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

Activation of parietal epithelial cells is in key factor in the development of scar tissue in the glomerulus of the kidney. Using this method, we can study the cellular processes involved in this activation and hopefully find treatment options to slow down or even stop disease progression. The main advantage of our technique is that we use primary cells that grow straight from the isolated glomeruli.

After freeing the kidneys, as outlined in the text manuscript, hold the kidney with the surgical forceps and use another pair of forceps to pull off the renal capsules. After this, place the kidneys into the wells of a six well culture plate that each contain two milliliters of HBSS and place the culture plate on ice. First, transfer the kidneys to a 100 millimeter Petri dish and use two scalpels to mince them into small pieces that are approximately one to two millimeters.

Keep the minced kidney pieces wet with HBSS. Next, place the kidney pieces on top of a 300 micrometer metal sieve and press the kidney through the sieve using the plunger from a 20 milliliters syringe. Repeatedly rinse the sieve with HBSS in between and use a serological pipette to collect the flow-through and transfer it to a clean Petri dish.

Use a scalpel to scrape off everything that remains in the bottom of the sieve and transfer it to the collected kidney homogenate. Rinse the kidney homogenate through a 75 and 53 micrometer sieve with HBSS. Then, wash both sieves with HBSS to remove all of the smaller structures.

Collect the kidney structures and material that remained in the sieves by washing the upper surface of each with DMEM supplemented with 20%fetal calf serum and transfer the material to the wells of a six well ultra-low attachment microplate. Bring the ultra-low attachment microplate to an inverted light microscope and use a 20 microliter pipette took collect single encapsulated and or decapsulated glomeruli. After catching a single glomerulus in the pipette tip, add fresh DMEM medium without collected kidney material into the same pipette tip to a volume of 20 microliters.

Transfer the single glomerulus with the DMEM medium to the center of a well of a 24 well cell culture plate and incubate at 37 degrees Celsius with 5%carbon dioxide for three hours to allow attachment of the glomerulus to the center of the well. After the incubation, the glomerulus will be attached to the center of the well. Carefully add 500 microliters of endothelial Basal medium supplemented with a growth factor kit and an additional 5%FBS and 1%penicillin and streptomycin to each well.

Culture the single glomeruli at 37 degrees Celsius with 5%carbon dioxide for six days. After six days of incubation, use a digital inverted light microscope to take images and analyze the glomerular outgrowth. Using an image analysis software, determine the surface area of glomerular outgrowth by opening one of the image files with a scale bar.

Draw a straight line on the scale bar and click on Analyze, then measure to determine the distance in pixels. To determine the scale, click Analyze and Set Scale and input the known distance in pixels, the known distance, and the unit of length. Click Okay when finished.

Click on Analyze and Set Measurements. Then, check the options for Area and Display Label. Click Okay when finished.

Next, draw a freehand selection around the glomerular outgrowth. Click Analyze and measure to display a results table, which contains the surface area of outgrowth in the earlier determined scale. in this study, the glomerular parietal epithelial cell outgrowths of encapsulated glomeruli are isolated from mouse kidneys, cultured, and analyzed.

Representative light microscopy images show the glomerular outgrowths at different time points during the culture after the glomerulus are isolated from the mouse kidneys. In order to validate that the outgrowing cells are parietal epithelial cells, decapsulated glomeruli are also isolated and cultured for six days. Decapsulated glomeruli show no cell outgrowth during this incubation period.

Immunofluorescent staining is then performed for different parietal epithelial cell markers, photo site specific markers, as well as endothelial cell markers. The results validate that the outgrowing cells, indeed, are parietal epithelial cells. Glomeruli associated from CD44 knockout mice show a decreased number of outgrowing cells, as well as a decreased surface area of glomerular outgrowth compared to the glomeruli isolated from wild type mice.

This suggests an important role for CD44 in parietal epithelial cell activation. Using this technique, it's also possible to study the effects on drugs on parietal epithelial cell activation. This can be done by treating the isolated glomeruli with the drug of your choice and to analyze the differences in cell growth and cell migration.