Glycogen synthase kinase-3β controls autophagy during renal ischemia/reperfusion in vivo

E Esposito, R Crupi, A Ahmad, M Campolo, I Paterniti, S Cuzzocrea. University of Messina, Dept. Clinical and Experimental Medicine and Pharmacology, 98125, Italy

Autophagy occurs at basal level in most cells and contributes to the turnover of long-lived proteins and organelles to maintain intracellular homeostasis. In response to cellular stress, autophagy is up-regulated and can provide an adaptive strategy for cell survival, but may also directly or indirectly lead to cell demise. With the dual role in life and death, autophagy is involved in various physiological processes, and linked to the pathogenesis of a wide array of diseases. However, the defined role of autophagy is further complicated by the cross talk and coordinated regulation between autophagy and apoptosis. The role of autophagy in the pathogenesis of renal diseases has not been identified. In this study, we determined the contribution of autophagy in renal tubular cell injury using in vivo models of renal ischemia/reperfusion (I/R). C57BL/6 mice (6 mice in each group) were anesthetized using a ketamine (150 mg/kg) and xylazine (15 mg/kg) mixture i.p., and subjected to sham operation or 30 minutes of bilateral renal ischemia, followed by 6 or 24 hours of reperfusion. Results are representative of at least three experiments performed on different experimental days. Statistical comparison was by ANOVA followed by post hoc Bonferroni. A P < 0.05 was considered as statistically significant. A basal level of LC3-II was shown by immunoblotting in the sham kidney homogenates. On reperfusion a significant amount of LC3-II accumulated in renal tissues in a time-dependent manner, starting at 6 hours and further increasing after 24 hours (2.1-fold over control). Thus, autophagy is induced in a time-dependent manner during renal I/R. Moreover, I/R increased mitochondrial cytochrome C release, renal O2(-*) level and 3-nitrotirosine accumulation, Beclin-1 expression (4-fold over control), Bax/Bcl-xL ratio, caspase 3 expression and poly-(ADP-ribose)-polymerase fragments. Treatment with SB216763 (0.6 mg/kg i.p., 5 minutes prior to reperfusion), an inhibitor of GSK-3β, decreased renal tubular dilatation, vacuolization and sloughing, blood urea nitrogen, creatinine and renal cell apoptosis. Reperfusion-induced autophagy was attenuated by GSK-3β inhibition. Our results suggest that GSK-3 inhibition protects against reperfusion injury through prevention of autophagy.