Our results provided the first evidence to our knowledge that long-term CR enhances autophagy with overexpression of Sirt1 in aged tissue of mammals and is a therapeutic target for age- and hypoxia-associated tissue damage including aged kidney. In this study, we focused on Sirt1, a survival molecule under CR (23
). Recent reports suggest that Sirt1
transgenic mice show a phenotype that resembles mice under CR (45
), and Sirt1
deficiency in mice failed to show lifespan prolongation under CR (46
). However, few studies have provided direct evidence for the involvement of Sirt1 in the pathogenesis of age-associated mitochondrial damage or in the mechanism underlying long-term CR-mediated mitochondrial protection. In the present study, Sirt1 expression was significantly decreased in aged kidney and was enhanced by long-term CR. Furthermore, the results showing acetylation state of Foxo3 emphasized the suppression of Sirt1 deacetylase activity in aged kidney and its enhancement by long-term CR. We believe we are the first to show that long-term CR can cause Sirt1 activation even in aged tissues.
The in vitro model of CR has been used to determine the effects of CR serum on cell proliferation (39
), apoptosis (38
), and mitochondria biogenesis (20
). Using this model, we showed that aging AL and CR serum affected hypoxia-induced autophagy through modification of the Sirt1-PI3K-Foxo3 axis. Sirt1 deficiency and enhanced PI3K during the aging process suppress both autophagy and cell cycle arrest even under hypoxic conditions, which results in accumulation of oxidative stress and subsequent apoptosis through acetylated Foxo3-mediated Bim expression (Figure A). In contrast, CR-mediated Sirt1 activation promotes cell adaptation to hypoxia through Bnip3-mediated autophagy and p27Kip1 expression via deacetylation of Foxo3 (Figure B). Furthermore, the results of in vivo study showing the effect of CR on age-associated kidney phenotypes in Sirt1+/–
mice were in agreement with this molecular mechanism. Our present results provide insight into the molecular mechanism of CR-mediated cell adaptation to age-associated hypoxia. Because stress-induced apoptosis and cell loss have been implicated in age-associated diseases (47
), Sirt1 deficiency–mediated apoptosis should be involved in age-dependent kidney dysfunction through loss of PTCs, and Sirt1-mediated cell adaptation to hypoxia is essential for the CR-mediated kidney protection.
Molecular mechanisms underlying CR-mediated cellular adaptation to hypoxia in aged kidney.
A recent study reported that Sirt1 regulates autophagy through deacetylation of certain molecules associated with autophagy under starvation, but not hypoxia (48
). However, how the autophagic machinery selectively destroys damaged mitochondria remains elusive. A study showed that Bnip3 selectively and positively regulates hypoxia-induced mitochondrial autophagy through the disruption of bcl2-beclin1 interaction (15
), suggesting that Sirt1-Foxo3 pathway–mediated Bnip3 overexpression may be the regulatory mechanism through which the autophagic machinery selectively destroys damaged mitochondria under hypoxia.
Nuclear translocation of Foxo3 is negatively regulated by PI3K-Akt–mediated phosphorylation (40
) and positively by oxidative stress (37
). Under AL serum, phosphorylated Foxo3 showed cytoplasmic distribution at the early phase of hypoxia, which suppressed hypoxia-induced autophagy and increased oxidative stress. In contrast, during the late phase of hypoxia under AL serum, Foxo3 translocated into the nucleus in response to oxidative stress, resulting in Foxo3-mediated apoptosis through Bim expression, and this process was enhanced by PI3K inhibition. Thus, under hypoxia in AL serum, the PI3K-Akt pathway plays a biphasic role, increasing mitochondrial oxidative stress through inhibition of autophagy and suppressing Foxo3-mediated apoptosis under oxidative stress. These findings are in agreement with the notion that acceleration of the insulin-PI3K-Akt pathway enhances the aging process (50
), but has an antiapoptotic effect against hypoxia (51
) and oxidative stress (40
). Furthermore, Sirt1 attenuated PI3K inhibition–mediated acceleration of apoptosis under AL serum, which suggests that Sirt1, rather than PI3K, plays a central role for cell survival in the aging process.
Foxo3 promotes resistance to certain stress (37
), whereas it promotes apoptosis under other stress (40
). However, how Foxo3 selects the exact target gene in response to a specific condition remains obscure. In this study, under CR serum, Foxo3 was deacetylated by Sirt1 and bound to p27Kip1 and Bnip3 promoter, resulting in cell adaptation to hypoxia. In contrast, acetylated Foxo3 enhanced hypoxia-related apoptosis through Bim overexpression under AL serum. A prior report suggested that Sirt1 regulates Foxo3 transcriptional activity under oxidative stress to promote cell survival (37
). In addition to this, our results provide further evidence that Sirt1-mediated deacetylation of Foxo3 is essential for cell survival and the mechanism by which Foxo3 selects a specific gene promoter.
The present study demonstrated that mitochondrial damage in aged kidney is associated with Sirt1 deficiency and that Sirt1 promotes cell adaptation to hypoxia through autophagy in aged kidney. Our results provide what we believe to be the first evidence that Sirt1, the PI3K-Akt pathway, and Foxo3 coordinately regulate age-dependent tissue dysfunction and autophagy in mammals, as previously reported in lower organisms (23
). The reduced autophagy in the kidney may be involved in the age-associated weakness of PTCs against various renal toxicities, such as drug toxicity and proteinuria in glomerular diseases. Autophagy is currently the focus of research in various fields, such as aging (55
), metabolic diseases (56
), and immune diseases (58
). Our concept should be helpful in the design of studies aiming to further explore autophagy-related diseases and lead to the establishment of new therapies that can delay the progression of hypoxia- and age-related tissue dysfunction, including CKD in elderly patients.