Name: use of enzymatic biosensors to quantify endogenous atp or h2o2 in the kidney
Uploaded: 2015-10-12T15:00:00
Description: Watch this Scientific Journal Video about Use of Enzymatic Biosensors to Quantify Endogenous ATP or H2O2 in the Kidney at JoVE.com

We appreciate Sarissa Biomedical for their work in developing the sensors used in the present manuscript. This research was supported by the National Heart, Lung, and Blood Institute grants HL108880 (A. Staruschenko),&#160;HL 116264 (A. Cowley) and HL 122662 (A. Staruschenko and A. Cowley),&#160;a project funded by the Medical College of Wisconsin Research Affairs Committee #9306830 (O. Palygin) and &#160;Advancing a Healthier Wisconsin Research and Education Program&#160;#9520217, and the Young Investigator Grant of the National Kidney Foundation (O. Palygin).

The following animal&#160;procedures &#160;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&#160;and prior to their use. Following these instructions will produce optimal results when using the&#160;sensors.
1. Sensor Calibration
<ol>
	<li>Prepare fresh stock solutions prior the sta...

<ol>
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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...

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 measurements1,2. 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 cultures4-10. 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&#160;combination with&#160;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&#160;driven by the presence of ATP. In the set of sensors used in the&#160;ex vivo studies, H2O2, the final enzymatic reaction product, is detected by&#160;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&#160;when paired with the corresponding Null sensor.
The ability&#160;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.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Sensor Kit</td>
			<td>Sarissa Biomedical</td>
			<td>SBK-ATP-05-125</td>
			<td>The kit includes storage bottle, rehydration chamber, electrode leads, and reference electrodes.&#160; Also included with the kit is the user&#39;s choice of sensors.</td>
		</tr>
		<tr>
			<td>Sarissaprobe ATP Biosensor 125 &#956;m</td>
			<td>Sarissa Biomedical</td>
			<td>SBS-ATP-05-125</td>
			<td>store at 2-8 C before use</td>
		</tr>
		<tr>
			<td>Sarissagold ATP Biosensor 50 &#956;m</td>
			<td>Sarissa Biomedical</td>
			<td>SGS-ATP-10-50</td>
			<td>store at 2-8 C before use</td>
		</tr>
		<tr>
			<td>Sarissaprobe null sensor 125&#956;m</td>
			<td>Sarissa Biomedical</td>
			<td>SBS-NUL-20-125</td>
			<td>store at 2-8 C before use</td>
		</tr>
		<tr>
			<td>Sarissagold null sensor 50 &#956;m</td>
			<td>Sarissa Biomedical</td>
			<td>SGS-NUL-10-50</td>
			<td>store at 2-8 C before use</td>
		</tr>
		<tr>
			<td>Sarissaprobe ATP Manual</td>
			<td>Sarissa Biomedical</td>
			<td></td>
			<td><a href="http://www.sarissa-biomedical.com/media/31563/instructions-atp.pdf">http://www.sarissa-biomedical.com/media/31563/instructions-atp.pdf</a></td>
		</tr>
		<tr>
			<td>Faraday cage&#160;</td>
			<td>TMC</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dual channel potentiostat</td>
			<td>Digi-Ivy</td>
			<td>DY2021</td>
			<td>Type II Faraday cage</td>
		</tr>
		<tr>
			<td>Data acquisition program</td>
			<td>Digi-Ivy</td>
			<td>DY2000</td>
			<td></td>
		</tr>
		<tr>
			<td>Perfusion pump</td>
			<td>Razel Scientific Instruments</td>
			<td>Model R99E</td>
			<td></td>
		</tr>
		<tr>
			<td>Fiber optic illuminator</td>
			<td>Schott</td>
			<td>ACE 1</td>
			<td></td>
		</tr>
		<tr>
			<td>micromanipulator</td>
			<td>Narishige</td>
			<td>MM-3</td>
			<td></td>
		</tr>
		<tr>
			<td>micromanipulator magnetic stand</td>
			<td>Narishige</td>
			<td>GJ-8</td>
			<td></td>
		</tr>
		<tr>
			<td>air table</td>
			<td>TMC</td>
			<td>63-500</td>
			<td></td>
		</tr>
		<tr>
			<td>isoflurane ventilator</td>
			<td>LEI Medical</td>
			<td>M2000</td>
			<td></td>
		</tr>
		<tr>
			<td>3 ml petri dish</td>
			<td>Fisher Scientific</td>
			<td>S3358OA</td>
			<td></td>
		</tr>
		<tr>
			<td>needle</td>
			<td>Santa Cruz</td>
			<td></td>
			<td>26-30 gauge</td>
		</tr>
		<tr>
			<td>pins</td>
			<td></td>
			<td></td>
			<td>Standard dissection pins</td>
		</tr>
		<tr>
			<td>catheter</td>
			<td></td>
			<td></td>
			<td>Polyethylene tubing (PE50)</td>
		</tr>
		<tr>
			<td>catheter tissue glue</td>
			<td>Vetbond</td>
			<td>1469SB</td>
			<td></td>
		</tr>
		<tr>
			<td>suture</td>
			<td>Look</td>
			<td>SP117</td>
			<td></td>
		</tr>
		<tr>
			<td>rubber bands</td>
			<td></td>
			<td></td>
			<td>any 2-4 mm wide rubber bands</td>
		</tr>
		<tr>
			<td>silicone</td>
			<td>Momentive</td>
			<td>RTV-615 Clear 1#</td>
			<td></td>
		</tr>
		<tr>
			<td>clamp</td>
			<td>Fine Science Tools</td>
			<td>18052-03</td>
			<td></td>
		</tr>
		<tr>
			<td>standard dissection kit</td>
			<td></td>
			<td></td>
			<td>Kit should include scalpel and dissection sissors&#160;</td>
		</tr>
		<tr>
			<td>Kidney Cup</td>
			<td></td>
			<td></td>
			<td>Of own design</td>
		</tr>
		<tr>
			<td>standard chemicals</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ATP</td>
			<td>Sigma-Aldrich</td>
			<td>A6559-25UMO</td>
			<td>100 mM ATP solution</td>
		</tr>
		<tr>
			<td>hydrogen peroxide</td>
			<td>Sigma-Aldrich</td>
			<td>216763</td>
			<td></td>
		</tr>
		<tr>
			<td>glycerol</td>
			<td>Sigma-Aldrich</td>
			<td>G9012</td>
			<td></td>
		</tr>
		<tr>
			<td>Apyrase</td>
			<td>Sigma-Aldrich</td>
			<td>A7646</td>
			<td></td>
		</tr>
		<tr>
			<td>Catalase</td>
			<td>Sigma-Aldrich</td>
			<td>C40</td>
			<td></td>
		</tr>
		<tr>
			<td>isoflurane</td>
			<td>Clipper</td>
			<td>10250</td>
			<td></td>
		</tr>
		<tr>
			<td>inactin</td>
			<td>Sigma-Aldrich</td>
			<td>T133</td>
			<td></td>
		</tr>
		<tr>
			<td>ketamine</td>
			<td>Clipper</td>
			<td>2010012</td>
			<td></td>
		</tr>
		<tr>
			<td>Hanks Balanced Salt Solution&#160;</td>
			<td>Gibco</td>
			<td>14025092</td>
			<td></td>
		</tr>
	</tbody>
</table>

use of enzymatic biosensors to quantify endogenous atp or h2o2 in the kidney

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 &#160;possible the measuring of&#160;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&#160;are coated with&#160;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&#160;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&#160;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&#160;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.

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...

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

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.

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

Enzymatic microelectrode biosensors have been widely used to measure extracellular signaling in real-time. Most of their use has been limited to brain ...

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