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AMP-activated Protein Kinase (AMPK) Negatively Regulates Nox4-dependent Activation of p53 and Epithelial Cell Apoptosis in Diabetes*

  • Assaad A. Eid
    Affiliations
    From the Department of Medicine, University of Texas Health Science Center, San Antonio, Texas 78229-3900
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  • Bridget M. Ford
    Affiliations
    From the Department of Medicine, University of Texas Health Science Center, San Antonio, Texas 78229-3900
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  • Karen Block
    Affiliations
    From the Department of Medicine, University of Texas Health Science Center, San Antonio, Texas 78229-3900

    Veterans Affairs Research, San Antonio, Texas 78229
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  • Balakuntalam S. Kasinath
    Affiliations
    From the Department of Medicine, University of Texas Health Science Center, San Antonio, Texas 78229-3900

    Veterans Affairs Research, San Antonio, Texas 78229
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  • Yves Gorin
    Affiliations
    From the Department of Medicine, University of Texas Health Science Center, San Antonio, Texas 78229-3900
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  • Goutam Ghosh-Choudhury
    Affiliations
    From the Department of Medicine, University of Texas Health Science Center, San Antonio, Texas 78229-3900

    Veterans Affairs Research, San Antonio, Texas 78229

    Geriatric Research Education and Clinical Center, South Texas Veterans Healthcare System, San Antonio, Texas 78229
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  • Jeffrey L. Barnes
    Affiliations
    From the Department of Medicine, University of Texas Health Science Center, San Antonio, Texas 78229-3900

    Veterans Affairs Research, San Antonio, Texas 78229
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  • Hanna E. Abboud
    Correspondence
    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;
    Affiliations
    From the Department of Medicine, University of Texas Health Science Center, San Antonio, Texas 78229-3900

    Veterans Affairs Research, San Antonio, Texas 78229
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  • Author Footnotes
    * 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.
Open AccessPublished:September 22, 2010DOI:https://doi.org/10.1074/jbc.M110.136796
      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.

      Introduction

      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 (
      • de Zeeuw D.
      • Remuzzi G.
      • Parving H.H.
      • Keane W.F.
      • Zhang Z.
      • Shahinfar S.
      • Snapinn S.
      • Cooper M.E.
      • Mitch W.E.
      • Brenner B.M.
      ). 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)
      The abbreviations used are: GBM
      glomerular basement membrane
      ROS
      reactive oxygen species
      HG
      high glucose
      AMPK
      AMP-activated protein kinase
      DN
      dominant-negative
      AICAR
      5-aminoimidazole-4-carboxamide-1-riboside
      ARA
      adenine 9-β-d-arabinofuranoside
      NG
      normal glucose.
      with decreased cell density (
      • Eid A.A.
      • Gorin Y.
      • Fagg B.M.
      • Maalouf R.
      • Barnes J.L.
      • Block K.
      • Abboud H.E.
      ,
      • Kanwar Y.S.
      • Liu Z.Z.
      • Kumar A.
      • Usman M.I.
      • Wada J.
      • Wallner E.I.
      ,
      • Wolf G.
      • Chen S.
      • Ziyadeh F.N.
      ). The mechanism(s) of podocyte depletion in diabetes are poorly understood.
      Expression of antioxidant enzymes in some animal models ameliorates diabetic kidney disease, thus establishing a role of reactive oxygen species (ROS) (
      • Brownlee M.
      ,
      • DeRubertis F.R.
      • Craven P.A.
      • Melhem M.F.
      ). 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 (
      • Eid A.A.
      • Gorin Y.
      • Fagg B.M.
      • Maalouf R.
      • Barnes J.L.
      • Block K.
      • Abboud H.E.
      ,
      • Gorin Y.
      • Block K.
      • Hernandez J.
      • Bhandari B.
      • Wagner B.
      • Barnes J.L.
      • Abboud H.E.
      ). 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 (
      • Lassègue B.
      • Griendling K.K.
      ). Along with Nox2, Nox1 and Nox4 are abundantly expressed in the renal cortex (
      • Gill P.S.
      • Wilcox C.S.
      ). We showed that Nox4 is expressed in rat and mouse glomeruli and contributes to matrix accumulation in diabetic kidney disease (
      • Eid A.A.
      • Gorin Y.
      • Fagg B.M.
      • Maalouf R.
      • Barnes J.L.
      • Block K.
      • Abboud H.E.
      ,
      • Gorin Y.
      • Block K.
      • Hernandez J.
      • Bhandari B.
      • Wagner B.
      • Barnes J.L.
      • Abboud H.E.
      ). Abundant expression of Nox4 in glomerular podocytes has been reported (
      • Eid A.A.
      • Gorin Y.
      • Fagg B.M.
      • Maalouf R.
      • Barnes J.L.
      • Block K.
      • Abboud H.E.
      ,
      • Sharma K.
      • Ramachandrarao S.
      • Qiu G.
      • Usui H.K.
      • Zhu Y.
      • Dunn S.R.
      • Ouedraogo R.
      • Hough K.
      • McCue P.
      • Chan L.
      • Falkner B.
      • Goldstein B.J.
      ). High glucose (HG) increases the expression of Nox4 and NADPH oxidase activity in podocytes (
      • Eid A.A.
      • Gorin Y.
      • Fagg B.M.
      • Maalouf R.
      • Barnes J.L.
      • Block K.
      • Abboud H.E.
      ). However, the mechanism by which glucose increases NADPH oxidase activity and the role of Nox4 in podocyte apoptosis are not known.
      AMP-activated protein kinase (AMPK), a serine/threonine kinase, is an energy sensor whose activity is regulated by glucose (
      • Long Y.C.
      • Zierath J.R.
      ). AMPK is a heterotrimeric protein consisting of a catalytic α-subunit and regulatory β- and γ-subunits (
      • Hardie D.G.
      • Carling D.
      ,
      • Hardie D.G.
      • Carling D.
      • Halford N.
      ,
      • Kemp B.E.
      • Mitchelhill K.I.
      • Stapleton D.
      • Michell B.J.
      • Chen Z.P.
      • Witters L.A.
      ,
      • Mitchelhill K.I.
      • Stapleton D.
      • Gao G.
      • House C.
      • Michell B.
      • Katsis F.
      • Witters L.A.
      • Kemp B.E.
      ). Seven AMPK genes encoding two α (α1 and α2), two β (β1 and β2), and three γ (γ1, γ2, and γ3) isoforms are present in the mammalian genome (
      • Rutter G.A.
      • Da Silva Xavier G.
      • Leclerc I.
      ,
      • Carling D.
      ,
      • Hardie D.G.
      ). The activity and subunit composition of AMPK are expressed in a cell- and tissue-specific manner, with the α1- and α2-subunits expressed in the kidney and in glomerular cells (
      • Cammisotto P.G.
      • Bendayan M.
      ). Activation of AMPK requires phosphorylation of a critical threonine residue (Thr172) in the activation loop of the α-subunit (
      • Hawley S.A.
      • Davison M.
      • Woods A.
      • Davies S.P.
      • Beri R.K.
      • Carling D.
      • Hardie D.G.
      ). 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 (
      • Kahn B.B.
      • Alquier T.
      • Carling D.
      • Hardie D.G.
      ). AMPK can also be activated independently of changes in the AMP/ATP ratio (
      • Woods A.
      • Dickerson K.
      • Heath R.
      • Hong S.P.
      • Momcilovic M.
      • Johnstone S.R.
      • Carlson M.
      • Carling D.
      ,
      • Hurley R.L.
      • Anderson K.A.
      • Franzone J.M.
      • Kemp B.E.
      • Means A.R.
      • Witters L.A.
      ,
      • Hawley S.A.
      • Pan D.A.
      • Mustard K.J.
      • Ross L.
      • Bain J.
      • Edelman A.M.
      • Frenguelli B.G.
      • Hardie D.G.
      ). AMPK signaling modulates multiple biological pathways, such as protein synthesis (
      • Shibata R.
      • Ouchi N.
      • Ito M.
      • Kihara S.
      • Shiojima I.
      • Pimentel D.R.
      • Kumada M.
      • Sato K.
      • Schiekofer S.
      • Ohashi K.
      • Funahashi T.
      • Colucci W.S.
      • Walsh K.
      ,
      • Tian R.
      • Musi N.
      • D'Agostino J.
      • Hirshman M.F.
      • Goodyear L.J.
      ,
      • Lee M.J.
      • Feliers D.
      • Mariappan M.M.
      • Sataranatarajan K.
      • Mahimainathan L.
      • Musi N.
      • Foretz M.
      • Viollet B.
      • Weinberg J.M.
      • Choudhury G.G.
      • Kasinath B.S.
      ), autophagy (
      • Matsui Y.
      • Takagi H.
      • Qu X.
      • Abdellatif M.
      • Sakoda H.
      • Asano T.
      • Levine B.
      • Sadoshima J.
      ,
      • Meijer A.J.
      • Codogno P.
      ), and apoptosis (
      • Hickson-Bick D.L.
      • Buja L.M.
      • McMillin J.B.
      ,
      • Russell 3rd, R.R.
      • Li J.
      • Coven D.L.
      • Pypaert M.
      • Zechner C.
      • Palmeri M.
      • Giordano F.J.
      • Mu J.
      • Birnbaum M.J.
      • Young L.H.
      ,
      • Kim J.
      • Ahn J.H.
      • Kim J.H.
      • Yu Y.S.
      • Kim H.S.
      • Ha J.
      • Shinn S.H.
      • Oh Y.S.
      ,
      • Riboulet-Chavey A.
      • Diraison F.
      • Siew L.K.
      • Wong F.S.
      • Rutter G.A.
      ).
      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.

      DISCUSSION

      Podocyte apoptosis is an early glomerular phenotype that contributes to podocyte depletion, albuminuria, and progression of renal disease (
      • Eid A.A.
      • Gorin Y.
      • Fagg B.M.
      • Maalouf R.
      • Barnes J.L.
      • Block K.
      • Abboud H.E.
      ,
      • Susztak K.
      • Raff A.C.
      • Schiffer M.
      • Böttinger E.P.
      ). 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).
      Figure thumbnail gr8
      FIGURE 8Proposed mechanism of HG/diabetes-induced glomerular epithelial cell (podocyte) apoptosis. See “Discussion” for details.
      ROS-generating NADPH oxidases play a dual role in regulating cellular apoptosis (
      • Lassègue B.
      • Griendling K.K.
      ,
      • Dong J.
      • Sulik K.K.
      • Chen S.Y.
      ,
      • Vaquero E.C.
      • Edderkaoui M.
      • Pandol S.J.
      • Gukovsky I.
      • Gukovskaya A.S.
      ,
      • Mochizuki T.
      • Furuta S.
      • Mitsushita J.
      • Shang W.H.
      • Ito M.
      • Yokoo Y.
      • Yamaura M.
      • Ishizone S.
      • Nakayama J.
      • Konagai A.
      • Hirose K.
      • Kiyosawa K.
      • Kamata T.
      ). NADPH-generated ROS play an important role in ethanol-induced apoptosis (
      • Dong J.
      • Sulik K.K.
      • Chen S.Y.
      ). However, NADPH oxidase-derived ROS, including Nox4, inhibit apoptosis in pancreatic cancer cells (
      • Vaquero E.C.
      • Edderkaoui M.
      • Pandol S.J.
      • Gukovsky I.
      • Gukovskaya A.S.
      ,
      • Mochizuki T.
      • Furuta S.
      • Mitsushita J.
      • Shang W.H.
      • Ito M.
      • Yokoo Y.
      • Yamaura M.
      • Ishizone S.
      • Nakayama J.
      • Konagai A.
      • Hirose K.
      • Kiyosawa K.
      • Kamata T.
      ). In this study, we have demonstrated that Nox4 induces podocyte apoptosis and identify inactivation of AMPK as a mechanism by which HG and hyperglycemia increase the expression of Nox4.
      AMPK activity is maintained through constitutive phosphorylation of Thr172 in the catalytic α-subunit by such upstream kinases as LKB1 and calcium/calmodulin kinase kinase-β (
      • Hawley S.A.
      • Davison M.
      • Woods A.
      • Davies S.P.
      • Beri R.K.
      • Carling D.
      • Hardie D.G.
      ,
      • Woods A.
      • Dickerson K.
      • Heath R.
      • Hong S.P.
      • Momcilovic M.
      • Johnstone S.R.
      • Carlson M.
      • Carling D.
      ,
      • Hurley R.L.
      • Anderson K.A.
      • Franzone J.M.
      • Kemp B.E.
      • Means A.R.
      • Witters L.A.
      ,
      • Hawley S.A.
      • Pan D.A.
      • Mustard K.J.
      • Ross L.
      • Bain J.
      • Edelman A.M.
      • Frenguelli B.G.
      • Hardie D.G.
      ). The interaction between ROS and AMPK is regulated in a cell stimulus- and tissue-specific manner. During hypoxia, mitochondria-generated ROS activate AMPK (
      • Emerling B.M.
      • Weinberg F.
      • Snyder C.
      • Burgess Z.
      • Mutlu G.M.
      • Viollet B.
      • Budinger G.R.
      • Chandel N.S.
      ). Similarly, during exercise, NADPH oxidase-derived ROS induce AMPK activation (
      • Moir H.
      • Hughes M.G.
      • Potter S.
      • Sims C.
      • Butcher L.R.
      • Davies N.A.
      • Verheggen K.
      • Jones K.P.
      • Thomas A.W.
      • Webb R.
      ). However, in β-cells exposed to HG, AMPK activation increases ROS production and potentiates β-cell apoptosis (
      • Kim W.H.
      • Lee J.W.
      • Suh Y.H.
      • Lee H.J.
      • Lee S.H.
      • Oh Y.K.
      • Gao B.
      • Jung M.H.
      ). AMPK plays a role in renal cell injury (
      • Sharma K.
      • Ramachandrarao S.
      • Qiu G.
      • Usui H.K.
      • Zhu Y.
      • Dunn S.R.
      • Ouedraogo R.
      • Hough K.
      • McCue P.
      • Chan L.
      • Falkner B.
      • Goldstein B.J.
      ,
      • Lee M.J.
      • Feliers D.
      • Mariappan M.M.
      • Sataranatarajan K.
      • Mahimainathan L.
      • Musi N.
      • Foretz M.
      • Viollet B.
      • Weinberg J.M.
      • Choudhury G.G.
      • Kasinath B.S.
      ). Type 1 diabetic rats treated with AICAR, a pharmacologic activator of AMPK, show significant attenuation of renal hypertrophy (
      • Lee M.J.
      • Feliers D.
      • Mariappan M.M.
      • Sataranatarajan K.
      • Mahimainathan L.
      • Musi N.
      • Foretz M.
      • Viollet B.
      • Weinberg J.M.
      • Choudhury G.G.
      • Kasinath B.S.
      ). Inhibition of AMPK activity in vitro alters localization of ZO-1 (zona occludens-1) in podocytes, an effect reversed by the pharmacologic activator AICAR and by the AMPK activator adiponectin (
      • Sharma K.
      • Ramachandrarao S.
      • Qiu G.
      • Usui H.K.
      • Zhu Y.
      • Dunn S.R.
      • Ouedraogo R.
      • Hough K.
      • McCue P.
      • Chan L.
      • Falkner B.
      • Goldstein B.J.
      ), indicating a role for this stress-sensing kinase in podocyte biology.
      AMPK exerts pro- or anti-apoptotic effects that are stimulus- or cell type-specific (
      • Hickson-Bick D.L.
      • Buja L.M.
      • McMillin J.B.
      ,
      • Russell 3rd, R.R.
      • Li J.
      • Coven D.L.
      • Pypaert M.
      • Zechner C.
      • Palmeri M.
      • Giordano F.J.
      • Mu J.
      • Birnbaum M.J.
      • Young L.H.
      ,
      • Kim J.
      • Ahn J.H.
      • Kim J.H.
      • Yu Y.S.
      • Kim H.S.
      • Ha J.
      • Shinn S.H.
      • Oh Y.S.
      ,
      • Riboulet-Chavey A.
      • Diraison F.
      • Siew L.K.
      • Wong F.S.
      • Rutter G.A.
      ). In pancreatic β-cells, HG activates AMPK and enhances the production of ROS, resulting in loss of mitochondrial membrane potential (
      • Kim W.H.
      • Lee J.W.
      • Suh Y.H.
      • Lee H.J.
      • Lee S.H.
      • Oh Y.K.
      • Gao B.
      • Jung M.H.
      ). 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 (
      • Kukidome D.
      • Nishikawa T.
      • Sonoda K.
      • Imoto K.
      • Fujisawa K.
      • Yano M.
      • Motoshima H.
      • Taguchi T.
      • Matsumura T.
      • Araki E.
      ), 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 (
      • Lee M.
      • Feliers D.
      • Sataranatarajan K.
      • Mariappan M.M.
      • Li M.
      • Barnes J.L.
      • Choudhury G.G.
      • Kasinath B.S.
      ).
      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 (
      • Chipuk J.E.
      • Green D.R.
      ). It plays a compelling role in the apoptotic intrinsic pathway by integrating the action of pro-apoptotic Bax (Bcl-2-associated X protein) (
      • Chipuk J.E.
      • Green D.R.
      ). This action of p53 is regulated mainly by enhanced transcription of Bax (
      • Vousden K.H.
      • Prives C.
      ). However, a direct transcription-independent role of p53 in the induction of apoptosis has also been reported (
      • Mihara M.
      • Erster S.
      • Zaika A.
      • Petrenko O.
      • Chittenden T.
      • Pancoska P.
      • Moll U.M.
      ,
      • Chipuk J.E.
      • Kuwana T.
      • Bouchier-Hayes L.
      • Droin N.M.
      • Newmeyer D.D.
      • Schuler M.
      • Green D.R.
      ). Also, p53 directly activates the pro-apoptotic protein Bax to initiate apoptosis through the mitochondrial pathway (
      • Chipuk J.E.
      • Kuwana T.
      • Bouchier-Hayes L.
      • Droin N.M.
      • Newmeyer D.D.
      • Schuler M.
      • Green D.R.
      ). 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 (
      • Vousden K.H.
      • Prives C.
      ). Phosphorylation at Ser46 correlates with the p53 transcriptional program that launches apoptosis (
      • D'Orazi G.
      • Cecchinelli B.
      • Bruno T.
      • Manni I.
      • Higashimoto Y.
      • Saito S.
      • Gostissa M.
      • Coen S.
      • Marchetti A.
      • Del Sal G.
      • Piaggio G.
      • Fanciulli M.
      • Appella E.
      • Soddu S.
      ,
      • Hofmann T.G.
      • Möller A.
      • Sirma H.
      • Zentgraf H.
      • Taya Y.
      • Dröge W.
      • Will H.
      • Schmitz M.L.
      ). Kinases such as PKCδ and p38 MAPK, which may phosphorylate this residue, are known to be activated by HG (
      • Vousden K.H.
      • Prives C.
      ,
      • Geraldes P.
      • Hiraoka-Yamamoto J.
      • Matsumoto M.
      • Clermont A.
      • Leitges M.
      • Marette A.
      • Aiello L.P.
      • Kern T.S.
      • King G.L.
      ,
      • Levine A.J.
      • Feng Z.
      • Mak T.W.
      • You H.
      • Jin S.
      ,
      • Yu J.
      • Zhang L.
      ). 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 (
      • Yu J.
      • Zhang L.
      ). PUMA-deficient mice show a requirement of p53-dependent PUMA expression for induction of apoptosis in many tissues (
      • Yu J.
      • Zhang L.
      ,
      • Villunger A.
      • Michalak E.M.
      • Coultas L.
      • Müllauer F.
      • Böck G.
      • Ausserlechner M.J.
      • Adams J.M.
      • Strasser A.
      ). Interestingly, this function of PUMA establishes that both transcription-dependent and transcription-independent activities of p53 are required for induction of apoptosis (
      • Vousden K.H.
      • Prives C.
      ). PUMA binds to the anti-apoptotic protein Bcl-xL, thus releasing cytosolic p53 to activate Bax (
      • Vousden K.H.
      • Prives C.
      ,
      • Chipuk J.E.
      • Kuwana T.
      • Bouchier-Hayes L.
      • Droin N.M.
      • Newmeyer D.D.
      • Schuler M.
      • Green D.R.
      ). 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 (
      • Okoshi R.
      • Ozaki T.
      • Yamamoto H.
      • Ando K.
      • Koida N.
      • Ono S.
      • Koda T.
      • Kamijo T.
      • Nakagawara A.
      • Kizaki H.
      ). In murine embryonic fibroblasts, low glucose also activates AMPK and induces the expression and phosphorylation of p53 (
      • Jones R.G.
      • Plas D.R.
      • Kubek S.
      • Buzzai M.
      • Mu J.
      • Xu Y.
      • Birnbaum M.J.
      • Thompson C.B.
      ). Furthermore, glucose starvation reduces p53 stability, which correlates with AMPK inactivation, indicating that AMPK may positively regulate the function of p53 (
      • Lee C.H.
      • Inoki K.
      • Karbowniczek M.
      • Petroulakis E.
      • Sonenberg N.
      • Henske E.P.
      • Guan K.L.
      ). 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 (
      • Liu B.
      • Chen Y.
      • St Clair D.K.
      ). p53 has been shown to regulate ROS generation, and conversely, ROS generation modulates selective transactivation of p53 target genes (
      • Liu B.
      • Chen Y.
      • St Clair D.K.
      ). 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 (
      • Gorin Y.
      • Block K.
      • Hernandez J.
      • Bhandari B.
      • Wagner B.
      • Barnes J.L.
      • Abboud H.E.
      ). More recently, we reported increased expression of Nox4 in glomeruli of diabetic OVE26 mice (
      • Eid A.A.
      • Gorin Y.
      • Fagg B.M.
      • Maalouf R.
      • Barnes J.L.
      • Block K.
      • Abboud H.E.
      ). 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 (
      • Forbes J.M.
      • Coughlan M.T.
      • Cooper M.E.
      ). 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.

      Acknowledgments

      We thank Andrea Barrantine, Sergio Garcia, and Fredyne Springer for technical assistance.

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