Journal Article

A novel method for isolating podocytes using magnetic activated cell sorting

Ayumi Murakami, Hisashi Oshiro, Seiichi Kanzaki, Akira Yamaguchi, Shoji Yamanaka, Mitsuko Furuya, Satoshi Miura, Hiroshi Kanno, Yoji Nagashima, Ichiro Aoki and Kiyotaka Nagahama

in Nephrology Dialysis Transplantation

Published on behalf of European Renal Association - European Dialysis and Transplant Assoc

Volume 25, issue 12, pages 3884-3890
Published in print December 2010 | ISSN: 0931-0509
Published online June 2010 | e-ISSN: 1460-2385 | DOI: http://dx.doi.org/10.1093/ndt/gfq323
A novel method for isolating podocytes using magnetic activated cell sorting

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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.

Keywords: MACS; nephrin; podocyte isolation

Journal Article.  3048 words.  Illustrated.

Subjects: Nephrology

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