Atractyloside nephrotoxicity: in vitro studies with suspensions of rat renal fragments and precision-cut cortical slices.
The consumption of plants containing atractyloside, a diterpenoid glycoside, causes selective proximal tubule injury leading to renal failure and death in humans. The underlying mechanisms responsible for its toxicity are still not well understood. The present study was therefore carried out to determine the mechanism and the exact sequence of events that lead to molecular toxic injury. A comparative study using renal cortical slices, suspension of freshly isolated renal proximal tubular fragments and glomeruli of male Wistar rat was made. These in vitro systems were exposed to 100-1000 mM atractyloside for 2-3 h at 37 degrees C. Atractyloside caused a significant alteration in various toxicity parameters in a concentration- and time-dependent manner in renal cortical slices and proximal tubular fragments, but not in glomeruli. The earliest change following exposure to atractyloside (1000 microM) was a significant reduction of intracellular adenosine 5'-triphosphate (ATP) content occurring within 1 h in the tubules and 2 h in slices. The significant depletion of reduced glutathione (GSH) inhibitor of p-aminohippuric (acid) (PAH) uptake and gluconeogenesis occurred simultaneously following loss of cellular energy. These events were only limited to the renal cortical slices and proximal tubular fragments. Increased severity of cellular injury resulted in cytotoxicity with the significant increase in the leakage of alkaline phosphatase (ALP) and lactate dehydrogenase (LDH) in proximal tubular fragments (occurring at 2 h) and renal cortical slices (occurring at 3 h). There were, however, no alterations in oxidized glutathione (GSSG) levels or in the ratio of GSH/GSSG. Only limited lipid peroxidation in proximal tubular fragments and glomeruli was observed at atractyloside concentrations of 500 microM and above. In all cases of toxicity, the glomeruli were unaffected. Pretreatment of slices or fragments with probenecid (1.0 mM) failed to completely abolish atractyloside toxicity. These data demonstrate dose- and time-dependent toxicity of atractyloside and clearly confirmed the proximal tubular fragments as the target tissue. Atractyloside exhibits a toxicity profile that indicates early alteration in mitochondrial function and consequently loss of cellular energy, followed by reduced metabolic function and transport processes and ultimately cell death. This appears to be the most likely mechanism by which atractyloside exerted its acute cytotoxicity. Renal cortical slices, which maintain proximal tubule and glomeruli in their anatomic relationship, responded similarly to atractyloside toxicity as the proximal tubular fragments, and might be suggested as the most suitable in vitro model system for studying the mechanisms of atractyloside toxicity as they are more likely to mirror changes seen in the whole organ.
Obatomi
D K
DK
Department of Life Sciences, Faculty of Science and Health, University of East London, London, United Kingdom. D.K.Obatomi@uel.ac.uk
Bach
P H
PH
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001278:Atractyloside / Q000633:toxicity*; D001692:Biological Transport / Q000187:drug effects; D016923:Cell Death / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D005943:Gluconeogenesis / Q000187:drug effects; D005978:Glutathione / Q000378:metabolism; D066298:In Vitro Techniques; D007672:Kidney Cortex / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism / Q000473:pathology; D007678:Kidney Glomerulus / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism / Q000473:pathology; D007687:Kidney Tubules, Proximal / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism / Q000473:pathology; D015227:Lipid Peroxidation / Q000187:drug effects; D008297:Male; D008315:Malondialdehyde / Q000378:metabolism; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D010085:Oxidative Phosphorylation / Q000187:drug effects; D018384:Oxidative Stress / Q000187:drug effects; D051381:Rats; D017208:Rats, Wistar; D051437:Renal Insufficiency / Q000139:chemically induced; D013997:Time Factors; D010130:p-Aminohippuric Acid / Q000378:metabolism
D001278:Atractyloside; D008315:Malondialdehyde; D000255:Adenosine Triphosphate; D005978:Glutathione; D010130:p-Aminohippuric Acid