Method Article

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

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DOI:

10.3791/53035

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September 1st, 2015

Ā ,Ā Ā ,Ā Ā ,Ā Ā ,Ā Ā ,Ā 
1Department of Physiology, Medical College of Wisconsin, 2Department of Integrative Biology & Pharmacology, University of Texas Health Science Center at Houston

In This Article

Summary

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.

Abstract

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.

Introduction

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+]iĀ 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.

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Protocol

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

  1. 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.
  2. Perform laparotomy and flush the kidneys with phosphate buffered saline (PBS) through the abdominal aorta17.
    1. 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 organsĀ 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.
    2. 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).
    3. 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.
    4. 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.
    5. 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.
  3. 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 Āµl of 0.01% sterile filtered Poly-L-Lysine solution onto each glass chip. Prepare saline and two pairs of watchmaker forceps for dissection.
  4. 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.
  5. 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).
  6. 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.
  7. 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).

2. Single Channel Patch-clamp Electrophysiology

  1. 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 MĪ© for reliable on-cell (cell-attached) measurements.
  2. 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).
  3. 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.
  4. 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.

3. Ratiometric Epifluorescence Measurements of Intracellular Calcium Concentration in the Epithelial Cells

  1. Use 5 mM Fura-2AM dissolved in DMSO. Aliquot stock solution into individual 500 Āµl conical tubes (approximately 10 Āµl). Protect from light and store in the freezer at -20 ĖšC for up to six months.
  2. 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 ĀµM 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.
  3. Change Fura-2 AM containing media to clean PSS after incubation and place the tissue under a microscope to further perform epifluorescence imaging.
  4. 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).
  5. 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.
  6. 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 40Ɨ/NA 1.3 or similar objective lens for proper resolution image.
  7. 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.
  8. 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.

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Results

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+Ā absorption is decreased in CD cells from the non-orthologous BPK mouse model of ARPKD29. We recently demonstrated that impaired sodium and water reabsorptio...

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Discussion

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...

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Disclosures

The authors have nothing to disclose.

Acknowledgements

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).

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
Fura-2 AMLife TechnologiesF-14185
Fluo-8AAT Bioquest21091
Poly-L-lysineSigma-AldrichP4707
Pluronic acidSigma-AldrichF-68Ā solution
ShakerBoekel Scientific260350
Light sourceSutter Instrument CoLambda XLwith integrated shutter/filter wheel driver
Neutral density filtersNikonND4, ND8
ObjectiveNikonSFluoĀ 40/1.3 DIC WD 0.22Ā Ā  oil
CameraAndor TechnologiesZyla sCMOS
NikonĀ  microscope (inverted)NikonNikon Eclipse TE2000-S
Cover GlassThermo Scientific6661B52
Diamond pencilFisher Scientific22268912
Image acquisition softwareNikonNikon NIS-ElementsĀ 
Image analysis softwareImageJhttp://imagej.nih.gov/ND Utility plugin allows to import images in the native Nikon Instruments .nd2 format
Recording/perfusion chamberWarner InstrumentsRC-26
Patch Clamp amplifierMolecular DevicesMultiClamp 700B
Data Acquisition SystemMolecular DevicesDigidata 1440AAxon DigidataĀ® System
Low Pass FilterWarner InstrumentsLPF-88 pole Bessel
Borosilicate glass capillariesWorld Precision Instruments1B150F-4
Micropipette PullerSutter Instrument CoP-97Flaming/Brown type micropipette puller
MicroforgeNarishigeMF-830Japan
Motorized MicromanipulatorSutter Instrument CoMP-225
Inverted microscopeNikonEclipse Ti
Microvibration isolation tableTMCequipped with Faraday cage
Multichannel valve perfusion systemAutoMake ScientificValve Bank II
Recording/perfusion chamberWarner InstrumentsRC-26
SoftwareMolecular DevicespClamp 10 . 2
Temperature controlled surgical tableĀ MCW corefor rodents
Binocular stereomicroscopeNikonSMZ745
Syringe pump-based perfusion systemHarvard Apparatus
polyethylene tubingSigma-AldrichPE50
Isofluorane anesthesiahttp://www.vetequip.com/911103
Other basic reagentsSigma-Aldrich

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Tags

Ion Channel ActivityIntracellular CalciumCystic Monolayer IsolationElectrophysiology RecordingCalcium ImagingConfocal MicroscopyFluorescent Dye Loading

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