Today, we will be demonstrating the technique of transdermal measurement of glomerular filtration or tGFR in mice. There are a number of features of this method that I think are worth mentioning. First of all, currently tGFR is the most accurate way of assessing renal function in mice.
Secondly, tGFR is performed in conscious, freely moving mice so it avoids the complications of anesthesia. Thirdly, once you have mastered the technique, it's reasonable to be able to perform 30 to 40 tGFR measurements in a single day. Finally, tGFR measurements can be performed repeatedly in an in vitro mice.
What this means is that we can perform serial measurements of renal function in acute and chronic disease settings. The main advantage of this technique is that it allows researchers to measure the Glomerular Filtration Rate or GFR in conscious, freely moving mice with high precision and sensitivity. One to two days before the tGFR measurement, after confirming a lack of response to toe pinch, place an anesthetized mouse in the prone position on a heat pad.
Using an electric shaver, trim against the direction of growth to remove most of the fur from one side of the mouse's back from the top of the hind legs up to the neck and across the ribs. Use a cotton bud to apply a thin layer of depilation cream to the exposed skin against the direction of growth to ensure that the cream is applied as close to the skin as possible and remove the cream after one to three minutes with clean cotton swabs and warm water. On the day of the procedure, trim a six by three centimeter adhesive patch to the size of the GFR device before peeling off the back of the patch and sticking the device to the adhesive side of the patch with the light-emitting diodes positioned immediately above the clear window.
Then stick a small piece of the patch to the battery. To attach the monitor, with the anesthetized mouse in the prone position, clean the pre-shaved skin with 70%ethanol and adjust the width of an approximately 12 centimeter piece of tape by tearing it lengthwise. Place the tape under the mouse with two centimeters on one side of the animal folding over one edge of the right side of the tape for easy placement and removal after the measurement.
Next, connect the battery to the device, removing the backing from the battery and securely placing the battery onto the device. The device is ready to use and the data acquisition begins when the blue LEDs start blinking. Remove the backing from the patch on the device and place the device onto the shaved skin such that the window exposing the LEDs is over the ribs securing the right side of the device with tape.
It's important to ensure secure attachment of the device without restricting the mouse's mobility or placing too much pressure on the skin. Then firmly wrap the left side of the tape around the mouse and device. Allow the device to record a steady background reading for three minutes.
In the meantime, warm the tail with a head pad and load an insulin syringe with the appropriate experimental volume of FITC-sinistrin for the injection rounded to the nearest 10 microliters. Administer the FITC-sinistrin intravenously in one smooth but rapid bolus. It's important that the user is comfortable with IV injections as it is necessary to successfully administer the FITC-sinistrin in one bolus rather than over several attempts which will result in multiple peaks in the clearance curve.
To measure the tGFR, place the mouse in its own cage with monitoring until full recumbency and allow the FITC-sinistrin clearance to be recorded for 1.5 hours. At the end of the measurement period, place the mouse on the wire rack on top of the cage and allowing the mouse to grip the metal bars, pull the tape from underneath the belly in one quick smooth motion. Then remove any tape from the device and the patch from the skin taking care that the battery does not disconnect from the device and return the mouse to its cage.
To evaluate the data, carefully disconnect the battery and use a USB cable to connect the device to the computer. Open the reading software and click Connect, Read, Rename and Save. Then close the program and process and evaluate the data in the analysis software according to the manufacturer's instructions.
While FITC-sinistrin is rapidly cleared from the circulation in healthy mice, this clearance is dramatically delayed in mice with acute kidney injury. In mice with very severe acute kidney injury, there may be very little or no clearance of the FITC-sinistrin fluorescence during the entire 90-minute measurement period indicating a complete absence of glomerular filtration. Transdermal GFR measurement is minimally invasive and therefore can be used to monitor changes in kidney function in individual animals over multiple time points.
Indeed in this representative experiment, sequential measurements demonstrated changes in kidney function at zero, one, two, and four days after ischemia reperfusion injury. In this mouse chronic kidney disease model, the inverse relationship of the FITC-sinistrin half-life to GFR can be directly correlated to the impaired renal function observed in these animals. Indeed, the FITC-sinistrin half-life correlates closely with the semi-quantitative histological assessment of tubular injury over the full range of GFR measurements in uninjured mice and in mice with different severities of ischemia reperfusion injury-induced acute kidney injury.
In contrast, serum creatinine and blood urea nitrogen demonstrate a positive but weaker correlation with FITC-sinistrin clearance indicating the transcutaneous GFR measurements provide a more reliable measure of renal injury than either serum creatinine or blood urea nitrogen. Transdermal measurement of GFR is a more sensitive measure of kidney function than traditional biomarkers such as creatinine and BUN. This provides a more accurate assessment of the efficacy of novel therapies for kidney disease in preclinical research.
The most critical steps in this method are the proper attachment of the device to the mouse's back and the successful IV administration of FITC-sinistrin. These steps are essential to obtain an accurate assessment of GFR in mice.
