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 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 µL). 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.
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 (5µL) 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.
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 für Natur, Umwelt und Verbraucherschutz [LANUV] reference number 84-02.04.2012.A397).
1. Preparation of instruments, solutions, and equipment
2. Preparation phase
3. Equilibration phase
4. Experimental phase
NOTE: In this phase, the effect of drugs on glomerular permselectivity can be investigated.
5. Urine analysis
6. Data analysis
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 phase. In this phase, the infusion of FITC-polysucrose 70 is continued and drugs or other substances can be tested for influencing glomerular permeability. Urine is collected at the end of each phase.
To investigate glomerular permeability within this tracer model, it is essential to place a urinary catheter properly and without injuring the mucosa. The placement of a urinary catheter into a female mouse bladder is demonstrated in Figure 1. The catheter is placed 3 mm into the urethral ostium, parallel to the craniocaudal urethral axis (Figure 1A). The catheter is turned 180° toward the tail of the mouse (Figure 1B) and introduced 7 mm further into the bladder, parallel to the murine spine (Figure 1C,D).
As described above, previous methods to test glomerular permeability in mice used HPLC to purify FITC-polysucrose 70 signals from urine samples5,6. As this method uses fluorescence measurement without a previous purification of the tracer, PBS, mouse urine, and FITC-polysucrose 70 in mouse urine were analyzed. Figure 3A shows the fluorescence scan of PBS with a fluorescence peak at 325 nm, which seems to be an effect of the excitation flash at 290 nm. The fluorescence scan of native mouse urine shows a fluorescence maximum at 395 nm (Figure 3B). FITC-polysucrose 70 dissolved in mouse urine displays a fluorescence maximum at 525 nm (Figure 3C,D). Figure 3C shows that mouse urine does not disturb the fluorescence measurement of FITC-polysucrose 70 in mouse urine. Increasing concentrations of FITC-polysucrose 70 show an increased fluorescence intensity (Figure 3E).
To prove that this method is capable of detecting differences in glomerular permeability, Ang II was applied in the experimental phase of the model. Ang II increased the glomerular permeability in mice 60 min after a continuous administration in nonblood-pressure-relevant dosages (Figure 4A,B). The increase in glomerular permeability due to Ang II could be blocked by an Ang II-receptor blocker (ARB), namely candesartan (Figure 4A). Ang II washout decreased glomerular permeability (Figure 4A). Blood pressure was measured via the tail-cuff method and did not show significant differences between the groups (Figure 4B). Polysucrose 70- and FITC-polysucrose 70-infused animals serve as controls for FITC-polysucrose 70- and Ang II-treated animals (Figure 4C).
Figure 1: Placement of a urinary catheter in female mice. Lateral view of the mouse abdomen. (A) The marked and lubricated catheter is introduced 3 mm into the external urethral ostium of a female mouse. The craniocaudal axis of the urethra is paralleled with the catheter. Careful tension on the lower abdomen with the left index finger facilitates the introduction of the catheter into the external urethral ostium. (B) The catheter is turned 180° toward the tail of the mouse. The tip of the catheter remains 3 mm within the external urethral ostium. (C) The catheter is now introduced 7 mm further into the bladder. The direction of the catheter is aligned with the murine spine. (D) Ventral view of the mouse abdomen. The catheter is placed 10 mm into the mouse. (E) Urine appears with a correct insertion into the bladder. Please click here to view a larger version of this figure.
Figure 2: Schematic illustration of the experimental procedures on a timeline. After narcosis of the mouse, a urinary catheter is placed. The central venous catheter is implanted and baseline urine is obtained (0 min). Afterward, the FITC-polysucrose 70 bolus is applied via the central venous catheter and a continuous infusion of FITC-polysucrose 70 is started. The equilibration phase for FITC-polysucrose 70 lasts 60 min. Urine is collected after the equilibration phase (0 min) and before the experimental phase starts. Within this phase, drugs or other substances (like angiotensin II) can be applied. At the end of the experimental phase, urine is obtained for analysis (60 min). Please click here to view a larger version of this figure.
Figure 3: Fluorescence of PBS and mouse urine with and without FITC-polysucrose 70. (A) Fluorescence frequency scan (excitation of 290 nm, emission of 325-700 nm) of PBS shows a signal at 325 nm, which seems to be an effect of the excitation flash at 290 nm. (B) In the frequency scan of mouse native urine (urine pool diluted at 1:10 and 1:20), there is a signal with a maximum at 395 nm, probably caused by urinary protein autofluorescence. (C) Mouse urine containing FITC-polysucrose 70 (40 µg/mL) shows a signal peak at 525 nm that does not interfere with the measurement of the urine autofluorescence. (D) Magnification of the FITC-polysucrose 70 fluorescence peak depending on the emission wavelength. Different concentrations of FITC-polysucrose 70 (1.25, 2.5, 5, 10, 20, and 40 µg/mL) are displayed. (E) Increasing FITC-polysucrose 70 concentrations show an increase of fluorescence intensity at the emission of 525 nm. Please click here to view a larger version of this figure.
Figure 4: Angiotensin II (Ang II) increases glomerular permeability. (A) Ang II significantly increases the glomerular permeability in mice, measured by FITC-polysucrose 70 detection in the mice's urine (mean + SEM; n = 5; p < 0.004, tested by Kruskal-Wallis test). FITC-polysucrose 70 concentrations in the urine were referenced to urine creatinine concentrations. The white columns (0 min) represent FITC-polysucrose 70 levels before the start of Ang II stimulation, the black columns (60 min) represent FITC-polysucrose 70 levels 60 min after the start of Ang II stimulation, and the grey columns (120 min or +60 min) after an additional 60 min of Ang II stimulation or 60 min of Ang II washout. (B) Systolic blood pressure was monitored by the tail-cuff method (mean + SEM). No significant blood pressure differences between the control and Ang II-treated groups were noted. (C) Polysucrose 70 and FITC-polysucrose 70 do not alter glomerular permeability in mice significantly. Ang II increases glomerular permeability in mice significantly (mean + SEM; n = 7, p < 0.005). This figure has been modified from Konigshausen et al.8. Please click here to view a larger version of this figure.
Supplemental Figure 1: Schematic diagram of the placement of a urinary catheter in female mice. Lateral view of the mouse abdomen. (A) The marked and lubricated catheter is introduced 3 mm into the external urethral ostium of a female mouse. The craniocaudal axis of the urethra is paralleled with the catheter. Careful tension on the lower abdomen with the left index finger facilitates the introduction of the catheter into the external urethral ostium. (B) The catheter is turned 180° toward the tail of the mouse. The tip of the catheter remains 3 mm within the external urethral ostium. (C) The catheter is now introduced 7 mm further into the bladder. The direction of the catheter is aligned with the murine spine. (D) Ventral view of the mouse abdomen. The catheter is introduced 3 mm into the external urethral ostium of a female mouse. (E) After the catheter is turned 180°, it is introduced 7 mm further into the mouse bladder. The direction of the catheter is aligned with the murine spine. This figure has been modified from Reis et al.7. Please click here to view a larger version of this figure.
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 with small volumes of samples.
When placing the central venous catheter, it is essential that the catheter does not penetrate the jugular vein. To avoid penetration, we recommend putting tension on the jugular vein with a distal ligature (see step 2.2.7) and to lift the jugular vein with fine tweezers to extend the lumen of the jugular vein. To keep the insertion site small, try to avoid gross movements while inserting the catheter and always ensure the best view of the surgical field by removing extra tissue. The most critical step in the placement of the urinary catheter is the final insertion into the bladder. If resistance is felt, we recommend starting over from the beginning in order not to cause false tracks and injury to the mucosa. Catheter rotation, lubrication, and gentle movements, as well as training, help in difficult cases7.
FITC-polysucrose 70 is a fluorescently labeled, branched, and cross-linked polymer of sucrose and epichlorohydrin9. It behaves in solution as a globular molecule with a spherical shape and with a low shape asymmetry but with a molecular deformability9. Like other sucrose molecules, FITC-polysucrose 70 is filtered by the glomerulus and not reabsorbed in the tubular system4. To avoid free FITC molecules in the infusion, we dialyzed the FITC-polysucrose 70 solution before application to the mice. It is possible to store dialyzed FITC-polysucrose 70 molecules at -20 °C for months. As FITC-polysucrose 70 is applied intravenously in this and other protocols, it is essential that no blood contaminates the urine samples. Therefore, the urinary catheter needs to be placed with caution and anticoagulant substances within the experiment should be avoided. The standard curve for FITC-polysucrose 70 is dissolved in mouse urine pool to get the most accurate results. Due to concentration differences in the urine at different experimental time points and between groups, FITC-polysucrose 70 concentrations need to be referenced to a marker in the urine that reflects urine concentration (e.g., creatinine). Interference of FITC-polysucrose 70 fluorescence with the measurement of creatinine in an enzymatic assay is unlikely.
The analysis of FITC-polysucrose 70 fluorescence should be performed in black 384-well plates as small urine volumes of 5 µL can be measured in these plates. We do not recommend reusing the same wells within the 384-well plates, even after washing the plates, as fluorescence is still measurable. As bubbles interfere with the fluorescence reading, the plates should be centrifuged before analysis. Alternatively, bubbles can be destroyed manually with the tip of a pipette.
The use of polysucroses to test glomerular permselectivity was described almost 40 years ago10. Fluorescently labeled polysucrose has been investigated in glomerular injury studies almost exclusively in rats in the past years4,11,12,13,14,15,16,17,18,19,20. This may have anatomical reasons due to easier surgical procedures in rats than in mice. In rats, urethrotomy is used to obtain urine samples4,5,9,12,13,14,21. To analyze plasma and urine samples, probes are subjected to high-performance size exclusion chromatography4,5,9,12,13,14,21. In addition, the glomerular filtration rate (GFR) is measured by radioactive 51Cr-ethylenediamine tetraacetic acid (EDTA)4,5,9,12,13,14,21. Two previous studies have applied FITC-polysucrose 70 in mice5,6. In mice, a suprapubic urinary catheter is introduced into the bladder6,20. Urinary and plasma probes are subjected to gel filtration before the analysis of FITC-polysucrose 70 fluorescence. Similar as in rats, GFR was analyzed via radioactivity6,20. The second publication concerning the application of FITC-polysucrose 70 in mice uses a model of peritoneal permeability and, therefore, did not analyze mouse urine for FITC-polysucrose 70 fluorescence22. The presented method was, therefore, modified to facilitate urine sampling and analysis. Urine samples can be easily obtained through a noninvasive transurethral urinary catheter. Urine analysis for FITC-polysucrose 70 fluorescence is performed without any previous high-performance size exclusion chromatography or gel filtration. By referencing FITC-polysucrose 70 fluorescence to mouse urine creatinine, the measurement of GFR with a radioactive assay is not needed.
Increases in blood pressure enhance glomerular permeability. Therefore, blood pressure monitoring is essential whilst investigating glomerular permeability. The most accurate blood pressure measurements in mice are performed via a central arterial catheter23 needing heparinization of the catheter to prevent clotting. We have experienced problems with hematuria after low-dose heparinization and urinary catheter placement. Therefore, we decided to perform the tail-cuff technique to measure blood pressure, which is noninvasive and does not need heparinization. Depending on the depth of anesthesia, blood pressure monitoring with the tail-cuff method is sometimes challenging. Blood pressure membranes need to be checked in advance and the position of the mouse should be optimized before blood pressure recording.
In addition to challenges in blood pressure measurement, this technique is also limited by producing arbitrary units of FITC-polysucrose 70. So far, this technique has only been investigated in animals and, therefore, its relevance to the human glomerular filter is still unknown. Time intervals to investigate increases in glomerular permeability depend on mouse urine production. Therefore, very short-term increases in glomerular permeability (in minutes) may be missed due to the lack of mouse urine production. In this protocol, FITC-polysucrose 70 is normalized to urine creatinine, which is removed from the blood mainly via glomerular filtration, but also by proximal tubular secretion. This will introduce an error when estimating glomerular permeability using this method, reducing the measured fractional clearance of polysucrose24.
The authors have nothing to disclose.
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.
Motic SMZ168 BL | Motic | SMZ168BL | microscope for mouse surgery |
KL1500LCD | Pulch and Lorenz microscopy | 150500 | light for mouse surgery |
Microfederschere | Braun, Aesculap | FD100R | fine scissors |
Durotip Feine Scheren | Braun, Aesculap | BC210R | for neck cut |
Anatomische Pinzette | Braun, Aesculap | BD215R | for surgery |
Präparierklemme | Aesculap | BJ008R | for surgery |
Seraflex | Serag Wiessner | IC108000 | silk thread |
Ketamine 10% | Medistar | anesthesia | |
Rompun (Xylazin) 2% | Bayer | anesthesia | |
Fine Bore Polythene Tubing ID 0.28mm OD 0.61mm | Portex | 800/100/100 | Catheter |
Fine Bore Polythene Tubing ID 0.58mm OD 0.96mm | Portex | 800/100/200 | Catheter |
Harvard apparatus 11 Plus | Harvard Apparatus | 70-2209 | syringe pump |
BD Insyte Autoguard | BD | 381823 | urinary catheter |
Multimode Detector DTX 880 | Beckman Coulter | plate reader | |
384 well microtiterplate | Nunc | 262260 | 384 well platte |
Creatinine Assay Kit | Sigma-Aldrich | MAK080 | to measure creatinine concentration |
96 well plate | Nunc | 260836 | for creatinine assay |
FITC-labeled polysuccrose 70 | TBD Consultancy | FP70 | FITC-ficoll |
Angiotensin II | Sigma-Aldrich | A9525 | used to test glomerular permeability |
BP-98A | Softron | for blood pressure measurement | |
OTS 40.3040 | Medite | 01-4005-00 | heating plate for mouse surgery |
Instillagel 6mL | Farco-Pharma GmbH | for urinary catheter | |
Exacta | Aesculap | GT415 | shaver |