For example, Ann and colleagues18 demonstrated that PKCmediated autophagy during acute hypoxic stress by phosphorylating/activating JNK1, whereas Ozpolat suppressed autophagy in pancreatic cancer cells by inducing tissue transglutaminase. In this study, we have identified PKCas a critical negative regulator of autophagy in both and experimental models of cisplatin. serine-473 by PKCinhibitor rottlerin with cisplatin protected against cisplatin nephrotoxicity in wild-type mice, but not in renal autophagyCdeficient mice. Together, these results reveal a pathway consisting of PKCmediates cisplatin nephrotoxicity at least in part by suppressing autophagy, and accordingly, PKCinhibition protects kidneys by upregulating autophagy. (PKCnot only protected kidneys but enhanced the chemotherapeutic effects of cisplatin in several tumor models, opening a new avenue for renoprotection during chemotherapy.8,9 However, the mechanism underlying the renoprotective effect of PKCinhibition is unclear. Autophagy is a highly regulated cellular process of catabolism that degrades cytoplasmic constituents the Taranabant racemate formation of autophagosome followed by its fusion with lysosome. Originally described as a cellular response to starvation, autophagy is now known to be crucial to the maintenance of cellular homeostasis and play important roles in animal development, physiology, and pathogenesis of a variety of diseases.10C12 In cisplatin nephrotoxicity, autophagy is rapidly activated in kidney tubular cells and tissues.13,14 Using renal Taranabant racemate tubuleCspecific Atg-knockout models, recent studies have further demonstrated autophagy as an important kidney protective mechanism.15,16 However, it Taranabant racemate remains elusive how autophagy is regulated during cisplatin nephrotoxicity. In view of these findings and questions, we hypothesized that PKCmay play a regulatory role in autophagy during cisplatin nephrotoxicity and inhibition of PKCmay protect kidney cells and tissues by upregulating autophagy. In support of this hypothesis, several studies have implicated PKCin the regulation of autophagy.17C20 Nonetheless, whether PKCpromotes or inhibits autophagy remains controversial. For example, Ann and colleagues18 demonstrated that PKCmediated autophagy during acute hypoxic stress by phosphorylating/activating JNK1, whereas Ozpolat suppressed autophagy in pancreatic cancer cells by inducing tissue transglutaminase. In this study, we have identified PKCas a critical negative regulator of autophagy in both and experimental models of cisplatin. Mechanistically, we show that PKCmay directly bind and phosphorylate AKT at Serine-473, resulting in the activation of mammalian target of rapamycin (mTOR) to suppress ULK1 and autophagy. Moreover, PKCinhibitors lost their renoprotective effect in autophagy-deficient mice, supporting a role of autophagy in the effect of PKCinhibition. Results Autophagy Is Induced during Cisplatin Treatment We first verified that cisplatin induced autophagy in cultured rat proximal tubular cells (RPTC). In this experiment, we also observed the effect of chloroquine (CQ), which accumulates in lysosomes to raise pH resulting in the inhibition of lysosomal enzymes and the suppression of autophagic degradation. By this property, CQ is frequently used to block autolysosomal degradation to reveal upstream Taranabant racemate autophagic activation upon stimulation. In immunoblot analysis, cisplatin treatment for 6 hours induced the conversion of LC3I to LC3II, which was further enhanced by the presence of CQ (Figure 1, A and B). To visualize autophagsosome formation, the cells were transfected with GFP-LC3 and then treated with cisplatin in the presence or absence of CQ. As shown in Figure 1, C and D, cisplatin treatment increased the number of GFP-LC3 puncta, which was further increased by CQ, confirming autophagy induction in this experimental condition. Open in a separate window Figure 1. Cisplatin-induced autophagy in RPTC cells. (A) LC3-II formation during cisplatin treatment. RPTC were incubated with 20 Is Activated during Cisplatin Treatment to Activate mTOR and Suppress Autophagy Our recent work demonstrated a rapid activation of PKCduring cisplatin treatment of RPTC and mice. Moreover, pharmacologic and genetic suppression of PKCafforded remarkable renoprotective effects.8 Because autophagy is an important mechanism of renoprotection in kidney injury including cisplatin nephrotoxicity,26 we hypothesized that PKCinhibition may protect autophagy. To test this possibility, we first confirmed PKCactivation during cisplatin treatment of RPTC by immunoblot analysis Rabbit Polyclonal to RIN3 of its phosphorylation (Figure 4A). To determine the involvement of PKCin cisplatin-induced autophagy, we examined the effects Taranabant racemate of dominant-negative PKC(PKC(PKCsuppressed autophagy through the activation of mTOR and consequent inhibitory.