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

Summary

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.

Abstract

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.

Introduction

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 cause^1,2,3,4.

Specifically, it was shown that glomerulosclerotic lesions are mainly composed of activated parietal epithelial cells^5,6. 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 lesions^5,7,8. 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 diseases^1,2,3,4,10.

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 diseases^11,12.

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.

Protocol

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

1. Sacrifice healthy WT mice or genetically altered mice by cervical dislocation.
2. 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.
3. Free the kidneys from connecting tissue and pull out the kidney using surgical forceps, cutting the renal artery, renal vein and ureter with scissors.
4. Remove the renal capsules from the kidneys by holding the kidney with surgical forceps and pull off the capsule using another pair of forceps.
5. Place the kidneys in a 6-well cell culture plate (2 kidneys/well) prepared with 2 mL of Hanks' balanced salt solution (HBSS) per well and place on ice.

2. Isolation of glomeruli from mouse kidney

1. Transfer the kidneys to a 100 mm Petri dish and mince the kidneys into small pieces of 1−2 mm using two scalpels. Keep the minced kidney pieces wet using 1−2 mL of HBSS.
2. Place the minced kidney pieces on top of a 300 µm 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).
3. Rinse the kidney homogenate through a 75 µm sieve with HBSS. Collect the flow-through and subsequently rinse this flow-through through a 53 µm 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 µm and 53 µm sieve. Washing with HBSS is necessary to remove debris and smaller structures on the sieves. Therefore normally 200−300 mL of HBSS are used in total.
4. Collect the kidney structures/material that remain on the 75 µm and 53 µm sieve by washing the upper surface of the sieves with Dulbecco's modified Eagle'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 (>45°) 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.

3. Culturing of glomerular outgrowth

1. Bring the ultra-low attachment microplate to an inverted light microscope and collect single encapsulated and/or decapsulated glomeruli with a 20 µL pipette. Avoid pipetting other structures and debris. After collecting 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 µL.
2. Transfer the single glomerulus with the 20 µL DMEM medium to the center of a well of a 24-well cell culture plate and incubate for 3 h at 37 °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.
3. After 3 h incubation, the glomerulus is attached to the center of the well. Carefully add 500 µL 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.
4. Culture the single glomeruli for 6 days at 37 °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.

4. Analysis of parietal epithelial cell proliferation

1. Analyze the glomerular outgrowth after 6 days. Take microscopic images using a digital inverted light microscope.
2. 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.
   1. 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).
   2. 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.
   3. 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.
   4. 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).

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.

1. Carefully remove the medium and gently wash the glomeruli twice using phosphate-buffered saline (PBS).
2. 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.
3. 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.
4. Remove the antibody solution and carefully wash 3x with PBS.
5. Incubate with secondary antibody (Table of Materials) diluted in PBS-BSA 1% (v/v) in the dark for 45 min at room temperature.
6. Carefully wash 3x with PBS and mount using 1−2 drops of aqueous mounting medium with 4′,6-diamidino-2-phenylindole (DAPI) to visualize nuclei and cover the well with a round cover slip.
7. Take microscopic images using a fluorescent microscope.

Representative Results

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.

Discussion

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.

Materials

Name	Company	Catalog Number	Comments
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