Isolation and characterization of conditionally immortalized mouse glomerular endothelial cell lines.
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.
Rops
Angelique L
AL
Division of Nephrology, Department of Pathology, Radboud University Nijmegen Medical Center, Nijmegen, The Netherlands.
van der Vlag
Johan
J
Jacobs
Cor W
CW
Dijkman
Henry B
HB
Lensen
Joost F
JF
Wijnhoven
Tessa J
TJ
van den Heuvel
Lambert P
LP
van Kuppevelt
Toin H
TH
Berden
Jo H
JH
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
D007375:Interleukin-1; D014409:Tumor Necrosis Factor-alpha; D019010:Vascular Cell Adhesion Molecule-1; D018799:Intercellular Adhesion Molecule-1