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  • Klotho is a multifunctional protein that is

    2019-07-30

    Klotho is a multifunctional protein that is well known for its anti-aging property [35]. Klotho is not only predominantly expressed in the kidney as a membrane protein but also exists as a circulating soluble form generated by proteolytic cleavage [12]. Soluble Klotho acts as a hormone involved in phosphate metabolism and vascular homeostasis [13]. It is generally accepted that Klotho is not expressed in the heart [24], [36], [37], [38], [39], suggesting that the pathological and functional changes of the heart are likely related to deficient circulating soluble Klotho. Thus, in our study we mainly investigated the cardioprotective effect of circulating soluble Klotho. Recently, it was found that circulating Klotho prevents lung oxidative damage by increasing the endogenous antioxidative capacity of pulmonary epithelial Heparin [40]. Further, Klotho has been shown to interact with inflammatory mediators and to modulate tissue inflammatory responses [41], [42]. Despite this evidence, the role of Klotho in DCM remains unexplored. In the current study, we elucidated the preventive effects of Klotho on cardiac histological abnormalities, oxidative stress, inflammatory responses and cell death pathways using a type 1 diabetic mouse model and HG-treated cardiac cells. Accumulating evidence suggests that DCM is a chronic inflammatory disease that is characterized by elevated pro-inflammatory cytokine levels [43], [44]. Previously, our group reported a clear association between DCM and inflammation [45]. In addition to its antioxidant properties, Klotho has been reported to exhibit anti-inflammatory activity under several pathological conditions [46], [47]. A recent study indicated that Klotho suppresses cardiac inflammatory responses in aging endotoxemic mice [23]. In the current study, we found that Klotho significantly inhibited cardiac NF-κB activation induced by HG/diabetes, thus alleviating inflammatory cytokine expression both in vitro and in vivo (Fig. 1, Fig. 5A–D and S1). NF-κB is a critical transcription factor regulating inflammatory responses [31], [48], [49]. It mainly consists of di09mers of the two subunits p50 and p65 (RelA). In response to stimuli via the canonical pathway, the IKK complex is activated, which causes IκBα phosphorylation and subsequent degradation. The dissociation of IκBα from the sequestered cytoplasmic complex results in the translocation of the active NF-κB subunit p65 from the cytoplasm to the nucleus, where it then binds to NF-κB promoter sequences and triggers inflammatory gene expression. In the current study, we found that Klotho interferes with HG/diabetes-induced IκBα degradation and p65 translocation. Our findings indicated that Klotho has a potent role in inhibiting HG/diabetes-related cardiac inflammation via inactivation of the canonical NF-κB pathway. This hypothesis that Klotho suppresses the canonical NF-κB pathway has also been supported by previous findings [17], [42], [46], [47]. However, our observations are slightly different from those of a previous report by Zhao et al. [18]. Although both studies revealed a modulatory role of Klotho in NF-κB activity, the previous report found that Klotho inhibited RelA Ser536 phosphorylation without affecting IκBα degradation or p65 nuclear translocation. Some possible reasons for this discrepancy include 1) different cell types (renal epithelial cells vs. the cardiac cells used in our study), 2) distinctive cell culture conditions and the unique expression of signaling molecules, and 3) the source of Klotho. Excessive ROS generation has been identified as an initial pathogenic factor of DCM [32], and enhancing the myocardial endogenous antioxidative capacity has been considered effective to attenuate DCM. Nrf2, by regulating numerous antioxidant genes (i.e., NQO1, HO-1 and GCLC) in response to oxidative damage, has been found to play a central role in DCM [50], [51]. Klotho is an anti-aging hormone that confers antioxidant and anti-apoptotic effects in endothelial and renal cells [15], [47]. Previous studies have revealed that Klotho activates the Nrf2 pathway to prevent oxidative injury in several models [52], [53]. Recently, it was found that Klotho acts as an antioxidant to delay Nrf2 consumption in airway epithelial cells [54]. However, whether Klotho could protect the heart from HG-induced damage via Nrf2 activation has not been investigated. We found that treatment with Klotho elevated Nrf2 expression and activity in HG-stimulated H9c2 cells (Fig. 2C–D), neonatal cardiomyocytes (Fig. S2B) as well as in diabetic myocardium (Fig. 5F). This study also revealed a remarkable enhancement in Nrf2 nuclear accumulation by Klotho pretreatment in vitro (Fig. 2E and S2B). Moreover, a marked increase in the mRNA expression of the Nrf2 downstream genes NQO-1, HO-1, and GCLC was also observed after Klotho treatment (Figs. 2F and 5G). Therefore, these data indicate that Klotho-induced Nrf2 activation is a potential mechanism that protects against HG-stimulated oxidative stress to alleviate cardiac injury.