Chronic Cyclosporine Nephropathy Is Characterized by Excessive Autophagosome Formation and Decreased Autophagic Clearance

BACKGROUND: The study was performed to investigate the influence of cyclosporine A (CsA)-induced renal injury on autophagy in an experimental model of chronic CsA nephropathy. METHODS: Three dosages of CsA (7.5, 15, and 30 mg/kg/day) were administered to mice for 4 weeks. The formation of autophagosomes was measured with microtubule-associated protein 1 light chain 3 phospholipid-conjugated form (LC3-II) and beclin-1, and the ability of autophagic clearance was examined with sequestosome-1 (p62). Autophagic vacuoles were visualized and counted using electron microscopy. Double immunolabeling of LC3-II and active caspase-3 was performed to evaluate the association between autophagy and apoptosis. Oxidative stress was evaluated by measuring urinary 8-hydroxy-2'-deoxyguanosine excretion, demonstrating oxidative DNA damage. Antioxidative drugs, pravastatin and N-acetylcysteine, were used to evaluate the role of CsA-induced oxidative stress on autophagy. RESULTS: CsA treatment increased the expressions of LC3-II and beclin-1 in the kidney in a dose-dependent manner. The number of p62-positive cells was also significantly increased in a CsA dose-dependent manner. Electron microscopy revealed excessive autophagic vacuoles in the CsA group compared with the vehicle group. Expression of active caspase-3 was increased in a CsA dose-dependent manner and was colocalized with LC3-II in the injured area of CsA-treated kidneys. Concurrent pravastatin or N-acetylcysteine treatment reduced urinary excretion of 8-hydroxy-2'-deoxyguanosine and subsequently decreased LC3-II expression and the number of p62-positive cells compared with the CsA group. CONCLUSIONS: Chronic CsA nephropathy is a state of excessive autophagic vacuoles and decreased autophagic clearance. Oxidative stress may play an importation role in the induction of autophagy.