Diverse effects of natural antioxidants on cyclosporin cytotoxicity in rat renal tubular cells.
BACKGROUND
As is well known, the use of the immunosuppressive drug cyclosporin A (CsA) is partially restricted by its nephrotoxic effects, which include early changes in haemodynamics followed by irreversible injuries to the renal tubules. Although the mechanisms responsible for these side effects are poorly understood, an involvement of reactive oxygen species (ROS) has been suggested. In this study, we selected three natural antioxidants, resveratrol, hydroxytyrosol and vitamin E, on the basis of their scavenging capabilities, and tested their protective effects against CsA toxicity.


METHODS
Immortalized rat tubular cells (RPTc) were used as the model system. Cell viability was checked with trypan blue assay, and free radical formation was measured using the fluorescent probe 2,7-dichlorofluorescein (DCF). We evaluated several oxidative stress parameters, including phospholipid peroxidation products, glutathione levels and oxygenase expression.


RESULTS
Incubation of RPTc with 25 muM CsA induced a significant decrease in cell viability paralleled by intracellular ROS formation and alterations in lipid peroxidation. There was also an imbalance of glutathione redox state as well as upregulation of heme oxygenase-1 (HO-1). The three antioxidants, at micromolar concentration, quantitatively prevented the ROS-activated DCF fluorescent signal and membrane lipid peroxidation. Both hydroxytyrosol and resveratrol strengthened the CsA induction of HO-1 expression. Moreover, vitamin E and resveratrol counteracted CsA-induced changes in the glutathione redox state via different mechanisms, whereas hydroxytyrosol was completely ineffective. Similarly, CsA-dependent nephrotoxicity was prevented by vitamin E, while resveratrol only exerted partial protection, and hydroxytyrosol showed no protective effects.


CONCLUSIONS
Our results indicate that the diverse cytoprotective effects of the antioxidants tested in these studies were not directly related to their scavenging capabilities. These findings confirm a key role for glutathione in protecting cells from CsA-induced adverse effects and do not support a direct link between CsA-mediated ROS generation and adverse renal effects.
Galletti
Patrizia
P
Department of Biochemistry and Biophysics F. Cedrangolo, Via Costantinopoli, 16, 80138, Napoli, Italia. patrizia.galletti@unina2.it
Di Gennaro
Chiara Iolanda
CI
Migliardi
Valentina
V
Indaco
Stefania
S
Della Ragione
Fulvio
F
Manna
Caterina
C
Chiodini
Paolo
P
Capasso
Giovanni
G
Zappia
Vincenzo
V
D000818:Animals; D000975:Antioxidants / Q000627:therapeutic use*; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D016572:Cyclosporine / Q000633:toxicity*; D004848:Epithelium / Q000187:drug effects; D005452:Fluoresceins; D005978:Glutathione / Q000378:metabolism; D007674:Kidney Diseases / Q000139:chemically induced / Q000378:metabolism / Q000517:prevention & control*; D007687:Kidney Tubules, Proximal / Q000187:drug effects* / Q000378:metabolism; D008054:Lipid Peroxides / Q000378:metabolism; D010626:Phenylethyl Alcohol / Q000031:analogs & derivatives* / Q000627:therapeutic use; D051381:Rats; D011921:Rats, Inbred WKY; D017382:Reactive Oxygen Species / Q000378:metabolism; D000077185:Resveratrol; D013267:Stilbenes / Q000627:therapeutic use*; D017392:Thiobarbituric Acid Reactive Substances / Q000378:metabolism; D014810:Vitamin E / Q000627:therapeutic use*
D000975:Antioxidants; D005452:Fluoresceins; D008054:Lipid Peroxides; D017382:Reactive Oxygen Species; D013267:Stilbenes; D017392:Thiobarbituric Acid Reactive Substances; C005975:3,4-dihydroxyphenylethanol; D014810:Vitamin E; C037631:2',7'-dichlorofluorescein; D016572:Cyclosporine; D005978:Glutathione; D010626:Phenylethyl Alcohol; D000077185:Resveratrol