Phenotypic profiles of cultured glomerular cells following repeated cycles of hydrocarbon injury

Napoleon F. Alejandro, Alan R. Parrish, Russell C. Bowes, Robert C. Burghardt, Kenneth Ramos

Research output: Contribution to journalArticle

7 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)1571-1580
Number of pages10
JournalKidney International
Volume57
Issue number4
DOIs
StatePublished - 2000
Externally publishedYes

Fingerprint

Hydrocarbons
Cultured Cells
Wounds and Injuries
Clone Cells
Podocytes
Mesangial Cells
Cell Culture Techniques
Cadherins
Fluorescent Antibody Technique
Smooth Muscle
Actins
DNA
Focal Segmental Glomerulosclerosis
Benzo(a)pyrene
Polycyclic Aromatic Hydrocarbons
Vimentin
Growth
Keratins
Population
Organism Cloning

Keywords

  • Endothelial cells
  • Focal segmental glomerulosclerosis
  • Glomerular mesangial cells
  • Hydrocarbon induced nephropathies
  • Nephrotoxicity
  • Visceral epithelial cells

ASJC Scopus subject areas

  • Nephrology

Cite this

Phenotypic profiles of cultured glomerular cells following repeated cycles of hydrocarbon injury. / Alejandro, Napoleon F.; Parrish, Alan R.; Bowes, Russell C.; Burghardt, Robert C.; Ramos, Kenneth.

In: Kidney International, Vol. 57, No. 4, 2000, p. 1571-1580.

Research output: Contribution to journalArticle

Alejandro, Napoleon F. ; Parrish, Alan R. ; Bowes, Russell C. ; Burghardt, Robert C. ; Ramos, Kenneth. / Phenotypic profiles of cultured glomerular cells following repeated cycles of hydrocarbon injury. In: Kidney International. 2000 ; Vol. 57, No. 4. pp. 1571-1580.
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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.

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KW - Nephrotoxicity

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