title: Isolation and characterization of conditionally immortalized mouse glomerular endothelial cell lines.
abstract: BACKGROUND
  The culture and establishment of glomerular cell lines has proven to be an important tool for the understanding of glomerular cell functions in glomerular physiology and pathology. Especially, the recent establishment of a conditionally immortalized visceral epithelial cell line has greatly boosted the research on podocyte biology.
  
  
  METHODS
  Glomeruli were isolated from H-2Kb-tsA58 transgenic mice that contain a gene encoding a temperature-sensitive variant of the SV40 large tumor antigen, facilitating proliferative growth at 33 degrees C and differentiation at 37 degrees C. Glomerular endothelial cells were isolated from glomerular outgrowth by magnetic beads loaded with CD31, CD105, GSL I-B4, and ULEX. Clonal cell lines were characterized by immunofluorescence staining with antibodies/lectins specific for markers of endothelial cells, podocytes, and mesangial cells. Putative glomerular endothelial cell lines were analyzed for (1) cytokine-induced expression of adhesion molecules; (2) tube formation on Matrigel coating; and (3) the presence of fenestrae.
  
  
  RESULTS
  As judged by immunostaining for Wilms tumor-1, smooth muscle actin (SMA), podocalyxin, and von Willebrand factor (vWF), we obtained putative endothelial, podocyte and mesangial cell lines. The mouse glomerular endothelial cell clone #1 (mGEnC-1) was positive for vWF, podocalyxin, CD31, CD105, VE-cadherin, GSL I-B4, and ULEX, internalized acetylated-low-density lipoprotein (LDL), and showed increased expression of adhesion molecules after activation with proinflammatory cytokines. Furthermore, mGEnC-1 formed tubes and contained nondiaphragmed fenestrae.
  
  
  CONCLUSIONS
  The mGEnC-1 represents a conditionally immortalized cell line with various characteristics of differentiated glomerular endothelial cells when cultured at 37 degrees C. Most important, mGEnC-1 contains nondiaphragmed fenestrae, which is a unique feature of glomerular endothelial cells.
authors.affiliation: Division of Nephrology, Department of Pathology, Radboud University Nijmegen Medical Center, Nijmegen, The Netherlands.
authors.forename: Angelique L
authors.initials: AL
authors.lastname: Rops
authors.forename: Johan
authors.initials: J
authors.lastname: van der Vlag
authors.forename: Cor W
authors.initials: CW
authors.lastname: Jacobs
authors.forename: Henry B
authors.initials: HB
authors.lastname: Dijkman
authors.forename: Joost F
authors.initials: JF
authors.lastname: Lensen
authors.forename: Tessa J
authors.initials: TJ
authors.lastname: Wijnhoven
authors.forename: Lambert P
authors.initials: LP
authors.lastname: van den Heuvel
authors.forename: Toin H
authors.initials: TH
authors.lastname: van Kuppevelt
authors.forename: Jo H
authors.initials: JH
authors.lastname: Berden
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods; D002455:Cell Division; D002461:Cell Line, Transformed / Q000166:cytology*; D002999:Clone Cells; D042783:Endothelial Cells / Q000166:cytology* / Q000502:physiology; D018799:Intercellular Adhesion Molecule-1 / Q000378:metabolism; D007375:Interleukin-1 / Q000494:pharmacology; D007678:Kidney Glomerulus / Q000166:cytology*; D051379:Mice; D008822:Mice, Transgenic; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D019010:Vascular Cell Adhesion Molecule-1 / Q000378:metabolism
chemical_list: D007375:Interleukin-1; D014409:Tumor Necrosis Factor-alpha; D019010:Vascular Cell Adhesion Molecule-1; D018799:Intercellular Adhesion Molecule-1