heart Rate in Mouse by Noninvasive M-mode Echocardiography

Ultra-high frequency ultrasound is a powerful live imaging tool to examine cardiac abnormalities in small animals. Its noninvasiveness allows maintaining the physiologic state of embryos. Herein, we show the use of M-mode ultrasound to measure the heart rates of embryos at E18.5 in utero.

비 침습 M-모드 초음파 심장 검진 법에 의한 마우스의 심장 박동의 자궁 측정

Congenital heart disease (CHD) is the most frequent noninfectious cause of death at birth. The incidence of CHD ranges from 4 to 50/1,000 births (Disease ...

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study model. As the use of mouse models are often a vital step in preclinical drug discovery and drug development1-8, it is necessary to design a system to introduce drugs into mice in a uniform, reproducible manner. Ideally, the system should permit the collection of blood samples at regular intervals over a set time course. The ability to measure drug concentrations by mass-spectrometry, has allowed investigators to follow the changes in plasma drug levels over time in individual mice1, 9, 10. In this study, paclitaxel was introduced into transgenic mice as a continuous arterial infusion over three hours, while blood samples were simultaneously taken by retro-orbital bleeds at set time points. Carotid artery infusions are a potential alternative to jugular vein infusions, when factors such as mammary tumors or other obstructions make jugular infusions impractical. Using this technique, paclitaxel concentrations in plasma and tissue achieved similar levels as compared to jugular infusion. In this tutorial, we will demonstrate how to successfully catheterize the carotid artery by preparing an optimized catheter for the individual mouse model, then show how to insert and secure the catheter into the mouse carotid artery, thread the end of the catheter out through the back of the mouse&#8217;s neck, and hook the mouse to a pump to deliver a controlled rate of drug influx. Multiple low volume retro-orbital bleeds allow for analysis of plasma drug concentrations over time.

This method was developed with the goal of delivering a steady drug solution via the carotid artery, to assess the pharmacokinetics of novel drugs in mouse models.

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...

Automated Measurement of Pulmonary Emphysema and Small Airway Remodeling in Cigarette Smoke-exposed Mice

COPD is projected to be the third most common cause of mortality world-wide by 2020(1). Animal models of COPD are used to identify molecules that contribute to the disease process and to test the efficacy of novel therapies for COPD. Researchers use a number of models of COPD employing different species including rodents, guinea-pigs, rabbits, and dogs(2). However, the most widely-used model is that in which mice are exposed to cigarette smoke. Mice are an especially useful species in which to model COPD because their genome can readily be manipulated to generate animals that are either deficient in, or over-express individual proteins. Studies of gene-targeted mice that have been exposed to cigarette smoke have provided valuable information about the contributions of individual molecules to different lung pathologies in COPD(3-5). Most studies have focused on pathways involved in emphysema development which contributes to the airflow obstruction that is characteristic of COPD. However, small airway fibrosis also contributes significantly to airflow obstruction in human COPD patients(6), but much less is known about the pathogenesis of this lesion in smoke-exposed animals. To address this knowledge gap, this protocol quantifies both emphysema development and small airway fibrosis in smoke-exposed mice. This protocol exposes mice to CS using a whole-body exposure technique, then measures respiratory mechanics in the mice, inflates the lungs of mice to a standard pressure, and fixes the lungs in formalin. The researcher then stains the lung sections with either Gill&#8217;s stain to measure the mean alveolar chord length (as a readout of emphysema severity) or Masson&#8217;s trichrome stain to measure deposition of extracellular matrix (ECM) proteins around small airways (as a readout of small airway fibrosis). Studies of the effects of molecular pathways on both of these lung pathologies will lead to a better understanding of the pathogenesis of COPD.

The goal of this protocol is to provide automated methods to quantify chronic lung pathologies in a murine model of COPD. The protocol includes exposing mice to cigarette smoke (CS), measuring pulmonary function, inflating the lungs, and using morphometry methods to measure emphysema and small airway remodeling in mice.

담배 연기에 노출 된 마우스의 폐 폐기종 및 작은기도 리모델링 자동 측정

COPD is projected to be the third most common cause of mortality world-wide by 2020(1).  Animal models of COPD are used to identify molecules that ...

Measurement of the Pressure-volume Curve in Mouse Lungs

In recent decades the mouse has become the primary animal model of a variety of lung diseases. In models of emphysema or fibrosis, the essential phenotypic changes are best assessed by measurement of the changes in lung elasticity. To best understand specific mechanisms underlying such pathologies in mice, it is essential to make functional measurements that can reflect the developing pathology. Although there are many ways to measure elasticity, the classical method is that of the total lung pressure-volume (PV) curve done over the whole range of lung volumes. This measurement has been made on adult lungs from nearly all mammalian species dating back almost 100 years, and such PV curves also played a major role in the discovery and understanding of the function of pulmonary surfactant in fetal lung development. Unfortunately, such total PV curves have not been widely reported in the mouse, despite the fact that they can provide useful information on the macroscopic effects of structural changes in the lung. Although partial PV curves measuring just the changes in lung volume are sometimes reported, without a measure of absolute volume, the nonlinear nature of the total PV curve makes these partial ones very difficult to interpret. In the present study, we describe a standardized way to measure the total PV curve. We have then tested the ability of these curves to detect changes in mouse lung structure in two common lung pathologies, emphysema and fibrosis. Results showed significant changes in several variables consistent with expected structural changes with these pathologies. This measurement of the lung PV curve in mice thus provides a straightforward means to monitor the progression of the pathophysiologic changes over time and the potential effect of therapeutic procedures.

Here we present a protocol to simply and reliably measure the lung pressure-volume curve in mice, showing that it is sufficiently sensitive to detect phenotypic parenchymal changes in two common lung pathologies, pulmonary fibrosis and emphysema. This metric provides a means to quantify the lung&#8217;s structural changes with developing pathology.

마우스 폐의 압력 볼륨 곡선의 측정

In recent decades the mouse has become the primary animal model of a variety of lung diseases. In models of emphysema or fibrosis, the essential ...

Long-term Blood Pressure Measurement in Freely Moving Mice Using Telemetry

During the development of new vasoactive agents, arterial blood pressure monitoring is crucial for evaluating the efficacy of the new proposed drugs. Indeed, research focusing on the discovery of new potential therapeutic targets using genetically altered mice requires a reliable, long-term assessment of the systemic arterial pressure variation. Currently, the gold standard for obtaining long-term measurements of blood pressure in ambulatory mice uses implantable radio-transmitters, which require&#160;artery cannulation. This technique eliminates the need for tethering, restraining, or anesthetizing the animals which introduce stress and artifacts during data sampling. However, arterial blood pressure monitoring in mice via catheterization can be rather challenging due to the small size of the arteries. Here we present a step-by-step guide to illustrate the crucial key passages for a successful subcutaneous implantation of radio-transmitters and carotid artery cannulation in mice. We also include examples of long-term blood pressure activity taken from freely moving mice after a period of post-surgery recovery. Following this procedure will allow reliable direct blood pressure recordings from multiple animals simultaneously.

The goal of this protocol is to assess systemic blood pressure in conscious freely moving mice using implantable radio-telemetry devices.

텔레 메 트리를 사용하여 자유롭게 이동 마우스의 장기 혈압 측정

During the development of new vasoactive agents, arterial blood pressure monitoring is crucial for evaluating the efficacy of the new proposed drugs. ...

Assessment of Kidney Function in Mouse Models of Glomerular Disease

The use of murine models to mimic human kidney disease is becoming increasingly common. Our research is focused on the assessment of glomerular function in diabetic nephropathy and podocyte-specific VEGF-A knock-out mice; therefore, this protocol describes the full kidney work-up used in our lab to assess these mouse models of glomerular disease, enabling a vast amount of information regarding kidney and glomerular function to be obtained from a single mouse. In comparison to alternative methods presented in the literature to assess glomerular function, the use of the method outlined in this paper enables the glomerular phenotype to be fully evaluated from multiple aspects. By using this method, the researcher can determine the kidney phenotype of the model and assess the mechanism as to why the phenotype develops. This vital information on the mechanism of disease is required when examining potential therapeutic avenues in these models. The methods allow for detailed functional assessment of the glomerular filtration barrier through measurement of the urinary albumin creatinine ratio and individual glomerular water permeability, as well as both structural and ultra-structural examination using the Periodic Acid Schiff stain and electron microscopy. Furthermore, analysis of the genes dysregulated at the mRNA and protein level enables mechanistic analysis of glomerular function. This protocol outlines the generic but adaptable methods that can be applied to all mouse models of glomerular disease.

This protocol describes a full kidney work-up that should be carried out in mouse models of glomerular disease. The methods allow for detailed functional, structural, and mechanistic analysis of glomerular function, which can be applied to all mouse models of glomerular disease.

신장 기능 사 질병 마우스 모델에서의 평가

The use of murine models to mimic human kidney disease is becoming increasingly common. Our research is focused on the assessment of glomerular function ...

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.

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.

플루오레세인 이소티오차네이트-폴리수크로오스 70을 가진 마우스에서 사구체 투과성의 고감도 측정

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