Isolation of Conditionally Immortalized Mouse Glomerular Endothelial Cells with Fluorescent Mitochondria

Summary

The article describes the method for isolating conditionally immortalized glomerular endothelial cells from the kidneys of transgenic mice expressing the thermolabile simian virus 40 and photo-activatable mitochondria, PhAM^excised. We describe the procedure for glomeruli isolation from whole kidneys using beads, digestion steps, seeding, and culturing of GECs-CD31 positive.

Abstract

Glomerular endothelial cell (GEC) dysfunction can initiate and contribute to glomerular filtration barrier breakdown. Increased mitochondrial oxidative stress has been suggested as a mechanism resulting in GEC dysfunction in the pathogenesis of some glomerular diseases. Historically the isolation of GECs from in vivo models has been notoriously challenging due to difficulties in isolating pure cultures from glomeruli. GECs have complex growth requirements in vitro and a very limited lifespan. Here, we describe the procedure for isolating and culturing conditionally immortalized GECs with fluorescent mitochondria, enabling the tracking of mitochondrial fission and fusion events. GECs were isolated from the kidneys of a double transgenic mouse expressing the thermolabile SV40 TAg (from the Immortomouse), conditionally promoting proliferation and suppressing cell differentiation, and a photo-convertible fluorescent protein (Dendra2) in all mitochondria (from the photo-activatable mitochondria [PhAM^excised] mouse). The stable cell line generated allows for cell differentiation after inactivation of the immortalizing SV40 TAg gene and photo-activation of a subset of mitochondria causing a switch in fluorescence from green to red. The use of mitoDendra2-GECs allows for live imaging of fluorescent mitochondria's distribution, fusion, and fission events without staining the cells.

Introduction

The glomerulus is critical for blood filtration by restricting the passage of large molecules through the glomerular filtration barrier^1,2. The glomerulus contains four cell types: parietal epithelial cells, podocytes (visceral epithelial cells), glomerular endothelial cells (GEC), and mesangial cells³. The glomerular endothelium is characterized by a unique vascular structure, as per the presence of fenestrae required for large filtration volumes⁴. The apical surface of the glomerular endothelium is covered with a negatively charged glycocalyx layer and a coat c....

Protocol

All animal procedures described here were approved by the IACUC at Icahn School of Medicine at Mount Sinai. We used three male mice (H-2Kb-tsA58 transgenic mice with photo-activatable mitochondria [PhAM]) purchased from Jackson lab and kept on a normal chow diet.

1. Working conditions and preparations

1. Clean the working space inside the laminar flow hood using 70% ethanol and UV-C light.
2. Prepare the bead's washing buffers. Make buffer-1 using 0.1 M sodium phosphate at pH 7.4-8.0, buffer-2 using 1x phosphate-buffered saline (PBS) without calcium and magnesium supplemented with 0.1% bovine serum antig....

Results

In this article, a detailed protocol for the isolation of conditionally immortalized glomerular endothelial cells with stable fluorescent mitochondria (mitoDendra2-GECs) is described (Figure 1). The use of young 6-10-week-old mice is essential to obtain a substantial number of healthy cells. After 3 days of culture, cells start growing slowly from the isolated glomeruli, as shown in Figure 3G. After 7 days, cells are heterogeneous, showing other glomerular cell .......

Discussion

Mitochondria are critical for cellular metabolism, homeostasis, and stress responses, and their dysfunction is linked to many diseases, including kidney disease. Mitochondria have a role in the pathologic generation of excessive reactive oxygen species (ROS), the regulation of intracellular calcium levels, cell death pathways, and cytoskeletal dynamics^21,22,23.

The isolation of murine GECs is challeng.......

Materials

Name	Company	Catalog Number	Comments
100 µm cell strainer	Fisher	22-363-549
1ml Insulin Syringes	BD	329424
25G butterfly	BD	367298
3 mm cutting edge scissors	F.S.T	15000-00
30ml syringe	BD Biooscience	309650
40 µm cell strainer	Fisher	22-363-547
40 µm nylon mesh
Bonn Scissors	F.S.T	14184-09
Bovine serum albumin	Fisher	BP1600-100
CD31	abcam	ab7388
Collagenase type I	Corning	354236
Collagenase type II	SIGMA	C6885	125CDU/mg
Collagene type IV	SIGMA	C5533-5M
Dnase-I	Qiagen	79254
Dynabeads 450	Thermofisher Scientific	14013
endothelial cells growth medium	Lonza	cc-3156
Extra fine graefe forceps	F.S.T	11150-10
FBS	Gemini	100-106	Heat inactivated
Fibronectin	Thermofisher	33016015
Fine forceps	F.S.T Dumont	E6511
HBSS	GIBCO	14065-056
IFNg	Cell Science	CRI001B
Immortomouse	Jackson laboratory	32619	Tg(H2-K1-tsA58)6Kio/LicrmJ
L-Glutamine 100x	Thermofisher Scientific	25030081
Magnetic particle concentrator	Thermofisher Scientific	12320D
mitotracker	Thermofisher Scientific	M7512
PBS 1X	Corning	46-013-CM
penecillin streptomycin 100x	Thermofisher Scientific	10378016
PhaM mice	Jackson laboratory	18397	B6;129S-Gt(ROSA)26Sortm1.1(CAG-COX8A/Dendra2)Dcc/J
Protease (10 mg/ml)	SIGMA	P6911
RPMI	GIBCO	3945
Sodium Pyruvate 100mM	Thermofisher Scientific	11360070
Standard pattern forceps	F.S.T	11000-12
Surgical Scissors - Sharp-Blunt	F.S.T	14008-14
synaptopodin	Santa Cruz	sc-515842
Trypsin 0.05%	Thermofisher Scientific	25300054