Characterization of a simian virus 40-transformed human podocyte cell line producing type IV collagen and exhibiting polarized response to atrial natriuretic peptide.
Biology of glomerular visceral epithelial cells ("podocytes") and their role in inflammatory process remain obscure, partly because of the lack of well-differentiated podocyte cultures. We have established a human cell line by transfecting with a replication-defective SV40 plasmid (pSVHB1), a primary culture of podocytes derived from an enriched preparation of unencapsulated glomeruli free of tubule and Bowman's capsule contaminants. Podocyte specificity of the primary culture was assessed by a dual immunomorphological and functional approach. The resulting cell line (HGVEC.SV1) was cloned and the clonal cells were adapted to hormonally defined medium supplemented with only 2% newborn bovine serum. Clone A4 has been exhibiting over 35 passages, a combination of markers unique to podocytes, including expression of vimentin, podocalyxin, ectoenzymes (CALLA antigen and mRNA), heparan-sulfate proteoglycans (molecular mass of core protein = 75 kDa), and production of type IV collagen (alpha 1 and alpha 5 chains) established by immunoprecipitation and Northern blot analysis. Cytokeratin was detected in rare cellular foci and the search of Von Willebrand factor was negative. This clonal cell line has been used to demonstrate: (1) that human podocytes are highly sensitive to atrial natriuretic peptide (ANP) which induced a dose-dependent increase in cGMP production (x20 at 0.5 microM ANP), and (2) that secretion of ANP-stimulated cGMP is dramatically polarized as 93% of extracellular cGMP were released in the apical medium when filter-grown HGVEC. SV1A4 cells were stimulated at their basal pole.
Ardaillou
N
N
INSERM U.64, Hôpital Tenon, Paris, France.
Lelongt
B
B
Turner
N
N
Piedagnel
R
R
Baudouin
B
B
Estrade
S
S
Cassingena
R
R
Ronco
P M
PM
D000943:Antigens, Differentiation / Q000032:analysis; D000951:Antigens, Neoplasm / Q000032:analysis; D009320:Atrial Natriuretic Factor / Q000494:pharmacology*; D002461:Cell Line, Transformed; D016764:Cell Polarity; D002999:Clone Cells; D003094:Collagen / Q000096:biosynthesis*; D003470:Culture Media; D000242:Cyclic AMP / Q000378:metabolism; D006152:Cyclic GMP / Q000378:metabolism*; D018819:Dipeptidyl Peptidase 4; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases / Q000032:analysis; D019812:Heparan Sulfate Proteoglycans; D006497:Heparan Sulfate / Q000032:analysis; D006801:Humans; D007678:Kidney Glomerulus / Q000737:chemistry / Q000166:cytology* / Q000378:metabolism; D015260:Neprilysin; D011509:Proteoglycans / Q000032:analysis; D013539:Simian virus 40; D014746:Vimentin / Q000032:analysis
D000943:Antigens, Differentiation; D000951:Antigens, Neoplasm; D009320:Atrial Natriuretic Factor; D003094:Collagen; D003470:Culture Media; D000242:Cyclic AMP; D006152:Cyclic GMP; D018819:Dipeptidyl Peptidase 4; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; D019812:Heparan Sulfate Proteoglycans; D006497:Heparan Sulfate; D015260:Neprilysin; D011509:Proteoglycans; D014746:Vimentin