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

Podsumowanie

Enzymatic microelectrode biosensors enable real-time measurements of extracellular cell signaling in biologically-relevant concentrations. The following protocols extend the applications of biosensors to the ex vivo and in vivo detection of ATP and H₂O₂ in the kidney.

Streszczenie

Enzymatic microelectrode biosensors have been widely used to measure extracellular signaling in real-time. Most of their use has been limited to brain slices and neuronal cell cultures. Recently, this technology has been applied to the whole organs. Advances in sensor design have made  possible the measuring of cell signaling in blood-perfused in vivo kidneys. The present protocols list the steps needed to measure ATP and H₂O₂ signaling in the rat kidney interstitium. Two separate sensor designs are used for the ex vivo and in vivo protocols. Both types of sensor are coated with a thin enzymatic biolayer on top of a permselectivity layer to give fast responding, sensitive and selective biosensors. The permselectivity layer protects the signal from the interferents in biological tissue, and the enzymatic layer utilizes the sequential catalytic reaction of glycerol kinase and glycerol-3-phosphate oxidase in the presence of ATP to produce H₂O₂. The set of sensors used for the ex vivo studies further detected analyte by oxidation of H₂O₂ on a platinum/iridium (Pt-Ir) wire electrode. The sensors for the in vivo studies are instead based on the reduction of H₂O₂ on a mediator coated gold electrode designed for blood-perfused tissue. Final concentration changes are detected by real-time amperometry followed by calibration to known concentrations of analyte. Additionally, the specificity of the amperometric signal can be confirmed by the addition of enzymes such as catalase and apyrase that break down H₂O₂ and ATP correspondingly. These sensors also rely heavily on accurate calibrations before and after each experiment. The following two protocols establish the study of real-time detection of ATP and H₂O₂ in kidney tissues, and can be further modified to extend the described method for use in other biological preparations or whole organs.

Wprowadzenie

Enzymatic microelectrode biosensors (also referenced as sensors in the present manuscript) have been a valuable tool for studying dynamic signaling processes in living cells and tissues. The sensors provide increased temporal and spatial resolution of cell signaling molecules in biologically relevant concentrations. Instead of sampling and analyzing extracellular fluids taken at intervals over long periods of time, these sensors respond as fast as their enzymes react to the analyte, thereby producing real-time measurements^1,2. Fast detection of interstitial concentrations of autocrine and paracrine factors, like purines or hydrogen peroxide, and the dynamics of their release can be used to establish a profile for the effects of drugs in normal and pathological conditions ³. Currently, the majority of applications using sensors have been in brain tissue slices and cell cultures^4-10. The protocols detailed in this manuscript aim to establish the means to accurately measure real-time concentrations of analytes in whole kidneys.

The following protocols were developed to study interstitial ATP and H₂O₂ signaling in kidneys. In the native environment of the kidney, extracellular ATP is rapidly catabolized by endogenous ectonucleotidases into its derivatives (ADP, AMP and adenosine). The sensors used here are highly selective to ATP over other purines or ATP degradation products¹¹. This offers a great advantage as it allows accurate monitoring of the constant and dynamic concentrations of ATP release and its signaling function. Interstitial ATP concentration is measured using the combination of two microelectrodes, an ATP sensor and a Null sensor. The Null sensor in combination with catalase applications is able to detect interstitial H₂O₂ concentrations¹². The following protocols use two different designs of sensors that have characteristics optimal for either ex vivo or in vivo applications.

Both designs are based on the sequential catalytic reaction of glycerol kinase and glycerol-3-phosphate oxidase contained in a sensor enzymatic layer and is driven by the presence of ATP. In the set of sensors used in the ex vivo studies, H₂O₂, the final enzymatic reaction product, is detected by oxidation on a platinum/iridium (Pt-Ir) wire electrode. Sensors for in vivo studies are instead based on H₂O₂ reduction on a mediator coated gold electrode designed for blood-perfused tissue. Shown in Figure 1 is a scheme of both protocols described in this manuscript. The Null sensor is identical to its corresponding ATP sensor except it lacks the bound enzymes. Therefore, in addition to the detection of H₂O₂ with the catalase enzyme, the Null sensor measures nonspecific interferences. ATP concentrations are calculated by subtracting the Null detected nonspecific interferences and background H₂O₂ from the ATP sensor signal. Several sensors are also commercially available to detect other analytes including adenosine, ionosine, hypoxanthine, acetylcholine, choline, glutamate, glucose, lactate, d-serine for ex vivo applications or adenosine, ionosine, and hypoxanthine for in vivo when paired with the corresponding Null sensor.

The ability of the sensor to accurately detect analytes depends on the proper pre- and post- calibrations¹³. This ensures that the analysis accounts for the drift in sensor sensitivity that occurs during use in biological tissues. The sensor holds a depot of glycerol that is used as a reagent in the sensor enzymatic reactions. If the sensor is not used in bath solutions containing glycerol, it will wash out over time. Shorter recording times are then necessary to minimize the sensor drift. Additionally sensor fouling by endogenous proteases and protein fragments can greatly diminish the sensitivity of the sensors¹⁴.

The present manuscript establishes the use of enzymatic microelectrode biosensors for ex vivo and in vivo kidney preparations. Real-time analyte quantification provides unprecedented detail of cellular signaling that may reveal novel insights into the mechanisms of kidney diseases and pharmacological agents.

Protokół

The following animal procedures  adhered to the NIH Guide for the Care and Use of Laboratory Animals. Prior approval was obtained from the Institutional Animal Care and Use Committee (IACUC).
NOTE: Review of the sensor manufacturer instructions should be done during the experiment design and prior to their use. Following these instructions will produce optimal results when using the sensors.

1. Sensor Calibration

1. Prepare fresh stock solutions prior the start of the experiment.
2. Create Buffer A containing 10 mM NaPi buffer, 100 mM NaCl, 1 mM MgCl₂, and 2 mM glycerol. Adjust the pH to 7.4 using NaOH. Store at 2-8 °C.
3. Using a stock ATP concentration (100 mM) stored at -20 °C, create a fresh 10 mM ATP calibration solution by adding 10 µl stock to 90 µl of Buffer A.
4. Rehydrate the ATP sensor by placing its tip (Figure 2) into the rehydration chamber containing Buffer A for at least 10 min at 2-8 °C.
   NOTE: After rehydration, the sensors should not be exposed to air for more than 20 sec or the sensor sensitivity may be reduced. If long exposures to air are anticipated, dip the sensor briefly into a solution of glycerol. The sensors may be used for multiple experiments but these must occur on the same day as sensor rehydration. The sensors can be stored in the rehydration chamber with Buffer A for up to 24 hr.
5. Turn on the dual channel potentiostat (Figure 3) and start the recording system software.
6. Prepare a calibration chamber with 3 ml of Buffer A. Place the reference electrode into the calibration chamber. Take each sensor from the rehydration chamber, then attach it to the manipulator and insert it into the calibration chamber solution.
   NOTE: Use a standard silicone coated petri dish as a calibration chamber. Carry out all calibrations and studies in a Faraday cage on a high-performance lab air table to reduce signal noise during the amperometry recordings (Figure 4). Calibrations are best done as close to the start of data collection as possible. For in vivo applications the optimal time for calibration is during the animal post-surgery recovery period.
7. Ex vivo calibration
   1. Perform cyclic voltammetry in the calibration chamber by cycling the sensors from -500 mV to +500 mV at a rate of 100 mV/sec for 10 cycles. This greatly improves the sensitivity of the sensors. See Figure 5 for the traces observed from the 10 cycles.
   2. Polarize the sensors to +600 mV after the last cycle. The sensor current will decay to an asymptote. A steady reading is achieved after a minimum of 5 min. Record the zero reading.
8. In vivo sensor calibration
   1. Do not perform the cyclic voltammetry on the in vivo sensors. Instead, polarize the sensor in the calibration chamber for 30 sec to +500 mV. Then set the potentiostat to 0 mV and allow the sensor current to rise to an asymptote. The sensor current will take at least 2 min to asymptote. Record the zero reading.
9. Consecutively add set amounts of ATP solution into the chamber to produce a calibration line encompassing a desired detection range. The ATP solution produces a sharp peak in the sensor signal initially, followed by a decay as the ATP diffuses evenly throughout the chamber. Record signal values once the signal level has stabilized after each ATP addition. Figures 6A and 7 show traces and suggested ATP concentrations for both ex vivo and in vivo studies, respectively.
   NOTE: It is important to confirm the selectivity of the electrodes by using scavengers of the analytes. The present protocol used apyrase to test the specificity of the ATP sensor and catalase for the H₂O₂ signal (Figures 6B). If drugs are to be administered, measurements of their reactivity with the sensors should be determined before the study.
10. Add 3 µl of apyrase from a stock of 2 mg/ml (89 UN/mg) to test specificity of the ATP sensor (the current produced by ATP application should reduce to the zero level (Figure 7).
11. Add 3 µl of catalase from a stock of 2mg/ml (100 UN/mg) to test the specificity of the null sensor (the current produced by H₂O₂ application should reduce to the zero reading).

2. Animal Surgery For Sensor Studies

1. Surgery for ex vivo
   1. Anesthetize the experimental animal with isoflurane (5% induction, 1.5 to 2.5% maintenance)/medical grade O₂ or another approved method. ¹⁵’ ¹² Animals must be continually monitored to ensure an adequate level of anesthesia. Stable respiratory rate and toe pinch reaction are used to confirm proper anesthesia.
      Note: Euthanize the animal according to approved IACUC protocols. At the completion of all non-survival procedures euthanize deeply anaesthetized animals by thoracotomy inducing pneumothorax to ensure the humane demise of the animal¹².
   2. Place the rat on a temperature-controlled surgical table in a supine position. While maintaining a proper anesthetic depth, make a midline incision of approximately 5 cm in line with the left kidney and expose the distal abdominal aorta.
   3. Wrap a ligature around the celiac and superior mesenteric arteries, and the abdominal aorta above these arteries but do not ligate. Wrap two ligatures around the abdominal aorta below the renal arteries.
   4. Clamp the abdominal aorta above the ligatures. Tie the lower ligature. Catheterize the abdominal aorta with polyethylene tubing (PE50). Secure the catheter with the second aorta ligature.
   5. Remove the clamp and ligate the mesenteric and celiac arteries. Perfuse the kidney at 6 ml/min with Hanks Balanced Salt Solution at RT for 2-3 min until the kidney is completely blanched.
   6. Excise the kidney and the catheter, connected portion of the aorta. Place the kidney into a 3 ml Petri dish filled with bath solution.
      Note: Experiment protocol can be performed at RT. The bath solution contains in mM: 145 NaCl, 4.5 KCl, 2 MgCl₂, 1 CaCl₂, 10 HEPES, pH 7.35 adjusted with NaOH.
2. Surgery for in vivo
   1. Anesthetize the rat using approved IACUC protocols. For in vivo analysis anesthetize the rats with ketamine (20 mg/kg i.m.) and inactin (50 mg/kg i.p.). Animals must be continually monitored to ensure an adequate level of anesthesia. A stable respiratory rate and toe pinch reaction are used to confirm proper anesthesia.
   2. After proper anesthesia depth is obtained, place the rat in a supine position on a temperature-controlled grounded surface located on the air table. The surface should be preheated and maintained at 36 °C.
   3. While maintaining proper anesthetic depth, make a midline incision approximately 5 cm in line with the kidney.
   4. Use a suture to deflect and anchor the cutaneous and subcutaneous tissue so that the entire kidney is visible. Place the kidney in a kidney cup to minimize any movement artifacts.
   5. Use an IV infusion of 2% BSA: 0.9% NaCl at 1 ml/100 g/hr via the jugular vein to maintain blood volume. Cannulate both ureters for urine collection. Place ties around the superior mesenteric and celiac arteries and the distal aortal for the manipulation of renal perfusion pressure (Figures 10A).
   6. If the application of pharmacological agents is required during the in vivo experiments, insertion of an interstitial catheter is recommended (Figures 10B).

3. Data Acquisition Setup

1. Open the data acquisition program and set its polarity for both ex vivo and in vivo experiments to Anodic Positive. Set the program to save data as ASCII code.
2. Position the micromanipulators for quick insertion of the sensors into the kidneys.
   NOTE: Alternatively, use a dummy probe attached to the micromanipulator to help achieve the desired placement of sensors.
3. Ex vivo data acquisition
   1. Perfuse the kidney with bath solution (from 2.1.6) via the cannulated aorta at a constant rate of 650 µl/min. Using surgical scissors, carefully remove the kidney capsule, which is necessary for sensor insertion.
   2. Secure the kidney with rubber bands strapped over the kidney and attached to the silicone-coated dish with pins.
   3. Place the reference electrode close to the kidney in the petri dish with its tip submerged in the buffer solution.
4. In vivo data acquisition
   1. Place the kidney in a kidney cup. Depending on the strain and age of the animal use a size of cup that holds the kidney loosely. Figure 8 shows two sizes of kidney cups. Similar cups are used for different physiological approaches focused on the analysis of kidney function such as micropuncture etc¹⁶.
      Note: The position of the kidney cup is critical to remove the mechanical noise produced by the animal breathing but should not interfere with or block kidney perfusion or urine flow.
   2. Allow 45 min of recovery time before performing data collection.
   3. Using a 26-30G needle, make a puncture hole at the desired location and depth of the sensor in the kidney. Blot the surface hole to remove exuded blood. Add glycerol solution to the surface of the kidney. This will prevent the kidney surface from drying out during the experiment.
   4. Remove the first sensor from the rehydration chamber and attach it to the micromanipulator. Quickly, within 20 sec, insert the electrode into the freshly created hole in the kidney. Repeat steps 3.5-3.9 for the null sensor.
   5. Insert the reference electrode into the kidney, approximately 1 cm from from the sensors.
5. Turn on the potentiostat and activate the recording program on the computer. Figure 9 shows the final setup of the ex vivo kidney data acquisition. Figure 10 shows the final setup of the in vivo kidney data acquisition with an inserted catheter.

4. Data Analysis

1. Import the ASCII data file into Origin or any other similar software.
2. Concentration current relation
   1. Use the linear fit/extrapolate function to build a linear concentration (x-axis) to current (y-axis) relationship for the ATP or H₂O₂ calibration points (Figures 6 and 7).
      linear fit line: y = mx + b
3. Equate the current to concentration
4. Subtract the measured traces of the null sensor from those of the ATP sensor to obtain the actual current produced by intracellular ATP.
5. Convert the ATP values in pA obtained by amperometry to nM using the calibration equation detailed in 4.2.1. Determine the concentration of the subtracted data trace of the ATP sensor by importing each adjusted current into the “x” value and solving for y (the concentration of analyte).
6. Similarly, calculate H₂O₂ concentration from its calibration equation if needed.

Wyniki

The design of the enzymatic microelectrode biosensor allows the real-time detection of analytes in whole kidneys. The general experiment design for either ex vivo or in vivo studies is illustrated in Figure 1.The sensors used and the surgical procedures differ depending on whether the study is ex vivo or in vivo.

To obtain reproducible results, accurate pre- and post- calibrations are critical. Figure 6A shows a rep...

Dyskusje

The present protocols were developed to provide enhanced temporal and spatial resolution of ATP and H₂O₂ signaling for ex vivo isolated, perfused and in vivo blood-perfused kidneys. The differences between the protocols and the sensors used here provide optimal data acquisition for either pharmacological agents or physiological studies. The protocols consist of 1) sensor calibration, 2) surgical procedure, 3) data acquisition setup, and 4) data analysis. They enable the real-time m...

Materiały

Name	Company	Catalog Number	Comments
Sensor Kit	Sarissa Biomedical	SBK-ATP-05-125	The kit includes storage bottle, rehydration chamber, electrode leads, and reference electrodes.  Also included with the kit is the user's choice of sensors.
Sarissaprobe ATP Biosensor 125 μm	Sarissa Biomedical	SBS-ATP-05-125	store at 2-8 oC before use
Sarissagold ATP Biosensor 50 μm	Sarissa Biomedical	SGS-ATP-10-50	store at 2-8 oC before use
Sarissaprobe null sensor 125 μm	Sarissa Biomedical	SBS-NUL-20-125	store at 2-8 oC before use
Sarissagold null sensor 50 μm	Sarissa Biomedical	SGS-NUL-10-50	store at 2-8 oC before use
Sarissaprobe ATP Manual	Sarissa Biomedical		http://www.sarissa-biomedical.com/media/31563/instructions-atp.pdf
Faraday cage	TMC
Dual channel potentiostat	Digi-Ivy	DY2021	Type II Faraday cage
Data acquisition program	Digi-Ivy	DY2000
Perfusion pump	Razel Scientific Instruments	Model R99E
Fiber optic illuminator	Schott	ACE 1
micromanipulator	Narishige	MM-3
micromanipulator magnetic stand	Narishige	GJ-8
air table	TMC	63-500
isoflurane ventilator	LEI Medical	M2000
3 ml petri dish	Fisher Scientific	S3358OA
needle	Santa Cruz		26-30 G
pins			Standard dissection pins
catheter			Polyethylene tubing (PE50)
catheter tissue glue	Vetbond	1469SB
suture	Look	SP117
rubber bands			any 2-4 mm wide rubber bands
silicone	Momentive	RTV-615 Clear 1#
clamp	Fine Science Tools	18052-03
standard dissection kit			Kit should include scalpel and dissection sissors
Kidney Cup			Of own design
standard chemicals	Sigma-Aldrich
ATP	Sigma-Aldrich	A6559-25UMO	100 mM ATP solution
hydrogen peroxide	Sigma-Aldrich	216763
glycerol	Sigma-Aldrich	G9012
Apyrase	Sigma-Aldrich	A7646
Catalase	Sigma-Aldrich	C40
isoflurane	Clipper	10250
inactin	Sigma-Aldrich	T133
ketamine	Clipper	2010012
Hanks Balanced Salt Solution	Gibco	14025092