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,

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....

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 out.......

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 e.......

Materials

List of materials used in this article

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