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Gentamicin-induced alterations in pig kidney epithelial (LLC-PK1) cells in culture
Abstract:
The effect of gentamicin exposure was investigated in LLC-PK1 cells in culture. Gentamicin (0.5-2.0 mM) was added to the medium of cells which had been grown to confluency in the absence of antibiotics and antimycotics. Exposure to gentamicin (1-4 days) did not effect total cellular protein or DNA levels, total cell number or the release of various marker enzymes to the medium. ATP levels in gentamicin-treated cells did not differ from control cells; however, medium from the gentamicin-treated cells contained significantly lower lactic acid levels. Morphological examination by electron microscopy revealed gentamicin-elicited myeloid body formation. Furthermore, total phospholipid level was elevated markedly in gentamicin-treated cells. Analysis of specific phospholipid classes showed only phosphatidylcholine, phosphatidylinositol and polyphosphoinositide phospholipid levels increased in a time-dependent manner. Phosphatidylinositol showed the highest percentage of increase. Raising the normal medium calcium concentration (0.2 mg/ml) 1.5-, 2.0- or 3.0-fold did not alter gentamicin-induced elevation in cellular phosphatidylinositol and phosphatidylcholine. Gentamicin exposure also resulted in a concentration-dependent increase in the turnover of LLC-PK1 cell-free fatty acids, monoglyceride, diglyceride and nonesterified cholesterol and a decrease in triglyceride turnover. Calcium transport into and through the cell monolayer was inhibited markedly by gentamicin despite the fact that 45Ca++ binding to gentamicin-treated cells was greater. These results demonstrate that manifestations of gentamicin toxicity in LLC-PK1 cells parallel those reported in the whole animal thus making the LLC-PK1 cell in culture a valid system for elucidating the mechanism of gentamicin-elicited alterations in renal epithelium.
Insights
Gentamicin exposure in LLC-PK1 cells caused myeloid body formation and altered phospholipid metabolism. Calcium transport was inhibited, validating this cell model for studying gentamicin toxicity in renal epithelium.
Area of Science:
- Cell Biology
- Toxicology
- Pharmacology
Background:
- Gentamicin is an aminoglycoside antibiotic with known nephrotoxicity.
- Understanding the cellular mechanisms of gentamicin toxicity is crucial for mitigating adverse effects.
Purpose of the Study:
- To investigate the effects of gentamicin exposure on LLC-PK1 cells in culture.
- To determine if LLC-PK1 cells serve as a valid model for studying gentamicin-induced renal epithelial alterations.
Main Methods:
- LLC-PK1 cells were exposed to varying concentrations of gentamicin.
- Cellular and biochemical analyses included protein, DNA, cell number, enzyme release, ATP, lactic acid, phospholipids, and calcium transport.
- Electron microscopy was used for morphological examination.
Main Results:
- Gentamicin exposure did not affect total protein, DNA, cell number, or marker enzyme release.
- Significant alterations included myeloid body formation, increased phospholipid levels (phosphatidylinositol, phosphatidylcholine), and inhibited calcium transport.
- Despite increased 45Ca++ binding, gentamicin markedly inhibited calcium transport into and through the cell monolayer.
Conclusions:
- LLC-PK1 cells exhibit gentamicin-induced toxicity mirroring effects seen in vivo.
- This cell culture system is a valid model for elucidating the mechanisms of gentamicin's effects on renal epithelium.
- Specific alterations in phospholipid metabolism and calcium transport pathways are key indicators of gentamicin toxicity.