ZHP was supported by the Marie-Curie project: NephroTools.

Necessary experiments to develop the protocol were performed according to national regulations and were approved by the local ethical science committee (Regierungspr&#228;sidium Karlsruhe).
1. Preparation of FITC-sinistrin Injection Solution
<ol>
	<li>Dissolve FITC-sinistrin in physiological saline to prepare a stock solution. The recommended dose in mice is 7.5 mg/100 g body weight (BW), while in rats 5 mg/100 g BW FITC-sinistrin is preferable. For mice, prepare a solution of FITC-sinistrin stock solution at 15 mg/ml, while for rats prepare at a concentration of 40 mg/ml. 
	Note: FITC-sinistrin stock solution can be prepared in advance and stored at -20 &#176;C protected from light.</li>
</ol>
2. Animal Preparation
<ol>
	<li>Acclimate animals for at least one week before their introduction in any experiment.</li>
	<li>Anesthetize the animal using 5% isoflurane at 5 L/min O2 flow for 2 min.</li>
	<li>Confirm that the animal is properly anesthetized by observing its breathing, which becomes slower and deeper, and verify unresponsiveness with a lack of a toe response.</li>
	<li>Once the animal is asleep, reduce the anesthesia to 2.5% isoflurane at 2 L/min O2 delivery rate and take the BW.</li>
	<li>Remove the fur from the flank of the back of the animal using an electric shaver. Shave an area slightly bigger than the area that will be occupied by the double-sided adhesive patch, which has dimensions of 3 cm x 6 cm.</li>
	<li>Subsequently apply depilation cream for a short period (2-3 min) to remove the remaining fur.</li>
	<li>Wash thoroughly the area until the cream has been completely removed, as the cream itself may be fluorescent. 
	Note: Avoid scratching the skin of the animal as it can produce irritation/edema. If the depilated area shows pigmentation on the skin, shave a bigger area until a non-pigmented spot for the optical part of the device is found. It is recommended to shave 24 hr in advance to minimize anesthesia exposure.</li>
</ol>
3. Device Preparation
<ol>
	<li>Place the optical device to measure renal function onto one side of the double-sided adhesive patch, positioning the optical part above the transparent window (Figure 1) leaving the opposite part with the protective foil. 
	Note: When working with small rodents, reduce the size of the patch as shown in the Figure 2.</li>
	<li>Attach a piece of double-sided adhesive patch of matching size to the battery as shown in Figure 3.</li>
</ol>
4. Fixation of the Device on the Animal
<ol>
	<li>Calculate the appropriate volume of injection based on the animal body weight.</li>
	<li>Anesthetize the animal with isoflurane as previously indicated on sections 2.1-2.3.</li>
	<li>Cut a piece of tubular elastic gauze bandage of about 1 cm longer than the width of the double-sided adhesive patch to be used.</li>
	<li>Pull the elastic gauze bandage over the head of the animal and place on its back, leaving the shaved area uncovered. 
	Note: Alternatively, fix the device using only adhesive tape, but be aware of undue pressure on the device.</li>
	<li>Connect the battery to the device by plugging the battery connector to the corresponding port on the device. After that remove the protective foil from the piece of patch and mount the battery on the upper surface of the device (Figure 4A). Ensure that the battery is properly plugged by checking that the device starts blinking. 
	Note: In rats, the battery can be also attached directly on the adhesive patch, next to the device (Figure 4B).</li>
	<li>Remove the protective cover from the patch with the operating device and place it onto the shaved area on the back of the animal holding its edges until it is correctly fixed.</li>
	<li>Cover the device with the tubular elastic gauze bandage adhering it to the adhesive surface of the patch (Figure 5).</li>
	<li>Properly stretch the tubular bandage on the abdomen of the animal, ensuring that the limbs can move freely.</li>
	<li>For a better fixation and protection of the device, apply a strip of adhesive tape following the shape of the device and covering the wires of the battery.</li>
	<li>Measure background for 1 to 3 min, without putting pressure on the device</li>
</ol>
5. FITC-sinistrin Administration and Measurement Procedure
<ol>
	<li>If necessary, warm up the tail of the animal using warm water before injection or a temperature-controlled&#160;heating plate along all the procedure.</li>
	<li>Inject through tail vein the appropriate volume of FITC-sinistrin stock solution. The volume of injection depends on animal BW and therefore must be calculated for each individual considering the desired dose and concentration of stock solution, aforementioned in section 1.1.</li>
	<li>Ensure that all the FITC-sinistrin solution is administered intravenously and not subcutaneously. 
	Note: Alternatively, FITC-sinistrin can be administered using a pre-implanted intravenous catheter exited at the nape of the neck in rodents or via retro orbital injection in mice.</li>
	<li>Carefully, return the animal to its home cage avoiding strong movements and any pressure on the device, as it would introduce movement artifacts.</li>
	<li>Place the home cage in a calm place to avoid that the animal is disturbed.</li>
	<li>Perform the measurement during at least 1 hr if working with mice and 2 hr when using rats. During that period, the optical device will measure through the skin the fluorescence emitted by FITC-sinistrin. 
	Note: During the recording period the animal must be hosted alone. Furthermore water supply, as well as protruding structures such as the wire lids, must be removed to avoid damaging of the electronic device and movement artifacts due to impacts with objects.</li>
</ol>
6. Device Removal
<ol>
	<li>Once the recording period is over, remove the device without anesthesia; however, if needed, anesthetize the animal briefly under 5% isoflurane at 5 L/min O2 delivery rate during 2 min.</li>
	<li>Carefully, pull off the adhesive tape first, then the tubular bandage.</li>
	<li>Gently detach the double-sided adhesive patch from the skin and return the animal to its normal home cage.</li>
</ol>
7. Read out of Data
<ol>
	<li>Disconnect the battery from the device and remove the adhesive patch. 
	Note: The data are stored on the device until a new measurement starts. When a battery is connected the stored data are overwritten with the new recordings. Therefore, do not reconnect the battery before downloading the data from the device. There is a 10 sec grace period intended for accidental reconnections of the battery.</li>
	<li>Connect the device to a PC using a micro USB cable and download the data using the software provided. The output file is a .csv file that can be opened and modified with a spreadsheet program.</li>
	<li>Open the data file with the specific software for the optical device and generate the elimination kinetics curve using the software protocols.</li>
	<li>Analyze the curve by following the instructions provided with the optical device. Shortly, for the evaluation of the data, set the background signal measured prior FITC-sinistrin administration and mark the beginning of the exponential excretion phase of the marker, which usually occurs 15 min and 45 min after the bolus injection, in mice and rats respectively. 
	Note: The software will automatically display the FITC-sinistrin half-life (t1/2) along with an R2 value, determined by a 1-compartment model. t1/2 can be utilized to calculate GFR by using a conversion factor9,10.</li>
</ol>

<ol>
	<li>Stevens, L. A., Levey, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measured+GFR+as+a+confirmatory+test+for+estimated+GFR.">Measured GFR as a confirmatory test for estimated GFR.</a> J Am Soc Nephrol. 20, 2305-2313 (2009).</li><li>Katayama, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calculation+of+glomerular+filtration+rate+in+conscious+rats+by+the+use+of+a+bolus+injection+of+iodixanol+and+a+single+blood+sample.">Calculation of glomerular filtration rate in conscious rats by the use of a bolus injection of iodixanol and a single blood sample.</a> J Pharmacol Toxicol Methods. 61, 59-64 (2010).</li><li>Reinhardt, C. 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=Functional+immunoassay+technology+(FIT),+a+new+approach+for+measuring+physiological+functions:+application+of+FIT+to+measure+glomerular+filtration+rate+(GFR).">Functional immunoassay technology (FIT), a new approach for measuring physiological functions: application of FIT to measure glomerular filtration rate (GFR).</a> Am J Physiol Renal Physiol. 295, F1583-F1588 (2008).</li><li>Rieg, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-throughput+method+for+measurement+of+glomerular+filtration+rate+in+conscious+mice.">A high-throughput method for measurement of glomerular filtration rate in conscious mice.</a> J Vis Exp. , e50330 (2013).</li><li>Yu, W., Sandoval, R. M., Molitoris, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+determination+of+renal+filtration+function+using+an+optical+ratiometric+imaging+approach.">Rapid determination of renal filtration function using an optical ratiometric imaging approach.</a> Am J Physiol Renal Physiol. 292, F1873-F1880 (2007).</li><li>Wang, E., Sandoval, R. M., Campos, S. B., Molitoris, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+diagnosis+and+quantification+of+acute+kidney+injury+using+fluorescent+ratio-metric+determination+of+glomerular+filtration+rate+in+the+rat.">Rapid diagnosis and quantification of acute kidney injury using fluorescent ratio-metric determination of glomerular filtration rate in the rat.</a> Am J Physiol Renal Physiol. 299, F1048-F1055 (2010).</li><li>Colson, 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=Does+choice+of+the+anesthetic+influence+renal+function+during+infrarenal+aortic+surgery?.">Does choice of the anesthetic influence renal function during infrarenal aortic surgery?.</a> Anesth Analg. 74, 481-485 (1992).</li><li>Fusellier, 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=Influence+of+three+anesthetic+protocols+on+glomerular+filtration+rate+in+dogs.">Influence of three anesthetic protocols on glomerular filtration rate in dogs.</a> Am J Vet Res. 68, 807-811 (2007).</li><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> Am J Physiol Renal Physiol. 303, F783-F788 (2012).</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+measurement+of+glomerular+filtration+rate+using+FITC-sinistrin+in+rats.">Transcutaneous measurement of glomerular filtration rate using FITC-sinistrin in rats.</a> Nephrol Dial Transplant. 24, 2997-3001 (2009).</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 Int. 79, 1254-1258 (2011).</li><li>Steinbach, 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=A+pilot+study+to+assess+the+feasibility+of+transcutaneous+glomerular+filtration+rate+measurement+using+fluorescence-labelled+sinistrin+in+dogs+and+cats.">A pilot study to assess the feasibility of transcutaneous glomerular filtration rate measurement using fluorescence-labelled sinistrin in dogs and cats.</a> PLoS One. 9, e111734 (2014).</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, e71519 (2013).</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, 85-90 (2013).</li><li>Giani, J. F., 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+angiotensin-converting+enzyme+is+essential+for+the+hypertension+induced+by+nitric+oxide+synthesis+inhibition.">Renal angiotensin-converting enzyme is essential for the hypertension induced by nitric oxide synthesis inhibition.</a> J Am Soc Nephrol. 25, 2752-2763 (2014).</li><li>Sadick, 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=Two+non-invasive+GFR-estimation+methods+in+rat+models+of+polycystic+kidney+disease:+3.0+Tesla+dynamic+contrast-enhanced+MRI+and+optical+imaging.">Two non-invasive GFR-estimation methods in rat models of polycystic kidney disease: 3.0 Tesla dynamic contrast-enhanced MRI and optical imaging.</a> Nephrol Dial Transplant. 26, 3101-3108 (2011).</li><li>Trudu, 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=Common+noncoding+UMOD+gene+variants+induce+salt-sensitive+hypertension+and+kidney+damage+by+increasing+uromodulin+expression.">Common noncoding UMOD gene variants induce salt-sensitive hypertension and kidney damage by increasing uromodulin expression.</a> Nat Med. 19, 1655-1660 (2013).</li><li>Zollner, F. 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=Simultaneous+measurement+of+kidney+function+by+dynamic+contrast+enhanced+MRI+and+FITC-sinistrin+clearance+in+rats+at+3+tesla:+initial+results.">Simultaneous measurement of kidney function by dynamic contrast enhanced MRI and FITC-sinistrin clearance in rats at 3 tesla: initial results.</a> PLoS One. 8, e79992 (2013).</li></ol>

NG holds a patent on the production of FITC-sinistrin.

The present paper describes the procedure to determine kidney function in conscious rodents using a miniaturized device and the exogenous renal marker FITC-sinistrin. As stated before, this method has several advantages in comparison with the traditional procedures of urinary and plasma clearance. One of them is the independence from blood and urine sampling which leads to great savings in terms of consumables, time and, of course, animal stress.
As shown in Table 2, the exogenous renal marker FITC-sinistrin and the measurement of its elimination kinetics through the skin allow the impact of renal pathologies on kidney function to be studied 9,10,14-18. The serial collection of blood samples required to assess plasma clearance or biomarkers of renal disease are stressful for the animal, cumbersome and time-consuming for the researchers. Therefore, sequential measurements within the same animal have been limited by the welfare of the animals and methodological reasons. The proved within-animal reproducibility of the transcutaneous measurement13 makes it an ideal method for detecting changes in kidney function due to disease progression or ageing. For the same reason, it can also be extremely useful to study the effectiveness of therapeutic approaches.
Two critical steps for a successful application of this technology are the fixation of the optical device on the animal and the correct administration of the marker. The device must be appropriately fixed to avoid movement artifacts. For that purpose, preventing the attachment and detachment of the double-sided adhesive patch is crucial as it can lead to the loss of its adhesive properties and ultimately an inappropriate fixation on the animal. As mentioned in the protocol, immobilization and protection of the optical device by covering it with adhesive tape is an effective approach to ensure an adequate fixation. When performing FITC-sinistrin bolus injection it is crucial to administer all the substance intravenously and not subcutaneously. The release of the marker in the subcutaneous tissue can be easily identified by a yellow ring circling the tail of the animal produced by FITC-sinistrin&#39;s own color. A subcutaneous injection typically manifests as a flatter curve and prolonged marker excretion time. The possible limitation of this method is more related to the marker used than with the technology per se. When an edema is present in the skin of an animal, the transcutaneous assessment of renal function would not be recommended, as there is an additional compartment which could alter the excretion dynamics of FITC-sinistrin. Similarly, pigmented skin can limit the use of the technique due to the overlapping excitation and emission spectra of FITC-sinistrin and melanin. However, in animals with skin pigmentation in patches, the problem is solved by placing the optical part of the device in an unpigmented area.
It is possible to modify different aspects of the transcutaneous assessment of renal function in order to adapt the procedure to different animal species and/or approaches. For example, the recommended FITC-sinistrin dosage or the length of the measurement can be adjusted depending on the goal of the experiment as well as for different species or animal models. This adaptability of the method has already been confirmed by its successful application in different species of rodents and several types of strains with varied health conditions. In addition, the transcutaneous assessment of renal function has been recently validated in dogs and cats12 with excellent results. Due to the undoubted differences between these animals and rodents, this study determined the appropriate dose of FITC-sinistrin for both, dogs and cats, and it performed a much longer measuring time, 4 hours instead of the 1 or 2 hours used in rodents.
Taken together, the use of this technique can help us to gain more accurate insights in nephrology, including a better understanding of renal disease progression and the effect of therapies. The characteristics of the transcutaneous assessment of renal function places it as a suitable technique to be applied in human and veterinary clinical fields in the future, and should be considered as a promising technology to replace the traditional approaches.

Glomerular filtration rate (GFR) is the best parameter to assess overall renal function. The gold standard to determine GFR relies on urinary or plasma clearance of exogenous renal markers, such as inulin1. However, these procedures are time consuming and complicated, requiring the collection of serial blood and/or urine samples and posterior laboratory assays for its analysis. In addition, these methods are invasive and stressful for the animals, limiting the number of times and frequency in which the measurements can be repeated. A number of alternative approaches have been developed to simplify the classical procedures to determine GFR, but they still rely on urine and plasma sampling2-4 and/or require deep anesthesia5,6, which is known to influence renal hemodynamics and function7,8. End-products of metabolism, such as creatinine, are also widely used to estimate kidney function. However, it is known that the accuracy of these endogenous markers is not optimal and, moreover, to analyze them, urine or blood sampling is also indispensable.
Here we describe a transcutaneous methodology to assess renal function in conscious animals. This method is simpler and faster than traditional methods as well as only minimally invasive. With this technique, using a miniaturized optical device and the exogenous renal marker fluorescein isothiocyanate (FITC)-sinistrin, it is possible to determine kidney function almost in real-time without a need for plasma or urine sampling and without surgical catheterization for substance administration. Due to these characteristics, the method is suitable to perform sequential measurements in the same animal.
FITC-sinistrin is a renal marker freely filtered by the glomerulus, with no tubular reabsorption or secretion. The optical part of the miniaturized device consists of two light-emitting diodes (LEDs), which excite the administered marker and a photodiode to detect the fluorescence emitted through the skin. The recorded data are stored in the internal memory integrated in the device and they can be used to generate the FITC-sinistrin elimination kinetics curve. Power is supplied by a small rechargeable lithium battery. For more detailed information on the device and its individual components refer to Schreiber et al.9. The device is easily mounted on the animal body using a double-sided adhesive patch carrying a transparent window for the optical components. The results are readily available after the recording period is over.
With the method and protocol provided here, we introduce a promising technology which could replace the current gold standard to determine GFR, not only in research but also in the clinical field.

<table><tbody><tr><td>NIC-Kidney device</td><td>Mannheim Pharma &amp;amp; Diagnostics GmbH</td><td></td><td>Software, batteries and chargers are provided together with the device/s. Orderings should be done through info@mapdiagnostics.com</td></tr><tr><td>FITC-sinistrin</td><td>Mannheim Pharma &amp;amp; Diagnostics GmbH</td><td></td><td>Orderings should be done through info@mapdiagnostics.com</td></tr><tr><td>Double-sided adhesive patch</td><td>Mannheim Pharma &amp;amp; Diagnostics GmbH</td><td></td><td>Orderings should be done through info@mapdiagnostics.com</td></tr><tr><td>Isis Rodent electric shaver</td><td>Braun Aesculap&amp;nbsp;</td><td>GT420</td><td></td></tr><tr><td>Isoflurane</td><td>Abbott GmbH&amp;nbsp;</td><td>PZN4831850</td><td></td></tr><tr><td>Leukosilk adhesive tape</td><td>BSN medical</td><td>102200</td><td></td></tr><tr><td>Tubular elastic gauze bandage&amp;nbsp;</td><td>MaiMed Medical GmbH</td><td>73012</td><td>There are different sizes available. Size 1 is recommended for mice.</td></tr><tr><td>Veet depilation cream</td><td>Reckitt Benckiser</td><td>PZN7768307</td><td>Sensitive skin formulation is recommended as is more gentle with the skin of the animals</td></tr><tr><td>Micro USB cable</td><td>Samsung</td><td>APCBU10BBE</td><td></td></tr><tr><td>Deltajonin Physiologic solution</td><td>AlleMan Pharma GmbH</td><td>3366954</td><td>Alternatively, it can be used saline or PBS&amp;nbsp;</td></tr></tbody></table>

transcutaneous assessment of renal function in conscious rodents

Glomerular filtration rate (GFR) is the gold standard to assess overall kidney function. However, traditional methods to evaluate GFR are cumbersome and time-consuming. In addition, serial blood or urine samples are required, with the associated stress for the experimental animals. A recent technique significantly reduces the investment in time and resources, minimizing the invasiveness and the animal stress, but being equally valid as the traditional approaches. The method measures transcutaneously renal function. Using an optical device and the exogenous renal marker fluorescein isothiocyanate (FITC)-sinistrin, this technique is capable of measuring the elimination kinetics of the marker through the skin. With neither blood nor urine samples nor the associated laboratory assays needed, the results of the transcutaneous measurement are almost instantaneously available. The method has been already validated in different species and successfully applied in several models of renal pathology. Moreover, due to its minimally invasive characteristics, it is suitable for sequential measurements within the same animal. Here is provided a detailed protocol to carry out the transcutaneous assessment of renal function in rodents.

The setup of the transcutaneous measurement is very simple and fast: the device is placed on the double-sided adhesive patch (Figure 1) and adjusted in size if necessary (Figure 2), the battery is prepared (Figure 3) and connected (Figure 4).
This method to assess renal function has been already validated in different species by comparison with the traditional approach of plasma clearance9,11,12. Following the protocol exposed here, Schreiber et al. demonstrated the validity of the technique in different mouse models, showing highly comparable results among the transcutaneous measurement and plasma clearance for all the groups studied (Table 1)9. In this work the obtained t1/2 was converted to GFR using a mouse specific semi-empirical conversion factor.
The consistency of the transcutaneous assessment of renal function has been also proved using different strains of mice. Sequential measurements within 3 days in the same animal showed a coefficient of variances of 3.0-6.2%13. In this study there was no conversion to GFR but the results were expressed and interpreted directly in terms of t1/2.
The possibility to have the results almost in real-time is one of the great advantages of this method. After the recording period, the results are immediately available for analysis and the provided software displays the excretion curve of FITC-sinistrin instantaneously (Figure 6). Within the same software the t1/2 of FITC-sinistrin can be obtained, which can be directly used as the parameter to evaluate renal function of converted in terms of GFR. Figure 7 shows how it looks the FITC-sinistrin excretion curve measured transcutaneously in an animal with renal impairment. When renal function is affected, FITC-sinistrin t1/2 increases due to the reduced excretion of the substance and the appearance of the curve changes. Typically, the measured curve does not come back to background level and presents an increased area under the curve. In the same animal, measurements from pre- to post-injury can experience an increase in the maximum fluorescence intensity due to accumulation of the marker caused by its reduced excretion. In presence of kidney failure, the transcutaneously measured curve of FITC-sinistrin can show a steady state due to severely impaired function (Figure 7B).
The use of the miniaturized device in several strains of rodents with different health status has shown that this technique is suitable and sensitive enough to detect changes due to renal disease and ageing. Table 2 shows a summary of the murine models studied to date with this method.
<img alt="Figure 1" src="/files/ftp_upload/53767/53767fig1.jpg" /> 
Figure 1.&#160;Placement of the device on the double-sided adhesive patch. The device is mounted on the adhesive patch positioning its optical part in the transparent window.
<img alt="Figure 2" src="/files/ftp_upload/53767/53767fig2.jpg" /> 
Figure 2.&#160;Adapting the adhesive patch for small-sized rodents. For use in small animals it is recommended to reduce the size of the patch. A: Use the device as a guide to cut the patch properly. B: Once the desired size has been obtained, the device can be placed on the sticky surface of the patch. <a href="https://www.jove.com/files/ftp_upload/53767/53767fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/53767/53767fig3.jpg" /> 
Figure 3.&#160;Preparation of the battery. To attach the battery to the device, cut a small piece of double-sided adhesive patch (A and B) and place it on the surface of the battery (C). <a href="https://www.jove.com/files/ftp_upload/53767/53767fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/53767/53767fig4.jpg" /> 
Figure 4.&#160;Connection and placement of the battery during the measurement.&#160;(A) In small rodents such as mice, due to the reduced space, the battery must be placed on top of the device. (B) In larger animals the battery can be placed next to the device. <a href="https://www.jove.com/files/ftp_upload/53767/53767fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/53767/53767fig5.jpg" /> 
Figure 5.&#160;Placement of the tubular elastic gauze bandage in a rat. The tubular bandage should cover the double-sided adhesive patch without interfering with the free movement of the limbs for the comfort of the animal. <a href="https://www.jove.com/files/ftp_upload/53767/53767fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" src="/files/ftp_upload/53767/53767fig6.jpg" /> 
Figure 6.&#160;Representative image of an elimination curve of FITC-sinistrin. The signal generated by FITC-sinistrin is detected transcutaneously and stored in the internal memory of the device. When the recorded data are downloaded onto a PC, the software generates a curve comparable to the one presented in the image. Y-axis shows the recorded fluorescence intensity [AU], emitted by the injected FITC-sinistrin marker, while X-axis represents the duration of the measurement in time [min]. <a href="https://www.jove.com/files/ftp_upload/53767/53767fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" src="/files/ftp_upload/53767/53767fig7.jpg" /> 
Figure 7.&#160;Representative image of an elimination curve of FITC-sinistrin in animals with impaired renal function.&#160;(A) FITC-sinistrin elimination curve in animals with reduced renal function typically shows an increased area under the curve and inability to reach the baseline within the normal measurement period. (B) In severely impaired animals the transcutaneously measured curve can show a steady state which indicates renal failure. <a href="https://www.jove.com/files/ftp_upload/53767/53767fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Table 1" src="/files/ftp_upload/53767/53767table1.jpg" /> 
Table 1.&#160;Validation of the transcutaneous measurement by comparing with the traditional plasma clearance. The transcutaneous measurement of renal function has been validated in different mouse models (healthy and unilaterally nephrectomized (UNX) C57BL/6-129 SV mice and mouse model of nephronophthisis (pcy)) by comparison with plasma clearance. Values are means &#177; SD. GFR, glomerular filtration rate9.
<img alt="Table 2" src="/files/ftp_upload/53767/53767table2.jpg" /> 
Table 2. Murine models studied using the transcutaneous measurement. UNX, unilaterally nephrectomized; ACE, angiotensin-converting enzyme; Tgumodwt/wt, transgenic uromodulin knockout, SD, Sprague Dawley rat; PKD, polycystic kidney disease.

Watch this Scientific Journal Video about Transcutaneous Assessment of Renal Function in Conscious Rodents at JoVE.com

Transcutaneous Assessment of Renal Function in Conscious Rodents

Determination of glomerular filtration rate (GFR) is the gold standard to assess overall kidney function. However, traditional procedures to measure this parameter are cumbersome and require a large investment of time. Here we describe a faster and minimally invasive method to determinate GFR transcutaneously.

Glomerular filtration rate (GFR) is the gold standard to assess overall kidney function. However, traditional methods to evaluate GFR are cumbersome and ...

transcutaneous-assessment-of-renal-function-in-conscious-rodents

Research

JoVE Journal

Medicine

12.4K Views.  Medical Faculty Mannheim, University of Heidelberg. Determination of glomerular filtration rate (GFR) is the gold standard to assess overall kidney function. However, traditional procedures to measure this parameter are cumbersome and require a large investment of time. Here we describe a faster and minimally invasive method to determinate GFR transcutaneously.

Video: Transcutaneous Assessment of Renal Function in Conscious Rodents

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.

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

Early Detection of Drug-Induced Renal Hemodynamic Dysfunction Using Sonographic Technology in Rats

The kidney normally functions to maintain hemodynamic homeostasis and is a major site of damage caused by drug toxicity. Drug-induced nephrotoxicity is estimated to contribute to 19- 25% of all clinical cases of acute kidney injury (AKI) in critically ill patients. AKI detection has historically relied on metrics such as serum creatinine (sCr) or blood urea nitrogen (BUN) which are demonstrably inadequate in full assessment of nephrotoxicity in the early phase of renal dysfunction. Currently, there is no robust diagnostic method to accurately detect hemodynamic alteration in the early phase of AKI while such alterations might actually precede the rise in serum biomarker levels. Such early detection can help clinicians make an accurate diagnosis and help in in decision making for therapeutic strategy. Rats were treated with Cisplatin to induce AKI. Nephrotoxicity was assessed for six days using high-frequency sonography, sCr measurement and upon histopathology of kidney. Hemodynamic evaluation using 2D and Color-Doppler images were used to serially study nephrotoxicity in rats, using the sonography. Our data showed successful drug-induced kidney injury in adult rats by histological examination. Color-Doppler based sonographic assessment of AKI indicated that resistive-index (RI) and pulsatile-index (PI) were increased in the treatment group; and peak-systolic velocity (mm/s), end-diastolic velocity (mm/s) and velocity-time integral (VTI, mm) were decreased in renal arteries in the same group. Importantly, these hemodynamic changes evaluated by sonography preceded the rise of sCr levels. Sonography-based indices such as RI or PI can thus be useful predictive markers of declining renal function in rodents. From our sonography-based observations in the kidneys of rats that underwent AKI, we showed that these noninvasive hemodynamic measurements may consider as an accurate, sensitive and robust method in detecting early stage kidney dysfunction. This study also underscores the importance of ethical issues associated with animal use in research.

Early stage hemodynamic dysfunction is critical to the development of kidney disease. Yet, detection methodologies are limited. Recent advances in sonography provide a noninvasive, accurate option for early detection of kidney injury. This study outlines a step-by-step, sonographic methodology for detecting kidney dysfunction using a drug-induced nephrotoxicity rat model.

The kidney normally functions to maintain hemodynamic homeostasis and is a major site of damage caused by drug toxicity. Drug-induced nephrotoxicity is ...

Novel Approach for Simultaneous Recording of Renal Sympathetic Nerve Activity and Blood Pressure with Intravenous Infusion in Conscious, Unrestrained Mice.

Renal sympathetic nerves contribute significantly to both physiological and pathophysiological phenomena. Evaluating renal sympathetic nerve activity (RSNA) is of great interest in many areas of research such as chronic kidney disease, hypertension, heart failure, diabetes and obesity. Unequivocal assessment of the role of the sympathetic nervous system is thus imperative for proper interpretation of experimental results and understanding of disease processes. RSNA has been traditionally measured in anesthetized rodents, including mice. However, mice usually exhibit very low systemic blood pressure and hemodynamic instability for several hours during anesthesia and surgery. Meaningful interpretation of RSNA is confounded by this non-physiological state, given the intimate relationship between sympathetic nervous tone and cardiovascular status. To address this limitation of traditional approaches, we developed a new method for measuring RSNA in conscious, freely-moving mice. Mice were chronically instrumented with radio-telemeters for continuous monitoring of blood pressure as well as a jugular venous infusion catheter and custom-designed bipolar electrode for direct recording of RSNA. Following a 48-72 hour recovery period, survival rate was 100% and all mice behaved normally. At this time-point, RSNA was successfully recorded in 80% of mice, with viable signals acquired up to 4 and 5 days post-surgery in 70% and 50% of mice, respectively. Physiological blood pressures were recorded in all mice (116&plusmn;2 mmHg; n=10). Recorded RSNA increased with eating and grooming, as well-established in the literature. Furthermore, RSNA was validated by ganglionic blockade and modulation of blood pressure with pharmacological agents. Herein, an effective and manageable method for clear recording of RSNA in conscious, freely-moving mice is described.

Anesthetized mice exhibit non-physiological systemic blood pressure, which precludes meaningful assessment of autonomic tone given the intimate relationship between blood pressure and the autonomic nervous system. Thus, a novel method to simultaneously record renal sympathetic nerve activity and blood pressure with intravenous infusion in conscious mice is outlined.

Renal sympathetic nerves contribute significantly to both physiological and pathophysiological phenomena. Evaluating renal sympathetic nerve activity ...

A High-throughput Method for Measurement of Glomerular Filtration Rate in Conscious Mice

The measurement of glomerular filtration rate (GFR) is the gold standard in kidney function assessment. Currently, investigators determine GFR by measuring the level of the endogenous biomarker creatinine or exogenously applied radioactive labeled inulin (3H or 14C). Creatinine has the substantial drawback that proximal tubular secretion accounts for ~50% of total renal creatinine excretion and therefore creatinine is not a reliable GFR marker. Depending on the experiment performed, inulin clearance can be determined by an intravenous single bolus injection or continuous infusion (intravenous or osmotic minipump). Both approaches require the collection of plasma or plasma and urine, respectively. Other drawbacks of radioactive labeled inulin include usage of isotopes, time consuming surgical preparation of the animals, and the requirement of a terminal experiment. Here we describe a method which uses a single bolus injection of fluorescein isothiocyanate-(FITC) labeled inulin and the measurement of its fluorescence in 1-2 &mu;l of diluted plasma. By applying a two-compartment model, with 8 blood collections per mouse, it is possible to measure GFR in up to 24 mice per day using a special work-flow protocol. This method only requires brief isoflurane anesthesia with all the blood samples being collected in a non-restrained and awake mouse. Another advantage is that it is possible to follow mice over a period of several months and treatments (i.e. doing paired experiments with dietary changes or drug applications). We hope that this technique of measuring GFR is useful to other investigators studying mouse kidney function and will replace less accurate methods of estimating kidney function, such as plasma creatinine and blood urea nitrogen.

Measurement of glomerular filtration rate (GFR) is the gold standard for kidney function assessment. Here we describe a high-throughput method which allows the determination of GFR in conscious mice by using a single bolus injection, determination of fluorescein isothiocyanate (FITC)-inulin in plasma and calculation of GFR by a two-phase exponential decay model.

The measurement of  glomerular filtration rate (GFR) is the gold standard in kidney function  assessment. Currently, investigators determine GFR by ...

Biology

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.

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 ...

Physiology Lab Demonstration: Glomerular Filtration Rate in a Rat

Measurements of glomerular filtration rate (GFR), and the fractional excretion of sodium (Na) and potassium (K) are critical in assessing renal function in health and disease. GFR is measured as the steady state renal clearance of inulin which is filtered at the glomerulus, but not secreted or reabsorbed along the nephron. The fractional excretion of Na and K can be determined from the concentration of Na and K in plasma and urine. The renal clearance of inulin can be demonstrated in an anesthetized animal which has catheters in the femoral artery, femoral vein and bladder. The equipment and supplies used for this procedure are those commonly available in a research core facility, and thus makes this procedure a practical means for measuring renal function. The purpose of this video is to demonstrate the procedures required to perform a lab demonstration in which renal function is assessed before and after a diuretic drug. The presented technique can be utilized to assess renal function in rat models of renal disease.

The purpose of this protocol is to demonstrate the principles and techniques for measuring and calculating glomerular filtration rate, urine flow rate, and excretion of sodium and potassium in a rat. This demonstration can be used to provide students with an overall conceptual understanding of how to measure renal function.

Measurements of glomerular filtration rate (GFR), and the fractional excretion of sodium (Na) and potassium (K) are critical in assessing renal function ...

Use of Enzymatic Biosensors to Quantify Endogenous ATP or H2O2 in the Kidney

Enzymatic microelectrode biosensors have been widely used to measure extracellular signaling in real-time. Most of their use has been limited to brain slices and neuronal cell cultures. Recently, this technology has been applied to the whole organs. Advances in sensor design have made &#160;possible the measuring of&#160;cell signaling in blood-perfused in vivo kidneys. The present protocols list the steps needed to measure ATP and H2O2 signaling in the rat kidney interstitium. Two separate sensor designs are used for the ex vivo and in vivo protocols. Both types of sensor&#160;are coated with&#160;a thin enzymatic biolayer on top of a permselectivity layer to give fast responding, sensitive and selective biosensors. The permselectivity layer protects the signal from the interferents in biological tissue, and the enzymatic layer utilizes&#160;the sequential catalytic reaction of glycerol kinase and glycerol-3-phosphate oxidase in the presence of ATP to produce H2O2. The set of sensors used for the ex vivo studies further detected analyte by oxidation of H2O2 on a platinum/iridium (Pt-Ir) wire electrode. The sensors for the&#160;in vivo studies are instead based on the reduction of H2O2 on a mediator coated gold electrode designed for blood-perfused tissue. Final concentration changes&#160;are detected by real-time amperometry followed by calibration to known concentrations of analyte. Additionally, the specificity of the amperometric signal can be confirmed by the addition of enzymes such as catalase and apyrase that break down H2O2 and ATP correspondingly. These sensors also rely heavily on accurate calibrations before and after each experiment. The following two protocols establish the study of real-time detection of ATP and H2O2 in kidney tissues, and can be further modified to extend the described method for use in other biological preparations or whole organs.

Use of Enzymatic Biosensors to Quantify Endogenous ATP or H2O2 in the Kidney

Enzymatic microelectrode biosensors enable real-time measurements of extracellular cell signaling in biologically-relevant concentrations. The following protocols extend the applications of biosensors to the ex vivo and in vivo detection of ATP and H2O2 in the kidney.

Enzymatic microelectrode biosensors have been widely used to measure extracellular signaling in real-time. Most of their use has been limited to brain ...

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 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 ...

Transdermal Measurement of Glomerular Filtration Rate in Mechanically Ventilated Piglets

Transdermal measurement of glomerular filtration rate (GFR) has been used to evaluate kidney function in conscious animals. This technique is well established in rodents to study acute kidney injury and chronic kidney disease. However, GFR measurement using the transdermal system has not been validated in pigs, a species with a similar renal system to humans. Hence, we investigated the effect of sepsis on transdermal GFR in anesthetized and mechanically ventilated neonatal pigs. Polymicrobial sepsis was induced by cecal ligation and puncture (CLP). The transdermal GFR measurement system consisting of a miniaturized fluorescence sensor was attached to the pig's shaved skin to determine the clearance of fluorescein-isothiocyanate (FITC) conjugated sinistrin, an intravenously injected GFR tracer. Our results show that at 12 h post-CLP, serum creatinine increased with a decrease in GFR. This study demonstrates, for the first time, the utility of the transdermal GFR approach in determining renal function in mechanically ventilated, neonatal pigs.

Glomerular filtration rate (GFR) is the ideal marker for assessing kidney function. However, the standard measurement method using inulin injection with serial blood and urine analysis is impractical. This article delineates a practical method to measure GFR transdermally in piglets.

Transdermal measurement of glomerular filtration rate (GFR) has been used to evaluate kidney function in conscious animals. This technique is well ...

Cystometric and External Urethral Sphincter Measurements in Awake Rats with Implanted Catheter and Electrodes Allowing for Repeated Measurements

Lower urinary tract function is mainly assessed by means of cystometric bladder function analysis in rodents. Conventional cystometries are usually performed as terminal analysis under urethane anesthesia. It is well known that anesthetic drugs can influence bladder function. Hence, the aim of this technique is to perform cystometric measurements of the urinary bladder and external urethral sphincter in lightly restrained awake rats. For this purpose, a bladder catheter is implanted into the bladder dome. Subsequently, two electrodes are implanted bilateral to the external urethral sphincter and a ground electrode is sutured to a non-responsive skeletal muscle. The bladder catheter and the three electrodes are finally tunneled subcutaneously to the neck region and affixed to a harness. With this technique, the lower urinary tract can be measured at multiple time points in the same animal to assess lower urinary tract function. The main application of this technique is the follow-up of simultaneous urinary bladder and external urethral sphincter function in awake healthy rats and after induction of a disease or injury. Moreover, subsequent lower urinary tract monitoring can be performed during evaluation of the disease/injury and to monitor treatment efficacy.

This protocol first describes the surgical procedure of the permanent implantation of a urinary bladder catheter combined with external urethral sphincter electrodes, and second, the measurement of the function of the urinary bladder and external urethral sphincter in implanted awake animals.

Lower urinary tract function is mainly assessed by means of cystometric bladder function analysis in rodents. Conventional cystometries are usually ...

Transcutaneous Assessment of Renal Function in Conscious Rodents

Samenvatting

Meer video's verkennen