A novel method for isolating podocytes using magnetic activated cell sorting.
BACKGROUND
A large body of accumulated data has now revealed that podocytes play a major role in the development of proteinuria. However, the mechanisms of podocyte injury, leading to foot process effacement and proteinuria, are still unclear partly due to the current lack of an appropriate strategy for preparing podocytes. In this study, we have developed a novel method of rapid isolation of podocytes from mice using magnetic activated cell sorting with an anti-nephrin antibody.


METHODS
After endothelial cell depletion using anti-CD31 antibody, nephrin-positive cells were prepared from mouse kidneys using magnetic activated cell sorting with polyclonal rabbit anti-nephrin antibody. Purity of the positively sorted cells was determined by confocal microscopy and fluorescence-activated cell sorting (FACS) analysis. Expression profiles of podocyte-specific molecules in the sorted fractions were characterized by qualitative PCR and immunoblot analysis.


RESULTS
Nephrin-positive cells, isolated from mouse kidneys within 6 h, showed dual positivity for synaptopodin and rabbit IgG on confocal microscopy. FACS analysis revealed that the purity of the positively sorted fractions was ∼75%. The nephrin-positive cells sorted by this approach showed a significantly higher expression of podocyte-specific molecules compared with nephrin-negative fractions.


CONCLUSIONS
These data strongly suggest that our novel method for isolating podocytes has great utility for various downstream applications such as genomic analysis, proteomics and transcriptomics to elucidate molecular profiling of podocyte biology in vivo compared with conventional methods as our approach requires only several hours to complete and no tissue culture.
Murakami
Ayumi
A
Department of Pathology, Yokohama City University School of Medicine 3-9 Fukuura, Kanazawa-ku, Yokohama 236-0004, Japan.
Oshiro
Hisashi
H
Kanzaki
Seiichi
S
Yamaguchi
Akira
A
Yamanaka
Shoji
S
Furuya
Mitsuko
M
Miura
Satoshi
S
Kanno
Hiroshi
H
Nagashima
Yoji
Y
Aoki
Ichiro
I
Nagahama
Kiyotaka
K
D000818:Animals; D000888:Antibodies, Anti-Idiotypic / Q000494:pharmacology; D002469:Cell Separation / Q000379:methods*; D005260:Female; D005434:Flow Cytometry / Q000379:methods*; D008280:Magnetics / Q000379:methods*; D008565:Membrane Proteins / Q000276:immunology / Q000378:metabolism; D051379:Mice; D008815:Mice, Inbred Strains; D019408:Platelet Endothelial Cell Adhesion Molecule-1 / Q000276:immunology / Q000378:metabolism; D050199:Podocytes / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism
D000888:Antibodies, Anti-Idiotypic; D008565:Membrane Proteins; D019408:Platelet Endothelial Cell Adhesion Molecule-1; C113267:nephrin