Stable expression of nephrin and localization to cell-cell contacts in novel murine podocyte cell lines.
BACKGROUND
Cell culture of podocytes has become an indispensable tool in the study of podocyte biology. To date, however, podocyte cell lines with stable expression of the crucial slit diaphragm protein nephrin and localization of nephrin to cell-cell contacts are not available.


METHODS
Conditionally immortalized cells were grown from isolated glomeruli of mice, harboring the temperature-sensitive SV40 large T antigen. About 60 clonal cell lines were generated by limiting dilution.


RESULTS
Among 30 Wilm's tumor (WT)-1- and podocalyxin-positive cell clones, two cell clones stably expressed nephrin as assessed by reverse transcription-polymerase chain reaction (RT-PCR), Northern and Western blotting, and immunofluorescence. In addition, expression of the following podocyte proteins was demonstrated: NEHP1, FAT, P-cadherin, podocin, CD2AP, ZO-1 (alpha(-) isoform), Lmx1b, podoplanin, synaptopodin, cortactin, and vimentin. The nephrin-positive podocyte cell lines formed a monolayer with abundant cell-cell contacts. Transmission electron microscopy revealed formation of primitive foot process-like interdigitations and slit diaphragm-like junctions. Nephrin colocalized with F-actin at cell-cell contacts as demonstrated by immunofluorescence. Intriguingly, nephrin and actin-associated proteins (synaptopodin, CD2AP, and cortactin) were recruited to and accumulated at the entire cell margin only in confluent cells, but not in dispersed cells.


CONCLUSIONS
We present novel murine podocyte cell lines with stable expression of nephrin and abundant formation of cell-cell contacts, possessing several features of in situ podocyte cell-cell contacts. Furthermore, our data suggest that the accumulation of certain proteins in podocyte foot processes is linked to formation of cell-cell contacts.
Schiwek
Daniel
D
Department of Anatomy and Cell Biology, University of Heidelberg, Heidelberg, Germany.
Endlich
Nicole
N
Holzman
Lawrence
L
Holthöfer
Harry
H
Kriz
Wilhelm
W
Endlich
Karlhans
K
D000818:Animals; D002450:Cell Communication; D002461:Cell Line, Transformed; D004847:Epithelial Cells / Q000378:metabolism / Q000502:physiology; D007678:Kidney Glomerulus / Q000166:cytology / Q000378:metabolism / Q000502:physiology* / Q000648:ultrastructure; D008565:Membrane Proteins; D051379:Mice; D008815:Mice, Inbred Strains; D008854:Microscopy, Electron; D011506:Proteins / Q000378:metabolism*; D012795:Sialoglycoproteins / Q000378:metabolism; D025721:WT1 Proteins / Q000378:metabolism
D008565:Membrane Proteins; D011506:Proteins; D012795:Sialoglycoproteins; D025721:WT1 Proteins; C113267:nephrin; C001643:podocalyxin