The overall goal of this procedure is to transcutaneously measure renal function without the need for blood samples or further experimental analysis. This method can help answer key questions in the nephrology field, such as, what is the impact of giving kidney damage or specific therapeutic interventions on renal function. The main advantages of this technique are that it is minimally invasive and that the results are ready to analyze immediately after the measurement without subsequent experimental manipulations.
This technique can be used to study virtually any renal condition in several different species. Also, it is useful for performing sequential measurements within one animal. Before beginning the procedure, confirm the proper level of anesthesia by toe pinch.
And use an electric shaver to remove a three by six centimeter area of fur from the back lower abdomen of the animal. Remove the remaining fur with depilatory cream. After two to three minutes, thoroughly wash the denuded skin as the cream may emit some background fluorescence.
Next, place the optical device for measuring renal function onto one side of a double sided adhesive patch. Positioning the optical part above the transparent window and leaving the opposite side covered in protective foil. Then, attach a size-matched piece of double sided adhesive patch to the battery.
Before fixing the device, weigh the animal. Then, cut a piece of tubular elastic gauze bandage about one centimeter longer than the width of the double sided adhesive patch. And pull the bandage over the head of the animal.
Next, place the animal in the supine position, leaving the shaved area uncovered. And plug the battery connector into the corresponding port on the device. If the battery has been properly connected, the device will begin blinking.
Remove the protective foil from the other side of the patch attached to the battery. And mount the battery on the upper surface of the device. Now, remove the protective cover from the operating device patch and holding the edges until it is correctly fixed, place the patch onto the shaved area on the back of the animal.
Cover the device with the tubular elastic gauze bandage, taking care to fully stretch the bandage over the abdomen while making sure that the limbs can move freely. A good fixation is crucial for avoiding movement artifacts during the measurement, so please take care that the device is attached properly before the measurement starts. When the bandage is in place, apply a strip of size matched adhesive tape over the device so that it covers the wires of the battery.
And measure the background reading for one to three minutes without putting pressure on the device. To administer the FITC-sinistrin, after warming up the animal's tail to dilate the tail veins, IV inject the appropriate volume of FITC-sinistrin stock solution. Next, carefully place the animal alone in its home cage without water or a wire lid.
Taking care not to introduce movement artifacts. Then record the fluorescence emitted by the FITC-sinistrin with the optical device for the appropriate experimental time period. At the end of the recording period, carefully remove the adhesive tape followed by the tubular bandage.
Then gently detach the double sided adhesive patch from the skin. And return the animal to its normal home cage. To read the data, disconnect the battery from the device and remove the adhesive patch.
Then use a micro USB cable to connect the optical recorder to a computer and download the data with the appropriate software. Finally, use the appropriate software to generate the elimination kinetics curve according to the software protocols. And open the dot CSV file containing the data.
When renal function is affected, the FITC-sinistrin half-life 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 the background level and an increased area under the curve is observed.
In the same animal, pre to post injury measurements demonstrate an increase in the maximum fluorescence intensity due to an accumulation of the marker caused by its reduced excretion. For example, in the presence of kidney failure, the transcutaneously measured FITC-sinistrin curve exhibits a steady state due to the severely impaired function of the renal tissue. Further, the miniaturized device has been used to successfully measure renal function in several strains of rodents with a variety of different health statuses.
Underscoring the suitability and sensitivity of the device for detecting changes due to renal disease and aging. Once mastered, the experiment can be completely set-up in 50 minutes with at least four animals per hour able to be evaluated by a single experimentator. While attempting this procedure, it is important to remember to protect the electronic device by removing the water supply and any protruding structure during the measurement period.
