Name: glomerular outgrowth as an ex vivo assay to analyze pathways involved in parietal epithelial cell activation
Uploaded: 2020-08-19T13:00:00.000+00:00
Duration: 6 min 39 s
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>
	<li>Dissect whole mouse kidneys directly after sacrificing the mice. For this, perform a median laparotomy using abdominal scissors, cutting the skin and then the abdominal muscles. Remove the intestine and place it next to the mouse.</li>
	<li>Free the kidneys from connecting tissue and pull out the kidney using surgical forceps, cutting the renal artery, renal vein and ureter with scissors.</li>
	<li>Remove the renal capsules from the kidneys by holding the kidney with surgical forceps and pull off the capsule using another pair of forceps.</li>
	<li>Place the kidneys in a 6-well cell culture plate (2 kidneys/well) prepared with 2 mL of Hanks&#39; balanced salt solution (HBSS) per well and place on ice.</li>
</ol>
2. Isolation of glomeruli from mouse kidney
<ol>
	<li>Transfer the kidneys to a 100 mm Petri dish and mince the kidneys into small pieces of 1&#8722;2 mm using two scalpels. Keep the minced kidney pieces wet using 1&#8722;2 mL of HBSS.</li>
	<li>Place the minced kidney pieces on top of a 300 &#181;m metal sieve and press the kidney through the sieve using a plunger of a 20 mL syringe. Repeatedly rinse the sieve with HBSS in between and collect the flow-through in a clean Petri dish using a serological pipet. Collect also everything that remains/sticks to the bottom side of the sieve by scraping it off with the scalpel and transfer it to the collected flow-through (kidney homogenate).</li>
	<li>Rinse the kidney homogenate through a 75 &#181;m sieve with HBSS. Collect the flow-through and subsequently rinse this flow-through through a 53 &#181;m sieve. Wash both sieves using HBSS to remove all smaller structures. 
	NOTE: In this step the flow-though is only rinsed but not pressed through the 75 &#181;m and 53 &#181;m sieve. Washing with HBSS is necessary to remove debris and smaller structures on the sieves. Therefore normally 200&#8722;300 mL of HBSS are used in total.</li>
	<li>Collect the kidney structures/material that remain on the 75 &#181;m and 53 &#181;m sieve by washing the upper surface of the sieves with Dulbecco&#39;s modified Eagle&#39;s medium (DMEM) supplemented with 20% fetal calf serum (FCS) and transfer the material into a 6-well ultra-low attachment microplate. 
	NOTE: To wash the upper surface of the sieve, rinse the sieve in a tilted position (&#62;45&#176;) and collect the kidney material at the edge of the sieve. The collected material is enriched for encapsulated as well as decapsulated glomeruli, showing only a few tubular fragments. Encapsulated glomeruli are very sticky. To collect single glomeruli, it is therefore important to prevent glomeruli to adhere to the surface of a Petri dish or well plate. To avoid adherence, use medium with 20% FCS and use ultra-low attachment plates for this step.</li>
</ol>
3. Culturing of glomerular outgrowth
<ol>
	<li>Bring the ultra-low attachment microplate to an inverted light microscope and collect single encapsulated and/or decapsulated glomeruli with a 20 &#181;L pipette. Avoid pipetting other structures and debris. After collecting&#160;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 &#181;L.</li>
	<li>Transfer the single glomerulus with the 20 &#181;L DMEM medium to the center of a well of a 24-well cell culture plate and incubate for 3 h at 37 &#176;C and 5% (v/v) CO2 to allow attachment of the glomerulus to the center of the well. Carefully move the plate to avoid floating of the glomerulus to the boarders of the well.</li>
	<li>After 3 h incubation, the glomerulus is attached to the center of the well. Carefully add 500 &#181;L of endothelial basal medium (EBM) supplemented with a growth factor kit containing hydrocortisone, human endothelial growth factor, bovine brain extract and gentamicin sulfate-amphotericin B (Table of Materials) and additional 5% (v/v) FBS and 1% (v/v) penicillin/streptomycin (pen/strep) to each well.</li>
	<li>Culture the single glomeruli for 6 days at 37 &#176;C, 5% (v/v) CO2. 
	NOTE: Within 6 days the outgrowth consisting of parietal epithelial cells are formed. If one aims to test the effects of specific compounds or drugs on the parietal epithelial cells it should be added to the medium within this six-day period.</li>
</ol>
4. Analysis of parietal epithelial cell proliferation
<ol>
	<li>Analyze the glomerular outgrowth after 6 days. Take microscopic images using a digital inverted light microscope.</li>
	<li>Use an image analysis software (e.g., ImageJ/FIJI) to determine the surface area and the diameter of the glomerular outgrowth, and the number of outgrowing cells or outgrowing glomeruli.
	<ol>
		<li>To determine the surface area of glomerular outgrowth, open the tif. file of the glomerular outgrowth with scale bar in ImageJ. Draw a straight line on the scale bar and determine the distance in pixels by clicking on analyze and measure (e.g., 1 mm = 460 pixel).</li>
		<li>Determine the scale by clicking on analyze, set scale and type the known distance in pixel (e.g., 460), also type the known distance (e.g., 1) and the unit of length (e.g., 1 mm). Click ok.</li>
		<li>Determine which results will show up in the results table by clicking on analyze and then set measurements. To determine the surface area of glomerular outgrowth, activate the options area and display label. Click ok.</li>
		<li>To determine the surface area of glomerular outgrowth, draw a freehand selection around the glomerular outgrowth. Click on analyze and then measure, a result table will pop up in ImageJ showing the surface area of outgrowth in the earlier determined scale (see step 4.4, e.g., mm2).</li>
	</ol>
	</li>
</ol>
5. Characterization of the glomerular cell outgrowth
NOTE: To assess the cellular composition of the outgrowth, immunofluorescence staining for cell-specific markers are performed on the glomerular outgrowths at t = 6 days.
<ol>
	<li>Carefully remove the medium and gently wash the glomeruli twice using phosphate-buffered saline (PBS).</li>
	<li>Fixate for 10 min at room temperature using 2% (w/v) paraformaldehyde (PFA) supplemented with 4% (w/v) sucrose in PBS and carefully wash 2x using PBS.</li>
	<li>Incubate with the primary antibody with appropriate concentration (Table of Materials) diluted in PBS-ovine serum albumin (BSA) 1% (v/v) for 1 h at room temperature.</li>
	<li>Remove the antibody solution and carefully wash 3x with PBS.</li>
	<li>Incubate with secondary antibody (Table of Materials) diluted in PBS-BSA 1% (v/v) in the dark for 45 min at room temperature.</li>
	<li>Carefully wash 3x with PBS and mount using 1&#8722;2 drops of aqueous mounting medium with 4&#8242;,6-diamidino-2-phenylindole (DAPI) to visualize nuclei and cover the well with a round cover slip.</li>
	<li>Take microscopic images using a fluorescent microscope.</li>
</ol>

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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>

The authors have nothing to disclose.

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 epithelial cells under different experimental conditions. The outcomes that can be analyzed after 6 days in culture are, for instance, the surface area or diameter of the outgrowth or the number of outgrowing cells of a single capsulated glomerulus. Another application for this assay could be to study the effects of drugs that induce or inhibit molecular pathways that may be involved in parietal epithelial cell activation.
Immunofluorescence staining confirmed that the outgrowing cells after 6 days in culture are parietal epithelial cells as they stained positive for the parietal epithelial cell markers (CD44, claudin-1, SSeCKS) but did not express the podocyte-specific marker synaptopodin, nor the endothelial cell marker CD31. In line with the results of the staining, no outgrowing cells could be observed 6 days after isolation and culture of single decapsulated glomeruli, indicating that there is limited contamination of other glomerular cells in the cell outgrowth within this 6-day period. Another study also analyzed glomerular outgrowth and showed that the fast proliferating cells derived from the glomerular outgrowth are indeed descendent from parietal epithelial cells14.
The staining protocol that was used to analyze the marker expression of the outgrowing cells can also be adapted to test other molecules of interest. The immunostaining was performed inside the wells of the plates in which glomeruli were incubated for 6 days. These wells were not coated but glomeruli attached to the wells during the first 2&#8722;3 h incubation. Incubation on glass inserts or a chamber slide system which would result in better imaging was not possible as glomeruli did not attach completely to the surface and glomerular outgrowth was impaired. This specific protocol was set up recently to study the parietal epithelia cell outgrowth from glomeruli of healthy WT and cd44-/-&#160;mice11. Using this method, it was shown that CD44-deficient parietal epithelial cells show a decreased proliferation rate, which is also demonstrated in Figure 4. This method can also be used for mice of other strains and also for other genetically altered mice. In a previous study for instance, a comparable approach was used to analyze the effects of glucocorticoid receptor signaling15.
The use of this technique to isolate glomeruli from mice and analyze the cellular outgrowths has many advantages towards the use of immortalized parietal epithelial cell lines for the analysis of pathways involved in parietal epithelial cell activation or drugs that could influence the process of epithelial cell proliferation. First, in this method, primary cells are used which directly grow out of the glomerulus and are only 6 days in culture. Therefore, the parietal epithelial cells from glomerular outgrowths underwent fewer changes in phenotype compared to immortalized cell lines, which need additional growth passages to create the cell line16. Furthermore, the method described here can be used to compare the effect of specific gene knockout on parietal cell proliferation also for pathways that are difficult to knock out in cell lines because of impaired cell growth or efficiency of gene knockout using silencing methods.
To adapt the protocol to other animal models or to human kidney tissue, the size of the sieves should be optimized to obtain the best result. This is because the glomerular size differs between species and therefore the size of the sieves on which the glomeruli can be collected varies. Also, it is important to isolate intact encapsulated glomeruli for the purpose of this method. Therefore, the glomeruli should not be pressed but gently rinsed through the smaller sieves.
Another critical step in the protocol is the collection of the encapsulated glomeruli after sieving. Here, it is important to use medium with 20% FCS to avoid attachment of glomeruli to each other. In addition, the solution enriched with glomeruli should be directly transferred to ultra-low adhesion plates because, otherwise, glomeruli will directly attach to the surface of regular cell culture plates and even to the surface of plastic tubes which makes it difficult to capture and isolate single glomeruli.
After collection of single encapsulated glomeruli, these should be cultured 3 h in a small volume of culturing medium at the center of the well to allow adherence. Floating of the glomeruli towards the boarder of the wells should be avoided to optimize the read-out during image analysis.
To obtain the best results using the protocol described here, we would recommend to culture single glomeruli and perform the read-out at day 6. At this time point, a homogenous cellular outgrowth consisting of parietal epithelial cells can be observed. At later time points, glomerular outgrowth becomes phenotypically heterogenous indicating outgrowth of other cell types. Therefore, the protocol does not seem to be suitable for very long incubation times. One should keep in mind that incubation times for parietal epithelial cell outgrowth could vary between species or between different mouse strains. Therefore, culture times should be tested and optimized for each mouse strain or species. In addition, the origin of glomerular outgrowth should always be validated by staining for parietal epithelial cell-specific markers.

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><tbody><tr><td>24-well cell culture plate</td><td>Corning Costar</td><td></td><td></td></tr><tr><td>anti-CD31</td><td>BD Pharmingen</td><td>Endothelial cell marker (used concentration 1:200)</td><td></td></tr><tr><td>chicken-anti-rat Alexa 647</td><td>Thermo Fisher</td><td>&amp;nbsp;(used concentration 1:200)</td><td></td></tr><tr><td>DAPI-Fluoromount G</td><td>Southern Biotech</td><td>Mounting medium containing DAPI</td><td></td></tr><tr><td>Digital inverted light microscope</td><td>Westburg, EVOS fl microscope</td><td></td><td></td></tr><tr><td>donkey-anti-goat Alexa 568</td><td>Thermo Fisher</td><td>&amp;nbsp;(used concentration 1:200)</td><td></td></tr><tr><td>donkey-anti-rabbit Alexa 568</td><td>Thermo Fisher</td><td>&amp;nbsp;(used concentration 1:200)</td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle&#039;s medium&amp;nbsp;</td><td>Lonza</td><td></td><td></td></tr><tr><td>EBM Medium</td><td>Lonza</td><td></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><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Lonza</td><td></td><td></td></tr><tr><td>Fetal Calf Serum</td><td>Lonza</td><td></td><td></td></tr><tr><td>Fluorescent microscope</td><td>Leica Microsystems GmbH</td><td></td><td></td></tr><tr><td>goat-anti-synaptopodin</td><td>Santa Cruz</td><td>Podocyte marker (used concentration 1:200)</td><td></td></tr><tr><td>Hanks&#039;Balanced Salt Solution</td><td>Gibco</td><td></td><td></td></tr><tr><td>ImageJ software</td><td>FIJI 1.51n</td><td></td><td></td></tr><tr><td>petri dish</td><td>Sarstedt</td><td>size 100</td><td></td></tr><tr><td>rabbit-anti-claudin1</td><td>Abcam</td><td>Parietal epithelial cell marker (used concentration 1:100)</td><td></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><td></td></tr><tr><td>rat-anti-CD44</td><td>BD Pharmingen</td><td>Parietal epithelial cell marker (used concentration 1:200)</td><td></td></tr><tr><td>scalpel</td><td>Dahlhausen</td><td>size 10</td><td></td></tr><tr><td>Sieves&amp;nbsp;</td><td>Endecotts Ltd</td><td>size 300 &amp;micro;m, 75 &amp;micro;m, 53 &amp;micro;m, steel</td><td></td></tr><tr><td>syringe</td><td>BD Plastipak</td><td>size: 20 ml</td><td></td></tr><tr><td>Ultra-Low Attachment Microplates&amp;nbsp;</td><td>Corning Costar</td><td>6-well plates</td><td></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 outgrowing cells are parietal epithelial cells, decapsulated glomeruli have also been isolated and cultured for 6 days as shown in Figure 2E,F. Decapsulated glomeruli showed no cell outgrowth during the incubation period within 6 days. In Figure 3, immunofluorescence staining was performed for different parietal epithelial cell markers, podocyte specific markers as well as endothelial cell markers. The results validate that the outgrowing cells indeed are parietal epithelial cells. Figure 4 shows the outgrowth of isolated encapsulated glomeruli from cd44-/- vs WT mice after 6 days in culture. Glomeruli isolated from cd44-/-&#160;mice showed a decreased number of outgrowing cells as well as a decreased surface area of glomerular outgrowth compared to the glomeruli isolated from WT mice, suggesting an important role for CD44 in parietal epithelial cell activation as published previously11. In Figure 5, an example is given, in which the surface area of the outgrowing parietal epithelial cells is determined using ImageJ.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60324/60324fig1.jpg" /> 
Figure 1: Schematic overview of the method to perform a glomerular outgrowth assay to analyze parietal epithelial cell proliferation. (1) Kidneys are dissected from sacrificed mouse and minced into small pieces. (2) Kidney tissue is pressed through the 300 &#181;m sieve and rinsed through the 75 &#181;m and 53 &#181;m sieves. (3) Glomeruli that remain on top of the sieves are collected using medium + 20% (v/v) FCS and are transferred to an ultra-low attachment plate. (4) Single glomeruli are collected&#160;using an inverted light microscope and are transferred to 24-well culture plates. (5) After incubation at 37 &#176;C, 5% (v/v) CO2 for 6 days, glomerular outgrowth can be analyzed using a digital inverted light microscope. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60324/60324fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60324/60324fig2.jpg" /> 
Figure 2: Glomerular outgrowth of isolated encapsulated glomeruli from kidneys dissected from WT mice. The outgrowth of encapsulated glomeruli incubated at 37 &#176;C is shown at different time points: (A) 0 days, (B) 2 days, (C) 4 days, and (D) 6 days. Decapsulated glomeruli were also isolated and cultured at 37 &#176;C and microscopic images were taken at (E) day 0 and (F) day 6 showing no outgrowing cells. Scale bars: (A,E,F) 200 &#181;m, (B) 400 &#181;m, (C,D) 1000 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60324/60324fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60324/60324fig3v2.jpg" /> 
Figure 3: Outgrowing glomerular cells show expression of parietal epithelial cell marker. Immunofluorescence staining was performed at day 6 after isolation of single encapsulated glomeruli to characterize the outgrowing epithelial cells. Outgrowing cells stained positive for parietal epithelial cell markers: (A) CD44, (B) SSeCKS, and (C) claudin-1, but did not show expression of (D) the podocyte-specific marker synaptopodin or (E) the endothelial cell-specific marker CD31, which were exclusively localized inside the glomerulus. Scale bars: (A,B,D,E) 100&#181;m, (C) 50 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60324/60324fig3v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/60324/60324fig4.jpg" /> 
Figure 4: Glomerular outgrowth is impaired in glomeruli isolated from CD44 knockout mice. Encapsulated glomeruli were isolated from dissected kidneys of (A) WT mice and (B) cd44-/-&#160;mice. Microscopic pictures were taken after 6 days in culture using a digital inverted light microscope. The number of outgrowing parietal epithelial cells as well as the surface area of outgrowth was increased in the glomeruli from WT mice compared to cd44-/-&#160;mice, suggesting an important role for CD44 in parietal epithelial cell activation. Scale bars: 1000 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60324/60324fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/60324/60324fig5.jpg" /> 
Figure 5: An example of the analysis of the surface area of the glomerular outgrowth as a marker for parietal epithelial cell proliferation using ImageJ (FIJI). (A) Glomerular outgrowth of an encapsulated glomerulus of a WT mouse after 6 days in culture at 37 &#176;C. (B) First, the scale is determined to analyze the surface area in mm2. Here: 1 mm = 460 pixel. (C) After setting the scale, a selection line is drawn around the area of glomerular outgrowth. (D) This selected area can then be measured (surface area in this example = 2.235 mm2). Scale bars: 1000 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60324/60324fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

glomerular-outgrowth-as-an-ex-vivo-assay-to-analyze-pathways-involved

Nanyang Technological University

Research

JoVE Journal

Biology

5.5K Views.  Radboud University Medical Center. Activering van pariëtale epitheelcellen is een belangrijke factor in de ontwikkeling van littekenweefsel in de glomerulus van de nier. Met behulp van deze methode kunnen we de cellulaire processen bestuderen die betrokken zijn bij deze activering en hopelijk behandelopties vinden om de progressie van de ziekte te vertragen of zelfs te stoppen. Het belangrijkste voordeel van onze techniek is dat we primaire cellen gebruiken die rechtstreeks uit de geïsoleerde glomeruli groeien.Na het ...

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

Isolation of Glomeruli and In Vivo Labeling of Glomerular Cell Surface Proteins

Proteinuria results from the disruption of the glomerular filter that is composed of the fenestrated endothelium, glomerular basement membrane, and podocytes with their slit diaphragms. The delicate structure of the glomerular filter, especially the slit diaphragm, relies on the interplay of diverse cell surface proteins. Studying these cell surface proteins has so far been limited to in vitro studies or histologic analysis. Here, we present a murine in vivo biotinylation labeling method, which enables the study of glomerular cell surface proteins under physiologic and pathophysiologic conditions. This protocol contains information on how to perfuse mouse kidneys, isolate glomeruli, and perform endogenous immunoprecipitation of a protein of interest. Semi-quantitation of glomerular cell surface abundance is readily available with this novel method, and all proteins accessible to biotin perfusion and immunoprecipitation can be studied. In addition, isolation of glomeruli with or without biotinylation enables further analysis of glomerular RNA and protein as well as primary glomerular cell culture (i.e., primary podocyte cell culture).

Isolation of Glomeruli and In Vivo Labeling of Glomerular Cell Surface Proteins

Here we present a protocol for murine in vivo labeling of glomerular cell surface proteins with biotin. This protocol contains information on how to perfuse mouse kidneys, isolate glomeruli, and perform endogenous immunoprecipitation of the protein of interest.

Proteinuria results from the disruption of the glomerular filter that is composed of the fenestrated endothelium, glomerular basement membrane, and ...

Biochemistry

Epithelial Cell Repopulation and Preparation of Rodent Extracellular Matrix Scaffolds for Renal Tissue Development

This protocol details the generation of acellular, yet biofunctional, renal extracellular matrix (ECM) scaffolds that are useful as small-scale model substrates for organ-scale tissue development. Sprague Dawley rat kidneys are cannulated by inserting a catheter into the renal artery and perfused with a series of low-concentration detergents (Triton X-100 and sodium dodecyl sulfate (SDS)) over 26 hr to derive intact, whole-kidney scaffolds with intact perfusable vasculature, glomeruli, and renal tubules. Following decellularization, the renal scaffold is placed inside a custom-designed perfusion bioreactor vessel, and the catheterized renal artery is connected to a perfusion circuit consisting of: a peristaltic pump; tubing; and optional probes for pH, dissolved oxygen, and pressure. After sterilizing the scaffold with peracetic acid and ethanol, and balancing the pH (7.4), the kidney scaffold is prepared for seeding via perfusion of culture medium within a large-capacity incubator maintained at 37 &#176;C and 5% CO2. Forty million renal cortical tubular epithelial (RCTE) cells are injected through the renal artery, and rapidly perfused through the scaffold under high flow (25 ml/min) and pressure (~230 mmHg) for 15 min before reducing the flow to a physiological rate (4 ml/min). RCTE cells primarily populate the tubular ECM niche within the renal cortex, proliferate, and form tubular epithelial structures over seven days of perfusion culture. A 44 &#181;M resazurin solution in culture medium is perfused through the kidney for 1 hr during medium exchanges to provide a fluorometric, redox-based metabolic assessment of cell viability and proliferation during tubulogenesis. The kidney perfusion bioreactor permits non-invasive sampling of medium for biochemical assessment, and multiple inlet ports allow alternative retrograde seeding through the renal vein or ureter. These protocols can be used to recellularize kidney scaffolds with a variety of cell types, including vascular endothelial, tubular epithelial, and stromal fibroblasts, for rapid evaluation within this system.

This protocol describes decellularization of Sprague Dawley rat kidneys by antegrade perfusion of detergents through the vasculature, producing acellular renal extracellular matrices that serve as templates for repopulation with human renal epithelial cells. Recellularization and use of the resazurin perfusion assay to monitor growth is performed within specially-designed perfusion bioreactors.

This protocol details the generation of acellular, yet biofunctional, renal extracellular matrix (ECM) scaffolds that are useful as small-scale model ...

Bioengineering

A Simplified Method for Generating Kidney Organoids from Human Pluripotent Stem Cells

Kidney organoids generated from hPSCs have provided an unlimited source of renal tissue. Human kidney organoids are an invaluable tool for studying kidney disease and injury, developing cell-based therapies, and testing new therapeutics. For such applications, large numbers of uniform organoids and highly reproducible assays are needed. We have built upon our previously published kidney organoid protocol to improve the overall health of the organoids. This simple, robust 3D protocol involves the formation of uniform embryoid bodies in minimum component medium containing lipids, insulin-transferrin-selenium-ethanolamine supplement and polyvinyl alcohol with GSK3 inhibitor (CHIR99021) for 3 days, followed by culture in knock-out serum replacement (KOSR)-containing medium. In addition, agitating assays allows for reduction in clumping of the embryoid bodies and maintaining a uniform size, which is important for reducing variability between organoids. Overall, the protocol provides a fast, efficient, and cost-effective method for generating large quantities of kidney organoids.

Here we describe a protocol to generate kidney organoids from human pluripotent stem cells (hPSCs). This protocol generates kidney organoids within two weeks. The resulting kidney organoids can be cultured in large-scale spinner flasks or multi-well magnetic stir plates for parallel drug-testing approaches.

Kidney organoids generated from hPSCs have provided an unlimited source of renal tissue. Human kidney organoids are an invaluable tool for studying kidney ...

Developmental Biology

Renal Cancer and Healthy Tissue Co-Culture Using Microphysiological Systems

Microfluidic Co-culture of Renal Healthy and Tumor Epithelium to Model Kidney Cancer Progression

Microphysiological systems (MPS) have enabled the introduction of more complex and relevant physiological elements into in vitro models, recreating intricate morphological features in three-dimensional environments with dynamic interactions lacking in conventional models. We implemented a renal cell carcinoma (RCC) co-culture model to recreate the cross-talk between healthy and malignant renal tissue. 
This model is based on the referenced multi-organ platform and consists of co-culturing a reconstructed renal proximal tubule with RCC spheroids. Custom-designed 3D-printed chambers were used to culture human renal epithelial proximal tubule cells (RPTEC) and facilitate their self-assembly into a renal tubule contained in a collagen type I matrix. Caki-1 RCC cells were embedded in an agar collagen matrix, subsequently forming cancer spheroids. Both collagen and agar/collagen gels were optimized to maintain their integrity during cyclic perfusion and withstand shear stress during a minimum culture period of 7 days. 
The gels also enable adequate nutrient supply and cell secretions. Moreover, the agar/collagen gels limit the overproliferation of RCC cells, ensuring relatively homogeneous spheroid size. The MPS chip microfluidic circuits comprise two independent culture chambers with the size of a standard 96-microplate well. The renal tubule and RCC gels populate separate chambers and share the same culture media, which is recirculated approximately twice per minute. Under these conditions, we observed upregulation of immune factor expression and secretion in the renal tubules (interleukin-8 and tumor necrosis factor-alpha). The renal tubules also shift their metabolic activity towards glycolysis under the influence of RCC. This novel approach demonstrates that a co-culture-based MPS can amplify the complexity of RCC in vitro and be employed to study the impact of cancer on non-tumor cells.

Here, we present protocols that detail instructions for implementing 3D cell cultures using collagen and collagen-agarose matrixes in a microphysiological system. These protocols support renal proximal tubule and renal cell carcinoma spheroids co-culture, simulating in vivo conditions and enabling advanced investigation of kidney cancer cell interactions.

Microphysiological systems (MPS) have enabled the introduction of more complex and relevant physiological elements into in vitro models, recreating ...

Cancer Research

Isogenic Kidney Glomerulus Chip Engineered from Human Induced Pluripotent Stem Cells

Chronic kidney disease (CKD) affects 15% of the U.S. adult population, but the establishment of targeted therapies has been limited by the lack of functional models that can accurately predict human biological responses and nephrotoxicity. Advancements in kidney precision medicine could help overcome these limitations. However, previously established in vitro models of the human kidney glomerulus-the primary site for blood filtration and a key target of many diseases and drug toxicities-typically employ heterogeneous cell populations with limited functional characteristics and unmatched genetic backgrounds. These characteristics significantly limit their application for patient-specific disease modeling and therapeutic discovery. 
This paper presents a protocol that integrates human induced pluripotent stem (iPS) cell-derived glomerular epithelium (podocytes) and vascular endothelium from a single patient to engineer an isogenic and vascularized microfluidic kidney glomerulus chip. The resulting glomerulus chip is comprised of stem cell-derived endothelial and epithelial cell layers that express lineage-specific markers, produce basement membrane proteins, and form a tissue-tissue interface resembling the kidney's glomerular filtration barrier. The engineered glomerulus chip selectively filters molecules and recapitulates drug-induced kidney injury. The ability to reconstitute the structure and function of the kidney glomerulus using isogenic cell types creates the opportunity to model kidney disease with patient specificity and advance the utility of organs-on-chips for kidney precision medicine and related applications.

Presented here is a protocol to engineer a personalized organ-on-a-chip system that recapitulates the structure and function of the kidney glomerular filtration barrier by integrating genetically matched epithelial and vascular endothelial cells differentiated from human induced pluripotent stem cells. This bioengineered system can advance kidney precision medicine and related applications.

Chronic kidney disease (CKD) affects 15% of the U.S. adult population, but the establishment of targeted therapies has been limited by the lack of ...

Assessment of Kidney Function in Mouse Models of Glomerular Disease

The use of murine models to mimic human kidney disease is becoming increasingly common. Our research is focused on the assessment of glomerular function in diabetic nephropathy and podocyte-specific VEGF-A knock-out mice; therefore, this protocol describes the full kidney work-up used in our lab to assess these mouse models of glomerular disease, enabling a vast amount of information regarding kidney and glomerular function to be obtained from a single mouse. In comparison to alternative methods presented in the literature to assess glomerular function, the use of the method outlined in this paper enables the glomerular phenotype to be fully evaluated from multiple aspects. By using this method, the researcher can determine the kidney phenotype of the model and assess the mechanism as to why the phenotype develops. This vital information on the mechanism of disease is required when examining potential therapeutic avenues in these models. The methods allow for detailed functional assessment of the glomerular filtration barrier through measurement of the urinary albumin creatinine ratio and individual glomerular water permeability, as well as both structural and ultra-structural examination using the Periodic Acid Schiff stain and electron microscopy. Furthermore, analysis of the genes dysregulated at the mRNA and protein level enables mechanistic analysis of glomerular function. This protocol outlines the generic but adaptable methods that can be applied to all mouse models of glomerular disease.

This protocol describes a full kidney work-up that should be carried out in mouse models of glomerular disease. The methods allow for detailed functional, structural, and mechanistic analysis of glomerular function, which can be applied to all mouse models of glomerular disease.

The use of murine models to mimic human kidney disease is becoming increasingly common. Our research is focused on the assessment of glomerular function ...

Medicine

An Efficient Sieving Method to Isolate Intact Glomeruli from Adult Rat Kidney

Preservation of glomerular structure and function is pivotal in the prevention of glomerulonephritis, a category of kidney disease characterized by proteinuria which can eventually lead to chronic and end-stage renal disease. The glomerulus is a complex apparatus responsible for the filtration of plasma from the body. In disease, structural integrity is lost and allows for the abnormal leakage of plasma contents into the urine. A method to isolate and examine glomeruli in culture is critical for the study of these diseases. In this protocol, an efficient method of retrieving intact glomeruli from adult rat kidneys while conserving structural and morphological characteristics is described. This process is capable of generating high yields of glomeruli per kidney with minimal contamination from other nephron segments. With these glomeruli, injury conditions can be mimicked by incubating them with a variety of chemical toxins, including protamine sulfate, which causes foot process effacement and proteinuria in animal models. Degree of injury can be assessed using transmission electron microscopy, immunofluorescence staining, and western blotting. Nephrin and Wilms Tumor 1 (WT1) levels can also be assessed from these cultures. Due to the ease and flexibility of this protocol, the isolated glomeruli can be utilized as described or in a way that best suits the needs of the researcher to help better study glomerular health and structure in diseased states.

The main focus of this protocol is to efficiently isolate viable primary glomeruli cultures with minimal contaminants for use in a variety of downstream applications. The isolated glomeruli retain structural relationships between component cell types and can be cultured ex vivo for a short time.

Preservation of glomerular structure and function is pivotal in the prevention of glomerulonephritis, a category of kidney disease characterized by ...

Manipulating the Murine Lacrimal Gland

The lacrimal gland (LG) secretes aqueous tears necessary for maintaining the structure and function of the cornea, a transparent tissue essential for vision. In the human a single LG resides in the orbit above the lateral end of each eye delivering tears to the ocular surface through 3 - 5 ducts. The mouse has three pairs of major ocular glands, the most studied of which is the exorbital lacrimal gland (LG) located anterior and ventral to the ear. Similar to other glandular organs, the LG develops through the process of epithelial branching morphogenesis in which a single epithelial bud within a condensed mesenchyme undergoes multiple rounds of bud and duct formation to form an intricate interconnected network of secretory acini and ducts. This elaborate process has been well documented in many other epithelial organs such as the pancreas and salivary gland. However, the LG has been much less explored and the mechanisms controlling morphogenesis are poorly understood. We suspect that this under-representation as a model system is a consequence of the difficulties associated with finding, dissecting and culturing the LG. Thus, here we describe dissection techniques for harvesting embryonic and post-natal LG and methods for ex vivo culture of the tissue.

The lacrimal gland (LG) is a branching organ that produces the aqueous components of tears necessary for maintaining vision and ocular health. Here we describe murine LG dissection and ex vivo culture techniques to decipher signaling pathways involved in LG development.

The lacrimal gland (LG) secretes aqueous tears necessary for maintaining the structure and function of the cornea, a transparent tissue essential for ...

An Ex Vivo Choroid Sprouting Assay of Ocular Microvascular Angiogenesis

Pathological choroidal angiogenesis, a salient feature of age-related macular degeneration, leads to vision impairment and blindness. Endothelial cell (EC) proliferation assays using human retinal microvascular endothelial cells (HRMECs) or isolated primary retinal ECs are widely used in vitro models to study retinal angiogenesis. However, isolating pure murine retinal endothelial cells is technically challenging and retinal ECs may have different proliferation responses than choroidal endothelial cells and different cell/cell interactions. A highly reproducible ex vivo choroidal sprouting assay as a model of choroidal microvascular proliferation was developed. This model includes the interaction between choroid vasculature (EC, macrophages, pericytes) and retinal pigment epithelium (RPE). Mouse RPE/choroid/scleral explants are isolated and incubated in growth-factor-reduced basal membrane extract (BME) (day 0). Medium is changed every other day and choroid sprouting is quantified at day 6. The images of individual choroid explant are taken with an inverted phase microscope and the sprouting area is quantified using a semi-automated macro plug-in to the ImageJ software developed in this lab. This reproducible ex vivo choroidal sprouting assay can be used to assess compounds for potential treatment and for microvascular disease research to assess pathways involved in choroidal micro vessel proliferation using wild type and genetically modified mouse tissue.

This protocol presents choroid sprouting assay, an ex vivo model of microvascular proliferation. This assay can be used to assess pathways involved in proliferating choroidal micro vessels and assess drug treatments using wild type and genetically modified mouse tissue.

Pathological choroidal angiogenesis, a salient feature of age-related macular degeneration, leads to vision impairment and blindness. Endothelial cell ...

Using Skin Biopsies to Generate Patient-Derived Podocytes

Generation of Patient-Derived Podocytes from Skin Biopsies

Podocytes are epithelial cells sitting on the urinary site of the glomerular filtration barrier that contribute to the selective filter function of the glomerulus. Mutations in podocyte-specific genes can cause focal segmental glomerulosclerosis (FSGS), and podocytes are also affected in many other primary and secondary nephropathies. Due to their differentiated nature, primary cell culture models are limited for podocytes.&#160;Therefore, commonly conditionally immortalized cells are used. However, these conditionally immortalized podocytes (ciPodocytes) have several limitations: the cells can dedifferentiate in culture, especially when they reach confluency, and several podocyte-specific markers are either only slightly or not expressed at all. This brings the use of ciPodocytes and their applicability for physiological, pathophysiological, and clinical reach into question. Here, we describe a protocol for the generation of human podocytes-including patient-specific podocytes-from a skin punch biopsy by episomal reprogramming of dermal fibroblasts into hiPSCs and subsequent differentiation into podocytes. These podocytes resemble in vivo podocytes much better in terms of morphological characteristics, like the development of foot processes and the expression of the podocyte-specific marker. Finally, yet importantly, these cells maintain patients&#39; mutations, resulting in an improved ex vivo model to study podocyte diseases and potential therapeutic substances in an individualized approach.

Author Spotlight: Generation of Patient-Derived Podocytes from Skin Biopsies  

This manuscript describes a two-step protocol to generate patient-specific podocytes from dermal fibroblasts via episomal reprogramming into human-induced pluripotent stem cells (hiPSCs) and subsequent differentiation into podocytes.

Podocytes are epithelial cells sitting on the urinary site of the glomerular filtration barrier that contribute to the selective filter function of the ...

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

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