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

The investigations were conducted according to the guidelines outlined in the Guide for Care and Use of Laboratory Animals (US National Institutes of Health Publication No. 85-23, revised 1996). All animal experiments were performed in accordance with the relevant institutional approvals (state government Landesamt f&#252;r Natur, Umwelt und Verbraucherschutz [LANUV] reference number 84-02.04.2012.A397).
1. Preparation of instruments, solutions, and equipment
<ol>
	<li>Reconstitute FITC-polysucrose 70 with 0.9% sterile sodium chloride (NaCl) to a final concentration of 10 mg/mL (i.e., 100 mg in 10 mL of NaCl).</li>
	<li>Dialyze FITC-polysucrose 70 solution to remove free FITC molecules overnight at 4 &#176;C (molecular weight cut-off [MWCO] at 10,000). Use 1 L of 0.9% sterile NaCl per 10 mL of FITC-polysucrose 70 under constant stirring. Protect from light. Aliquot the dialyzed FITC-polysucrose 70 and store it at -20 &#176;C.</li>
	<li>For the FITC-polysucrose 70 bolus, add 4 &#181;L of 10 mg/mL FITC-polysucrose 70 solution to 996 &#181;L of 0.9% NaCl (the final concentration of FITC-polysucrose 70: 40 &#181;g/mL).</li>
	<li>For the equilibration infusion solution, add 20 &#181;L of a 10 mg/mL FITC-polysucrose 70 solution to 9.98 mL of 0.9% sterile NaCl yielding to a final concentration of 20 &#181;g/mL.</li>
	<li>For the experimental solution, add drugs or substances to the infusion solution (e.g., for angiotensin II [Ang II] [100 ng/kg/min] for a 25 g mouse, add 3 &#181;L of Ang II of a 1 mM solution).</li>
	<li>For the surgery, prepare one shaver, two surgical clamps, one pair of surgical scissors, two tweezers, two fine tweezers, one pair of fine scissors, and swabs. Prepare two 10 cm silk threads (4-0 to 6-0) for ligation procedures.</li>
	<li>For the placement of a central venous catheter, prepare a 10 mL syringe with a 21 G needle. Place the tip of the needle in a 30 cm-long catheter (with an inner diameter [ID] of 0.58 mm). Connect the 0.58 mm catheter to a 10 cm catheter (with an ID of 0.28 mm). Cut the tip of the smaller catheter oblique to create a sharp tip that is introduced into the jugular vein.</li>
	<li>Prepare the anesthesia (i.e., intraperitoneal anesthesia ketamine, 100 mg/kg of body weight, and xylazine, 5 mg/kg of body weight).</li>
	<li>Prepare a 22 G angiocatheter by discarding the needle and marking the catheter 1 cm from the tip. Preheat the heating pad to 37 &#176;C.</li>
	<li>Prepare a blood pressure device and change the blood-pressure-measuring membrane if necessary.</li>
</ol>
2. Preparation phase
<ol>
	<li>Urinary catheter 
	NOTE: Section 2.1 follows the protocol as described by Reis et al.7. Figure 1 and Supplemental Figure 1 show the placement of a urinary catheter in female mice.
	<ol>
		<li>Anesthetize the mouse with ketamine/xylazine (see step 1.8). Use the toe-pinch test to confirm proper anesthesia. To maintain anesthesia, repeat the anesthesia (e.g., with half the dosage) after 60 min.&#160;Proper anesthesia is confirmed if toe pinch test does not result in reflex withdrawl. 
		NOTE: Female FVB mice are used in this protocol.</li>
		<li>Position the mouse in dorsal recumbency on a 37 &#176;C heating pad. Tighten the lower abdomen and find the urethral ostium (e.g., under a microscope).</li>
		<li>Use the plastic part of the catheter of a 22 G angiocatheter and lubricate it with xylocaine gel. Introduce it carefully 3 mm into the urethral ostium while paralleling the distal urethral axis (Figure 1A).</li>
		<li>Turn the top of the angiocatheter 180&#176; by keeping the tip within the urethral ostium and maintaining the axis of the urethra (Figure 1B).</li>
		<li>Introduce the catheter 7 mm further into the mouse so that it is placed within the bladder (Figure 1C). Do not force the catheter beyond resistance. Correct the position of the catheter from the beginning if resistance is felt. Take note that if the position of the urinary catheter is correct, urine might already appear within the catheter.</li>
		<li>Place a 1.5 mL brown tube over the top of the angiocatheter to collect the urine. Apply 1 mL of 0.9% NaCl subcutaneously to enhance urine production.</li>
	</ol>
	</li>
	<li>Central venous catheter 
	<ol>
		<li>Shave the neck of the mouse and place it in recumbency with the head toward the surgeon. Hyperextend the head of the mouse with a tape.</li>
		<li>Disinfect the neck with 70% isopropanol. Make a small skin incision (5 mm) below the jawline, using a tweezer and a pair of scissors. Cut the skin approximately 1 cm in the direction of the sternum until the middle of the sternum is reached.</li>
		<li>Carefully dissect the skin on the right side of the neck, using a pair of scissors. Make a rectangular incision of the skin to the right side of the mouse to expose the soft tissue of the neck. Use a pair of scissors and a tweezer. Fix the skin flaps with two clamps. 
		NOTE: The jugular vein runs along the left side of the thyroid gland or is slightly covered by the right lobe of the thyroid gland. After this step, use a microscope for surgery.</li>
		<li>Carefully expose the jugular vein by blunt preparation, using the tip of the fine tweezer. Avoid injury to vein branches. 
		NOTE: It might be necessary to remove tissue with fine scissors. Be careful when using scissors to remove tissue as it increases the risk of bleeding.</li>
		<li>Place and close a ligature with a silk thread (4-0 to 6-0) at the distal part of the visible jugular vein (toward the head of the mouse). Put tension on the ligature by fixing the silk thread with a tape to ensure slight tension on the jugular vein. Prepare a ligature around the proximal part of the jugular vein.</li>
		<li>Fill the catheter with the equilibration infusion solution (see step 1.4) and fix the catheter with a tape so that the catheter is aligning the jugular vein. Control for bubbles to avoid air embolism.</li>
		<li>Lift the jugular vein with fine tweezers at the site of insertion (the insertion site is 1&#8211;2 mm proximal of the ligature). Align the tubing parallel to the jugular vein. Puncture the jugular vein, aiming for the lumen, and insert for approximately 2&#8211;4 mm, paralleling the axis of the jugular vein. Avoid any brisk movements.</li>
		<li>Close the ligature to fix the catheter. Control for tightness of the ligature by carefully viewing through the microscope. Put a damp swab over the site of surgery.</li>
	</ol>
	</li>
	<li>Blood pressure measurement
	<ol>
		<li>Place the tail-cuff at the bottom of the mouse tail while the mouse lays in dorsal recumbency. Start the measurements and repeat them 10x per time point. Build a mean from the measurements.</li>
		<li>Adjust the position of the tail-cuff if blood pressure measurements do not seem correct and false data are produced.</li>
	</ol>
	</li>
</ol>
3. Equilibration phase
<ol>
	<li>Change the urinary collection tube before the equilibration phase starts and place it on ice.</li>
	<li>Introduce a 10 mL syringe filled with FITC equilibration phase solution (see step 1.4) with a 21 G needle and the larger catheter (with an ID of 0.58 mm) and place it into the syringe pump.</li>
	<li>Fold a swab and put it around the small-vessel catheter (with an ID of 0.28 mm) that is introduced into the jugular vein. Lay the catheter inside the swab. Place a clamp over the swab to abolish retrograde blood flow or air embolism. Connect a 27 G needle with a 1 mL syringe filled with the FITC bolus to the end of the small-vessel catheter.</li>
	<li>Open the clamp and apply the FITC bolus (100 &#181;L). Close the clamp again and connect the larger catheter with the smaller catheter. Start the syringe pump with 0.008 mL/min (0.480 mL/h). Continue the infusion for 60 min.</li>
</ol>
4. Experimental phase
NOTE: In this phase, the effect of drugs on glomerular permselectivity can be investigated.
<ol>
	<li>Change the urinary tube (time point 0 min) to another 1.5 mL brown tube. Put a swab around the small-vessel catheter and close a clamp as indicated in step 3.3.</li>
	<li>Disconnect the large catheter from the small-vessel catheter. Change the syringes to the experimental phase solutions (see step 1.5). Let the infusion pump run so that the large catheter is filled with the equilibration solution.</li>
	<li>Reconnect the catheters while avoiding air embolism and reopen the clamp. Start the syringe pump at 0.008 mL/min.</li>
	<li>Continue running the syringe pump for 60 min and collect the urine within the urinary tube thereafter (urine 60 min). Place the urine tube on ice.</li>
	<li>Sacrifice the mouse by cervical dislocation during anesthesia. Prior to disposal the mouse is observed for 5 minutes to ensure it is dead.</li>
</ol>
5. Urine analysis
<ol>
	<li>Fluorescence measurement
	<ol>
		<li>Thaw the urinary pool and dilute it 1:10 with phosphate-buffered saline (PBS). 
		NOTE: The urinary pool is a pool of urine that has been collected from healthy mice in a 12&#8211;24 h urine collection in a metabolic cage.</li>
		<li>Prepare standards for the fluorescence measurement. Take 10 brown 1.5 mL tubes and label them for blank (1:10 diluted urine pool) and the following FITC-polysucrose 70 concentrations (0.625, 1.25, 2.5, 5, 10, 20, 40, 80, and 160 &#181;g/mL).</li>
		<li>Pipet 246 &#181;L of diluted urine pool into a 1.5 mL brown tube and add 4 &#181;L of the FITC-polysucrose 70 stock concentration (see step 1.1) to get a final concentration of 160 &#181;g/mL FITC-polysucrose 70. Pipet 100 &#181;L of the diluted urine pool into all other tubes.</li>
		<li>Dilute 1:2 by pipetting 100 &#181;L of the 160 &#181;g/mL FITC-polysucrose 70 tube into the 80 &#181;g/mL tube and continue with the other concentrations. Assure proper mixture of the diluted concentrations (e.g., by vortexing the tube before continuing).</li>
		<li>Dilute the mouse urine samples (0 min, 60 min) 1:10 with diluted urinary pool.</li>
		<li>Pipette 5 &#181;L of each standard FITC-polysucrose 70 concentration and of the mouse urine samples as triplicates in a black 384-well plate. Centrifuge the plate at 1,000 x g for 15 s to avoid bubbles.</li>
		<li>Analyze the 384-well plate in a plate reader. Perform excitation at 496 nm and measure the fluorescence at 525 nm.</li>
	</ol>
	</li>
	<li>Creatinine measurement
	<ol>
		<li>Follow the manufacturer&#8217;s instructions. In brief, prepare creatinine standards by diluting 10 &#181;L of the 100 mM creatinine standard solution with 990 &#181;L of creatinine assay buffer to prepare a 1 mM standard solution.</li>
		<li>Add 0, 2, 4, 6, 8, and 10 &#181;L of the 1 mM creatinine standard solution into a 96-well plate to generate 0, 2, 4, 6, 8, and 10 nmol/well standards, respectively. Add creatinine assay buffer to each well to bring the volume to 50 &#181;L.</li>
		<li>Prepare the reaction mixes by adding 42&#8211;44 &#181;L of creatinine assay buffer, 2 &#181;L of creatinase, 2 &#181;L of creatininase, 2 &#181;L of creatinine enzyme mix, and 2 &#181;L of creatinine probe. For the blank, use 44 &#181;L of creatinine assay buffer, 2 &#181;L of creatinase, 2 &#181;L of creatinine enzyme mix, and 1&#8211;2 &#181;L of creatinine probe.</li>
		<li>Add 50 &#181;L of the appropriate reaction mix to each well in the 96-well plate and incubate it for 60 min on a horizontal shaker at 37 &#176;C. Protect the plate from light during the incubation. Measure the absorbance at 570 nm on a plate reader.</li>
		<li>Calculate the creatinine concentrations by subtracting the blank value from all readings. The result will have the unit nanomoles/microliter. Multiply the concentration of creatinine by the molecular weight of creatinine (113.12 ng/nmol) to receive the unit nanograms/microliter.</li>
	</ol>
	</li>
</ol>
6. Data analysis
<ol>
	<li>Calculate the FITC-polysucrose 70 urine concentration from the standard curve.</li>
	<li>Reference the FITC-polysucrose 70 urine concentrations to creatinine concentrations in the representative urine sample.</li>
	<li>Reference the urine 60 min samples to urine 0 min samples of controls and treated mice.</li>
</ol>

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The authors have nothing to disclose.

The presented method enables the investigator to test glomerular permeability in mice in a very sensitive manner using a tracer. With this method, short-term increases in glomerular permeability can be diagnosed using only small amounts of urine. The most critical steps for successfully mastering this technique are 1) developing manual expertise in mouse surgery, especially in the cannulation of a central vein, 2) placing the urinary catheter without harming the mucosa, and 3) manual expertise in handling 384-well plates...

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

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

highly sensitive measurement of glomerular permeability in mice with fluorescein isothiocyanate-polysucrose 70

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

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

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

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

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

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

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JoVE Journal

Medicine

6.8K Views.  Heinrich-Heine-University. 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.

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

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

Tumor heterogeneity represents a fundamental feature supporting tumor robustness and presents a central obstacle to the development of therapeutic strategies1. To overcome the issue of tumor heterogeneity, it is essential to develop assays and tools enabling phenotypic, (epi)genetic and functional identification and characterization of tumor subpopulations that drive specific disease pathologies and represent clinically relevant targets. It is now well established that tumors exhibit distinct sub-fractions of cells with different frequencies of cell division, and that the functional criteria of being slow cycling is positively associated with tumor formation ability in several cancers including those of the brain, breast, skin and pancreas as well as leukemia2-8. The fluorescent dye carboxyfluorescein succinimidyl ester (CFSE) has been used for tracking the division frequency of cells in vitro and in vivo in blood-borne tumors and solid tumors such as glioblastoma2,7,8. The cell-permeant non-fluorescent pro-drug of CFSE is converted by intracellular esterases into a fluorescent compound, which is retained within cells by covalently binding to proteins through reaction of its succinimidyl moiety with intracellular amine groups to form stable amide bonds9. The fluorescent dye is equally distributed between daughter cells upon divisions, leading to the halving of the fluorescence intensity with every cell division. This enables tracking of cell cycle frequency up to eight to ten rounds of division10. CFSE retention capacity was used with brain tumor cells to identify and isolate a slow cycling subpopulation (top 5% dye-retaining cells) demonstrated to be enriched in cancer stem cell activity2. 
This protocol describes the technique of staining cells with CFSE and the isolation of individual populations within a culture of human glioblastoma (GBM)-derived cells possessing differing division rates using flow cytometry2. The technique has served to identify and isolate a brain tumor slow-cycling population of cells by virtue of their ability to retain the CFSE labeling.

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

This video protocol demonstrates the application of the fluorescent dye carboxyfluorescein succinimidyl ester (CFSE) for the identification and separation of different sub-populations of cells in human glioblastoma based on frequency of cell division.

Tumor heterogeneity represents a fundamental feature supporting tumor robustness and presents a central obstacle to the development of therapeutic ...

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 ...

An in vivo Assay to Test Blood Vessel Permeability

This method is based on the intravenous injection of Evans Blue in mice as the test animal model. Evans blue is a dye that binds albumin. Under physiologic conditions the endothelium is impermeable to albumin, so Evans blue bound albumin remains restricted within blood vessels. In pathologic conditions that promote increased vascular permeability endothelial cells partially lose their close contacts and the endothelium becomes permeable to small proteins such as albumin. This condition allows for extravasation of Evans Blue in tissues. A healthy endothelium prevents extravasation of the dye in the neighboring vascularized tissues. Organs with increased permeability will show significantly increased blue coloration compared to organs with intact endothelium. The level of vascular permeability can be assessed by simple visualization or by quantitative measurement of the dye incorporated per milligram of tissue of control versus experimental animal/tissue. Two powerful aspects of this assay are its simplicity and quantitative characteristics. Evans Blue dye can be extracted from tissues by incubating a specific amount of tissue in formamide. Evans Blue absorbance maximum is at 620 nm and absorbance minimum is at 740 nm. By using a standard curve for Evans Blue, optical density measurements can be converted into milligram dye captured per milligram of tissue. Statistical analysis should be used to assess significant differences in vascular permeability.

An in vivo Assay to Test Blood Vessel Permeability

We are presenting an in vivo assay to test blood vessel permeability. This assay is based on intravenous injection of a dye and subsequent visualization of its diffusion into interstitial spaces.

This method is based on the intravenous injection of Evans Blue in mice as the test animal model. Evans blue is a dye that binds albumin. Under ...

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.

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 ...

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.

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

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 ...

Using 2-Photon Microscopy to Quantify the Effects of Chronic Unilateral Ureteral Obstruction on Glomerular Processes

Applying novel microscopy methods to suitable animal disease models to explore the dynamic physiology of the kidney remains a challenge. Rats with surface glomeruli provide a unique opportunity to investigate physiological and pathophysiological processes using intravital 2-photon microscopy. Quantification of glomerular capillary blood flow and vasoconstriction and dilatation in response to drugs, permeability, and inflammation are just some of the processes that can be studied. In addition, transgenic rats, i.e., podocytes labeled with fluorescent dyes and other molecular biomarker approaches, provide increased resolution to directly monitor and quantify protein-protein interactions and the effects of specific molecular alterations.
In mice, which lack surface glomeruli after four weeks of age, unilateral ureteral obstruction (UUO) for several weeks has been used to induce surface glomeruli. As this induction model does not allow for baseline studies, we quantified the effects of UUO on glomerular processes in the UUO model in Munich Wistar Fr&#246;mter (MWF) rats, which have surface glomeruli under physiologic conditions. The UUO model for five weeks or more induced significant alterations to gross renal morphology, the peritubular and glomerular microvasculature, as well as the structure and function of tubular epithelia. Glomerular and peritubular red blood cell (RBC) flow decreased significantly (p &#60; 0.01), probably due to the significant increase in the adherence of white blood cells (WBCs) within glomerular and peritubular capillaries. The glomerular sieving coefficient of albumin increased from 0.015 &#177; 0.002 in untreated MWFs to 0.045 &#177; 0.05 in 5-week-old UUO MWF rats. Twelve weeks of UUO resulted in further increases in surface glomerular density and glomerular sieving coefficient (GSC) for albumin. Fluorescent albumin filtered across the glomeruli was not reabsorbed by the proximal tubules. These data suggest that using UUO to induce surface glomeruli limits the ability to study and interpret normal glomerular processes and disease alterations.

Here, we present a protocol using 2-photon microscopy in Munich Wistar Fromter rats with surface glomeruli to quantifythe effects of prolonged ureteral obstruction on glomerular dynamics and function.

Applying novel microscopy methods to suitable animal disease models to explore the dynamic physiology of the kidney remains a challenge. Rats with surface ...

Assessment of Gut Barrier Integrity in Mice Using Fluorescein-Isothiocyanate-Labeled Dextran

Gut barrier integrity is a hallmark of intestinal health. While gut barrier integrity can be assessed using indirect markers such as the measurement of plasma inflammatory markers and bacterial translocation to the spleen and lymph nodes, the gold standard directly quantifies the ability of selected molecules to traverse the gut mucosal layer toward systemic circulation. This article uses a non-invasive, cost-effective, and low-burden technique to quantify and follow in real time the intestinal permeability in mice using fluorescein-isothiocyanate-labeled dextran (FITC-dextran). Prior to oral supplementation with FITC-dextran, the mice are fasted. They are then gavaged with FITC-dextran diluted in phosphate-buffered saline (PBS). One hour after the gavage, the mice are subjected to general anesthesia using isoflurane, and the in vivo fluorescence is visualized in an imaging chamber. This technique aims to assess residual fluorescence in the abdominal cavity and the hepatic uptake, which is suggestive of portal migration of the fluorescent probe. Blood and stool samples are collected 4 h after oral gavage, and the mice are sacrificed. Plasma and fecal samples diluted in PBS are then plated, and the fluorescence is recorded. The concentration of FITC-dextran is then calculated using a standard curve. In previous research, in vivo imaging has shown that fluorescence rapidly spreads to the liver in mice with a weaker gut barrier induced by a low-fiber diet, while in mice supplemented with fiber to strengthen the gut barrier, the fluorescent signal is retained mostly in the gastrointestinal tract. In addition, in this study, control mice had elevated plasma fluorescence and reduced fluorescence in the stool, while inversely, inulin-supplemented mice had higher levels of fluorescence signals in the gut and low levels in the plasma. In summary, this protocol provides qualitative and quantitative measurements of intestinal permeability as a marker for gut health.

In the present study, fluorescein-isothiocyanate-labeled (FITC) dextran is administered to mice via oral gavage to evaluate intestinal permeability both in vivo and in plasma and fecal samples. As gut barrier function is affected in many disease processes, this direct and quantitative assay can be used in diverse research areas.

Gut barrier integrity is a hallmark of intestinal health. While gut barrier integrity can be assessed using indirect markers such as the measurement of ...

Fluorescent Labeling of Glomeruli in Cleared Mouse Kidneys for Microscopy

An Efficient and Fast Method for Labeling and Analyzing Mouse Glomeruli

The glomeruli are fundamental units in the kidney; hence, studying the glomeruli is pivotal for understanding renal function and pathology. Biological imaging provides intuitive information; thus, it is of great significance to label and observe the glomeruli. However, the glomeruli observation methods currently in use require complicated operations, and the results may lose label details or three-dimensional (3D) information. The clear, unobstructed brain imaging cocktails and computational analysis (CUBIC) tissue clearing technology has been widely used in renal research, allowing for more accurate detection and deeper detection depth. We found that mouse glomeruli can be rapidly and effectively labeled by tail vein injection of medium molecular weight FITC-Dextran followed by the CUBIC clearing method. The cleared mouse kidney could be scanned by a light-sheet microscope (or a confocal microscope when sliced) to obtain three-dimensional image stacks of all the glomeruli in the entire kidney. Processed with appropriate software, the glomeruli signals could be easily digitized and further analyzed to measure the number, volume, and frequency of the glomeruli.

Author Spotlight: Aiding Research in Kidney Biology by Labeling Glomeruli in Cleared Tissues 

This study presents an easy-to-use, complete, and simple set of methods to label and analyze glomeruli from CUBIC-cleared mouse kidneys. Data such as glomerulus number and volume can be obtained easily and reliably using fluorescein isothiocyanate (FITC)-Dextran, light sheet fluorescence microscopy (LSFM), or common confocal microscopy and software such as Imaris.

The glomeruli are fundamental units in the kidney; hence, studying the glomeruli is pivotal for understanding renal function and pathology. Biological ...

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

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Chapters in this video

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

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

An in vivo Assay to Test Blood Vessel Permeability

Transcutaneous Assessment of Renal Function in Conscious Rodents

Assessment of Kidney Function in Mouse Models of Glomerular Disease

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

Transdermal Measurement of Glomerular Filtration Rate in Mice

Using 2-Photon Microscopy to Quantify the Effects of Chronic Unilateral Ureteral Obstruction on Glomerular Processes

Assessment of Gut Barrier Integrity in Mice Using Fluorescein-Isothiocyanate-Labeled Dextran

An Efficient and Fast Method for Labeling and Analyzing Mouse Glomeruli