Name: implementing patch clamp and live fluorescence microscopy to monitor functional properties of freshly isolated pkd epithelium
Uploaded: 2015-09-01T12:00:00.000+00:00
Duration: 8 min 46 s
Description: Watch this Scientific Journal Video about Implementing Patch Clamp and Live Fluorescence Microscopy to Monitor Functional Properties of Freshly Isolated PKD Epithelium at JoVE.com

The authors would like to thank Glen Slocum (Medical College of Wisconsin) and Colleen A. Lavin (Nikon Instruments Inc) for excellent technical assistance with microscopy experiments. This study was supported by the National Institutes of Health grants R01 HL108880 (to AS), R01 DK095029 (to OPo) and K99 HL116603 (to TSP), National Kidney Foundation IG1724 (to TSP), American Heart Association 13GRNT16220002 (to OPo) and the Ben J. Lipps Research Fellowship from the American Society of Nephrology (to DVI).

The experimental procedures described below were approved by the Institutional Animal Care and Use Committee at the Medical College of Wisconsin and University of Texas Health Science Center at Houston and were in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Figure 1 demonstrates main steps of the tissue isolation and processing procedure. Briefly, kidneys from PCK or SD rats are used for manual isolation of epithelial monolayers of collecting ducts either from healthy non-dialed tubules or cysts. Here we studied kidneys from 4-16 weeks old PCK rats10,13.
1. Isolation of Renal Cysts and Connecting Tubules (CNT)/Collecting Ducts (CD) Segments
<ol>
	<li>Anesthetize experimental animal with isoflurane (5% induction, 1.5 to 2.5% maintenance)/medical grade O2 or another approved method. Animals must be continually monitored to ensure adequate level of anesthesia. Stable respiratory rate and toe pinch reaction are used to confirm proper anesthesia.</li>
	<li>Perform laparotomy and flush the kidneys with phosphate buffered saline (PBS) through the abdominal aorta17.
	<ol>
		<li>Prepare a polyethylene tubing catheter (PE50) and connect it to a syringe pump filled with PBS. Cut the skin and abdominal wall along the linea alba, then make a transverse abdominal incision across the abdomen from side to side and shift the abdominal organs&#160;with cotton swabs to get access to the descending aorta with branching mesenteric and celiac arteries. Moisten the internal organs with warm saline during further procedures to prevent their dryness.</li>
		<li>Place one ligature (#1) around the mesenteric and celiac arteries and another ligature (#2) around the aorta under the diaphragm (do not tie). Gently separate a. abdominalis by blunt dissecting connective tissues around it with thin forceps and place two ligatures ~3 mm below the left renal artery (#3) and above the iliac arteries bifurcation (#4).</li>
		<li>Tie ligature #4 and attach a vessel clamp around the aorta between the left renal artery and ligature #3. Make an incision between the clamp and ligature #4 to catheterize the aorta, use ligature #3 to firmly fix the catheter.</li>
		<li>Release the clamp and make sure that the blood pulse is visible in the catheter to ensure proper installation. Start perfusion at a rate of 6 ml/min, tie ligatures #1 and #2, and cut the left renal vein. Continue flushing for 1-2 min until the organs are completely blanched.</li>
		<li>Cut renal blood vessels, urethra and surrounding connective and adipose tissues with scissors to collect the kidneys. Then make a short tear in kidney capsule and peel the kidneys to decapsulate them. Place the kidneys into ice-cold PBS. Euthanasia is confirmed by a thoracotomy.</li>
	</ol>
	</li>
	<li>Prepare 5 x 5 mm cover glass chips coated with Poly-L-Lysine. Cut a cover glass with a diamond pencil and place approximately 20 &#181;l of 0.01% sterile filtered Poly-L-Lysine solution onto each glass chip. Prepare saline and two pairs of watchmaker forceps for dissection.</li>
	<li>Cut the kidneys with a razor blade along the frontal plane into slices of ~1-2 mm thickness. Place one of the slices under a stereomicroscope. Isolation of tissues should be done in ice-cold saline.</li>
	<li>Locate cysts under a stereomicroscope as round-shape cavities (Figure 2A); their walls often contain prominent network of proliferated blood vessels. Using fine-tipped forceps dissect the internal epithelial layer of a cyst as thin as possible to obtain a monolayer area. Attach it to a glass chip covered with Poly-L-Lysine. Sticky Poly-L-Lysine surface allows the researcher to firmly position the internal cyst layer on glass. Expose the cyst with the apical side up to provide access for the pipettes (Figure 2B).</li>
	<li>Use PCK or SD rat kidney central slices containing papilla for isolation of non-dilated CNT/CCDsegments18-21. 
	NOTE: Papillary tissues are more durable than cortex and by holding papillary tubular bands with forceps and tearing along radial axis the slice can be cleaved into thin sectors. Individual tubules are visible and can be pulled out to be placed on cover glass chips.</li>
	<li>Identify the CNT/CCD segments by bifurcations, higher transparency than proximal tubules and large prominently visible cells (Figure 2C). Place the cover glass chip with attached tubules under a microscope equipped with micromanipulators suitable for driving micropipettes. Using sharp micropipettes split-open tubules and attach their edges to the glass to make the apical surface accessible (Figure 2D).</li>
</ol>
2. Single Channel Patch-clamp Electrophysiology
<ol>
	<li>Fill the patch-clamp chamber with bath solution and transfer the cover glass chip with isolated tissues to the chamber. Ensure that the patch-clamp micropipettes have resistance of 7-10 M&#937; for reliable on-cell (cell-attached) measurements.</li>
	<li>For the cell-attached measurements, set the amplifier gain ratio to 20x and low-pass the currents at 300 Hz by an eight-pole Bessel filter. For ENaC channels monitoring, use a bath solution, in mM: 150 NaCl, 1 CaCl2, 2 MgCl2, 10 HEPES (pH 7.4); pipette: 140 LiCl, 2 MgCl2 and 10 HEPES (pH 7.4).</li>
	<li>Conduct a conventional patch-clamp experiment in a cell-attached mode10. Select a cell in the epithelial monolayer and approach the pipette to the apical membrane. Form a high-resistance seal between a pipette and cell membrane by applying gentle suction. Once a high resistant gigaOhm seal is formed, a gap-free protocol at a holding potential should be started for monitoring activity of the channel of interest.</li>
	<li>Store gap-free single channel current data from gigaOhm seals for subsequent analysis. Analyze the channel events using a software package generally supplied with patch-clamp setups18. Calculate channel activity as NPo where N refers to the number of active channels in the patch and Po is average open probability of the channels. 
	NOTE: Use isolated cysts or tubules in patch-clamp experiments for no more than 30 min.</li>
</ol>
3. Ratiometric Epifluorescence Measurements of Intracellular Calcium Concentration in the Epithelial Cells
<ol>
	<li>Use 5 mM Fura-2AM dissolved in DMSO. Aliquot stock solution into individual 500 &#181;l conical tubes (approximately 10 &#181;l). Protect from light and store in the freezer at -20 &#730;C for up to six months.</li>
	<li>Incubate isolated cysts and split-open tubules for 30-40 min in PSS (in mM: 145 NaCl, 4.5 KCl, 2 MgCl2, 2 CaCl2 and 10 HEPES at pH 7.35) containing 5 &#181;M Fura-2 AM dye and 0.05% pluronic acid to help disperse the acetoxymethyl esters. For that, place tissues in the 3.5 cm dish containing loading cocktail, protect from light and incubate on a slow shaker at room temperature.</li>
	<li>Change Fura-2 AM containing media to clean PSS after incubation and place the tissue under a microscope to further perform epifluorescence imaging.</li>
	<li>Turn on CCD camera and stable light source (monochromator system) equipped with filter wheel (each filter position can be associated with its own attenuation level, selected every time the filter is called).</li>
	<li>Find the tissue in bright field. Switch to detection of the fluorescence signal and adjust the intensity of the light source using neutral density filters and lamp power to avoid saturation of the signal.</li>
	<li>Monitor Fura-2 AM fluorescence in the tissue sample with ratiometric excitation at 340/380 nm with frequency of 0.125 Hz or higher. Use a 40&#215;/NA 1.3 or similar objective lens for proper resolution image.</li>
	<li>For image processing and calculations import the image sequence. Make sure to split the channels and use a hyperstack grayscale mode. Select several regions of interest (single cyst cells or selected areas of cystic tissue) and calculate intensity values for each channel (340 and 380 nm) into preferred data analysis software; subtract background intensity values from each data point.</li>
	<li>For each time point calculate the ratio of intensities of the Fura-2 AM 340 to 380 channels. Plot scatter/line point-time changes of Ca2+ transient for each region and calculate mean/SE values.</li>
</ol>

The authors have nothing to disclose.

We described here applications of conventional patch-clamp technique and epifluorescence calcium imaging to cystic epithelial monolayers derived from a murine genetic model of ARPKD. The protocol consist of three steps, of which the most attention should be paid to the isolation of the cysts (step 1.5 of the protocols section) and to the electrophysiological studies. These key procedures require extensive training and patience, and the reader should not be frustrated at once.
First of all, the most attention should be paid to the process of the cyst monolayer isolation. This part of preparation requires manual skills and significantly impacts further work, as thickness of the specimen and its even attachment to a glass chip is critical for the visibility of the cells. Thickness of isolated cyst walls varies in different parts of the specimen, and it is recommended to focus on larger cysts with bigger monolayer areas convenient for work. To help clean the specimen and access the monolayer, surrounding tissues should be peeled with forceps under a stereomicroscope. It should be emphasized that the technique implies utilization of a high quality stereomicroscope characterized by a significant field depth and a capability to change magnification in a wide range to accommodate varying sizes of objects during dissection. Another limitation of the method is that such sophisticated techniques as patch-clamp and calcium imaging require personnel familiar with these methods. We suggest that initial experience can be obtained on collecting duct cell cultures such as commercially available cortical M1 and medullar IMCD cells as they form epithelial monolayer similar to cysts.
Our approach allows measuring ion channels activity and [Ca2+]i levels in the native environment of freshly isolated tissues. The main advantage of this procedure is that preparation of specimens for single-channel analysis or dye loading does not require enzymatic treatment, damaging mechanical procedures or other potentially detrimental steps. It is performed manually with forceps in saline and provides large undamaged specimens. Such specimens can be used not only for the described techniques. In fact, isolated cystic epithelium represents thin film of pure tubular cells and maintains native monolayer properties, which makes it a valuable intact object for real-time experiments which produces physiologically relevant observations. If the isolation of cysts seems to hard a procedure for the researcher or a large amount of cystic tissue is required at once, enzymatic treatment (for instance, with dispase and collagenase, as described here38 for the cortical collecting duct tubules) can be used to simplify the isolation process. However, the researcher should be very careful while performing this treatment, as enzymes, especially proteases, can significantly affect ion channels&#8217; activity or damage the membrane receptors. This is, for instance, very important for the studies of purinergic signaling, as these membrane proteins are very well known to quickly degrade under enzymatic treatment39,40.
Additionally, cystic epithelium can be used for other purposes, such as immunohistochemistry or staining of a fixed monolayer with dyes (e.g. rhodamine phalloidin), or loading the fresh tissue with intracellular substance markers, such as DAF-FM for NO detection or various dyes to monitor reactive oxygen species production. It is suggested to use the pure fraction of isolated cystic epithelium for western blotting to eliminate impact of non-specific factors present in total kidney lysates. We also suggest that cystic epithelium can be similarly isolated from mice or other species that are available for the studies of various models of not only ARPKD, but also ADPKD. Freshly isolated cyst specimens have undeniable advantages for the molecular biology approaches compared to the cultured cystic cells, as they better sustain the properties of the cystic tissue within the organ, and undoubtedly provide more justified data regarding the in vivo disease processes.
One of the significant advantages of this technique is a possibility to use normal non-dilated collecting ducts from the same kidneys, or SD rats tubules (these rats share genetic background with PCK rats), as control tissues. Similar patch-clamp and calcium measurements in nephron segments are widely used on non-cystic kidneys in many laboratories41-43. Depending ion channels type, different modifications of patch-clamp method can be used (whole cell, inside-out, outside-out); for instance, ENaC and ROMK (renal outer medullary potassium channel) activity can be assessed by whole-cell configuration44,45. The proposed Fura-2 AM calcium imaging in wide-field epifluorescence with monochromator can be also performed with any other similar modification of calcium imaging such as analysis with confocal or two-photon microscopy with other calcium dyes. These microscope systems are less affordable but provide higher quality imaging and are more sensitive than wide-field microscopy. Similar approach was also applied to study TRPC channels activity and calcium signaling in podocytes of freshly isolated glomeruli17,46,47. Other calcium imaging that could be applied include utilization of Fluo-8 as demonstrated on Figure 4B or ratiometric confocal measurement with Fluo4/FuraRed fluorescent dyes as described in Ilatovskaya et al.46 Technically, it is possible to perform both patch-clamp and calcium imaging simultaneously on the same cell if microscope is equipped with both setups. Thus, the described technique is a feasible approach for high-quality observations in the field of PKD, which can be flexibly modified according to the needs of your specific research and availability of the equipment.&#160; Furthermore, various modifications of calcium imaging can be used here, including ratiometric confocal measurements with Fluo4/FuraRed fluorescent dyes &#160;(as described in Ilatovskaya et al.46) or Fluo-8 as demonstrated on Figure 4B.

Ion channels play a significant role in many physiological functions, including cell growth and differentiation. Autosomal dominant and recessive polycystic kidney diseases (ADPKD and ARPKD, respectively) are genetic disorders characterized by the development of renal fluid-filled cysts of the tubular epithelial cell origin. ADPKD is caused by mutations of PKD1 or PKD2 genes encoding polycystins 1 and 2, membrane proteins involved in the regulation of cell proliferation and differentiation. PKD2 by itself or as a complex with PKD1 also function as a Ca2+-permeable cation channel1. Mutations of the PKHD1 gene encoding fibrocystin (a cilia-associated receptor-like protein involved in the tubulogenesis and/or maintenance of polarity of epithelium) are the genetic impetus of ARPKD2. Cyst growth is a complex phenomenon accompanied with disturbed proliferation3,4, angiogenesis5, dedifferentiation and loss of polarity of tubular cells6-8.
Defective reabsorption and augmented secretion in cystic epithelium contribute to fluid accumulation in the lumen and cyst expansion9,10. Impaired flow-dependent [Ca2+]i&#160;signaling has been also linked to cystogenesis during PKD11-15.
Here, we describe a method suitable for patch-clamp measurements of single channel activity and intracellular Ca2+ levels in cystic epithelial monolayers isolated from PCK rats. This method was successfully applied by us to characterize of activity of the epithelial Na+ channel (ENaC)10 and [Ca2+]i-dependent processes induced by Ca2+-permeable TRPV4 and purinergic signaling cascade13.
In these studies we used PCK rats, a model of ARPKD caused by a spontaneous mutation in the PKHD1 gene. The PCK strain was originally derived from Sprague-Dawley (SD) rats16 thereby SD rats are used as an appropriate control for comparison with the PCK strain. As a result, both SD rat nephron segments and non-dilated collecting ducts isolated from same PCK rats can serve as two different comparison groups for experiments on cystic epithelium.

<table><tbody><tr><td>Fura-2 AM</td><td>Life Technologies</td><td>F-14185</td><td></td></tr><tr><td>Fluo-8</td><td>AAT Bioquest</td><td>21091</td><td></td></tr><tr><td>Poly-L-lysine</td><td>Sigma-Aldrich</td><td>P4707</td><td></td></tr><tr><td>Pluronic acid</td><td>Sigma-Aldrich</td><td>F-68&amp;nbsp;</td><td>solution</td></tr><tr><td>Shaker</td><td>Boekel Scientific</td><td>260350</td><td></td></tr><tr><td>Light source</td><td>Sutter Instrument Co</td><td>Lambda XL</td><td>with integrated shutter/filter wheel driver</td></tr><tr><td>Neutral density filters</td><td>Nikon</td><td></td><td>ND4, ND8</td></tr><tr><td>Objective</td><td>Nikon</td><td>SFluo&amp;nbsp;</td><td>40/1.3 DIC WD 0.22&amp;nbsp;&amp;nbsp; oil</td></tr><tr><td>Camera</td><td>Andor Technologies</td><td>Zyla sCMOS</td><td></td></tr><tr><td>Nikon&amp;nbsp; microscope (inverted)</td><td>Nikon</td><td>Nikon Eclipse TE2000-S</td><td></td></tr><tr><td>Cover Glass</td><td>Thermo Scientific</td><td>6661B52</td><td></td></tr><tr><td>Diamond pencil</td><td>Fisher Scientific</td><td>22268912</td><td></td></tr><tr><td>Image acquisition software</td><td>Nikon</td><td>Nikon NIS-Elements&amp;nbsp;</td><td></td></tr><tr><td>Image analysis software</td><td>ImageJ</td><td>http://imagej.nih.gov/</td><td>ND Utility plugin allows to import images in the native Nikon Instruments .nd2 format</td></tr><tr><td>Recording/perfusion chamber</td><td>Warner Instruments</td><td>RC-26</td><td></td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>Patch Clamp amplifier</td><td>Molecular Devices</td><td>MultiClamp 700B</td><td></td></tr><tr><td>Data Acquisition System</td><td>Molecular Devices</td><td>Digidata 1440A</td><td>Axon Digidata&amp;reg; System</td></tr><tr><td>Low Pass Filter</td><td>Warner Instruments</td><td>LPF-8</td><td>8 pole Bessel</td></tr><tr><td>Borosilicate glass capillaries</td><td>World Precision Instruments</td><td>1B150F-4</td><td></td></tr><tr><td>Micropipette Puller</td><td>Sutter Instrument Co</td><td>P-97</td><td>Flaming/Brown type micropipette puller</td></tr><tr><td>Microforge</td><td>Narishige</td><td>MF-830</td><td>Japan</td></tr><tr><td>Motorized Micromanipulator</td><td>Sutter Instrument Co</td><td>MP-225</td><td></td></tr><tr><td>Inverted microscope</td><td>Nikon</td><td>Eclipse Ti</td><td></td></tr><tr><td>Microvibration isolation table</td><td>TMC</td><td></td><td>equipped with Faraday cage</td></tr><tr><td>Multichannel valve perfusion system</td><td>AutoMake Scientific</td><td>Valve Bank II</td><td></td></tr><tr><td>Recording/perfusion chamber</td><td>Warner Instruments</td><td>RC-26</td><td></td></tr><tr><td>Software</td><td>Molecular Devices</td><td>pClamp 10 . 2</td><td></td></tr><tr><td>Temperature controlled surgical table&amp;nbsp;</td><td>MCW core</td><td></td><td>for rodents</td></tr><tr><td>Binocular stereomicroscope</td><td>Nikon</td><td>SMZ745</td><td></td></tr><tr><td>Syringe pump-based perfusion system</td><td>Harvard Apparatus</td><td></td><td></td></tr><tr><td>polyethylene tubing</td><td>Sigma-Aldrich</td><td>PE50</td><td></td></tr><tr><td>Isofluorane anesthesia</td><td>&lt;a href=&quot;http://www.vetequip.com/&quot;&gt;http://www.vetequip.com/&lt;/a&gt;</td><td>911103</td><td></td></tr><tr><td>Other basic reagents</td><td>Sigma-Aldrich</td><td></td><td></td></tr></tbody></table>

implementing patch clamp and live fluorescence microscopy to monitor functional properties of freshly isolated pkd epithelium

Cyst initiation and expansion during polycystic kidney disease is a complex process characterized by abnormalities in tubular cell proliferation, luminal fluid accumulation and extracellular matrix formation. Activity of ion channels and intracellular calcium signaling are key physiologic parameters which determine functions of tubular epithelium. We developed a method suitable for real-time observation of ion channels activity with patch-clamp technique and registration of intracellular Ca2+ level in epithelial monolayers freshly isolated from renal cysts. PCK rats, a genetic model of autosomal recessive polycystic kidney disease (ARPKD), were used here for ex vivo analysis of ion channels and calcium flux. Described here is a detailed step-by-step procedure designed to isolate cystic monolayers and non-dilated tubules from PCK or normal Sprague Dawley (SD) rats, and monitor single channel activity and intracellular Ca2+ dynamics. This method does not require enzymatic processing and allows analysis in a native setting of freshly isolated epithelial monolayer. Moreover, this technique is very sensitive to intracellular calcium changes and generates high resolution images for precise measurements. Finally, isolated cystic epithelium can be further used for staining with antibodies or dyes, preparation of primary cultures and purification for various biochemical assays.

Potential ENaC involvement in cystogenesis has been demonstrated by several studies that observed disrupted epidermal growth factor (EGF) signaling in PKD progression22-25 and abnormal sodium reabsorption in ARPKD murine models and tissue cultures26-28. For example, Veizis et al. showed that amiloride-sensitive Na+&#160;absorption is decreased in CD cells from the non-orthologous BPK mouse model of ARPKD29. We recently demonstrated that impaired sodium and water reabsorption in cysts is an important factor in aggravating cystogenesis10. Specifically, we employed electrophysiological and immunohistochemical analysis and found that cysts exert blunted sodium reabsorption. A pharmacological approach demonstrated that selective ENaC blockade markedly exacerbates cyst progression10.
We further demonstrated the effect of administration of benzamil, an ENaC blocker, on activity of the channels in the cyst wall. 4-weeks old PCK rats were supplied with vehicle or drinking water containing benzamil (15 mg/ml) ad libitum. After 12 weeks of treatment the animals were processed according to the protocol described above. Patch-clamp was performed on the cystic monolayers isolated from vehicle and benzamil treated groups. Figure 3 shows representative current traces of ENaC activity recorded from apical membranes. The summary graph demonstrates that average ENaC activity (NPo) was 0.91&#177;0.1510 in the cysts of control group, whereas benzamil administration decreased the activity of the channel to 0.32&#177;0.05. We conclude that in ARPKD (characterized by ecstatic distension of CDs to form numerous spindle-shaped renal cysts30) benzamil given in drinking water reaches renal cysts with urine flow30. This effect allows benzamil to decrease sodium reuptake from cyst fluid, contributing to cystogenesis10.
In addition to the EGF pathway, adenosine triphosphate (ATP) and other purines were also identified as a critical paracrine signaling component that is inappropriately modulated in PKD models and in patients with this disease. It was reported that purinergic signaling plays an important role in cystogenesis during both ADPKD and ARPKD31,32. Cyst cells of PCK rats have previously been shown to exhibit low basal [Ca2+]i and loss of flow-mediated [Ca2+]i signaling compared to healthy non-dilated collecting ducts of SD rats13. P2 receptor agonists modulate the development of renal cysts in an in vitro model of cyst formation derived from the cpk/cpk mouse33. High ATP concentration was found in cystic liquid from ADPKD patients34 and in media conditioned by cystic kidney epithelia cultured from cpk/cpk mice35.
We tested calcium flux in response to exogenous ATP administration. PCK cysts and normal cortical ducts from SD rats were isolated for the calcium measurements before and after application of 10 &#181;M ATP. Data illustrated in&#160;Figure 4&#160;reveal the effect of 10 &#181;M ATP in the cystic tubules of PCK rats studied with two different approaches: Figure 4A shows measurements of Fura-2AM fluorescence intensity at 340 and 380 nM in an epifluorescence setup equipped with a monochromator, and Figure 4B represents registration of Fluo-8 dye staining with confocal microscopy. Both techniques demonstrate similar kinetics of calcium transient response to ATP application in cystic epithelium10.
<img alt="Figure 1" src="/files/ftp_upload/53035/53035fig1.jpg" /> 
Figure 1: Schematic representation of the experimental protocol. After the animal is properly anaesthetized and prepared for the surgery, the kidneys are flushed with PBS to remove blood. Then, the kidneys are excised, decapsulated, and cystic monolayers or non-dilated tubules are isolated manually with forceps under a stereomicroscope. Non-dilated tubules are split-open with micropipettes driven by micromanipulators whereas cystic epithelium as internal surface of the cysts would be open to have access to the apical side.
<img alt="Figure 2" src="/files/ftp_upload/53035/53035fig2.jpg" /> 
Figure 2: Isolation and preparation of cystic and collecting duct monolayers. (A) A representative kidney slice from 16 week old PCK rat. (B) A cystic monolayer on a cover glass chip transferred to a microscope for patch-clamp analysis or calcium imaging (60X). (C) A CNT/CCD segment with bifurcation isolated from an SD rat. (D) A CNT/CCD tubule split-open with micropipettes to gain access to the apical surface.
<img alt="Figure 3" src="/files/ftp_upload/53035/53035fig3.jpg" /> 
Figure 3: Effect of benzamil treatment on ENaC activity in cystic epithelium. (A) Representative current traces for ENaC activity measured in cell attached patches of cysts freshly isolated from 16 week old PCK rats administered 12 weeks of benzamil in drinking water. These patches were held at a test potential of Vh = &#8722;Vp = -40 mV. Inward Li+ currents are downward. Dashed lines indicate the respective current state with a &#8220;c&#8221; and &#8220;on&#8221; denoting the closed and open states. (B) Summary graph of observed ENaC activity (NPo) (partially reproduced from10 with permission). *P &#60;0.05 versus vehicle.
<img alt="Figure 4" src="/files/ftp_upload/53035/53035fig4.jpg" /> 
Figure 4: Effects of ATP on calcium influx in the cystic cells of the PCK rats. (A) Representative images of cystic monolayer loaded with Fura-2 AM before and after application of 10 &#181;M ATP. Graph summarizing the effect of ATP on calcium levels in the cell monolayer of PCK rats and SD collecting ducts (N = 38 cells). (B) Cystic monolayer loaded with Fluo-8 dye before and after application of 10 &#181;M ATP and summary graph of intracellular calcium response.

Watch this Scientific Journal Video about Implementing Patch Clamp and Live Fluorescence Microscopy to Monitor Functional Properties of Freshly Isolated PKD Epithelium at JoVE.com

Implementing Patch Clamp and Live Fluorescence Microscopy to Monitor Functional Properties of Freshly Isolated PKD Epithelium

Ion channels expressed in renal tubular epithelium play a significant role in the pathology of polycystic kidney disease. Here we describe experimental protocols used to perform patch-clamp analysis and intracellular calcium level measurements in cystic epithelium freshly isolated from rodent kidneys.

Cyst initiation and expansion during polycystic kidney disease is a complex process characterized by abnormalities in tubular cell proliferation, luminal ...

implementing-patch-clamp-live-fluorescence-microscopy-to-monitor

Nanyang Technological University

Research

JoVE Journal

Biology

9.7K Views.  Medical College of Wisconsin.  Ionenkanalen tot expressie in renale tubulaire epitheel spelen een belangrijke rol in de pathologie van polycystische nierziekte. Hier beschrijven we de experimentele protocollen die worden gebruikt om patch-clamp-analyse en intracellulaire calciumgehalte metingen uit te voeren in cystic epitheel vers geïsoleerd van knaagdieren nieren. 

Video: Implementing Patch Clamp and Live Fluorescence Microscopy to Monitor Functional Properties of Freshly Isolated PKD Epithelium

Live-imaging of PKC Translocation in Sf9 Cells and in Aplysia Sensory Neurons

Protein kinase Cs (PKCs) are serine threonine kinases that play a central role in regulating a wide variety of cellular processes such as cell growth and learning and memory. There are four known families of PKC isoforms in vertebrates: classical PKCs (&alpha;, &beta;I, &beta;II and &gamma;), novel type I PKCs (&#949; and &eta;), novel type II PKCs (&delta; and &theta;), and atypical PKCs (&zeta; and &iota;). The classical PKCs are activated by Ca2+ and diacylclycerol (DAG), while the novel PKCs are activated by DAG, but are Ca2+-independent. The atypical PKCs are activated by neither Ca2+ nor DAG. In Aplysia californica, our model system to study memory formation, there are three nervous system specific PKC isoforms one from each major class, namely the conventional PKC Apl I, the novel type I PKC Apl II and the atypical PKC Apl III. PKCs are lipid-activated kinases and thus activation of classical and novel PKCs in response to extracellular signals has been frequently correlated with PKC translocation from the cytoplasm to the plasma membrane. Therefore, visualizing PKC translocation in real time in live cells has become an invaluable tool for elucidating the signal transduction pathways that lead to PKC activation. For instance, this technique has allowed for us to establish that different isoforms of PKC translocate under different conditions to mediate distinct types of synaptic plasticity and that serotonin (5HT) activation of PKC Apl II requires production of both DAG and phosphatidic acid (PA) for translocation 1-2. Importantly, the ability to visualize the same neuron repeatedly has allowed us, for example, to measure desensitization of the PKC response in exquisite detail 3. In this video, we demonstrate each step of preparing Sf9 cell cultures, cultures of Aplysia sensory neurons have been described in another video article 4, expressing fluorescently tagged PKCs in Sf9 cells and in Aplysia sensory neurons and live-imaging of PKC translocation in response to different activators using laser-scanning microscopy.

In this video, we demonstrate visualization of PKC translocation in living cells using fluorescently tagged PKCs.

Protein kinase Cs (PKCs) are serine threonine kinases that play a central role in regulating a wide variety of cellular processes such as cell growth and ...

Neuroscience

Electrophysiological Characterization of GFP-Expressing Cell Populations in the Intact Retina

Studying the physiological properties and synaptic connections of specific neurons in the intact tissue is a challenge for those cells that lack conspicuous morphological features or show a low population density. This applies particularly to retinal amacrine cells, an exceptionally multiform class of interneurons that comprise roughly 30 subtypes in mammals1. Though being a crucial part of the visual processing by shaping the retinal output2, most of these subtypes have not been studied up to now in a functional context because encountering these cells with a recording electrode is a rare event.
Recently, a multitude of transgenic mouse lines is available that express fluorescent markers like green fluorescent protein (GFP) under the control of promoters for membrane receptors or enzymes that are specific to only a subset of neurons in a given tissue3,4. These pre-labeled cells are therefore accessible to directed microelectrode targeting under microscopic control, permitting the systematic study of their physiological properties in situ. However, excitation of fluorescent markers is accompanied by the risk of phototoxicity for the living tissue. In the retina, this approach is additionally hampered by the problem that excitation light causes appropriate stimulation of the photoreceptors, thus inflicting photopigment bleaching and transferring the retinal circuits into a light-adapted condition. These drawbacks are overcome by using infrared excitation delivered by a mode-locked laser in short pulses of the femtosecond range. Two-photon excitation provides energy sufficient for fluorophore excitation and at the same time restricts the excitation to a small tissue volume minimizing the hazards of photodamage5. Also, it leaves the retina responsive to visual stimuli since infrared light (&gt;850 nm) is only poorly absorbed by photopigments6.
In this article we demonstrate the use of a transgenic mouse retina to attain electrophysiological in situ recordings from GFP-expressing cells that are visually targeted by two-photon excitation. The retina is prepared and maintained in darkness and can be subjected to optical stimuli which are projected through the condenser of the microscope (Figure 1). Patch-clamp recording of light responses can be combined with dye filling to reveal the morphology and to check for gap junction-mediated dye coupling to neighboring cells, so that the target cell can by studied on different experimental levels.

This article depicts the recording of individual cells from fluorescently tagged neuronal populations in the intact mouse retina. By using two-photon infrared excitation transgenetically labeled cells were targeted for patch-clamp recording to study their light responses, receptive field properties, and morphology.

Studying the physiological properties and synaptic connections of specific neurons in the intact tissue is a challenge for those cells that lack ...

Whole-cell Patch-clamp Recordings of Isolated Primary Epithelial Cells from the Epididymis

The epididymis is an essential organ for sperm maturation and reproductive health. The epididymal epithelium consists of intricately connected cell types that are distinct not only in molecular and morphological features but also in physiological properties. These differences reflect their diverse functions, which together establish the necessary microenvironment for the post-testicular sperm development in the epididymal lumen. The understanding of the biophysical properties of the epididymal epithelial cells is critical for revealing their functions in sperm and reproductive health, under both physiological and pathophysiological conditions. While their functional properties have yet to be fully elucidated, the epididymal epithelial cells can be studied using the patch-clamp technique, a tool for measuring the cellular events and the membrane properties of single cells. Here, we describe the methods of cell isolation and whole-cell patch-clamp recording to measure the electrical properties of primary dissociated epithelial cells from the rat cauda epididymides.

We present a protocol that combines cell isolation and whole-cell patch-clamp recording to measure the electrical properties of the primary dissociated epithelial cells from the rat cauda epididymides. This protocol allows for investigation of the functional properties of primary epididymal epithelial cells to further elucidate the physiological role of the epididymis.

The epididymis is an essential organ for sperm maturation and reproductive health. The epididymal epithelium consists of intricately connected cell types ...

Developmental Biology

Optical Control of Living Cells Electrical Activity by Conjugated Polymers

Hybrid interfaces between organic semiconductors and living tissues represent a new tool for in-vitro and in-vivo applications. In particular, conjugated polymers display several optimal properties as substrates for biological systems, such as good biocompatibility, excellent mechanical properties, cheap and easy processing technology, and possibility of deposition on light, thin and flexible substrates. These materials have been employed for cellular interfaces like neural probes, transistors for excitation and recording of neural activity, biosensors and actuators for drug release. Recent experiments have also demonstrated the possibility to use conjugated polymers for all-optical modulation of the electrical activity of cells. Several in-vitro study cases have been reported, including primary neuronal networks, astrocytes and secondary line cells. Moreover, signal photo-transduction mediated by organic polymers has been shown to restore light sensitivity in degenerated retinas, suggesting that these devices may be used for artificial retinal prosthesis in the future. All in all, light sensitive conjugated polymers represent a new approach for optical modulation of cellular activity.
In this work, all the steps required to fabricate a bio-polymer interface for optical excitation of living cells are described. The function of the active interface is to transduce the light stimulus into a modulation of the cell membrane potential. As a study case, useful for in-vitro studies, a polythiophene thin film is used as the functional, light absorbing layer, and Human Embryonic Kidney (HEK-293) cells are employed as the biological component of the interface. Practical examples of successful control of the cell membrane potential upon stimulation with light pulses of different duration are provided. In particular, it is shown that both depolarizing and hyperpolarizing effects on the cell membrane can be achieved depending on the duration of the light stimulus. The reported protocol is of general validity and can be straightforwardly extended to other biological preparations.

A method for stimulation of in-vitro cell cultures electrical activity with visible light, based on the use of organic semiconducting polymers is described.

Hybrid interfaces between organic semiconductors and living tissues represent a new tool for in-vitro and in-vivo applications. In particular, conjugated ...

Bioengineering

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4) - we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.

We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may also be used to study the structure-function relationship for other ion channels and neurotransmitter receptors1. BK channels are widely expressed in different tissues and have been implicated in many physiological functions, including regulation of smooth muscle contraction, frequency tuning of inner hair cells and regulation of neurotransmitter release2-6. BK channels are activated by membrane depolarization and by intracellular Ca2+ and Mg2+ 6-9. Therefore, the protocol is designed to control both the membrane voltage and the intracellular solution. In this protocol, messenger RNA of BK channels is injected into Xenopus laevis oocytes (stage V-VI) followed by 2-5 days of incubation at 18&deg;C10-13. Membrane patches that contain single or multiple BK channels are excised with the inside-out configuration using patch clamp techniques10-13. The intracellular side of the patch is perfused with desired solutions during recording so that the channel activation under different conditions can be examined. To summarize, the mRNA of BK channels is injected into Xenopus laevis oocytes to express channel proteins on the oocyte membrane; patch clamp techniques are used to record currents flowing through the channels under controlled voltage and intracellular solutions.

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

Ionic current of BK channels is recorded using patch clamp techniques. BK channels are expressed in Xenopus oocytes by injecting messenger RNA. The intracellular solution during patch clamp recordings is controlled by a perfusion system.

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may ...

One-channel Cell-attached Patch-clamp Recording

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate depends on properties intrinsic to each channel protein as well as the mechanism by which it is generated and controlled and represents an important area of current research. Information about the operational dynamics of ion channel proteins can be obtained by observing long stretches of current produced by a single molecule. Described here is a protocol for obtaining one-channel cell-attached patch-clamp current recordings for a ligand gated ion channel, the NMDA receptor, expressed heterologously in HEK293 cells or natively in cortical neurons. Also provided are instructions on how to adapt the method to other ion channels of interest by presenting the example of the mechano-sensitive channel PIEZO1. This method can provide data regarding the channel&#8217;s conductance properties and the temporal sequence of open-closed conformations that make up the channel&#8217;s activation mechanism, thus helping to understand their functions in health and disease.

Described here is a procedure for obtaining long stretches of current recording from one ion channel with the cell-attached patch-clamp technique. This method allows for observing, in real time, the pattern of open-close channel conformations that underlie the biological signal. These data inform about channel properties in undisturbed biological membranes.

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate ...

Single-channel Analysis and Calcium Imaging in the Podocytes of the Freshly Isolated Glomeruli

Podocytes (renal glomerular epithelial cells) are known to regulate glomerular permeability and maintain glomerular structure; a key role for these cells in the pathogenesis of various renal diseases has been established since podocyte injury leads to proteinuria and foot process effacement. It was previously reported that various endogenous agents may cause a dramatic overload in intracellular Ca2+ concentration in podocytes, presumably leading to albuminuria, and this likely occurs via calcium-conducting ion channels. Therefore, it appeared important to study calcium handling in the podocytes both under normal conditions and in various pathological states. However, available experimental approaches have remained somewhat limited to cultured and transfected cells. Although they represent a good basic model for such studies, they are essentially extracted from the native environment of the glomerulus. Here we describe the methodology of studying podocytes as a part of the freshly isolated whole glomerulus. This preparation retains the functional potential of the podocytes, which are still attached to the capillaries; therefore, podocytes remain in the environment that conserves the major parts of the glomeruli filtration apparatus. The present manuscript elaborates on two experimental approaches that allow 1) real-time detection of calcium concentration changes with the help of ratiometric confocal fluorescence microscopy, and 2) the recording of the single ion channels activity in the podocytes of the freshly isolated glomeruli. These methodologies utilize the advantages of the native environment of the glomerulus that enable researchers to resolve acute changes in the intracellular calcium handling in response to applications of various agents, measure basal concentration of calcium within the cells (for instance, to evaluate disease progression), and assess and manipulate calcium conductance at the level of single ion channels.

Changes in the intracellular calcium levels in the podocytes are one of the most important means to control the filtration function of glomeruli. Here we explain a high-throughput approach that allows detection of real-time calcium handling and single ion channels activity in the podocytes of the freshly isolated glomeruli.

Podocytes (renal glomerular epithelial cells) are known to regulate glomerular permeability and maintain glomerular structure; a key role for these cells ...

Isolating and Imaging Live, Intact Pacemaker Regions of Mouse Renal Pelvis by Vibratome Sectioning

The renal pelvis (RP) is a funnel-shaped, smooth muscle structure that facilitates normal urine transport from the kidney to the ureter by regular, propulsive contractions. Regular RP contractions rely on pacemaker activity, which originates from the most proximal region of the RP at the pelvis-kidney junction (PKJ). Due to the difficulty in accessing and preserving intact preparations of the PKJ, most investigations on RP pacemaking have focused on single-cell electrophysiology and Ca2+ imaging experiments. Although important revelations on RP pacemaking have emerged from such work, these experiments have several intrinsic limitations, including the inability to accurately determine cellular identity in mixed suspensions and the lack of in situ imaging of RP pacemaker activity. These factors have resulted in a limited understanding of the mechanisms that underlie normal rhythmic RP contractions. In this paper, a protocol is described to prepare intact segments of mouse PKJ using a vibratome sectioning technique. By combining this approach with mice expressing cell-specific reporters and genetically encoded Ca2+ indicators, investigators may be able to more accurately study the specific cell types and mechanisms responsible for peristaltic RP contractions that are vital for normal urine transport.

The goal of this protocol is to isolate intact pacemaker regions of the mouse renal pelvis using vibratome sectioning. These sections can then be used for in situ Ca2+ imaging to elucidate Ca2+ transient properties of pacemaker cells and other interstitial cells in vibratome slices.

The renal pelvis (RP) is a funnel-shaped, smooth muscle structure that facilitates normal urine transport from the kidney to the ureter by regular, ...

Optogenetic Electrophysiology Recording of Cells with Light-Dependent Ion Channels

Source: Grimm, C., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/55497">Whole-cell patch-clamp recordings for electrophysiological determination of ion selectivity in channelrhodopsins.</a> J. Vis. Exp. (2017)
In this video, embryonic kidney cell cultures expressing light-dependent ion channel proteins fused with a fluorescent reporter demonstrate the effect of light on ion movement. Whole-cell patch clamp is used to monitor the movement of ions.

1. Setup Prior to Recordings

	Prepare the recording solutions with ion concentrations as indicated in Table 1.&#8203;
	
		Prepare 15 mL of 2 M stock ...

Implementing Patch Clamp and Live Fluorescence Microscopy to Monitor Functional Properties of Freshly Isolated PKD Epithelium

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