title: Isolation and epithelial co-culture of mouse renal peritubular endothelial cells.
abstract: BACKGROUND
  Endothelial-mesenchymal transition (EndoMT) has been shown to be a major source of myofibroblasts, contributing to kidney fibrosis. However, in vitro study of endothelial cells often relies on culture of isolated primary endothelial cells due to the unavailability of endothelial cell lines. Our recent study suggested that peritubular endothelial cells could contribute to kidney fibrosis through EndoMT. Therefore, successful isolation and culture of mouse peritubular endothelial cells could provide a new platform for studying kidney fibrosis. This study describes an immunomagnetic separation method for the isolation of mouse renal peritubular endothelial cells using anti-CD146 MicroBeads, followed by co-culture with mouse renal proximal tubular epithelial cells to maintain endothelial phenotype.
  
  
  RESULTS
  Flow cytometry showed that after isolation and two days of culture, about 95% of cells were positive for endothelial-specific marker CD146. The percentage of other cells, including dendritic cells (CD11c) and macrophages (F4/80), was less than 1%. Maintenance of endothelial cell phenotype required vascular endothelial growth factor (VEGF) and co-culture with mouse proximal tubular epithelial cells.
  
  
  CONCLUSIONS
  In this study, we established a method for the isolation of mouse renal peritubular endothelial cells by using immunomagnetic separation with anti-CD146 MicroBeads, followed by co-culture with mouse renal proximal tubular epithelial cells to maintain phenotype.
authors.affiliation: Centre for Transplant and Renal Research, Westmead Millennium Institute, The University of Sydney, Sydney, NSW, Australia. yzha7726@uni.sydney.edu.au.
authors.forename: Ye
authors.initials: Y
authors.lastname: Zhao
authors.affiliation: Centre for Transplant and Renal Research, Westmead Millennium Institute, The University of Sydney, Sydney, NSW, Australia. zhaohong.syd@gmail.com.|Department of Biochemistry and Molecular Biology, Shanxi Medical University, Taiyuan, PR China. zhaohong.syd@gmail.com.
authors.forename: Hong
authors.initials: H
authors.lastname: Zhao
authors.affiliation: Centre for Transplant and Renal Research, Westmead Millennium Institute, The University of Sydney, Sydney, NSW, Australia. zhangyun2003190@hotmail.com.|Experimental Centre of Science and Research, The First Clinical Hospital of Shanxi Medical University, Taiyuan, PR China. zhangyun2003190@hotmail.com.
authors.forename: Yun
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Centre for Transplant and Renal Research, Westmead Millennium Institute, The University of Sydney, Sydney, NSW, Australia. tania.tsatralis@sydney.edu.au.
authors.forename: Tania
authors.initials: T
authors.lastname: Tsatralis
authors.affiliation: Centre for Transplant and Renal Research, Westmead Millennium Institute, The University of Sydney, Sydney, NSW, Australia. qi.cao@sydney.edu.au.
authors.forename: Qi
authors.initials: Q
authors.lastname: Cao
authors.affiliation: Centre for Transplant and Renal Research, Westmead Millennium Institute, The University of Sydney, Sydney, NSW, Australia. ya.wang@sydney.edu.au.
authors.forename: Ya
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Centre for Transplant and Renal Research, Westmead Millennium Institute, The University of Sydney, Sydney, NSW, Australia. yiping.wang@sydney.edu.au.
authors.forename: Yiping
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Centre for Kidney Research, Children's Hospital at Westmead, Sydney, NSW, Australia. YuanW@chw.edu.au.
authors.forename: Yuan Min
authors.initials: YM
authors.lastname: Wang
authors.affiliation: Centre for Kidney Research, Children's Hospital at Westmead, Sydney, NSW, Australia. stephena@chw.edu.au.
authors.forename: Steve I
authors.initials: SI
authors.lastname: Alexander
authors.affiliation: Centre for Transplant and Renal Research, Westmead Millennium Institute, The University of Sydney, Sydney, NSW, Australia. david.harris@sydney.edu.au.
authors.forename: David C
authors.initials: DC
authors.lastname: Harris
authors.affiliation: Centre for Transplant and Renal Research, Westmead Millennium Institute, The University of Sydney, Sydney, NSW, Australia. guoping.zheng@sydney.edu.au.
authors.forename: Guoping
authors.initials: G
authors.lastname: Zheng
mesh_terms: D000818:Animals; D051929:CD146 Antigen; D002469:Cell Separation / Q000379:methods*; D018920:Coculture Techniques / Q000379:methods*; D042783:Endothelial Cells / Q000166:cytology* / Q000187:drug effects; D004847:Epithelial Cells / Q000166:cytology*; D005434:Flow Cytometry / Q000379:methods*; D007672:Kidney Cortex / Q000166:cytology*; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008863:Microspheres; D010641:Phenotype; D042461:Vascular Endothelial Growth Factor A / Q000494:pharmacology
chemical_list: D051929:CD146 Antigen; D042461:Vascular Endothelial Growth Factor A