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 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.
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'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 isolated 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.
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 12−16 weeks. Both male and female mice were used. All mice were on the C57Bl/6 background.
1. Mouse kidney dissection
2. Isolation of glomeruli from mouse kidney
3. Culturing of glomerular outgrowth
4. Analysis of parietal epithelial cell proliferation
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.
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-/- 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.
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 µm sieve and rinsed through the 75 µm and 53 µm 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 using an inverted light microscope and are transferred to 24-well culture plates. (5) After incubation at 37 °C, 5% (v/v) CO2 for 6 days, glomerular outgrowth can be analyzed using a digital inverted light microscope. Please click here to view a larger version of this figure.
Figure 2: Glomerular outgrowth of isolated encapsulated glomeruli from kidneys dissected from WT mice. The outgrowth of encapsulated glomeruli incubated at 37 °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 °C and microscopic images were taken at (E) day 0 and (F) day 6 showing no outgrowing cells. Scale bars: (A,E,F) 200 µm, (B) 400 µm, (C,D) 1000 µm. Please click here to view a larger version of this figure.
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) 100µm, (C) 50 µm. Please click here to view a larger version of this figure.
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-/- 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-/- mice, suggesting an important role for CD44 in parietal epithelial cell activation. Scale bars: 1000 µm. Please click here to view a larger version of this figure.
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 °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 µm. Please click here to view a larger version of this figure.
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 2−3 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-/- 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.
The authors have nothing to disclose.
This research was supported by Dutch Kidney foundation (grant 14A3D104) and The Netherlands Organization for Scientific Research (NWO VIDI grant: 016.156.363).
24-well cell culture plate | Corning Costar | |
anti-CD31 | BD Pharmingen | Endothelial cell marker (used concentration 1:200) |
chicken-anti-rat Alexa 647 | Thermo Fisher | (used concentration 1:200) |
DAPI-Fluoromount G | Southern Biotech | Mounting medium containing DAPI |
Digital inverted light microscope | Westburg, EVOS fl microscope | |
donkey-anti-goat Alexa 568 | Thermo Fisher | (used concentration 1:200) |
donkey-anti-rabbit Alexa 568 | Thermo Fisher | (used concentration 1:200) |
Dulbecco's Modified Eagle's medium | Lonza | |
EBM Medium | Lonza | |
EBM-MV Single Quots kit | Lonza | containing hydrocortisone, hEGF, GA-1000, FBS and BBE |
Fetal Bovine Serum | Lonza | |
Fetal Calf Serum | Lonza | |
Fluorescent microscope | Leica Microsystems GmbH | |
goat-anti-synaptopodin | Santa Cruz | Podocyte marker (used concentration 1:200) |
Hanks'Balanced Salt Solution | Gibco | |
ImageJ software | FIJI 1.51n | |
petri dish | Sarstedt | size 100 |
rabbit-anti-claudin1 | Abcam | Parietal epithelial cell marker (used concentration 1:100) |
rabbit-anti-SSeCKS | Roswell Park Comprehensive Cancer Center,Buffalo, NY, USA | kindly provided by Dr. E. Gelman, Parietal epithelial cell marker |
rat-anti-CD44 | BD Pharmingen | Parietal epithelial cell marker (used concentration 1:200) |
scalpel | Dahlhausen | size 10 |
Sieves | Endecotts Ltd | size 300 µm, 75 µm, 53 µm, steel |
syringe | BD Plastipak | size: 20 ml |
Ultra-Low Attachment Microplates | Corning Costar | 6-well plates |