Name: ex vivo analysis of mechanically activated ca2+ transients in urothelial cells
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This work was supported by NIH grants R01DK119183 (to G.A. and M.D.C.) and S10OD028596 (to G.A.) and by the Cell Physiology and Model Organisms Kidney Imaging Cores of the Pittsburgh Center for Kidney Research (P30DK079307).

Care and handling of the animals were carried out in accordance with the University of Pittsburgh Institutional Animal Care and Use Committee. Female, 2-4-month-old C57Bl/6J mice were used for the present study. The mice were obtained commercially (see Table of Materials).
1. Equipment assembly and setup
<ol>
	<li>Perform Ca2+ imaging with an upright microscope equipped with a high-resolution camera and a stable light source (see Table of ...

<ol>
	<li>Kunau, R. T., Webb, H. L., Borman, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characteristics+of+the+relationship+between+the+flow+rate+of+tubular+fluid+and+potassium+transport+in+the+distal+tubule+of+the+rat.">Characteristics of the relationship between the flow rate of tubular fluid and potassium transport in the distal tubule of the rat.</a> Journal of Clinical Investigation. 54 (6), 1488-1495 (1974).</li><li>Engbretson, B. G., Stoner, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow-dependent+potassium+secretion+by+rabbit+cortical+collecting+tubule+in+vitro.">Flow-dependent potassium secretion by rabbit cortical collecting tubule in vitro.</a> American Journal of Physiology. 253 (5), 896-903 (1987).</li><li>Satlin, L. M., Sheng, S., Woda, C. B., Kleyman, T. 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Pfl&#252;gers Archiv.</a> European Journal of Physiology. 391 (2), 85-100 (1981).</li><li>Akerboom, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+a+GCaMP+calcium+indicator+for+neural+activity+imaging.">Optimization of a GCaMP calcium indicator for neural activity imaging.</a> The Journal of Neuroscience. 32 (40), 13819-13840 (2012).</li><li>Akerboom, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetically+encoded+calcium+indicators+for+multi-color+neural+activity+imaging+and+combination+with+optogenetics.">Genetically encoded calcium indicators for multi-color neural activity imaging and combination with optogenetics.</a> Frontiers in Molecular Neuroscience. 6, 2 (2013).</li><li>Sun, X. 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All organisms, and seemingly most cell types, express ion channels that respond to mechanical stimuli20,33,34,35,36,37. The function of these mechanically activated channels has been predominantly assessed with the patch-clamp technique. However, due to accessibility issues, patch-clamp studies of mechanically activated ion c...

Epithelial cells in the urinary tract are subjected to mechanical forces as the urinary filtrate travels through the nephrons, and urine is pumped out of the renal pelvis and travels through the ureters to be stored in the urinary bladder. It has been long recognized that mechanical forces (e.g., shear stress and stretch) exerted by fluids on the epithelial cells that line the urinary tract regulate the reabsorption of protein in the proximal tubule and of solutes in the distal nephron1,2,3,4,5,6,7,8,9,10,11,12,13, as well as the storage of urine in the urinary bladder and micturition14,15,16,17.
The conversion of mechanical stimuli into electrical and biochemical signals, a process referred to as mechanotransduction, is mediated by proteins that respond to the deformation of cellular structures or the associated extracellular matrix18,19,20,21. Mechanically activated ion channels are unique in the sense that they transition from a closed state to an open permeable state in response to changes in membrane tension, pressure, or shear stress18,19,20,21,22. In addition, Ca2+ transients can be initiated by&#160;integrin-mediated mechanotransduction or by activation of mechanoresponsive adhesion systems at cell-cell junctions23,24,25,26. Ion channel function is usually assessed with the patch-clamp technique, which involves the formation of a gigaohm seal between the cell membrane and the patch pipette27. However, cells located in deep tissue layers with a dense extracellular matrix (e.g., kidney tubules) or surrounded by a physical barrier (e.g., glycocalyx) are difficult to access with a glass micropipette. Likewise, cells embedded or that are integral parts of tissues with poor mechanical stability (e.g., the urothelium) can not be readily studied with the patch-clamp technique. Because many mechanically activated ion channels are permeable to Ca2+, an alternative approach is to assess their activity by fluorescent microscopy using a Ca2+-sensitive dye or genetically encoded calcium indicators (GECIs) such as GCaMP. Recent efforts in protein engineering have significantly increased the dynamic range, sensitivity, and response of GECIs28,29,30, and advances in genetics have allowed their expression in specific cell populations, making them ideally suited to study mechanotransduction.
The urothelium, the stratified epithelium that covers the interior of the urinary bladder, functions as a barrier, preventing the diffusion of urinary solutes into the bladder interstitium, but also functions as a transducer, sensing bladder fullness and communicating these events to the underlying nerves and musculature16. Previous studies have shown that the communication between the urothelium and underlying tissues requires the mechanically activated ion channels Piezo1 and Piezo231. To assess mechanically induced Ca2+ transients in urothelial cells, a new technique described that uses adenoviral gene transfer to express the Ca2+ sensor GCaMP5G in urothelial cells was developed. This technique employs a mucosal sheet preparation that provides easy access to the outermost umbrella cell layer and a computer-assisted system for the simultaneous mechanical stimulation of individual cells with a closed glass micropipette and recording of changes in fluorescence over time.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>20x Objective</td>
			<td>Olympus</td>
			<td>UMPlanFL N</td>
			<td></td>
		</tr>
		<tr>
			<td>24 G &#190;&#8221; catheter</td>
			<td>Medline&#160;</td>
			<td>Suresite IV slide&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>4x Objective</td>
			<td>Olympus</td>
			<td>UPlanFL N</td>
			<td></td>
		</tr>
		<tr>
			<td>Analog/digital converter</td>
			<td>Molecular Devices</td>
			<td>Digidata 1440A</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-GFP antibody</td>
			<td>Abcam&#160;</td>
			<td>Ab6556</td>
			<td></td>
		</tr>
		<tr>
			<td>Beam splitter</td>
			<td>Chroma</td>
			<td>T495lpxr</td>
			<td></td>
		</tr>
		<tr>
			<td>Bipolar temperature controller&#160;</td>
			<td>Warner Instruments</td>
			<td>TC-344B</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Fluka</td>
			<td>21114-1L</td>
			<td>1 M solution</td>
		</tr>
		<tr>
			<td>cellSens software</td>
			<td>Olympus</td>
			<td></td>
			<td>Imaging software</td>
		</tr>
		<tr>
			<td>CMOS camera</td>
			<td>Hamamatsu</td>
			<td>ORCA fusion</td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey anti-rabbit conjugated to Alexa Fluor 488&#160;</td>
			<td>Jackson ImmunoResearch</td>
			<td>711-545-152</td>
			<td></td>
		</tr>
		<tr>
			<td>Excel</td>
			<td>Microsoft Corporation</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Filter&#160;</td>
			<td>Chroma&#160;</td>
			<td>ET470/40X</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass capillaries Corning 8250 glass</td>
			<td>Warner Instruments&#160;</td>
			<td>G85150T-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma</td>
			<td>G8270</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES&#160;</td>
			<td>Sigma</td>
			<td>H4034</td>
			<td></td>
		</tr>
		<tr>
			<td>Inline heater&#160;</td>
			<td>Warner Instruments</td>
			<td>SH-27B</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma</td>
			<td>793590</td>
			<td></td>
		</tr>
		<tr>
			<td>Light source</td>
			<td>Sutter Instruments</td>
			<td>Lambda XL&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Manifold pump tubing</td>
			<td>Fisherbrand</td>
			<td>14-190-510</td>
			<td>ID 1.52 mm</td>
		</tr>
		<tr>
			<td>Manifold pump tubing</td>
			<td>Fisherbrand</td>
			<td>14-190-533</td>
			<td>ID 2.79 mm</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma</td>
			<td>M9272</td>
			<td></td>
		</tr>
		<tr>
			<td>Mice&#160;</td>
			<td>Jackson Lab</td>
			<td>664</td>
			<td>2-4 months old female C57BL/6J</td>
		</tr>
		<tr>
			<td>Microforge</td>
			<td>Narishige&#160;</td>
			<td>MF-830</td>
			<td></td>
		</tr>
		<tr>
			<td>Micromanipulator</td>
			<td>Sutter Instruments</td>
			<td>MP-285</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Olympus</td>
			<td>BX51W</td>
			<td></td>
		</tr>
		<tr>
			<td>Mounting media with DAPI</td>
			<td>Invitrogen</td>
			<td>S36964&#160;</td>
			<td>Slowfade Diamond Antifade with DAPI</td>
		</tr>
		<tr>
			<td>NaCl&#160;</td>
			<td>Sigma</td>
			<td>S7653</td>
			<td></td>
		</tr>
		<tr>
			<td>pClamp software</td>
			<td>Molecular Devices</td>
			<td>Version 10.4</td>
			<td>Patch-clamp electrophysiology data acquisition and analysis software</td>
		</tr>
		<tr>
			<td>Peristaltic pump</td>
			<td>Gilson</td>
			<td>Minipuls 3</td>
			<td></td>
		</tr>
		<tr>
			<td>Piezoelectric actuator</td>
			<td>Thorlabs</td>
			<td>PAS005</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette holder</td>
			<td>World Precision Instruments</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette puller</td>
			<td>Narishige</td>
			<td>PP-830</td>
			<td></td>
		</tr>
		<tr>
			<td>Quick exchange heated base with perfusion and adapter ring kit</td>
			<td>Warner Instruments</td>
			<td>QE-1</td>
			<td>Quick exchange platform fits 35 mm dish&#160;&#160;</td>
		</tr>
		<tr>
			<td>Rhodamine-phalloidin&#160;</td>
			<td>Invitrogen</td>
			<td>R415</td>
			<td></td>
		</tr>
		<tr>
			<td>Sigma-Plot</td>
			<td>Systat Software Inc</td>
			<td>Version 14.0</td>
			<td>Scientific graphing and data analysis software&#160;&#160;</td>
		</tr>
		<tr>
			<td>Silicone elastomer</td>
			<td>Dow</td>
			<td>Sylgard 184</td>
			<td></td>
		</tr>
		<tr>
			<td>Single channel open-loop piezo controller</td>
			<td>Thorlabs</td>
			<td>MDT694B</td>
			<td></td>
		</tr>
		<tr>
			<td>Square grid holder pad</td>
			<td>Ted Pella</td>
			<td>10520</td>
			<td></td>
		</tr>
		<tr>
			<td>Suture</td>
			<td>AD Surgical</td>
			<td>S-S618R13</td>
			<td>6-0 Sylk</td>
		</tr>
		<tr>
			<td>Teflon mounting rod</td>
			<td>Custom made</td>
			<td></td>
			<td>Use to mount the piezoelectric actuator in the micromanipulator</td>
		</tr>
		<tr>
			<td>Tubing</td>
			<td>Fisher Scientific</td>
			<td>14171129</td>
			<td>Tygon S3 ID 1/16 IN, OD 1/8 IN</td>
		</tr>
		<tr>
			<td>USB Digital I/O device&#160;</td>
			<td>National Instruments</td>
			<td>NI USB-6501</td>
			<td></td>
		</tr>
	</tbody>
</table>

ex vivo analysis of mechanically activated ca2+ transients in urothelial cells

Mechanically activated ion channels are biological transducers that convert mechanical stimuli such as stretch or shear forces into electrical and biochemical signals. In mammals, mechanically activated channels are essential for the detection of external and internal stimuli in processes as diverse as touch sensation, hearing, red blood cell volume regulation, basal blood pressure regulation, and the sensation of urinary bladder fullness. While the function of mechanically activated ion channels has been extensively studied in the in vitro setting using the patch-clamp technique, assessing their function in their native environment remains a difficult task, often because of limited access to the sites of expression of these channels (e.g., afferent terminals, Merkel cells, baroreceptors, and kidney tubules) or difficulties applying the patch-clamp technique (e.g., the apical surfaces of urothelial umbrella cells). This protocol describes a procedure to assess mechanically evoked Ca2+ transients using the fluorescent sensor GCaMP5G in an ex vivo urothelial preparation, a technique that could be readily adapted for the study of mechanically evoked Ca2+ events in other native tissue preparations.

The present protocol describes a technique to assess mechanically evoked Ca2+ transients in umbrella cells using the fluorescent Ca2+ sensor GCaMP5G. Adenoviral transduction was employed to express GCaMP5G in urothelial cells due to its high efficiency and because it produces an elevated level of expression. Fluorescent images of stained cryosections from a transduced bladder are shown in Figure 2D. For these experiments, GCaMP5G expression is highest in the umbrella ce...

Watch this Scientific Journal Video about Ex Vivo Analysis of Mechanically Activated Ca2+ Transients in Urothelial Cells at JoVE.com

Ex Vivo Analysis of Mechanically Activated Ca2+ Transients in Urothelial Cells

This protocol describes a methodology to assess the function of mechanically activated ion channels in native urothelial cells using the fluorescent Ca2+ sensor GCaMP5G.

Ex Vivo Analysis of Mechanically Activated Ca2+ Transients in Urothelial Cells

Mechanically activated ion channels are biological transducers that convert mechanical stimuli such as stretch or shear forces into electrical and ...

This protocol describes a technique to study mechanically-evoked calcium event in an ex vivo urothelial preparation. The technique is relatively easy to execute and it could be adapted to study mechanically-evoked calcium event in other native tissue preparations. Fabricate the micropipettes by placing the capillary glass in the puller and adjusting it with the capillary-retaining knobs.Pull the glass capillary in two steps as per the manufacturer's instructions. Close the micropipettes'tip using a microforge with the heater-adjustment knob set at 60. After exposing the bladder and the pelvic bone of the euthanized mouse, crack the pelvic bone to expose the urethra and cannulate the urethra with a 24-gauge catheter.Use a 6-0 suture to secure the catheter to the urethra. Harvest the bladder and urethra and affix them to a silicone rubber holder pad bathed in the recording solution. Separate the bladder mucosa from the underlying muscular layer with fine forceps.Cut open the bladder mucosa and pin it down with the urothelium facing up to a silicone elastomer insert in the bottom of a 35 millimeter diameter tissue culture dish. Mount the tissue culture dish with the pinned bladder mucosa in the microscope stage equipped with a culture dish incubator with resistive heating elements. Perfuse the cell culture dish continuously at a rate of 1.7 milliliter per minute with a recording solution warmed at 37 degrees Celsius with an inline heater.Maintain the temperature of the tissue dish incubator and solutions at approximately 37 degrees Celsius with a dual channel bipolar temperature controller. To record mechanically-induced calcium ion transients, submerge the micropipette in the recording solution. Under bright-field illumination, using a low magnification scanning objective, move the micropipette to the center of the field.Move the micromanipulator in the vertical plane and adjust the focus to move the pipette near the surface of the urothelial tissue. Switch to the objective with a higher magnification and numerical aperture suitable for immunofluorescence. Set the micromanipulator to fine and move the pipette near the top of the target cell.If necessary, adjust the focus and the position of the pipette. Open the stimulation protocol in the electrophysiology software and set it to play. Adjust the focus and press start in the experiment manager to initiate data acquisition.This drives the piezoelectric actuator and generates a file with the images of the experiment. When the cell is stimulated, an increase in fluorescence is observed. To quantify the fluorescent intensity, open the image file in the imaging software and select the count and measure window.Play the movie to identify the stimulated cell. Select the polygon tool and draw an ROI on the boundaries of the cell that was poked. Go to the measure window, select intensity profile, set the measurement to overtime, results to average, background subtraction to none, and then press execute.The mean fluorescence intensity will be computed over time. To export the intensity profile data, click on the Excel icon in the intensity profile results window and perform data analysis using scientific graphing and data analysis software. In this study, the umbrella cell was poked to assess mechanically-evoked calcium transients.The image captured at 10 seconds shows a fluorescent view of the stimulating pipette positioned on top of an umbrella cell expressing a fluorescent calcium sensor GCaMP5G. Mechanical stimulation of the umbrella cell expressing calcium sensor causes a deformation followed by a rapid increase in fluorescence emission. The graph demonstrates a change in the fluorescence intensity plotted as a function of time for several cells.Poking evokes a calcium response in most umbrella cells transduced with a fluorescent calcium sensor. The most important step is harvesting the bladder and urethra, followed by affixing them to a silicone rubber holder pad. Avoid tissue damage when separating the bladder mucosa from the underlying muscular layer.Imaging methods such as the one described here can provide unique insight into how mechano-activated channels sends changes in their native environment and generate physiological responses.

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