Name: glomerular outgrowth as an ex vivo assay to analyze pathways involved in parietal epithelial cell activation
Uploaded: 2020-08-19T13:00:00
Description: Watch this Scientific Journal Video about Glomerular Outgrowth as an Ex Vivo Assay to Analyze Pathways Involved in Parietal Epithelial Cell Activation at JoVE.com

This research was supported by Dutch Kidney foundation (grant 14A3D104) and The Netherlands Organization for Scientific Research (NWO VIDI grant: 016.156.363).

All animal experiments were performed according to the guidelines of the Animal Ethics Committee of the Radboud University Nijmegen.
NOTE: Untreated, healthy wild type (WT) mice (n = 4) and cd44-/- (n = 4) mice were sacrificed at the age of 12&#8722;16 weeks. Both male and female mice were used. All mice were on the C57Bl/6 background.
1. Mouse kidney dissection
<ol>
	<li>Sacrifice healthy WT mice or genetically altered mice by cervical dislocation.</li>
...

<ol>
	<li>Fatima, H., 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=Parietal+epithelial+cell+activation+marker+in+early+recurrence+of+FSGS+in+the+transplant.">Parietal epithelial cell activation marker in early recurrence of FSGS in the transplant.</a> Clinical journal of the American Society of Nephrology: CJASN. 7 (11), 1852-1858 (2012).</li><li>Dijkman, H. 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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parietal+epithelial+cells+and+podocytes+in+glomerular+diseases.">Parietal epithelial cells and podocytes in glomerular diseases.</a> Seminars in Nephrology. 32 (4), 357-367 (2012).</li><li>Ryu, M., 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=Plasma+leakage+through+glomerular+basement+membrane+ruptures+triggers+the+proliferation+of+parietal+epithelial+cells+and+crescent+formation+in+non-inflammatory+glomerular+injury.">Plasma leakage through glomerular basement membrane ruptures triggers the proliferation of parietal epithelial cells and crescent formation in non-inflammatory glomerular injury.</a> The Journal of Pathology. 228 (4), 482-494 (2012).</li><li>Eymael, J., Smeets, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Origin+and+fate+of+the+regenerating+cells+of+the+kidney.">Origin and fate of the regenerating cells of the kidney.</a> European Journal of Pharmacology. 790, 62-73 (2016).</li><li>Smeets, B., 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=Renal+progenitor+cells+contribute+to+hyperplastic+lesions+of+podocytopathies+and+crescentic+glomerulonephritis.">Renal progenitor cells contribute to hyperplastic lesions of podocytopathies and crescentic glomerulonephritis.</a> Journal of the American Society of Nephrology: JASN. 20 (12), 2593-2603 (2009).</li><li>Eymael, 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=CD44+is+required+for+the+pathogenesis+of+experimental+crescentic+glomerulonephritis+and+collapsing+focal+segmental+glomerulosclerosis.">CD44 is required for the pathogenesis of experimental crescentic glomerulonephritis and collapsing focal segmental glomerulosclerosis.</a> Kidney International. 93 (3), 626-642 (2018).</li><li>Roeder, S. S., 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=Activated+ERK1/2+increases+CD44+in+glomerular+parietal+epithelial+cells+leading+to+matrix+expansion.">Activated ERK1/2 increases CD44 in glomerular parietal epithelial cells leading to matrix expansion.</a> Kidney International. 91 (4), 896-913 (2017).</li><li>Appel, D., 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=Recruitment+of+podocytes+from+glomerular+parietal+epithelial+cells.">Recruitment of podocytes from glomerular parietal epithelial cells.</a> Journal of the American Society of Nephrology: JASN. 20 (2), 333-343 (2009).</li><li>Yaoita, E., 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=Visceral+epithelial+cells+in+rat+glomerular+cell+culture.">Visceral epithelial cells in rat glomerular cell culture.</a> European Journal of Cell Biology. 67 (2), 136-144 (1995).</li><li>Kuppe, C., 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=Investigations+of+Glucocorticoid+Action+in+GN.">Investigations of Glucocorticoid Action in GN.</a> Journal of the American Society of Nephrology: JASN. 28 (5), 1408-1420 (2017).</li><li>Ohse, 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=Establishment+of+conditionally+immortalized+mouse+glomerular+parietal+epithelial+cells+in+culture.">Establishment of conditionally immortalized mouse glomerular parietal epithelial cells in culture.</a> Journal of the American Society of Nephrology: JASN. 19 (10), 1879-1890 (2008).</li></ol>

Using the protocol described in this article, one can use single encapsulated glomeruli to evaluate parietal epithelial cell proliferation which is a consequence of parietal epithelial cell activation. This ex vivo model will enable us to study in detail the molecular pathways, which are involved in parietal epithelial cell activation. The described method relies on the simple concept of kidney dissection and sieving to isolate and culture encapsulated glomeruli and to compare proliferation and/or migration of parietal e...

Glomerular diseases are an important group of kidney disorders and represent a major cause of end stage renal disease (ESRD). Unfortunately, specific treatment options are limited and progression to ESRD is inevitable. Glomerular diseases are defined by the presence of glomerular injury and can be grouped in inflammatory and non-inflammatory diseases. Although the initial insult is different, recent studies have shown that a common cellular mechanism leads to glomerular epithelial cell hyperplasia and ultimately to glomerulosclerosis in all glomerular diseases, irrespective of the underlying cause1,2,3,4.
Specifically, it was shown that glomerulosclerotic lesions are mainly composed of activated parietal epithelial cells5,6. Under physiological conditions, parietal epithelial cells are flat quiescent epithelial cells that line the Bowman&#39;s capsule of the glomerulus. However, any glomerular injury either due to genetic mutations (e.g., podocyte specific or mitochondrial cytopathies), inflammation or hyperfiltration (e.g., caused by reduced renal mass, hypertension, obesity or diabetic mellitus) can trigger the activation of parietal epithelial cells. Activated parietal epithelial cells proliferate and deposit extracellular matrix which results in the formation of cellular crescents or sclerotic lesions5,7,8. Progression of these processes results in loss of renal function9. Therefore, parietal epithelial cell activation is a key factor in the development and progression of glomerulosclerosis in both inflammatory and non-inflammatory glomerular diseases1,2,3,4,10.
The molecular processes mediating parietal epithelial cell activation are still largely unknown. Recent studies show that activated parietal epithelial cells de novo express CD44, a receptor that is important for the activation of different pathways involved in cellular proliferation and migration. Furthermore, inhibition of CD44 was shown to inhibit parietal epithelial cell activation and attenuate the progression of crescent formation and glomerulosclerosis in animal models of inflammatory as well as non-inflammatory glomerular diseases11,12.
As parietal epithelial cell activation is a key player for the development of glomerulosclerosis and crescent formation, inhibition of these cells could slow down the progression of glomerular diseases. Elucidation of the molecular pathways driving parietal epithelial cell activation may lead to the development of specific therapeutic interventions that attenuate the formation of the hyperplastic and glomerulosclerotic lesions in glomerular disease.
In experimental animal models, it is frequently difficult to provide evidence for a direct effect of an altered gene expression (knock-out models or transgenic mouse models) or drug treatment on the parietal epithelial cells. In a conventional knock-out mouse the observed in vivo changes might be explained by direct changes in parietal epithelial cells. However, since the gene expression is also altered in other cell types within the mouse, one cannot exclude indirect effects mediated by other cell types. The development of conditional cre-lox mice driven by promoters mainly active in parietal epithelial cells has provided a solution in some cases13. Nevertheless, conditional transgenic models are complex and although more conditional lines become available, for many of the conventional knock-out or transgenic mouse lines there is not yet a conditional substitute.
To study the direct effects on parietal epithelial cells, our group has developed an ex vivo assay using single encapsulated glomeruli&#160;isolated&#160;from mouse kidneys to measure and analyze parietal epithelial cell proliferation and migration. This method will enable us to determine parietal epithelial cell specific effects and to find responsible pathways for parietal epithelial cell activation and test treatment options to inhibit this activation.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >24-well cell culture plate</td>
			<td >Corning Costar</td>
			<td ></td>
		</tr>
		<tr >
			<td >anti-CD31</td>
			<td>BD Pharmingen</td>
			<td>Endothelial cell marker (used concentration 1:200)</td>
		</tr>
		<tr >
			<td >chicken-anti-rat Alexa 647</td>
			<td>Thermo Fisher</td>
			<td>&#160;(used concentration 1:200)</td>
		</tr>
		<tr >
			<td >DAPI-Fluoromount G</td>
			<td>Southern Biotech</td>
			<td>Mounting medium containing DAPI</td>
		</tr>
		<tr >
			<td >Digital inverted light microscope</td>
			<td>Westburg, EVOS fl microscope</td>
			<td></td>
		</tr>
		<tr >
			<td >donkey-anti-goat Alexa 568</td>
			<td>Thermo Fisher</td>
			<td>&#160;(used concentration 1:200)</td>
		</tr>
		<tr >
			<td >donkey-anti-rabbit Alexa 568</td>
			<td>Thermo Fisher</td>
			<td>&#160;(used concentration 1:200)</td>
		</tr>
		<tr >
			<td >Dulbecco&#39;s Modified Eagle&#39;s medium&#160;</td>
			<td>Lonza</td>
			<td></td>
		</tr>
		<tr >
			<td >EBM Medium</td>
			<td>Lonza</td>
			<td></td>
		</tr>
		<tr >
			<td >EBM-MV Single Quots kit</td>
			<td>Lonza</td>
			<td>containing hydrocortisone, hEGF, GA-1000, FBS and BBE</td>
		</tr>
		<tr >
			<td >Fetal Bovine Serum</td>
			<td>Lonza</td>
			<td></td>
		</tr>
		<tr >
			<td >Fetal Calf Serum</td>
			<td>Lonza</td>
			<td></td>
		</tr>
		<tr >
			<td >Fluorescent microscope</td>
			<td>Leica Microsystems GmbH</td>
			<td></td>
		</tr>
		<tr >
			<td >goat-anti-synaptopodin</td>
			<td>Santa Cruz</td>
			<td>Podocyte marker (used concentration 1:200)</td>
		</tr>
		<tr >
			<td >Hanks&#39;Balanced Salt Solution</td>
			<td>Gibco</td>
			<td></td>
		</tr>
		<tr >
			<td >ImageJ software</td>
			<td >FIJI 1.51n</td>
			<td></td>
		</tr>
		<tr >
			<td >petri dish</td>
			<td >Sarstedt</td>
			<td>size 100</td>
		</tr>
		<tr >
			<td >rabbit-anti-claudin1</td>
			<td>Abcam</td>
			<td>Parietal epithelial cell marker (used concentration 1:100)</td>
		</tr>
		<tr >
			<td >rabbit-anti-SSeCKS</td>
			<td>Roswell Park Comprehensive Cancer Center,Buffalo, NY, USA</td>
			<td>kindly provided by Dr. E. Gelman, Parietal epithelial cell marker</td>
		</tr>
		<tr >
			<td >rat-anti-CD44</td>
			<td>BD Pharmingen</td>
			<td>Parietal epithelial cell marker (used concentration 1:200)</td>
		</tr>
		<tr >
			<td >scalpel</td>
			<td >Dahlhausen</td>
			<td>size 10</td>
		</tr>
		<tr >
			<td >Sieves&#160;</td>
			<td >Endecotts Ltd</td>
			<td>size 300 &#181;m, 75 &#181;m, 53 &#181;m, steel</td>
		</tr>
		<tr >
			<td >syringe</td>
			<td >BD Plastipak</td>
			<td>size: 20 ml</td>
		</tr>
		<tr >
			<td >Ultra-Low Attachment Microplates&#160;</td>
			<td>Corning Costar</td>
			<td>6-well plates</td>
		</tr>
	</tbody>
</table>

glomerular outgrowth as an ex vivo assay to analyze pathways involved in parietal epithelial cell activation

Parietal epithelial cell (PEC) activation is one of the key factors involved in the development and progression of glomerulosclerosis. Inhibition of pathways involved in parietal epithelial cell activation could therefore be a tool to attenuate the progression of glomerular diseases. This article describes a method to culture and analyze parietal epithelial cell outgrowth of encapsulated glomeruli isolated from mouse kidney. After dissecting isolated mouse kidneys, the tissue is minced, and glomeruli are isolated by sieving. Encapsulated glomeruli are collected, and single glomeruli are cultured for 6 days to obtain glomerular outgrowth of parietal epithelial cells. During this period, parietal epithelial cell proliferation and migration can be analyzed by determining the cell number or the surface area of outgrowing cells. This assay can therefore be used as a tool to study the effects of an altered gene expression in transgenic- or knockout-mice or the effects of culture conditions on parietal epithelial cell growth characteristics and signaling. Using this method, important pathways involved in the process of parietal epithelial cell activation and consequently in glomerulosclerosis can be studied.

A systematic diagram of the method to perform the glomerular outgrowth assay is shown in Figure 1. Figure 2A-D shows glomerular outgrowths of encapsulated glomeruli at different time points as observed using light microscopy. Outgrowths are shown at day 2, 4 and 6 (Figure 2B-D) in culture after glomerulus isolation from mouse kidney. In order to validate that the out...

Watch this Scientific Journal Video about Glomerular Outgrowth as an Ex Vivo Assay to Analyze Pathways Involved in Parietal Epithelial Cell Activation at JoVE.com

Glomerular Outgrowth as an Ex Vivo Assay to Analyze Pathways Involved in Parietal Epithelial Cell Activation

This article describes a method for culturing and analyzing glomerular parietal epithelial cell outgrowths of encapsulated glomeruli isolated from mouse kidney. This method can be used to study pathways involved in parietal epithelial cell proliferation and migration.

Parietal epithelial cell (PEC) activation is one of the key factors involved in the development and progression of glomerulosclerosis. Inhibition of ...

Activation of parietal epithelial cells is in key factor in the development of scar tissue in the glomerulus of the kidney. Using this method, we can study the cellular processes involved in this activation and hopefully find treatment options to slow down or even stop disease progression. The main advantage of our technique is that we use primary cells that grow straight from the isolated glomeruli.After freeing the kidneys, as outlined in the text manuscript, hold the kidney with the surgical forceps and use another pair of forceps to pull off the renal capsules. After this, place the kidneys into the wells of a six well culture plate that each contain two milliliters of HBSS and place the culture plate on ice. First, transfer the kidneys to a 100 millimeter Petri dish and use two scalpels to mince them into small pieces that are approximately one to two millimeters.Keep the minced kidney pieces wet with HBSS. Next, place the kidney pieces on top of a 300 micrometer metal sieve and press the kidney through the sieve using the plunger from a 20 milliliters syringe. Repeatedly rinse the sieve with HBSS in between and use a serological pipette to collect the flow-through and transfer it to a clean Petri dish.Use a scalpel to scrape off everything that remains in the bottom of the sieve and transfer it to the collected kidney homogenate. Rinse the kidney homogenate through a 75 and 53 micrometer sieve with HBSS. Then, wash both sieves with HBSS to remove all of the smaller structures.Collect the kidney structures and material that remained in the sieves by washing the upper surface of each with DMEM supplemented with 20%fetal calf serum and transfer the material to the wells of a six well ultra-low attachment microplate. Bring the ultra-low attachment microplate to an inverted light microscope and use a 20 microliter pipette took collect single encapsulated and or decapsulated glomeruli. After catching a single glomerulus in the pipette tip, add fresh DMEM medium without collected kidney material into the same pipette tip to a volume of 20 microliters.Transfer the single glomerulus with the DMEM medium to the center of a well of a 24 well cell culture plate and incubate at 37 degrees Celsius with 5%carbon dioxide for three hours to allow attachment of the glomerulus to the center of the well. After the incubation, the glomerulus will be attached to the center of the well. Carefully add 500 microliters of endothelial Basal medium supplemented with a growth factor kit and an additional 5%FBS and 1%penicillin and streptomycin to each well.Culture the single glomeruli at 37 degrees Celsius with 5%carbon dioxide for six days. After six days of incubation, use a digital inverted light microscope to take images and analyze the glomerular outgrowth. Using an image analysis software, determine the surface area of glomerular outgrowth by opening one of the image files with a scale bar.Draw a straight line on the scale bar and click on Analyze, then measure to determine the distance in pixels. To determine the scale, click Analyze and Set Scale and input the known distance in pixels, the known distance, and the unit of length. Click Okay when finished.Click on Analyze and Set Measurements. Then, check the options for Area and Display Label. Click Okay when finished.Next, draw a freehand selection around the glomerular outgrowth. Click Analyze and measure to display a results table, which contains the surface area of outgrowth in the earlier determined scale. in this study, the glomerular parietal epithelial cell outgrowths of encapsulated glomeruli are isolated from mouse kidneys, cultured, and analyzed.Representative light microscopy images show the glomerular outgrowths at different time points during the culture after the glomerulus are isolated from the mouse kidneys. In order to validate that the outgrowing cells are parietal epithelial cells, decapsulated glomeruli are also isolated and cultured for six days. Decapsulated glomeruli show no cell outgrowth during this incubation period.Immunofluorescent staining is then performed for different parietal epithelial cell markers, photo site specific markers, as well as endothelial cell markers. The results validate that the outgrowing cells, indeed, are parietal epithelial cells. Glomeruli associated from CD44 knockout mice show a decreased number of outgrowing cells, as well as a decreased surface area of glomerular outgrowth compared to the glomeruli isolated from wild type mice.This suggests an important role for CD44 in parietal epithelial cell activation. Using this technique, it's also possible to study the effects on drugs on parietal epithelial cell activation. This can be done by treating the isolated glomeruli with the drug of your choice and to analyze the differences in cell growth and cell migration.

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