TY - JOUR
T1 - Phenotypic profiles of cultured glomerular cells following repeated cycles of hydrocarbon injury
AU - Alejandro, Napoleon F.
AU - Parrish, Alan R.
AU - Bowes, Russell C.
AU - Burghardt, Robert C.
AU - Ramos, Kenneth S.
N1 - Funding Information:
This work was supported in part by NIEHS Superfund Program Project Grant 04917 to K.S.R. A.R.P. and R.C.B. III were predoctoral fellows in NIEHS Toxicology Training Grant ES 07273-02. N.F.A. is the recipient of a minority graduate fellowship from Texas A&M University. The authors thank Dr. Rick Metz for helpful comments during manuscript preparation.
PY - 2000
Y1 - 2000
N2 - Background. The glomerulus has been implicated as a target of hydrocarbon injury in vitro and in vivo. In the present studies, the phenotypic profiles of cultured rat glomerular cells (GCs) following repeated cycles of hydrocarbon injury were evaluated. Cultured GCs were incubated for 24 hours with benzo[a]pyrene (BaP; 3 μmol/L), a prototypical polycyclic aromatic hydrocarbon, and were allowed to recover overnight before two additional cycles of chemical challenge during serial propagation in vitro. At the end of this regimen, control cultures were characterized by predominance of fusiform cells that grew in 'hills and valleys,' while GCs subjected to hydrocarbon injury displayed an epithelial morphology characterized by a rounded, polygonal shape clearly distinct from that normally exhibited by glomerular mesangial cells (GMCs) in culture. Methods. Indirect immunofluorescent detection of cell markers was conducted to identify cells of mesenchymal or epithelial origin. Measurements of DNA synthesis and cell number were performed to determine proliferative capacities of the different cell types in response to hydrocarbon challenge. Results. Immunofluorescence studies revealed that control GC cultures contained mostly α-smooth muscle (SM) actin-positive cells, with a few (5.1.% ± 2.6) E-cadherin-positive cells occasionally identified. In contrast, BaP-treated cultures exhibited a mixed cell population in which E- cadherin-positive cells were predominant (66.6% ± 4.1). Single-cell cloning of naive cultures of GCs yielded four clones, three of which exhibited a fusiform morphology and were α-SM actin positive (SCC 1 through SCC 3) and one (SCC 4E) that exhibited epithelial characteristics similar to those found in hydrocarbon-treated cultures. Immunofluorescence studies showed that epithelial cells in hydrocarbon-treated cultures, as well as SCC 4E-derived clones, were vimentin positive and cytokeratin negative, characteristics similar to glomerular visceral epithelial cells (GVECs). DNA synthesis and cell proliferation in clone SCC 1 were decreased following acute BaP challenge, while growth rates in SCC 4E-derived clones were unaffected by hydrocarbon injury. Repeated cycles of hydrocarbon challenge in clonal populations yielded different profiles of DNA synthesis, with significant decreases in SCC 1 and no changes in SCC 4E. Conclusions. These observations suggest that hydrocarbon injury induces differential responses in cells of the glomerulus, resulting in inhibition of GMCs and selective growth advantage of GVECs. These alterations are reminiscent of critical events described in the pathogenesis of focal segmental glomerulosclerosis and raise important questions about the pathogenesis of hydrocarbon-induced nephropathies.
AB - Background. The glomerulus has been implicated as a target of hydrocarbon injury in vitro and in vivo. In the present studies, the phenotypic profiles of cultured rat glomerular cells (GCs) following repeated cycles of hydrocarbon injury were evaluated. Cultured GCs were incubated for 24 hours with benzo[a]pyrene (BaP; 3 μmol/L), a prototypical polycyclic aromatic hydrocarbon, and were allowed to recover overnight before two additional cycles of chemical challenge during serial propagation in vitro. At the end of this regimen, control cultures were characterized by predominance of fusiform cells that grew in 'hills and valleys,' while GCs subjected to hydrocarbon injury displayed an epithelial morphology characterized by a rounded, polygonal shape clearly distinct from that normally exhibited by glomerular mesangial cells (GMCs) in culture. Methods. Indirect immunofluorescent detection of cell markers was conducted to identify cells of mesenchymal or epithelial origin. Measurements of DNA synthesis and cell number were performed to determine proliferative capacities of the different cell types in response to hydrocarbon challenge. Results. Immunofluorescence studies revealed that control GC cultures contained mostly α-smooth muscle (SM) actin-positive cells, with a few (5.1.% ± 2.6) E-cadherin-positive cells occasionally identified. In contrast, BaP-treated cultures exhibited a mixed cell population in which E- cadherin-positive cells were predominant (66.6% ± 4.1). Single-cell cloning of naive cultures of GCs yielded four clones, three of which exhibited a fusiform morphology and were α-SM actin positive (SCC 1 through SCC 3) and one (SCC 4E) that exhibited epithelial characteristics similar to those found in hydrocarbon-treated cultures. Immunofluorescence studies showed that epithelial cells in hydrocarbon-treated cultures, as well as SCC 4E-derived clones, were vimentin positive and cytokeratin negative, characteristics similar to glomerular visceral epithelial cells (GVECs). DNA synthesis and cell proliferation in clone SCC 1 were decreased following acute BaP challenge, while growth rates in SCC 4E-derived clones were unaffected by hydrocarbon injury. Repeated cycles of hydrocarbon challenge in clonal populations yielded different profiles of DNA synthesis, with significant decreases in SCC 1 and no changes in SCC 4E. Conclusions. These observations suggest that hydrocarbon injury induces differential responses in cells of the glomerulus, resulting in inhibition of GMCs and selective growth advantage of GVECs. These alterations are reminiscent of critical events described in the pathogenesis of focal segmental glomerulosclerosis and raise important questions about the pathogenesis of hydrocarbon-induced nephropathies.
KW - Endothelial cells
KW - Focal segmental glomerulosclerosis
KW - Glomerular mesangial cells
KW - Hydrocarbon induced nephropathies
KW - Nephrotoxicity
KW - Visceral epithelial cells
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U2 - 10.1046/j.1523-1755.2000.01001.x
DO - 10.1046/j.1523-1755.2000.01001.x
M3 - Article
C2 - 10760092
AN - SCOPUS:0034074534
SN - 0085-2538
VL - 57
SP - 1571
EP - 1580
JO - Kidney International
JF - Kidney International
IS - 4
ER -