Name: single-channel analysis and calcium imaging in the podocytes of the freshly isolated glomeruli
Uploaded: 2015-06-27T14:00:00
Description: Watch this Scientific Journal Video about Single-channel Analysis and Calcium Imaging in the Podocytes of the Freshly Isolated Glomeruli 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. Gregory Blass is acknowledged for critical proofreading of the manuscript. This research was supported by the National Institutes of Health grant HL108880 and American Diabetes Association grant 1-15-BS-172 (to AS), and the Ben J. Lipps Research Fellowship from the American Society of Nephrology (to DVI).

Animal use and welfare should adhere to the NIH Guide for the Care and Use of Laboratory Animals following protocols reviewed and approved by the Institutional Animal Care and Use Committee (IACUC).
1. Kidney Flush
<ol>
	<li>Use 8 to 12 week old male rat (suggested is a Sprague Dawley strain, however other strains of different age and gender can be used with appropriate changes).</li>
	<li>Anesthetize the animal according to the procedure allowed by IACUC protocol; monitor depth of anesthesi...

<ol>
	<li>Machuca, E., Benoit, G., Antignac, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetics+of+nephrotic+syndrome:+connecting+molecular+genetics+to+podocyte+physiology.">Genetics of nephrotic syndrome: connecting molecular genetics to podocyte physiology.</a> Hum. Mol. Genet. 18, R185-R194 (2009).</li><li>Haraldsson, B., Nystrom, J., Deen, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Properties+of+the+glomerular+barrier+and+mechanisms+of+proteinuria.">Properties of the glomerular barrier and mechanisms of proteinuria.</a> Physiol. Rev. 88, 451-487 (2008).</li><li>Patrakka, J., Tryggvason, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+insights+into+the+role+of+podocytes+in+proteinuria.">New insights into the role of podocytes in proteinuria.</a> Nat. Rev. Nephrol. 5, 463-468 (2009).</li><li>Savin, V. J., Sharma, R., Lovell, H. B., Welling, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+albumin+reflection+coefficient+with+isolated+rat+glomeruli.">Measurement of albumin reflection coefficient with isolated rat glomeruli.</a> J. Am. Soc. Nephrol. 3, 1260-1269 (1992).</li><li>Gloy, 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=Angiotensin+II+depolarizes+podocytes+in+the+intact+glomerulus+of+the+Rat.">Angiotensin II depolarizes podocytes in the intact glomerulus of the Rat.</a> J. Clin. Invest. 99, 2772-2781 (1997).</li><li>Nitschke, R., 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=Angiotensin+II+increases+the+intracellular+calcium+activity+in+podocytes+of+the+intact+glomerulus.">Angiotensin II increases the intracellular calcium activity in podocytes of the intact glomerulus.</a> Kidney Int. 57, 41-49 (2000).</li><li>Ilatovskaya, D., Staruschenko, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-channel+analysis+of+TRPC+channels+in+the+podocytes+of+freshly+isolated+glomeruli.">Single-channel analysis of TRPC channels in the podocytes of freshly isolated glomeruli.</a> Methods Mol Biol. 998, 355-369 (2013).</li><li>Snitsarev, V. A., McNulty, T. J., Taylor, C. 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J. 71, 1048-1056 (1996).</li><li>Fukuda, A., Fujimoto, S., Iwatsubo, S., Kawachi, H., Kitamura, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+mineralocorticoid+and+angiotensin+II+receptor+blockers+on+proteinuria+and+glomerular+podocyte+protein+expression+in+a+model+of+minimal+change+nephrotic+syndrome.">Effects of mineralocorticoid and angiotensin II receptor blockers on proteinuria and glomerular podocyte protein expression in a model of minimal change nephrotic syndrome.</a> Nephrology (Carlton). 15, 321-326 (2010).</li><li>Abramowitz, J., Birnbaumer, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiology+and+pathophysiology+of+canonical+transient+receptor+potential+channels).">Physiology and pathophysiology of canonical transient receptor potential channels).</a> FASEB J. 23, 297-328 (2009).</li><li>Heeringa, S. F., 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=A+novel+TRPC6+mutation+that+causes+childhood+FSGS.">A novel TRPC6 mutation that causes childhood FSGS.</a> PLoS ONE. 4, e7771 (2009).</li><li>Zhang, X., Song, Z., Guo, Y., Zhou, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+novel+role+of+TRPC6+in+vitamin+D+ameliorating+podocyte+injury+in+STZ-induced+diabetic+rats.">The novel role of TRPC6 in vitamin D ameliorating podocyte injury in STZ-induced diabetic rats.</a> Mol. Cell. Biochem. 399, 155-165 (2015).</li><li>Bohrer, M. P., 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=Mechanisms+of+the+puromycin-induced+defects+in+the+transglomerular+passage+of+water+and+macromolecules.">Mechanisms of the puromycin-induced defects in the transglomerular passage of water and macromolecules.</a> J. Clin. Invest. 60, 152-161 (1977).</li><li>Olson, J. L., Rennke, H. G., Venkatachalam, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alterations+in+the+charge+and+size+selectivity+barrier+of+the+glomerular+filter+in+aminonucleoside+nephrosis+in+rats.">Alterations in the charge and size selectivity barrier of the glomerular filter in aminonucleoside nephrosis in rats.</a> Lab. Invest. 44, 271-279 (1981).</li><li>Schiessl, I. M., Castrop, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiotensin+II+AT2+receptor+activation+attenuates+AT1+receptor-induced+increases+in+the+glomerular+filtration+of+albumin:+a+multiphoton+microscopy+study.">Angiotensin II AT2 receptor activation attenuates AT1 receptor-induced increases in the glomerular filtration of albumin: a multiphoton microscopy study.</a> Am J Physiol Renal Physiol. 305, F1189-F1200 (2013).</li><li>Ilatovskaya, D. V., Levchenko, V., Ryan, R. P., Cowley, A. W., Staruschenko, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NSAIDs+acutely+inhibit+TRPC+channels+in+freshly+isolated+rat+glomeruli.">NSAIDs acutely inhibit TRPC channels in freshly isolated rat glomeruli.</a> Biochem. Biophys. Res. Commun. 408, 242-247 (2011).</li><li>Peti-Peterdi, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+wave+of+tubuloglomerular+feedback.">Calcium wave of tubuloglomerular feedback.</a> Am. J. Physiol. Renal Physiol. 291, F473-F480 (2006).</li><li>Peti-Peterdi, J., Warnock, D. G., Bell, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiotensin+II+directly+stimulates+ENaC+activity+in+the+cortical+collecting+duct+via+AT(1)+receptors.">Angiotensin II directly stimulates ENaC activity in the cortical collecting duct via AT(1) receptors.</a> J. Am. Soc. Nephrol. 13, 1131-1135 (2002).</li><li>Ilatovskaya, D. V., Palygin, O., Levchenko, V., Staruschenko, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacological+characterization+of+the+P2+receptors+profile+in+the+podocytes+of+the+freshly+isolated+rat+glomeruli.">Pharmacological characterization of the P2 receptors profile in the podocytes of the freshly isolated rat glomeruli.</a> Am. J. Physiol. Cell Physiol. 305, C1050-C1059 (2013).</li><li>Ilatovskaya, D. V., 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=Angiotensin+II+has+acute+effects+on+TRPC6+channels+in+podocytes+of+freshly+isolated+glomeruli.">Angiotensin II has acute effects on TRPC6 channels in podocytes of freshly isolated glomeruli.</a> Kidney Int. 305, C1050-C1059 (2014).</li><li>Schaldecker, T., 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=Inhibition+of+the+TRPC5+ion+channel+protects+the+kidney+filter.">Inhibition of the TRPC5 ion channel protects the kidney filter.</a> J. Clin. Invest. 123, 5298-5309 (2013).</li><li>Roshanravan, H., Dryer, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ATP+acting+through+P2Y+receptors+causes+activation+of+podocyte+TRPC6+channels:+role+of+podocin+and+reactive+oxygen+species.">ATP acting through P2Y receptors causes activation of podocyte TRPC6 channels: role of podocin and reactive oxygen species.</a> Am. J. Physiol. Renal Physiol. 306, F1088-F1097 (2014).</li><li>Anderson, M., Roshanravan, H., Khine, J., Dryer, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiotensin+II+activation+of+TRPC6+channels+in+rat+podocytes+requires+generation+of+reactive+oxygen+species.">Angiotensin II activation of TRPC6 channels in rat podocytes requires generation of reactive oxygen species.</a> J. Cell. Physiol. 229, 434-442 (2014).</li></ol>

The approach described here allows for the analysis of calcium handling by the podocytes of the rodent glomeruli. This technique allows application of patch-clamp single channel electrophysiology and fluorescence ratiometric confocal imaging. However, both approaches can be used separately, on their own. The proposed protocol has several relatively simple steps, including 1) kidney flush; 2) isolation of the glomeruli by differential sieving; 3) performing patch-clamp electrophysiological experiments, or incubation of th...

Kidneys maintain homeostatic balance for various substances and regulate blood volume in a way that determines total blood pressure. Disturbances in the renal filtration, reabsorption or secretion lead to or accompany pathological states, ranging from hyper- or hypotension to end stage renal disease that eventually requires kidney transplantation. The renal filtering unit (glomerulus) consists of three layers &#8211; the capillary endothelium, basement membrane and a single-cell layer of epithelial cells &#8211; podocytes, which play a major role in the maintenance of the slit-diaphragm integrity and function1. Dysfunction in the permselective glomerular filter causes urinary loss of macromolecules, such as proteinuria. Various agents may affect the structure of the podocytes and their foot processes, which determine the integrity of the glomeruli filtration barrier.
The podocytes are involved in the maintenance of the glomeruli filtration function. It has been established that improper calcium handling by the podocyte leads to cell injury and plays an important role in the progression of various forms of nephropathies2,3. Therefore, development of a model which allows for direct measuring of intracellular calcium concentration changes will be instrumental for studies of podocyte function. Isolated glomeruli were previously used in a numerous studies including measurement of albumin reflection coefficient changes4 and assessment of integral cellular currents in the whole-cell electrophysiological patch-clamp measurements5,6. In the present paper we describe the protocol that allows the researcher to measure intracellular calcium concentration changes in response to applications of pharmacological agents, estimate basal levels of calcium within the cells, and assess individual calcium channels activity. Ratometric calcium concentration measurements and patch-clamp electrophysiology were used to determine changes in the intracellular calcium concentration within the podocyte and channel activity, respectively.

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	<tbody>
		<tr>
			<td>Fluo4 AM</td>
			<td>Life Technologies</td>
			<td>F14217</td>
			<td>500&#181;l in DMSO</td>
		</tr>
		<tr>
			<td>FuraRed AM</td>
			<td>Life Technologies</td>
			<td>F-3020</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&#160;</td>
			<td>solution</td>
		</tr>
		<tr>
			<td>Ionomycin</td>
			<td>Sigma-Aldrich</td>
			<td>I3909-1ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Tube rotator</td>
			<td>Miltenyi Biotec GmbH</td>
			<td>130-090-753</td>
			<td>Germany</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon confocal microscope (inverted)</td>
			<td>Nikon</td>
			<td>Nikon A1R</td>
			<td>&#160;Laser exitation 488nm. Emission filters 500-550nm and 570-620nm</td>
		</tr>
		<tr>
			<td>Objective</td>
			<td>Nikon</td>
			<td>Plan Apo 60x/NA 1.4 Oil</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover Glass</td>
			<td>Thermo Scientific</td>
			<td>6661B52</td>
			<td></td>
		</tr>
		<tr>
			<td>High vacuum grease</td>
			<td>Dow Corning</td>
			<td></td>
			<td>Silicone Compound</td>
		</tr>
		<tr>
			<td>Software</td>
			<td>Nikon</td>
			<td>Nikon NIS-Elements&#160;</td>
			<td></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&#174; 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>Nicardipine</td>
			<td>Sigma-Aldrich</td>
			<td>N7510</td>
			<td></td>
		</tr>
		<tr>
			<td>Iberiotoxin</td>
			<td>Sigma</td>
			<td>I5904-5UG</td>
			<td></td>
		</tr>
		<tr>
			<td>Niflumic acid</td>
			<td>Sigma-Aldrich</td>
			<td>N0630</td>
			<td></td>
		</tr>
		<tr>
			<td>DIDS</td>
			<td>Sigma-Aldrich</td>
			<td>D3514-25MG</td>
			<td></td>
		</tr>
		<tr>
			<td>TEA chloride</td>
			<td>Tocris</td>
			<td>T2265</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640</td>
			<td>Life Technologies</td>
			<td>11835030</td>
			<td>without antibiotics</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma-Aldrich</td>
			<td>A8327</td>
			<td>30% albumin solution</td>
		</tr>
		<tr>
			<td>Temperature controlled surgical table&#160;</td>
			<td>MCW core</td>
			<td></td>
			<td>for rodents</td>
		</tr>
		<tr>
			<td>Steel sieves:</td>
			<td></td>
			<td></td>
			<td>#100 (150 &#956;m), 140 (106 &#956;m)</td>
		</tr>
		<tr>
			<td>Gilson, Inc&#160; SIEVE 3 SS FH NO200</td>
			<td>Fisher Sci</td>
			<td>50-871-316</td>
			<td></td>
		</tr>
		<tr>
			<td>Gilson, Inc&#160; SIEVE 3 SS FH NO270</td>
			<td>Fisher Sci</td>
			<td>50-871-318</td>
			<td></td>
		</tr>
		<tr>
			<td>Gilson, Inc&#160; SIEVE 3 SS FH NO400</td>
			<td>Fisher Sci</td>
			<td>50-871-320</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;mesh 200&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>s4145</td>
			<td>screen for CD-1</td>
		</tr>
		<tr>
			<td>Binocular microscope</td>
			<td>Nikon</td>
			<td>Eclipse TS100</td>
			<td></td>
		</tr>
		<tr>
			<td>Binocular microscope</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><a href="http://www.vetequip.com/">http://www.vetequip.com/</a></td>
			<td>911103</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Other basic reagents</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Here we addressed the problem of measuring acute changes in the calcium levels in the podocytes. Figure 1 shows a schematic representation of the experimental protocol designed in order to perform high resolution live fluorescence confocal imaging and single ion channel activity recordings in the podocytes of the freshly isolated rodent glomeruli. Briefly, after the rat is anaesthetized, the kidneys should be flushed with PBS to clear them of blood. Then, the kidneys are excised and decapsulated, and glo...

Watch this Scientific Journal Video about Single-channel Analysis and Calcium Imaging in the Podocytes of the Freshly Isolated Glomeruli at JoVE.com

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

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 ...

single-channel-analysis-calcium-imaging-podocytes-freshly-isolated

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Biology

Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT

Brown adipose tissue (BAT) differs from white adipose tissue (WAT) by its discrete location and a brown-red color due to rich vascularization and high density of mitochondria. BAT plays a major role in energy expenditure and non-shivering thermogenesis in newborn mammals as well as the adults 1. BAT-mediated thermogenesis is highly regulated by the sympathetic nervous system, predominantly via &beta; adrenergic receptor 2, 3. Recent studies have shown that BAT activities in human adults are negatively correlated with body mass index (BMI) and other diabetic parameters 4-6. BAT has thus been proposed as a potential target for anti-obesity/anti-diabetes therapy focusing on modulation of energy balance 6-8. While several cold challenge-based positron emission tomography (PET) methods are established for detecting human BAT 9-13, there is essentially no standardized protocol for imaging and quantification of BAT in small animal models such as mice. Here we describe a robust PET/CT imaging method for functional assessment of BAT in mice. Briefly, adult C57BL/6J mice were cold treated under fasting conditions for a duration of 4 hours before they received one dose of 18F-Fluorodeoxyglucose (FDG). The mice were remained in the cold for one additional hour post FDG injection, and then scanned with a small animal-dedicated micro-PET/CT system. The acquired PET images were co-registered with the CT images for anatomical references and analyzed for FDG uptake in the interscapular BAT area to present BAT activity. This standardized cold-treatment and imaging protocol has been validated through testing BAT activities during pharmacological interventions, for example, the suppressed BAT activation by the treatment of &beta;-adrenoceptor antagonist propranolol 14, 15, or the enhanced BAT activation by &beta;3 agonist BRL37344 16. The method described here can be applied to screen for drugs/compounds that modulate BAT activity, or to identify genes/pathways that are involved in BAT development and regulation in various preclinical and basic studies.

Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT

A method of functional imaging of mouse brown adipose tissue (BAT) is described in which cold-stimulated uptake of 18F-Fluorodeoxyglucose (FDG) in BAT is non-invasively assessed with a standardized micro-PET/CT protocol. This method is robust and sensitive to detect differences in BAT activities in mouse models.

Brown adipose tissue (BAT) differs from white adipose  tissue (WAT) by its discrete location and a brown-red color due to rich  vascularization and high ...

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare 'professional' phagocytes - macrophages and dendritic cells to 'non-professional' - tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: 'professional' macrophages and 'semi-professional' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Here we describe an effective method for studying dynamics of apoptotic cell clearance in vivo. This method employs live Drosophila embryos as a powerful model for monitoring phagocytosis of apoptotic cells using specific labeling of apoptotic cells and phagocytes.

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

Assessment of Calcium Sparks in Intact Skeletal Muscle Fibers

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in the striated muscle fibers that appear as highly localized Ca2+ release events mediated by ryanodine receptor (RyR) Ca2+ release channels on the sarcoplasmic reticulum (SR) membrane. Proper assessment of muscle Ca2+ sparks could provide information on the intracellular Ca2+&#160;handling properties of healthy and diseased striated muscles. Although Ca2+ sparks events are commonly seen in resting cardiomyocytes, they are rarely observed in resting skeletal muscle fibers; thus there is a need for methods to generate and analyze sparks in skeletal muscle fibers.
Detailed here is an experimental protocol for measuring Ca2+ sparks in isolated flexor digitorm brevis (FDB) muscle fibers using fluorescent Ca2+ indictors and laser scanning confocal microscopy. In this approach, isolated FDB fibers are exposed to transient hypoosmotic stress followed by a return to isotonic physiological solution. Under these conditions, a robust Ca2+ sparks response is detected adjacent to the sarcolemmal membrane in young healthy FDB muscle fibers. Altered Ca2+ sparks response is detected in dystrophic or aged skeletal muscle fibers. This approach has recently demonstrated that membrane-delimited signaling involving cross-talk between inositol (1,4,5)-triphosphate receptor (IP3R) and RyR contributes to Ca2+ spark activation in skeletal muscle. In summary, our studies using osmotic stress induced Ca2+ sparks showed that this intracellular response reflects a muscle signaling mechanism in physiology and aging/disease states, including mouse models of muscle dystrophy (mdx mice) or amyotrophic lateral sclerosis (ALS model).

Described here is a method to directly measure calcium sparks, the elementary units of Ca2+ release from sarcoplasmic reticulum&#160;in intact skeletal muscle fibers. This method utilizes osmotic-stress-mediated triggering of Ca2+ release from ryanodine receptor in isolated muscle fibers. The dynamics and homeostatic capacity of intracellular Ca2+ signaling can be employed to assess muscle function in health and disease.

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in ...

Quantifying Single Microvessel Permeability in Isolated Blood-perfused Rat Lung Preparation

The isolated blood-perfused lung preparation is widely used to visualize and define signaling in single microvessels. By coupling this preparation with real time imaging, it becomes feasible to determine permeability changes in individual pulmonary microvessels. Herein we describe steps to isolate rat lungs and perfuse them with autologous blood. Then, we outline steps to infuse fluorophores or agents via a microcatheter into a small lung region. Using these procedures described, we determined permeability increases in rat lung microvessels in response to infusions of bacterial lipopolysaccharide. The data revealed that lipopolysaccharide increased fluid leak across both venular and capillary microvessel segments. Thus, this method makes it possible to compare permeability responses among vascular segments and thus, define any heterogeneity in the response. While commonly used methods to define lung permeability require postprocessing of lung tissue samples, the use of real time imaging obviates this requirement as evident from the present method. Thus, the isolated lung preparation combined with real time imaging offers several advantages over traditional methods to determine lung microvascular permeability, yet is a straightforward method to develop and implement.

The isolated blood-perfused lung preparation makes it feasible to visualize microvessel networks on the lung surface. Here we describe an approach to quantify permeability of single microvessels in isolated lungs using real time fluorescence imaging.

The isolated blood-perfused lung preparation is widely used to visualize and define signaling in single microvessels. By coupling this preparation with ...

Measuring the Osmotic Water Permeability Coefficient (Pf) of Spherical Cells: Isolated Plant Protoplasts as an Example

Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is a simple and very efficient method for the determination of the osmotic water permeability coefficient (Pf) in plant protoplasts, applicable in principle also to other spherical cells such as frog oocytes. The first step of the assay is the isolation of protoplasts from the plant tissue of interest by enzymatic digestion into a chamber with an appropriate isotonic solution. The second step consists of an osmotic challenge assay: protoplasts immobilized on the bottom of the chamber are submitted to a constant perfusion starting with an isotonic solution and followed by a hypotonic solution. The cell swelling is video recorded. In the third step, the images are processed offline to yield volume changes, and the time course of the volume changes is correlated with the time course of the change in osmolarity of the chamber perfusion medium, using a curve fitting procedure written in Matlab (the &#8216;PfFit&#8217;), to yield Pf.

Measuring the Osmotic Water Permeability Coefficient Pf of Spherical Cells: Isolated Plant Protoplasts as an Example

Measuring the osmotic water permeability coefficient (Pf) of cells can help understand the regulatory mechanisms of aquaporins (AQPs). Pf determination in spherical plant cell protoplasts presented here involves protoplasts isolation and numerical analysis of their initial rate of volume change as a result of an osmotic challenge during constant bath perfusion.

Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is ...

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.

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 ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

The Mouse Isolated Perfused Kidney Technique

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. This is a prerequisite for studying the physiology of the isolated organ and for many innovative applications that may be possible in the future, including perfusion decellularization for kidney bioengineering or the administration of anti-rejection or genome-editing drugs in high doses to prime the kidney for transplantation. During the time of the perfusion, the kidney can be manipulated, renal function can be assessed, and various pharmaceuticals administered. After the procedure, the kidney can be transplanted or processed for molecular biology, biochemical analysis, or microscopy.
This paper describes the perfusate and the surgical technique needed for the ex vivo perfusion of mouse kidneys. Details of the perfusion apparatus are given and data are presented showing the viability of the kidney&#39;s preparation: renal blood flow, vascular resistance, and urine data as functional, transmission electron micrographs of different nephron segments as morphological readouts, and western blots of transport proteins of different nephron segments as molecular readout.

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. The buffers and surgical technique are described in detail.

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. This is a ...

Analysis of Beta-cell Function Using Single-cell Resolution Calcium Imaging in Zebrafish Islets

Pancreatic beta-cells respond to increasing blood glucose concentrations by secreting the hormone insulin. The dysfunction of beta-cells leads to hyperglycemia and severe, life-threatening consequences. Understanding how the beta-cells operate under physiological conditions and what genetic and environmental factors might cause their dysfunction could lead to better treatment options for diabetic patients. The ability to measure calcium levels in beta-cells serves as an important indicator of beta-cell function, as the influx of calcium ions triggers insulin release. Here we describe a protocol for monitoring the glucose-stimulated calcium influx in zebrafish beta-cells by using GCaMP6s, a genetically encoded sensor of calcium. The method allows monitoring the intracellular calcium dynamics with single-cell resolution in ex vivo mounted islets. The glucose-responsiveness of beta-cells within the same islet can be captured simultaneously under different glucose concentrations, which suggests the presence of functional heterogeneity among zebrafish beta-cells. Furthermore, the technique provides high temporal and spatial resolution, which reveals the oscillatory nature of the calcium influx upon glucose stimulation. Our approach opens the doors to use the zebrafish as a model to investigate the contribution of genetic and environmental factors to beta-cell function and dysfunction.

Beta-cell functionality is important for blood-glucose homeostasis, which is evaluated at single-cell resolution using a genetically encoded reporter for calcium influx.

Pancreatic beta-cells respond to increasing blood glucose concentrations by secreting the hormone insulin. The dysfunction of beta-cells leads to ...

Simultaneous Measurements of Intracellular Calcium and Membrane Potential in Freshly Isolated and Intact Mouse Cerebral Endothelium

Cerebral arteries and their respective microcirculation deliver oxygen and nutrients to the brain via blood flow regulation. Endothelial cells line the lumen of blood vessels and command changes in vascular diameter as needed to meet the metabolic demand of neurons. Primary endothelial-dependent signaling pathways of hyperpolarization of membrane potential (Vm) and nitric oxide typically operate in parallel to mediate vasodilation and thereby increase blood flow. Although integral to coordinating vasodilation over several millimeters of vascular length, components of endothelium-derived hyperpolarization (EDH) have been historically difficult to measure. These components of EDH entail intracellular Ca2+ [Ca2+]i increases and subsequent activation of small- and intermediate conductance Ca2+-activated K+ (SKCa/IKCa) channels.
Here, we present a simplified illustration of the isolation of fresh endothelium from mouse cerebral arteries; simultaneous measurements of endothelial [Ca2+]i and Vm using Fura-2 photometry and intracellular sharp electrodes, respectively; and a continuous superfusion of salt solutions and pharmacological agents under physiological conditions (pH 7.4, 37 &#176;C). Posterior cerebral arteries from the Circle of Willis are removed free of the posterior communicating and the basilar arteries. Enzymatic digestion of cleaned posterior cerebral arterial segments and subsequent trituration facilitates removal of adventitia, perivascular nerves, and smooth muscle cells. Resulting posterior cerebral arterial endothelial &#34;tubes&#34; are then secured under a microscope and examined using a camera, photomultiplier tube, and one to two electrometers while under continuous superfusion. Collectively, this method can simultaneously measure changes in endothelial [Ca2+]i and Vm in discrete cellular locations, in addition to the spreading of EDH through gap junctions up to millimeter distances along the intact endothelium. This method is expected to yield a high-throughput analysis of the cerebral endothelial functions underlying mechanisms of blood flow regulation in the normal and diseased brain.

Demonstrated here are protocols for (1) freshly isolating intact cerebral endothelial "tubes" and (2) simultaneous measurements of endothelial calcium and membrane potential during endothelium-derived hyperpolarization. Further, these methods allow for pharmacological tuning of endothelial cell calcium and electrical signaling as individual or interactive experimental variables.

Cerebral arteries and their respective microcirculation deliver oxygen and nutrients to the brain via blood flow regulation. Endothelial cells line the ...