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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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, PhAMexcised. 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 [PhAMexcised] 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 barrier1,2. 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....

Representative 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 dynamics21,22,23.

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

Disclosures

Authors have no competing financial interests to declare.

Acknowledgements

The authors thank Professor Cijiang He and Dr. Fu Jia for their insights in mice endothelial cell isolation and thank Professor Mone Zaidi for providing the PhAMexcised mice and valuable discussions. The authors would also like to acknowledge the Microscopy CORE at the Icahn School of Medicine at Mount Sinai and staff for the guidance we received. This work was supported by grants from National Institutes of Health grant R01DK097253 and Department of Defense CDMRP grant E01 W81XWH2010836 to I.S.D.

....

Materials

NameCompanyCatalog NumberComments
100 µm cell strainerFisher22-363-549
1ml Insulin SyringesBD329424
25G butterflyBD367298
3 mm cutting edge scissorsF.S.T15000-00
30ml syringeBD Biooscience309650
40 µm cell strainerFisher22-363-547
40 µm nylon mesh
Bonn ScissorsF.S.T14184-09
Bovine serum albuminFisherBP1600-100
CD31abcamab7388
Collagenase type ICorning354236
Collagenase type IISIGMAC6885125CDU/mg
Collagene type IVSIGMAC5533-5M
Dnase-IQiagen79254
Dynabeads 450Thermofisher Scientific14013
endothelial cells growth mediumLonzacc-3156
Extra fine graefe forcepsF.S.T11150-10
FBSGemini100-106Heat inactivated
FibronectinThermofisher33016015
Fine forcepsF.S.T DumontE6511
HBSSGIBCO14065-056
IFNgCell ScienceCRI001B
ImmortomouseJackson laboratory32619Tg(H2-K1-tsA58)6Kio/LicrmJ
L-Glutamine 100xThermofisher Scientific25030081
Magnetic particle concentratorThermofisher Scientific12320D
mitotrackerThermofisher ScientificM7512
PBS 1XCorning46-013-CM
penecillin streptomycin 100xThermofisher Scientific10378016
PhaM miceJackson laboratory18397B6;129S-Gt(ROSA)26Sortm1.1(CAG-COX8A/Dendra2)Dcc/J
Protease (10 mg/ml)SIGMAP6911
RPMIGIBCO3945
Sodium Pyruvate 100mMThermofisher Scientific11360070
Standard pattern forceps F.S.T11000-12
Surgical Scissors - Sharp-BluntF.S.T14008-14
synaptopodinSanta Cruzsc-515842
Trypsin 0.05%Thermofisher Scientific25300054

References

  1. Daehn, I. S., Duffield, J. S. The glomerular filtration barrier: A structural target for novel kidney therapies. Nature Reviews. Drug Discovery. 20 (10), 770-788 (2021).
  2. Fu, J., Lee, K., Chuang, P. Y., Liu, Z., He, J. C.

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