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From the Department of Medicine, University of Texas Health Science Center, San Antonio, Texas 78229-3900Veterans Affairs Research, San Antonio, Texas 78229Geriatric Research Education and Clinical Center, South Texas Veterans Healthcare System, San Antonio, Texas 78229
To whom correspondence should be addressed: Dept. of Medicine, Div. of Nephrology, MC 7882, University of Texas Health Science Center, 7703 Floyd Curl Dr., San Antonio, TX 78229-3900. Tel.: 210-567-4700; Fax: 210-567-4712;
* This work was supported, in whole or in part, by National Institutes of Health Grants CA131272 (to K. B.), R01 DK079996 (to Y. G.), R01 DK50190 (to G. G.-C.), R01 DK080106 (to J. L. B.), and DK-R01-078971 (to H. E. A.) and George O'Brien Kidney Center-Morphology Core Grant DK061597 (to J. L. B.). This work was also supported by a National Kidney Foundation postdoctoral fellowship grant and a Juvenile Diabetes Research Foundation grant (to A. A. E.), a Juvenile Diabetes Research Foundation regular research grant (to Y. G.), and a Juvenile Diabetes Research Foundation grant and a Veterans Affairs merit review grant (to H. E. A.). The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S3.
Diabetes and high glucose (HG) increase the generation of NADPH oxidase-derived reactive oxygen species and induce apoptosis of glomerular epithelial cells (podocytes). Loss of podocytes contributes to albuminuria, a major risk factor for progression of kidney disease. Here, we show that HG inactivates AMP-activated protein kinase (AMPK), up-regulates Nox4, enhances NADPH oxidase activity, and induces podocyte apoptosis. Activation of AMPK blocked HG-induced expression of Nox4, NADPH oxidase activity, and apoptosis. We also identified the tumor suppressor protein p53 as a mediator of podocyte apoptosis in cells exposed to HG. Inactivation of AMPK by HG up-regulated the expression and phosphorylation of p53, and p53 acted downstream of Nox4. To investigate the mechanism of podocyte apoptosis in vivo, we used OVE26 mice, a model of type 1 diabetes. Glomeruli isolated from these mice showed decreased phosphorylation of AMPK and enhanced expression of Nox4 and p53. Pharmacologic activation of AMPK by 5-aminoimidazole-4-carboxamide-1-riboside in OVE26 mice attenuated Nox4 and p53 expression. Administration of 5-aminoimidazole-4-carboxamide-1-riboside also prevented renal hypertrophy, glomerular basement thickening, foot process effacement, and podocyte loss, resulting in marked reduction in albuminuria. Our results uncover a novel function of AMPK that integrates metabolic input to Nox4 and provide new insight for activation of p53 to induce podocyte apoptosis. The data indicate the potential therapeutic utility of AMPK activators to block Nox4 and reactive oxygen species generation and to reduce urinary albumin excretion in type 1 diabetes.
One of the major early features of diabetic kidney disease is injury to glomerular epithelial cells or podocytes, which contribute to the increased urinary albumin losses and accelerated sclerosis of the glomerular microvascular bed (
). Podocyte injury manifests as phenotypic changes that range from foot process effacement and altered localization or abundance of specific slit diaphragm proteins to frank apoptosis with detachment of the cells from the glomerular basement membrane (GBM)
). More recently, along with ROS generated from mitochondrial respiratory chains, NADPH oxidase-derived ROS have been shown to play a significant role in injury to various organs, including the kidney (
). A number of homologs of the phagocyte NADPH oxidase catalytic subunit (Nox2) have been identified. These enzymes participate in a number of biological processes, including proliferation, migration, contraction, cytoskeletal organization, fibrosis, and apoptosis (
). In energy depletion states, AMPK activation slows metabolic reactions that consume ATP and stimulates reactions that produce ATP, thereby restoring the AMP/ATP ratio and the normal cellular energy stores (
In this study, we provide the first evidence that inactivation of AMPK by HG increases the expression of Nox4 and that the increased expression of Nox4 mediates podocyte apoptosis. Additionally, we demonstrate that Nox4 increases the abundance of p53 protein concomitant with an increase in its phosphorylation at Ser46 to increase the expression of the pro-apoptotic protein PUMA (p53-up-regulated modulator of apoptosis). In type 1 diabetic mice, AMPK is inactivated and up-regulates Nox4 to induce podocyte apoptosis. Furthermore, we show that pharmacologic activation of AMPK prevents these changes in vitro in podocytes and in vivo in diabetic mice and attenuates albuminuria.
Podocyte apoptosis is an early glomerular phenotype that contributes to podocyte depletion, albuminuria, and progression of renal disease (
). In this study, we have provided the first evidence that podocyte apoptosis in diabetes is mediated through inactivation of AMPK, up-regulation of Nox4, and an increase in NADPH oxidase-mediated ROS production. Furthermore, we have demonstrated that AMPK and Nox4 regulate the expression/phosphorylation of p53 and the pro-apoptotic protein PUMA. We have also shown that the AMPK/Nox4-driven pro-apoptotic pathway is operative in glomeruli of diabetic mice and that activation of AMPK by the administration of AICAR attenuates Nox4 and p53 expression, reduces albuminuria, and protects mice against podocyte loss and glomerular injury (Fig. 8).
ROS-generating NADPH oxidases play a dual role in regulating cellular apoptosis (
). On the other hand, in umbilical vein endothelial cells, activation of AMPK increases the expression of the antioxidant manganese superoxide dismutase and inhibits HG-induced intracellular and mitochondrial ROS production (
), suggesting that activated AMPK may suppress oxidative stress. The data in our study demonstrate that HG inactivates AMPK and significantly increases the expression of Nox4 and NADPH oxidase activity. Activation of AMPK by AICAR or expression of the AMPKα2 subunit prevents these effects of HG. Our data also suggest that inhibition of AMPK by HG is likely due to reduction in the phosphorylation and activity of LKB1. These results are consistent with recently published data showing that AMPK inactivation by HG is due to reduced LKB1 activity (
In this study, we also established that inactivation of AMPK increases Nox4 and NADPH oxidase activity and mediates the pro-apoptotic effect of HG on podocytes. In fact, our results using the pharmacologic activator AICAR or the inhibitor ARA indicate that AMPK inactivation is necessary for podocyte apoptosis. This conclusion is further substantiated using exogenous AMPKα2 and dominant-negative AMPKα2. To our knowledge, this is the first report in which inactivation of AMPK is linked to increased expression of Nox4 and NADPH oxidase activity, resulting in cell apoptosis in the HG environment. However, the mechanism by which AMPK regulates Nox4 protein expression and whether it involves transcription or stabilization of the mRNA need to be explored.
The active tumor suppressor transcription factor p53 is induced by genotoxic stress and energy starvation, both of which promote cell death (
). In this study, HG induced caspase-3 activation and DNA fragmentation and apoptosis of podocytes in culture. The induction of apoptosis by HG was associated with a significant increase in p53 mRNA and protein. Furthermore, down-regulation of p53 blocked HG-induced apoptosis of podocytes. These results represent a mechanism of HG-induced podocyte apoptosis involving p53 plausibly through the intrinsic pathway.
Phosphorylation of p53 at multiple serine residues is required for its transcriptional activity (
). Our results demonstrate that HG increases p53 phosphorylation at Ser46, suggesting enhancement of its transcriptional activity. We also observed an increase in p53 protein levels in cells exposed to HG. Therefore, it is likely that the increase in p53 phosphorylation is an indirect effect resulting from increased p53 levels.
The transcription-dependent pro-apoptotic function of p53 is mediated principally by the pro-apoptotic protein Bax and the BH3-only protein PUMA, either of which can carry out apoptosis through the mitochondrial pathway (
). In this study, we have shown a HG-mediated increase in the expression of PUMA mRNA concomitant with enhanced accumulation and phosphorylation of p53. Also, we have shown that PUMA expression is dependent upon HG-induced expression of p53. Furthermore, HG significantly enhanced the expression of Bax in podocytes (data not shown). These results suggest that HG stimulates accumulation of p53, which contributes to apoptosis of podocytes likely through the intrinsic pathway.
AMPK has been shown to regulate p53 activity and phosphorylation in a stimulus- and tissue-specific manner. Nutrient-deprived thymocytes show enhanced apoptosis associated with an increase in AMPK activity. In this model, AMPK activation results in enhanced levels of p53 and its Ser46 phosphorylation (
). In this study, we found that podocytes cultured in NG had low levels of p53. Treatment with HG significantly increased p53 mRNA and protein levels and p53 phosphorylation. We also showed that pharmacologic and genetic activation of AMPK inhibited the accumulation of p53 and its phosphorylation in response to HG. In addition, our data demonstrate that inactivation of AMPK by ARA exerts effects similar to HG and induces the expression and phosphorylation of p53, resulting in enhanced expression of PUMA mRNA and protein. Collectively, our data indicate that in podocytes, AMPK negatively regulates p53 expression/phosphorylation and expression of the pro-apoptotic protein PUMA.
The interaction between p53 and ROS is well described (
). In our study, we have presented evidence that Nox4 regulates the expression and phosphorylation of p53 in response to HG and that Nox4 mediates HG-induced expression of PUMA. These results conclusively demonstrate a positive regulatory role of inactivated AMPK and up-regulated Nox4 in the increased expression and phosphorylation of p53, leading to apoptosis of podocytes exposed to HG.
We previously reported glomerular hypertrophy and increased matrix protein expression in type 1 diabetic rats concomitant with increased Nox4 expression; inhibition of Nox4 ameliorates glomerular hypertrophy and matrix expansion (
). In this study, we have provided evidence that increased expression of Nox4 and augmented NADPH oxidase activity in glomeruli of these diabetic mice are associated with podocyte loss and severe albuminuria. In vitro, our results show involvement of AMPK inactivation in the up-regulation of Nox4, which results in enhanced expression of p53 necessary for podocyte apoptosis in cells exposed to HG (FIGURE 2, FIGURE 3, FIGURE 4, FIGURE 5). In line with these data, we found that pharmacologic activation of AMPK in diabetic OVE26 mice resulted in attenuation of Nox4 expression and a decrease in NADPH oxidase activity. AMPK inactivation in diabetic OVE26 mice also increased the expression of p53 in the glomeruli and enhanced the expression of the pro-apoptotic PUMA mRNA and protein (Fig. 6, E and F). These results indicate that Nox4-mediated up-regulation of p53 and PUMA may contribute to the loss of podocytes in the diabetic glomeruli (Fig. 7, D and E) and that activation of AMPK by the administration of AICAR attenuates Nox4 expression and podocyte loss and ameliorates albuminuria.
Although the contribution of ROS to the complications of diabetic kidney disease is established, the administration of antioxidants has not been associated with potent protection against apoptosis in human diabetic nephropathy (
). Our data in this study identify a previously unrecognized direct target, Nox4, for treating diabetic kidney disease. Our observations suggest that AMPK activators or Nox4 inhibitors may represent an adjunct therapy in addition to metabolic control to reduce kidney damage in type 1 diabetes.
We thank Andrea Barrantine, Sergio Garcia, and Fredyne Springer for technical assistance.