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

The overall goal of these procedures is to measure real-time endogenous interstitial concentrations of a TP and hydrogen peroxide in whole kidneys. This is accomplished by first rehydrating and calibrating sensors for ex vivo and in vivo applications to known concentrations of a TP and hydrogen peroxide. For ex vivo sensor installation, the kidney was surgically instrumented and cleaned from the blood.

This procedure is performed by flushing the kidney through the aorta. Then the rat kidney is surgically isolated, placed in a recording chamber and perfused with saline. A TP and hydrogen peroxide are measured using ex vivo sensors for the in vivo procedure.

An incision is made to allow visualization of the blood perfused kidney. The kidney is placed in the staleness still cap and an interstitial catheter can be installed for drug applications. Further renal interstitial A TP and hydrogen peroxide concentrations are measured using in vivo sensors.

Ultimately, these protocols facilitate the use of enzymatic micro electrode biosensors for real-time a TP and hydrogen peroxide measurements in the kidney interstitium. The main advantage of enzymatic biosensors is their ability to directly measure endogenous interstitial concentrations in biological tissues. Here this technology is used to detect a TP and hydrogen peroxide concentrations in the interstitium of isolated seline perfused kidney, or in vivo.

The enzymatic biosensors precision enables real-time measurements of basal and dynamic regulation of extracellular A TP or hydrogen peroxide concentrations. These measurements can establish drug application or disease profiles for functionally distinct kidney regions. Rehydrate both the A TP and null sensors by placing their tips into separate rehydration chambers containing buffer A for at least 10 minutes at two to eight degrees Celsius.

Turn on the dual channel POTENTIA stat and start the recording system software. Set the program to save data as A-S-C-I-I code and sensor polarity to anodic positive. Prepare a calibration chamber with three milliliters of buffer, a lower the reference electrode into the chamber.

Remove each sensor from the rehydration. Attach them to micro manipulators and insert them into the calibration chamber solution. Perform all calibrations and studies in a Faraday cage on a high performance lab air table to reduce signal noise.

During the pyrometry recordings. Perform cyclic vol telemetry for the ex vivo sensors in the calibration chamber. By cycling the sensors from minus 500 millivolts to plus 500 millivolts at a rate of 100 millivolts per second for 10 cycles, this greatly improves the sensitivity of the sensors, polarize the sensors to plus 600 millivolts.

After the last cycle, the sensor current will decay to an asim tote. A study reading is achieved after a minimum of five minutes. Record the zero reading consecutively add set amounts of a TP solution into the chamber for producing a calibration line encompassing a desired detection range of the A TP sensor.

The A TP solution will produce a sharp peak in the sensor signal initially followed by a decay as the A TP diffuses evenly throughout the chamber. Signal values should be recorded once the signal level has stabilized. For each A TP addition, add three microliters evaporates from a stock of two milligrams per milliliter to test specificity of the A TP sensor.

The current produced by a TP application should reduce to the zero level consecutively add set amounts of hydrogen peroxide solution into the chamber for producing a calibration line encompassing a detection range of the null sensor signal values should be recorded once the signal level is stabilized. For each hydrogen peroxide addition, add three microliters of cattle lace from a stock of two milligrams per milliliter to test the specificity of the null sensor. The current produced by hydrogen peroxide application should reduce to the zero reading for ex vivo studies.

Rapa ligature around the celiac and superior mesenteric arteries and the abdominal aorta above these arteries. But do not ligate rep two ligatures around the abdominal aorta below the renal artery. Ligate the abdominal aorta distally below the renal artery.

Clamp the aorta at least one centimeter above the ligature and below the renal artery. Catheterize the abdominal aorta with polyethylene tubing between the lowest ligature and the clamp. Secure the catheter with the second aorta ligature.

Remove the clamp and ligate the mesenteric and celiac arteries and the aorta above these arteries. Perfuse the kidney at six milliliters per minute with Hank's balance salt solution at room temperature for two to three minutes until the kidney is completely blanched. In addition, the renal vein can be catheterized for collection of outflow fluid.

Using surgical tweezers, carefully remove the kidney capsule, which is necessary for sensor insertion. Excise the kidney, including the catheter connected portion of the aorta. Place the kidney into a three milliliter Petri dish filled with bath solution.

Perfuse the kidney with bath solution via the cannulated aorta at a constant rate of 650 microliters per minute. Secure the kidney with rubber bands strapped over the kidney and attached to the silicone coated dish with pins. Place the reference electrode close to the kidney in the Petri dish with its tips submerged in the bath solution.

Position the micro manipulators for quick insertion of the sensors into the kidney. Alternatively, use a dummy probe attached to each micro manipulator to help achieve the desired placement of sensors for each sensor and lasting no more than 20 seconds. Remove the sensor from the rehydration chamber, attach to the micro manipulator and insert the sensor into the dish bath solution.

If a longer exposure to air is anticipated, dip the sensor briefly into a solution of glycerol for each exposure. Polarize the sensors with the potentia stat to plus 600 millivolts and allow the current to Asim tote. Advance each sensor to the same depth in the kidney.

Perform data analysis according to the text protocol. The design of the enzymatic micro electrode biosensor allows real-time detection of analytes and whole kidneys to obtain reproducible results. Accurate pre and post calibrations are critical.

This figure shows a representative trace of the signal produced by the X pvo, a TP and null sensors during a TP sensor calibration. The calibration procedure produces a linear fit that is used to calculate the dynamic changes of a TP.The null sensor is calibrated with addition of known hydrogen peroxide concentrations to the bath solution and the asim to am parametric values are recorded. Addition of catalysts to the bath solution results in rapid current decay.

The in vivo sensor produces a similar trace but detects reductive rather than oxidative currents, and therefore the current is negative Calibration also produces a linear fit in the 0.3 to 80 micromolar range. The specificity of the in vivo sensor to a TP over other purine products is shown here. Demonstrated in this figure angiotensin two induced interstitial endogenous hydrogen peroxide concentration changes in freshly isolated kidneys from salt resistant and salt sensitive rats.

Angiotensin two induces an acute release of hydrogen peroxide from both kidneys. However, the maximum amplitudes were significantly elevated in the kidneys from salt sensitive rats, especially when fed a high salt diet. While attempting this procedure, it's important to remember that the sensor tip and the surface of renal cortex are delicate and as such, damaging either will impair accurate recordings.

After watching this video, you should have a good understanding of how to measure endogenous substances and whole organs such as the kidney. You will be also able to accurately perform calibrations and the surgical procedures necessary for successful recordings. We have applied this method to study a TP and hydrogen peroxide basil levels, and they release in the kidney.

However, the same approach could be used to determine concentrations of adenosine glutamate, glucose, and some other substances when correspondent sensors are applied.