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Multigenerational study of chemically induced cytotoxicity and proliferation in cultures of human proximal tubular cells.

Lash LH, Putt DA, Benipal B - Int J Mol Sci (2014)

Bottom Line: Primary cultures of human proximal tubular (hPT) cells are a useful experimental model to study transport, metabolism, cytotoxicity, and effects on gene expression of a diverse array of drugs and environmental chemicals because they are derived directly from the in vivo human kidney.Mitochondrial activity was lower in P2-P4 cells than in either P0 or P1 cells.P1 and P2 cells were most sensitive to DCVC-induced apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Wayne State University School of Medicine, 540 East Canfield Avenue, Detroit, MI 48201, USA. l.h.lash@wayne.edu.

ABSTRACT
Primary cultures of human proximal tubular (hPT) cells are a useful experimental model to study transport, metabolism, cytotoxicity, and effects on gene expression of a diverse array of drugs and environmental chemicals because they are derived directly from the in vivo human kidney. To extend the model to investigate longer-term processes, primary cultures (P0) were passaged for up to four generations (P1-P4). hPT cells retained epithelial morphology and stained positively for cytokeratins through P4, although cell growth and proliferation successively slowed with each passage. Necrotic cell death due to the model oxidants tert-butyl hydroperoxide (tBH) and methyl vinyl ketone (MVK) increased with increasing passage number, whereas that due to the selective nephrotoxicant S-(1,2-dichlorovinyl)-l-cysteine (DCVC) was modest and did not change with passage number. Mitochondrial activity was lower in P2-P4 cells than in either P0 or P1 cells. P1 and P2 cells were most sensitive to DCVC-induced apoptosis. DCVC also increased cell proliferation most prominently in P1 and P2 cells. Modest differences with respect to passage number and response to DCVC exposure were observed in expression of three key proteins (Hsp27, GADD153, p53) involved in stress response. Hence, although there are some modest differences in function with passage, these results support the use of multiple generations of hPT cells as an experimental model.

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Chemically induced MTT reduction in P0–P4 hPT cells. hPT cells (approximately 80% confluent) at either primary culture stage (P0; A) or after one through four passages (P1–P4; B–E) were grown on collagen-coated 6-well plates and incubated for up to 24 h with either medium (=Control) or 100 µM DCVC, tBH, or MVK. At the indicated times, samples were incubated with MTT and fluorescence was read on a SpectraMax 2 plate reader. Results are means ± SEM of measurements from three separate cell cultures. *, Significantly different (p < 0.05) from the corresponding control.
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ijms-15-21348-f003: Chemically induced MTT reduction in P0–P4 hPT cells. hPT cells (approximately 80% confluent) at either primary culture stage (P0; A) or after one through four passages (P1–P4; B–E) were grown on collagen-coated 6-well plates and incubated for up to 24 h with either medium (=Control) or 100 µM DCVC, tBH, or MVK. At the indicated times, samples were incubated with MTT and fluorescence was read on a SpectraMax 2 plate reader. Results are means ± SEM of measurements from three separate cell cultures. *, Significantly different (p < 0.05) from the corresponding control.

Mentions: To further assess the cytotoxicity of DCVC in hPT cells at different passage numbers, reduction of MTT was measured (Figure 3). MTT reduction requires mitochondrial activity and is generally considered an indicator of cell proliferation. The fluorescence of control cells, while remaining relatively constant over the 24-h incubation period during a given passage, tended to decrease after P1. Because MTT reduction occurs in mitochondria, this suggests that the density and/or activity of mitochondria in P2–P4 cells is lower than that in P0 or P1 cells. Based on changes in MTT reduction as an index of cytotoxicity or proliferation, 100 µM DCVC did not elicit any toxicity in P0 cells but was slightly toxic to P1–P3 cells. Both tBH and MVK produced a decrease in MTT reduction that was greater in P2–P4 cells than in either P0 or P1 cells.


Multigenerational study of chemically induced cytotoxicity and proliferation in cultures of human proximal tubular cells.

Lash LH, Putt DA, Benipal B - Int J Mol Sci (2014)

Chemically induced MTT reduction in P0–P4 hPT cells. hPT cells (approximately 80% confluent) at either primary culture stage (P0; A) or after one through four passages (P1–P4; B–E) were grown on collagen-coated 6-well plates and incubated for up to 24 h with either medium (=Control) or 100 µM DCVC, tBH, or MVK. At the indicated times, samples were incubated with MTT and fluorescence was read on a SpectraMax 2 plate reader. Results are means ± SEM of measurements from three separate cell cultures. *, Significantly different (p < 0.05) from the corresponding control.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4264229&req=5

ijms-15-21348-f003: Chemically induced MTT reduction in P0–P4 hPT cells. hPT cells (approximately 80% confluent) at either primary culture stage (P0; A) or after one through four passages (P1–P4; B–E) were grown on collagen-coated 6-well plates and incubated for up to 24 h with either medium (=Control) or 100 µM DCVC, tBH, or MVK. At the indicated times, samples were incubated with MTT and fluorescence was read on a SpectraMax 2 plate reader. Results are means ± SEM of measurements from three separate cell cultures. *, Significantly different (p < 0.05) from the corresponding control.
Mentions: To further assess the cytotoxicity of DCVC in hPT cells at different passage numbers, reduction of MTT was measured (Figure 3). MTT reduction requires mitochondrial activity and is generally considered an indicator of cell proliferation. The fluorescence of control cells, while remaining relatively constant over the 24-h incubation period during a given passage, tended to decrease after P1. Because MTT reduction occurs in mitochondria, this suggests that the density and/or activity of mitochondria in P2–P4 cells is lower than that in P0 or P1 cells. Based on changes in MTT reduction as an index of cytotoxicity or proliferation, 100 µM DCVC did not elicit any toxicity in P0 cells but was slightly toxic to P1–P3 cells. Both tBH and MVK produced a decrease in MTT reduction that was greater in P2–P4 cells than in either P0 or P1 cells.

Bottom Line: Primary cultures of human proximal tubular (hPT) cells are a useful experimental model to study transport, metabolism, cytotoxicity, and effects on gene expression of a diverse array of drugs and environmental chemicals because they are derived directly from the in vivo human kidney.Mitochondrial activity was lower in P2-P4 cells than in either P0 or P1 cells.P1 and P2 cells were most sensitive to DCVC-induced apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Wayne State University School of Medicine, 540 East Canfield Avenue, Detroit, MI 48201, USA. l.h.lash@wayne.edu.

ABSTRACT
Primary cultures of human proximal tubular (hPT) cells are a useful experimental model to study transport, metabolism, cytotoxicity, and effects on gene expression of a diverse array of drugs and environmental chemicals because they are derived directly from the in vivo human kidney. To extend the model to investigate longer-term processes, primary cultures (P0) were passaged for up to four generations (P1-P4). hPT cells retained epithelial morphology and stained positively for cytokeratins through P4, although cell growth and proliferation successively slowed with each passage. Necrotic cell death due to the model oxidants tert-butyl hydroperoxide (tBH) and methyl vinyl ketone (MVK) increased with increasing passage number, whereas that due to the selective nephrotoxicant S-(1,2-dichlorovinyl)-l-cysteine (DCVC) was modest and did not change with passage number. Mitochondrial activity was lower in P2-P4 cells than in either P0 or P1 cells. P1 and P2 cells were most sensitive to DCVC-induced apoptosis. DCVC also increased cell proliferation most prominently in P1 and P2 cells. Modest differences with respect to passage number and response to DCVC exposure were observed in expression of three key proteins (Hsp27, GADD153, p53) involved in stress response. Hence, although there are some modest differences in function with passage, these results support the use of multiple generations of hPT cells as an experimental model.

Show MeSH
Related in: MedlinePlus