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Mol. Cells 2015; 38(4): 285-296

Published online March 31, 2015

https://doi.org/10.14348/molcells.2015.0010

© The Korean Society for Molecular and Cellular Biology

Upstream Regulators and Downstream Effectors of NADPH Oxidases as Novel Therapeutic Targets for Diabetic Kidney Disease

Yves Gorin*, and Fabien Wauquier

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Department of Medicine, University of Texas Health Science Center, San Antonio, Texas, USA

Correspondence to : *Correspondence: gorin@uthscsa.edu

Received: January 9, 2015; Accepted: January 12, 2015

Abstract

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Oxidative stress has been linked to the pathogenesis of diabetic nephropathy, the complication of diabetes in the kidney. NADPH oxidases of the Nox family, and in particular the homologue Nox4, are a major source of reactive oxygen species in the diabetic kidney and are critical mediators of redox signaling in glomerular and tubulointerstitial cells exposed to the diabetic milieu. Here, we present an overview of the current knowledge related to the understanding of the role of Nox enzymes in the processes that control mesangial cell, podocyte and tubulointerstitial cell injury induced by hyperglycemia and other predominant factors enhanced in the diabetic milieu, including the renin-angiotensin system and transforming growth factor-β. The nature of the upstream modulators of Nox enzymes as well as the downstream targets of the Nox NADPH oxidases implicated in the propagation of the redox processes that alter renal biology in diabetes will be highlighted.

Keywords diabetic nephropathy, downstream effectors of Nox NADPH oxidases, Nox family of NADPH oxidases, oxidative stress, upstream regulators of Nox NADPH oxidases

INTRODUCTION

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Oxidative stress plays a critical role in the initiation and development of diabetic nephropathy (DN) (Baynes, 1991; Forbes et al., 2008; Giacco and Brownlee, 2010; Hinokio et al., 1999; Kashihara et al., 2010; Schnackenberg, 2002; Singh et al., 2011; Son et al., 2004; Stanton, 2011). Diabetes is associated with an increase in generation of reactive oxygen species (ROS) in the glomerular and tubulointerstitial compartments of the kidney (Forbes et al., 2008; Kanwar et al., 2008; 2011; Kashihara et al., 2010; Koya et al., 2003; Singh et al., 2011; Stanton, 2011; Vasavada and Agarwal, 2005). Although chronic hyperglycemia is sufficient to promote renal cell injury, data from experimental animal models as well as cultured cells indicate that a combination of growth factors, hormones and cytokines, in addition to high glucose concentrations, alter the redox status of the cells and contribute to cell damages in the diabetic kidney (Abboud, 1997; Campbell et al., 2011; Gorin and Block, 2013a; 2013b; Kanwar et al., 2008; 2011; Kashihara et al., 2010; Rincon-Choles et al., 2002; Singh et al., 2011). While multiple pathways lead to ROS generation, i.e., mitochondrial electron transport chain, xanthine oxidase or uncoupled nitric oxide synthase, recent evidence demonstrates that the Nox family of NADPH oxidases constitutes a major source of ROS in nonphagocytic cells, including renal cells (Ago et al., 2010; Barnes and Gorin, 2011; Bedard and Krause, 2007; Block et al., 2009; Brown and Griendling, 2009; Brownlee, 2005; Coughlan et al., 2009; Craven et al., 2001; Dikalov, 2011; Gill and Wilcox, 2006; Gorin and Block, 2013a; 2013b; Gorin et al., 2005; Griendling and FitzGerald, 2003; Hwang et al., 2012; Kiritoshi et al., 2003; Kitada et al., 2011; Lambeth, 2007; Lambeth et al., 2007; Lassegue and Clempus, 2003; Lassegue and Griendling, 2010; Lassegue et al., 2012; Nishikawa et al., 2000; Octavia et al., 2012; Paravicini and Touyz, 2008; Rivera et al., 2010; Sedeek et al., 2012). The present review will focus on the upstream regulators controlling the expression and activity of the Nox enzymes as well as the downstream effectors targeted by Nox-derived ROS to initiate and propagate diabetes-induced glomerular and tubulointerstitial cell injury.

NADPH OXIDASES OF THE NOX FAMILY PREDOMINANTLY EXPRESSED IN THE KIDNEY

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To date, the Nox family comprises seven members: Nox1-5, and the dual oxidases (Duox) Duox1 and -2 (Bedard and Krause, 2007; Geiszt, 2006; Lambeth et al., 2007; Selemidis et al., 2008). Nox4, Nox1, Nox2 (a.k.a. gp91phox), and Nox5 are the Nox homologues that are predominantly expressed in glomerular cells (mesangial cells, glomerular epithelial cells or podocytes), glomerular endothelial cells and tubulointerstitial cells (tubular epithelial cells and interstitial fibroblasts) (Gorin and Block, 2013a; 2013b). These Nox homologues consist of six membrane-spanning regions with binding sites for NADPH, flavin adenine dinucleotide (FAD), and heme. The latter comprise electron transfer centers that pass electrons from NADPH to oxygen forming superoxide (O2?) and hydrogen peroxide (H2O2) (Bedard and Krause, 2007; Brown and Griendling, 2009; Geiszt, 2006; Geiszt et al., 2000; Gorin and Block, 2013a; Lassegue and Griendling, 2010; Lassegue et al., 2012; Selemidis et al., 2008; Shiose et al., 2001). The calcium-dependent homolog Nox5 is found in the human kidney tissue and cells, but is not present in mice and rats (Brandes et al., 2010; Lassegue and Griendling, 2010).

The isoforms Nox2 and Nox1 require p22phox as an activating, stabilizing and/or regulatory subunit for binding to p47phox or NoxO1 (Bedard and Krause, 2007; Geiszt, 2006; Selemidis et al., 2008). Activation mechanisms for Nox1 are similar to those of Nox2, and involve complex formation with regulatory subunits upon agonist stimulation. Although Nox1 seems to primarily interact with the p47phox homolog NoxO1 (Nox organizer 1), the p67phox homolog NoxA1 (Nox activator 1), and small GTPase Rac upon activation, it was reported that p47phox and p67phox can partially replace NoxO1 and NoxA1, respectively (Bedard and Krause, 2007; Brandes and Schroder, 2008; Brandes et al., 2010; Geiszt, 2006; Lambeth, 2007; Lassegue and Griendling, 2010; Selemidis et al., 2008;). Nox4 heterodimerization with p22phox enhances the enzyme activity but the oxidase does not required the regulatory subunits essential to other Nox isoforms and is a constitutively active enzyme regulated primarily at the level of its expression in response to various stimuli, including mediators of DN such as high glucose (HG) (Block et al., 2009; Eid et al., 2009; 2013b) transforming growth factor-β (TGF-β) (Bondi et al., 2010; Cucoranu et al., 2005), angiotensin II (Ang II) (Block et al., 2008), insulin, insulin-like growth factor (IGF-I), and advanced glycation end products (AGEs) or advanced oxidation protein products (AOPPs) (Bedard and Krause, 2007; Lassegue and Clempus, 2003; Lassegue and Griendling, 2010; Lassegue et al., 2012; Lee et al., 2013a; Mahadev et al., 2004; Meng et al., 2008; Menini et al., 2007; New et al., 2012; Selemidis et al., 2008; Thallas-Bonke et al., 2008). As a consequence, the overall ROS output of Nox4 is directly governed by its expression level. Importantly, it is suggested that transcriptional events are implicated in the chronic control of Nox4 protein expression (Chai et al., 2008; Cucoranu et al., 2005; Eid et al., 2009; 2010; 2013a; Hecker et al., 2009; Moe et al., 2006; Mittal et al., 2007; Pedruzzi et al., 2004; Serrander et al., 2007; Wingler et al., 2001; Yamagishi et al., 2005), whereas acute regulation of Nox4 occurs via translational mechanisms without change in its mRNA levels (Block et al., 2008; 2009; Bondi et al., 2010; Meng et al., 2008; New et al., 2012; Peshavariya et al., 2009). Regulatory proteins that enhance Nox4 activity like polymerase delta-interacting protein 2 (Poldip2) and the p47phox-related adaptor protein, Tks (tyrosine kinase substrate with five SH3 domains) 5, have recently been identified (Diaz et al., 2009; Lyle et al., 2009; Sedeek et al., 2009). A potential implication of Rac in the control of Nox4 function was suggested in endothelial and mesangial cells (Chai et al., 2008; Gorin et al., 2003; Wu et al., 2007); however, it remains a controversial topic because, unlike Nox2 or Nox1, Rac1 does not activate Nox4 in transfected cells (Bedard and Krause, 2007). The activity of Nox5 is not controlled by any other subunits and is strictly dependent on calcium binding to the enzyme through its EF hand domains (Bedard and Krause, 2007).

It is important to mention that Nox4 seems to produce a higher hydrogen peroxide to superoxide ratio than Nox1, Nox2 and Nox5. Recent studies strongly suggest that hydrogen peroxide formation occurs through Nox4 third extracytosolic loop (E-loop) and that the structure of the E-loop may obstruct superoxide release as well as provide a source for protons, thus permitting rapid dismutation of superoxide to generate hydrogen peroxide (Takac et al., 2011). Although Nox4 predominantly produces hydrogen peroxide, numerous studies in vascular or renal cells and tissue detected Nox4-dependent superoxide production (Ago et al., 2010; Block et al., 2009; Clempus et al., 2007; Cucoranu et al., 2005; Eid et al., 2009; 2010; Gorin et al., 2005; Kuroda et al., 2005; 2010; Liu et al., 2010; Maalouf et al., 2012; Peshavariya et al., 2007; Shiose et al., 2001). Figure 1 shows the structure and molecular organization of the renal Nox NADPH oxidases.

UPSTREAM REGULATORS AND DOWNSTREAM EFFECTORS OF NOX NADPH OXIDASES IN DIABETES-INDUCED GLOMERULAR MESANGIAL CELL INJURY

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The importance of Nox4 in mesangial cell injury is supported by studies in experimental rodent models of diabetes as well as in vitro work in cultured cells exposed to HG. Nox4 protein expression increases in the glomeruli, including the mesangium, and Nox4-derived ROS contribute to oxidative stress during the initial and chronic stages of diabetes (Eid et al., 2009; 2010; Etoh et al., 2003; Fujii et al., 2007; 2010; Gorin et al., 2005; Maeda et al., 2010; Sonta et al., 2005). The elevation in Nox4 protein and ROS generation are reversed by insulin treatment, confirming that hyperglycemia and hyperglycemia-induced mediators are responsible for these effects (Etoh et al., 2003; Gorin et al., 2005). Our group provided the initial evidence that Nox4-dependent ROS generation mediates glomerular hypertrophy and mesangial matrix accumulation (Gorin et al., 2005). We showed that inhibition of Nox4 oxidase by administration of antisense oligonucleotides for Nox4 significantly reduced glomerular enlargement as well as fibronectin accumulation in glomeruli from type 1 diabetic rats (Gorin et al., 2005). Recent studies using ApoE/Nox4 double knockout mouse or Nox4 knockout mice on C57BL6/J background made type 1 diabetic with streptozotocin showed that genetic deletion of Nox4 markedly attenuated diabetes-induced oxidative stress, mesangial matrix expansion as well as extracellular matrix protein fibronectin and collagen IV accumulation in the glomeruli (Jha et al., 2014; Thallas-Bonke et al., 2014). It should be mentioned that both Nox4 and Nox5 expression are increased in human diabetic glomeruli (Holterman et al., 2014).

In cultured mesangial cells, glucose elicits a rapid upregulation in Nox4 protein levels, including in the mitochondrial fraction, which is associated with an increase in cellular and mitochondrial ROS production (Block et al., 2009; Eid et al., 2013b; Papadimitriou et al., 2014; Shah et al., 2013). Moreover, prolonged exposure of mesangial cells to HG has also been described to augment Nox4 mRNA and protein expression (Etoh et al., 2003; Fu et al., 2010; Jeong et al., 2012). Nox4 is required for HG-induced (acute or chronic) increase in ROS production and accumulation of fibronectin in these cells (Gorin et al., 2005). Furthermore, Nox4 participates to HG-mediated mitochondrial ROS generation in mesangial cells (Block et al., 2009), suggesting that Nox4-derived ROS may affect mitochondrial function. This contention is supported by the recent observation that ROS generated by overexpression of Nox4 are able to oxidize and affect the activity of mitochondrial proteins in cardiac myocytes (Ago et al., 2010). Moreover, Nox4-derived ROS have been reported to decrease mitochondrial function via disruption of complex I in endothelial cells (Koziel et al., 2013). These findings suggest that mitochondrial electron transport chain may be a downstream effector of Nox4. A short paracrine loop may exist, by which ROS production by mitochondrial Nox4 alters mitochondrial respiratory chain activity, thereby leading to more ROS generation by the dysfunctional mitochondrial electron transport chain and alteration of mitochondrial function.

Recent work from our group identified important downstream targets of Nox4-derived ROS in the pathway linking HG to mesangial cell fibrotic injury (Eid et al., 2013b). The study revealed the role of Nox4 as a critical mediator of endothelial nitric oxide synthase (eNOS) uncoupling and decrease in nitric oxide (NO) bioavailability induced by HG in cultured mesangial cells and in diabetes in vivo (Eid et al., 2013b). We demonstrate that ROS derived from dysfunctional eNOS contribute to fibronectin expression in mesangial cells exposed to HG. The molecular mechanisms underlying this process involve the reaction of Nox4-derived superoxide with NO generated constitutively by functional eNOS resulting in the formation of peroxynitrite that subsequently uncouples eNOS, further promoting superoxide generation (Eid et al., 2013b). In the diabetic milieu, Nox4-dependent eNOS uncoupling, not only eliminates the protective effect of eNOS-derived NO, but also converts the enzyme to a phlogistic mediator that further enhances ROS generation and mesangial cell fibrotic response. A role for Nox4 in peroxynitrite production and NOS (eNOS or neuronal NOS) uncoupling have been confirmed in other systems (de Mochel et al., 2010; Ito et al., 2013; Siu et al., 2015) as well as in the diabetic kidney where Nox4 deletion reduced peroxynitrite production in the glomeruli (Jha et al., 2014; Thallas-Bonke et al., 2014). Protein kinase C (PKC) is also a downstream target of Nox4 in diabetic glomeruli. This is supported by the findings that the major isoforms of PKC that are increased with diabetes in the glomeruli can be normalized in Nox4 knockout mice (Thallas-Bonke et al., 2014).

Novel upstream regulators of Nox4-dependent ROS generation were also recently characterized. We demonstrated that Sestrin 2 attenuates HG-induced ROS generation and MC mesangial cell fibrotic injury through blockade of Nox4-dependent eNOS dysfunction/decline in NO levels (Eid et al., 2013b). Specifically, Sestrin 2 counteracts Nox4-mediated ROS production via impeding the rapid upregulation of Nox4 protein elicited by HG. The stress-inducible protein Sestrin 2 is known to suppress ROS and protect cells from oxidative stress but the mechanisms by which Sestrin 2 exerts its antioxidant properties remain unclear. The characterization of Sestrin 2 as an upstream negative regulator of Nox4 unveiled a novel molecular mechanism by which Sestrin 2 is able to inhibit oxidative stress. Importantly, AMP-activated protein kinase (AMPK) functions as a mediator of Sestrin 2 inhibitory effects on HG-stimulated and Nox4-dependent eNOS dysfunction and extracellular matrix protein accumulation in mesangial cells (Eid et al., 2013b). The function of AMPK as a negative modulator of HG-mediated increase in Nox4 expression in mesangial cells is further supported by a recent report (Papadimitriou et al., 2014). It should be mentioned that the protective effects of Sestrin 2/AMPK is reciprocally blunted by HG. Hence, HG promotes AMPK inactivation via downregulation of Sestrin 2, which in turn results in increased Nox4 and Nox4-dependent ROS production followed by eNOS uncoupling and decrease in NO bioavailability and enhancement of mesangial cell fibrotic response (Eid et al., 2013b).

Other studies have demonstrated that statins and Rho kinase inhibitors prevent increased Nox4 expression and oxidative stress in diabetic kidney, including glomeruli, concomitantly to the amelioration of mesangial matrix expansion and renal function, indicate that small GTPases Rho and Rac pathways (known targets of statins) may act as upstream modulators of Nox4 expression and Nox4-dependent ROS release (Fujii et al., 2007; Gojo et al., 2007). microRNAs also play a role in Nox4 expression and serves as an endogenous silencer for Nox4 gene in mesangial cells (Fu et al., 2010). miR-25 seems to negatively regulate Nox4 expression by directly targeting its 3’ untranslated region (3′-UTR) (Fu et al., 2010). The downregulation of microRNA-25 (miR-25) by HG in mesangial cells or by hyperglycemia in diabetic kidney results in the relief of Nox4 gene silencing that lead to increased Nox4 expression and ROS production (Fu et al., 2010). Similar functional relationship between miR-25 and Nox4 was reported in the heart where downregulation of miR-25 results in increased Nox4 expression and subsequent oxidative and nitrative stress (Varga et al., 2013). Thioredoxin-interacting protein (TxNIP) has been recently identified as an upstream regulator of Nox4 protein expression in mesangial cells exposed to HG. TxNIP mediates HG-induced ROS generation by Nox4 in mesangial cells (Shah et al., 2013).

The mRNA and protein expression of p22phox, the subunit required for full activation of Nox4, are upregulated by glucose in mesangial cells as well as in glomeruli from diabetic animals (Maeda et al., 2010; Whiteside et al., 2009; Xia et al., 2006; Zhang et al., 2012). Interestingly, Nox4 and p22phox both contribute to HG-dependent oxidative stress and fibronectin or collagen IV accumulation as well as the expression of other markers of fibrotic injury in mesangial cells (Xia et al., 2006; 2008; Zhang et al., 2012). Equivalent to what is seen for Nox4, AMPK activation is also able to counteract the induction of ROS production by HG via blockade of a p22phox upregulation in mesangial cells (Whiteside et al., 2009). These observations are consistent with the concept proposing that AMPK act as a suppressor of oxidative stress via inhibition of NADPH oxidase subunits expression in various biological systems including vascular tissues (McCarty et al., 2009; Schuhmacher et al., 2011; Wang et al., 2010). Because it is known that p22phox interacts with Nox4 and enhances its activity, it is reasonable to think that in these cells, Nox4 and p22phox may form a complex that accounts for HG-induced ROS generation and the subsequent fibrotic response.

Nox4 also confers Ang II-mediated harmful effects in mesangial cells. Ang II elicits an acute increase in Nox4 protein expression as well as a chronic prolonged upregulation of Nox4 mRNA and protein levels associated with enhanced ROS production in these cells (Block et al., 2008; Fujii et al., 2010). Ang II also elicits an increase in mitochondrial abundance of Nox4 protein and the oxidase contributes to ROS production in mitochondria (Lee et al., 2013a). Ang II-induced ROS generation in mesangial cells acts principally through Nox4 as an upstream activator of extracellular signal-regulated kinase 1/2 (ERK1/ERK2), proline-rich tyrosine kinase-2 (Pyk-2)/Src/3-phosphoinositide-dependent protein kinase-1 (PDK-1), Akt/protein kinase B (Akt/PKB), and/or p70 S6 kinase (p70S6K) pathways that lead to cell hypertrophy and increased protein synthesis and/or fibronectin expression (Barnes and Gorin, 2011; Block et al., 2008; Gorin et al., 2003; 2004). More specifically, Pyk-2 appears to act as a molecular scaffold binding to both PDK-1 and Src, thereby allowing Src to tyrosine phosphorylate and activate PDK-1, which in turn activate its downstream effectors, Akt/PKB and p70S6K (Block et al., 2008). Interestingly, PDK-1/Src/Pyk-2 complex may favor the formation of signaling platforms bringing key intermediates into proximity, thereby facilitating the contact between Nox4-derived ROS and downstream effectors (Block et al., 2008; Ushio-Fukai, 2006). Importantly, the most proximal event physically tethering Nox4 activation by Ang II to the rest of the signaling cascade is a redox-dependent posttranslational modification of Src on cysteine residues induced by Nox4-derived ROS (Block et al., 2008). It was reported that activation of Rac1 by phospholipase A2 (PLA2)-mediated arachidonic acid (AA) release appears to be implicated in Nox4-dependent ROS production and subsequent Akt/PKB-mediated cellular hypertrophy in mesangial cell treated with Ang II (Gorin et al., 2003; 2004). Our recent work also placed Nox4 as central mediator that control Ang II-induced redox signaling that lead to peroxynitrite-dependent eNOS dysfunction, decline in NO bioavailability and MC fibrotic injury (Lee et al., 2013a). The p22phox subunit is also required for the hypertrophic and fibrotic actions of Ang II in mesangial cells (Block et al., 2006).

Comparable to what was observed in a myriad of cell types (Barnes and Gorin, 2011; Bondi et al., 2010; Cucoranu et al., 2005), TGF-β specifically increases the expression of Nox4 and ROS production in mesangial cells. Moreover, a role for Nox4 in TGF-β-mediated ROS generation and mesangial cell fibrotic injury was established (Jha et al., 2014; Papadimitriou et al., 2014). p22phox is required for TGF-β-induced ROS production in mesangial cells (Xia et al., 2008). As described above for HG, AMPK also acts as a negative regulator of TGF-β-dependent Nox4 upregulation and subsequent ROS production in these cells.

PKC isozymes are positioned upstream and downstream of p22phox-containing NADPH oxidases and ROS production in mesangial cells exposed to HG or TGF-β (Kwan et al., 2005; Xia et al., 2006). Furthermore, in mesangial cells, HG-induced oxidative stress involves autocrine TGF-β stimulation of PKC and resultant generation of ROS by p22phox-based Nox oxidase via p22phox protein upregulation (Xia et al., 2008). Other studies with mesangial cells exposed to AOPPs place an NADPH oxidase, distal to PKC activation, resulting in extracellular matrix protein overproduction and upregulation of TGF-β (Wei et al., 2009).

The role of other Nox catalytic isoforms or regulatory subunits in mesangial cell injury in the diabetic kidney has been less studied. A role for Nox2 or Nox1 is indirectly suggested by the observation that administration of apocynin significantly reduced glomerular fibronectin and collagen accumulation in type 1 diabetic rats (Asaba et al., 2005). However, The involvement of Nox2 in the mesangium is not clear as some studies indicate that Nox2 is not detected in cultured human and rat mesangial cells whereas Nox2 is detected in isolated mouse mesangial cells and in diabetic glomeruli (Jones et al., 1995; Liu et al., 2012; Miyata et al., 2005). Nox2 regulatory subunits p47phox and p67phox are systematically found in mesangial cells (Jones et al., 1995; Pleskova et al., 2006) and the expression and translocation of p47phox and p67phox to the membrane is increased in the diabetic glomeruli (Kitada et al., 2003). HG causes an increase in p47phox protein expression in mesangial cells and downregulation of p47phox blunts high glucose-induced ROS and extracellular matrix accumulation (Hua et al., 2003; Kwan et al., 2005; Xia et al., 2006). Importantly, diabetes-induced oxidative stress and glomerular injury are attenuated in p47phox-deficient type 1 diabetic Akita mice (Liu et al., 2012). Moreover, deletion of p47phox attenuates diabetes-induced and HG-induced Nox2 expression in glomeruli and mesangial cells, respectively (Liu, et al., 2012). This indicates that p47phox-dependent activation of Nox oxidase, likely Nox2, is determinant for the promotion of DN. However, a recent study showed that glomerular mesangial matrix expansion and albuminuria are not attenuated in type 1 diabetic Nox2 knockout mice (You et al., 2013). Interestingly, an upregulation of Nox4 is observed in these mice (You et al., 2013). Together, these findings warrant a reassessment of the role of Nox2 in DN. While Nox1 is thought to promote the deleterious effects of glucose or Ang II in the vasculature (Lassegue et al., 2001; Lavrentyev and Malik, 2009), its function in mesangial cell injury in the diabetic milieu has not been reported. Indeed, a role for Nox1 as a source of renal ROS and mediator of DN has been challenged by the recent report showing that deletion of Nox4, but not Nox1, in a mice model of type 1 diabetes in ApoE knockout mice resulted in renal protection from glomerular injury (Jha et al., 2014). However, p47phox and p67phox recruitment to the membrane contributes to Ang II-mediated oxidative stress and mesangial cell growth (Ding et al., 2007) and TGF-β was reported to upregulate the expression of Nox2, p22phox, p47phox and p67phox in mesangial cells (Lee et al., 2003). AOPPs also increase p22phox, p47phox and Nox4 protein expression (Wei et al., 2009). AOPPs promote an increase in p47phox expression and translocation as well as its interaction with p22phox expression (Wei et al., 2009). These events are associated with enhanced extracellular matrix protein synthesis. Although PKC-dependent NADPH oxidase activation mediates AGEs-induced mesangial cell fibrotic injury (Thallas-Bonke et al., 2008), the Nox subunits implicated in these processes remain to be identified. In vivo studies suggested that induction of glomerular injury by AGEs is due to Src homology 2 domain-containing transforming protein C1 (Shc1) isoform p66Shc-mediated Nox4 expression (Menini et al., 2007).

Figure 2 is an overview of the major redox pathways and signaling intermediates positioned upstream and downstream of Nox enzymes that are stimulated by diabetic stimuli to promote glomerular mesangial cell injury.

UPSTREAM REGULATORS AND DOWNSTREAM EFFECTORS OF NOX NADPH OXIDASES IN DIABETES-INDUCED GLOMERULAR EPITHELIAL CELL/PODOCYTE INJURY

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Upregulation of Nox4 protein expression in diabetic glomeruli including podocytes is accompanied by increased oxidative stress, loss of podocyte or foot process effacement as well as albuminuria in OVE26 type 1 diabetic mice (Eid et al., 2009; 2010; 2013a; Khazim et al., 2013; Sharma et al., 2008). In cultured podocytes exposed to HG for prolonged time period, Nox4 mRNA and protein are augmented and Nox4-derived ROS play a key role in apoptotic cell death (Eid et al., 2009; 2010; 2013a; Jha et al., 2014; Khazim et al., 2013; Piwkowska et al., 2011; Sharma et al., 2008). In human podocytes where both Nox4 and Nox5 are present, HG had no effect on Nox5 mRNA expression but significantly increased Nox4 mRNA levels (Jha et al., 2014). Transgenic mice overexpressing human Nox5 in a podocyte-specific manner exhibited early onset of albuminuria and podocyte foot process effacement (Holterman et al., 2014). Subjecting the mice to streptozotocin-induced type 1 diabetes further exacerbated these changes (Holterman et al., 2014).

A sequential regulation of Nox oxidases by cytochrome P450 of the 4A family (CYP4A) was recently identified in podocytes in which 20-hydroxyeicosatetraenoic acid (20-HETE) generation by CYP4A mediates the stimulatory effect of HG on Nox4 and Nox1 expression and the resultant ROS production at later time points (Eid et al., 2009). In the presence of HG concentrations, Nox4 promotes podocyte cell death via activation of p53- and PUMA-dependent apoptotic pathway (Eid et al., 2010). The oxidative stress triggered by HG appears to be exacerbated by the fact that the ROS generated by Nox4 affects the balance between oxidants and antioxidants by decreasing the activity of key antioxidant enzymes such as glutathione peroxidase (GPx) and catalase (Piwkowska et al., 2011). Importantly, inactivation of AMPK-activated protein kinase (AMPK) by HG accounts for the increase in Nox4 mRNA and protein expression as well as subsequent ROS production and podocyte apoptosis (Eid et al., 2010). AMPK activators, e.g. AICAR (5-aminoimidazole-4-carboxamide-1-riboside) or adiponectin, significantly reduced Nox4 expression, oxidative stress and podocyte injury in vitro or in vivo (Eid et al., 2010; Piwkowska et al., 2010; Sharma et al., 2008). A recent report indicates that mammalian target of rapamycin complex 1 (mTORC1) is an upstream positive regulator of Nox4 expression and subsequent ROS production and injury in podocytes exposed to HG or in vivo in glomeruli from OVE26 type 1 diabetic mice (Eid et al., 2013a). The same report showed that AMPK exerts its protective actions by inhibiting mTORC1 pathway via tuberin activation (Eid et al., 2013a).

Although podocytes also express Nox2, p22phox, p47phox and p67phox (Greiber et al., 1998; Nistala et al., 2008), there is no evidence of regulation of these subunits by HG. Similar to Nox4, Nox1 protein levels are increased in glomeruli from OVE26 mice (Eid et al., 2009; 2010). Nox1 protein is upregulated by HG in cultured podocytes and, as for Nox4, mTORC1 positively regulates Nox1 expression (Eid et al., 2013a). While Ang II- or TGF-β-induced oxidative stress mediates podocyte injury (Campbell et al., 2011; Nistala et al., 2008; Ziyadeh and Wolf, 2008), very little is known regarding the role of the Nox oxidases in the podocyte dysfunction promoted by these agonists or the other major mediators of DN. Similar to what is observed in MCs, Ang II-dependent increase in NADPH oxidase activity is associated with the upregulation of Nox4, Nox2, Rac and p22phox expression in podocytes (Nistala et al., 2008; Whaley-Connell et al., 2008). Stimulation with Ang II upregulates Nox5 expression in human podocytes and Nox5 is required for Ang II-induced ROS production as well as altered podocyte cytoskeletal dynamics (Holterman et al., 2014). Nox4 was detected within the mitochondria in podocytes and a recent report suggested that TGF-β-induced mitochondrial Nox4 upregulation via the Sma and Mad homologue (Smad) 2/3 pathway is responsible for ROS production, mitochondrial dysfunction, and apoptosis in podocytes (Block et al., 2009; Das et al., 2014; Yu et al., 2014a). Note that in human podocytes, Nox5 mRNA expression is enhanced following treatment with TGF-β (Jha et al., 2014).

Figure 3 is an overview of the major redox pathways and signaling intermediates positioned upstream and downstream of Nox enzymes that are stimulated by diabetic stimuli to promote glomerular mesangial cell and podocyte injury.

UPSTREAM REGULATORS AND DOWNSTREAM EFFECTORS OF NOX NADPH OXIDASES IN DIABETES-INDUCED TUBULAR CELL INJURY

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Similar to glomeruli, tubules from type 1 diabetic rats show an increase in Nox4 mRNA and protein expression and downregulation of tubular Nox4 levels with in vivo administration of antisense oligonucleotides reduces diabetes-mediated ROS production and extracellular matrix protein synthesis in the renal cortex that is mainly composed of tubular epithelial cells (Etoh et al., 2003; Fujii et al., 2010; Gorin et al., 2005). The importance of Nox4 as a major source of ROS in the tubular compartment was further supported by a recent study in type 1 diabetic ApoE/Nox4 double knockout mice or Nox4 knockout mice on C57BL6/J background showing that Nox4 genetic deletion markedly reduced ROS production in renal cortex (Jha et al., 2014; Thallas-Bonke et al., 2014). Noteworthy, Nox4 was also shown to be responsible for diabetes-induced ROS production in mitochondria isolated from cortex (Jha et al., 2014). Importantly, the function of Nox1 as a generator of ROS in tubules is weakened by the observations that ROS production is not affected in the renal cortex of type 1 diabetic ApoE/Nox1 knockout mice (Jha et al., 2014). Interestingly, Nox4 protein expression is increased in renal cortex but is unchanged in medulla from type 2 diabetic mice (Sedeek et al., 2010). Increased Nox4 expression in diabetic tubules correlates with an augmentation in p22phox levels (Etoh et al., 2003; Sedeek et al., 2010). Whilst the levels of Nox2 and p47phox are not affected in renal cortex from type 2 diabetic mice (Sedeek et al., 2010), Nox2 is increased in the cortex from type 1 diabetic rats (Gorin et al., 2005). A role for Nox2 is challenged by a recent report showing that tubulointerstitial injury is not ameliorated in type 1 diabetic Nox2 knockout mice (You et al., 2013). Exposure of cultured renal proximal tubular epithelial cells to HG leads to the upregulation of Nox4 protein expression but seems to have no effect on Nox2, Nox1, p22phox or p47phox expression (Ford et al., 2013; Sedeek et al., 2010). Furthermore, Nox4-dependent ROS production is required for glucose-induced increase in fibronectin accumulation and TGF-β expression in these cells (Ford et al., 2013; Sedeek et al., 2010). The profibrotic action of the oxidase is corroborated by the finding that overexpression of Nox4 in tubular cells causes a robust increase in fibronectin synthesis (New et al., 2012). In human tubular epithelial cells, Nox5 has been shown to be express together with Nox1, Nox4 and Nox2 but its role in HG-induced ROS production and cell injury was not studied (Yu et al., 2014b).

A potential upstream regulator of Nox4 expression in the tubules is protein kinase C-β since diabetes-induced Nox4 expression and oxidative stress are attenuated in renal cortex from PKC-β-null mice kidney (Ohshiro et al., 2006). Interestingly, PKC seems to be positioned both upstream and downstream of Nox4 since it was recently reported that the increase in the major isoforms of PKC in tubules was blunted in the renal cortex from diabetic Nox4 knockout mice (Thallas-Bonke et al., 2014). A role for matrix metalloprotease a disintegrin and metalloprotease1 7 (ADAM17) as upstream regulator of Nox4 was recently established in vivo and in vitro. Pharmacological inhibition of ADAM17 attenuated diabetes-mediated Nox4 upregulation, oxidative stress and matrix protein accumulation in OVE26 mice kidney cortex (Ford et al., 2013). In cultured tubular epithelial cells, ADAM17 activity is required for HG-induced increase in Nox4 protein expression, ROS generation and fibrotic injury (Ford et al., 2013). Similar to podocytes, AMPK activation attenuates HG-mediated Nox4 protein upregulation in tubular epithelial cells, supporting the concept that the kinase acts as a suppressor of oxidative stress (Lee et al., 2013b). p38MAPK has been also identified as a critical downstream effector of Nox4 in the signaling pathway linking HG and tubular cell injury (Sedeek et al., 2010).

Ang II has been shown to utilize Nox4 as a mediator of its injurious effects in tubular epithelial cells. Chronic angiotensin II treatment upregulates Nox4 expression and induces epithelial-to-mesenchymal transition in renal epithelial cells through Nox4-dependent ROS production and resultant Src/caveolin-mediated activation of epidermal growth factor receptor/ERK signaling pathway (Chen et al., 2012; Peng et al., 2009; Xu et al., 2010). Furthermore, Ang II upregulates Nox4 expression in the mitochondrial fraction of renal tubular cells and Nox4 is required for Ang II-mediated ROS mitochondrial production (Kim et al., 2012). ROS derived from Nox4 contribute to Ang II-dependent apoptosis in these cells (Kim et al., 2012). Ang II enhances the expression of Nox2, p22phox and p47phox subunits along with Nox4 in tubular cells (Hannken et al., 1998; Xu et al., 2010). The effects on p22phox, p47phox and Nox4 appear to be mediated by lectin-like oxidized low-density lipoprotein receptor-1 (Hannken et al., 1998; Xu et al., 2010). Another report shows that HG induces p22phox expression and oxidative stress via angiotensin II type 1 (AT1) receptor activation, further underlining the important role of Ang II as a regulator of Nox signaling in tubular cells (Takao et al., 2011). Furthermore, a p22phox-containing NADPH oxidase is critical for Ang II-mediated tubular cell hypertrophy through induction of p27Kip1, a factor promoting cell cycle arrest (Hannken et al., 1998). As in mesangial cells, Src and ERK pathways appears to be a primary target of Nox4-derived ROS and plays a key role in the resultant fibrotic or hypertrophic response in tubular cells. AMPK also inhibits Ang II-induced increase in Nox4 expression and tubular epithelial-to-mesenchymal transition (EMT) (Lee et al., 2013b).

Although it is clear that oxidative stress is implicated in TGF-β-mediated tubular cell injury (Rhyu et al., 2005), there is a deficit of causal evidence supporting the role of the oxidase or other Nox enzymes in TGF-β signaling in tubular cells. Stimulation of tubular cells by TGF-β results in an increase in Nox4 protein expression, an effect prevented by AMPK activation (Lee et al., 2013b). Conflicting results exist concerning the regulation of other Nox subunits by TGF-β in these cells. On one hand, TGF-β has no effect on Nox2, p22phox and p47phox expression and that only p67phox seems to be required for the deleterious actions of the cytokine in renal tubular cells (Zhang et al., 2009). On the other hand, TGF-β was shown to upregulate p22phox (Lee et al., 2003). Nox4-derived ROS also serve as signal transducer for the fibrotic effects of IGF-I in renal tubular epithelial cells via Akt/PKB and mTORC1/p70S6K signaling pathways activation (New et al., 2012).

Figure 4 is an overview of the major redox pathways and signaling intermediates positioned upstream and downstream of Nox enzymes that are stimulated by diabetic stimuli to promote tubulointerstitial cell injury.

UPSTREAM REGULATORS AND DOWNSTREAM EFFECTORS OF NOX NADPH OXIDASES IN DIABETES-INDUCED INTERSTITIAL CELL INJURY

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The information regarding the roles of hyperglycemia-mediated oxidative stress or Nox-derived ROS in interstitial cell injury, particularly in the activation of renal fibroblast into myofibroblasts, remains very limited. There is indication that exposure of kidney fibroblasts to HG upregulates Nox4 and Nox2 mRNA and protein expression and that this is associated with an increase in ROS generation and hypertrophy (Williams and Gooch, 2014). Calcineurin A-β (CnAβ) maintains basal Nox4 and Nox2 expression and is required for HG-induced increase in Nox4 and Nox2 expression in these cells (Williams and Gooch, 2014). It seems that CnAβ promotes Nox4 and Nox2 transcription via activation of the nuclear factor of activated T cells (NFAT) (Williams and Gooch, 2014). A functional link between Nox4 or Nox2 and ROS generation and hypertrophy is suggested by the fact that expression of Nox4 or Nox2 in CnAβ deficient kidney fibroblasts (where Nox4 and Nox2 are downregulated) is able to restore the stimulatory effects of HG on these biological outcomes (Williams and Gooch, 2014).

Nox4 is the predominant Nox homolog implicated in kidney myofibroblast differentiation and expression of fibronectin and other fibrotic markers in response to TGF-β (Barnes and Gorin, 2011; Bondi et al., 2010). However, unlike cardiac and lung myofibroblasts where Nox4 acts upstream of transcription factor Smad2/3, Nox4 is positioned downstream of Smad3 and proximal to ERK in the pathway linking TGF-β to the fibrotic response in renal fibroblasts (Barnes and Gorin, 2011; Cucoranu et al., 2005; Hecker et al., 2009). These results are in agreement with what is reported in pulmonary vascular smooth muscle cells, where TGF-β-induced proliferation occurs through a Nox4-dependent pathway downstream of Smad3 (Sturrock et al., 2006). Small GTPase RhoA and its downstream target Rho kinase (ROCK) have been identified as upstream positive regulators of Nox4 expression and activity in the pathway linking TGF-β to kidney myofibroblast differentiation (Manickam et al., 2014). Interestingly, RhoA/ROCK seems to act via the increasing the expression of Poldip2, a newly discovered Nox4 enhancer protein (Manickam et al., 2014). Moreover, the data suggest that besides enhancing Nox4 activity Poldip2 is also able to regulate the expression of the oxidase.

CONCLUSION

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It is apparent from this review that the in vivo and in vitro experimental evidence support a fundamental role for NADPH oxidases of the Nox family, and especially the homologue Nox4, in the pathogenesis and pathophysiology of DN. The consequence of these observations is the consideration of the Nox homologs and their associated subunits as relevant therapeutic targets for the treatment of DN. There has been recently a considerable effort for the generation and development of agents able to inhibit the Nox enzymes in a homolog-specific manner (Altenhofer et al., 2012; Drummond et al., 2011; Gaggini et al., 2011; Jaquet et al., 2009; Kim et al., 2011; Laleu et al., 2010; Lambeth et al., 2008). Among these inhibitors, the small-molecule dual Nox4 and Nox1 inhibitors from the Pyrazolo pyridine chemical series, referred as to GKT136901 and GKT-137831, have drawn considerable attention. Preclinical studies performed with these inhibitors in experimental animal models indicate that they effectively attenuate the pathological changes observed in renal complication of type 1 and type 2 diabetes, atherosclerosis, ischemic retinopathy, liver fibrosis and idiopathic pulmonary fibrosis (Aoyama et al., 2012; Carnesecchi et al., 2011; Di Marco et al., 2014; Gorin and Block, 2013a; 2013b; Gray et al., 2013; Hecker et al., 2014; Jha et al., 2014; Jiang et al., 2012; 2014; Laleu et al., 2010; Sedeek et al., 2013; Vendrov et al., 2010; Wilkinson-Berka et al., 2014). GKT137831 have recently been successfully used in a phase 1 clinical trial and is currently being evaluated in a phase 2 clinical trial in patients with type 2 diabetes and albuminuria (Clinical-Trials.gov reference number NCT02010242).

Beside the direct inhibition of Nox4 and others Nox oxidases, the present review suggests that adjunct therapies targeting the agonists or signaling intermediates that regulate the expression or function of Nox subunits and subsequent ROS production as well as the downstream targets of Nox oxidases implicated in the pathological processes should be considered for the treatment of diabetic complications. In regards to the observation reported above, such strategies could involve the use of PKC inhibitors, agents that disrupt the AGE signaling, inhibitors of the renin-angiotensin system (angiotensin-converting enzyme inhibitors and Ang II receptor blockers), statins (that inhibit Rac1 and Rho), Rho/Rho kinase inhibitors, AMPK activators (i.e. metformin), Sestrin-mimicking small molecules or peptides Sestrin analogues/agents that stimulate Sestrin activity or expression. Importantly, the small molecules Nox inhibitors that are currently available affect Nox activity without altering the expression of the oxidases. Therefore, the utilization of agents that modulate Nox expression or that target downstream effectors of the oxidases as adjunct therapy to Nox allosteric inhibitors may allow a more effective neutralization of the enzyme due to the blockade of the sustained increase in Nox protein levels promoted by chronic hyperglycemia or other mediators of DN.

FIGURES

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Fig. 1. Structure and molecular organization of the renal Nox NADPH oxidases. The top right left panel illustrates the topology of and the enzymatic reaction catalyzed by the Nox enzymes. The other panels represent the molecular structure of the Nox oxidase homologues predominantly expressed in the kidney, Nox2 (a.k.a. gp91phox), Nox1, Nox4, and Nox5. The regulatory subunits differ from a Nox homologue to another.
Fig. 2. Upstream and downstream effectors of Nox oxidases implicated in glomerular mesangial cell injury triggered by diabetic stimuli. See text for detail.
Fig. 3. Upstream and downstream effectors of Nox oxidases implicated in podocyte injury triggered by diabetic stimuli. See text for detail.
Fig. 4. Upstream and downstream effectors of Nox oxidases implicated in tubular cell injury triggered by diabetic stimuli. See text for detail.

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Article

Minireview

Mol. Cells 2015; 38(4): 285-296

Published online April 30, 2015 https://doi.org/10.14348/molcells.2015.0010

Copyright © The Korean Society for Molecular and Cellular Biology.

Upstream Regulators and Downstream Effectors of NADPH Oxidases as Novel Therapeutic Targets for Diabetic Kidney Disease

Yves Gorin*, and Fabien Wauquier

Department of Medicine, University of Texas Health Science Center, San Antonio, Texas, USA

Correspondence to:*Correspondence: gorin@uthscsa.edu

Received: January 9, 2015; Accepted: January 12, 2015

Abstract

Oxidative stress has been linked to the pathogenesis of diabetic nephropathy, the complication of diabetes in the kidney. NADPH oxidases of the Nox family, and in particular the homologue Nox4, are a major source of reactive oxygen species in the diabetic kidney and are critical mediators of redox signaling in glomerular and tubulointerstitial cells exposed to the diabetic milieu. Here, we present an overview of the current knowledge related to the understanding of the role of Nox enzymes in the processes that control mesangial cell, podocyte and tubulointerstitial cell injury induced by hyperglycemia and other predominant factors enhanced in the diabetic milieu, including the renin-angiotensin system and transforming growth factor-β. The nature of the upstream modulators of Nox enzymes as well as the downstream targets of the Nox NADPH oxidases implicated in the propagation of the redox processes that alter renal biology in diabetes will be highlighted.

Keywords: diabetic nephropathy, downstream effectors of Nox NADPH oxidases, Nox family of NADPH oxidases, oxidative stress, upstream regulators of Nox NADPH oxidases

INTRODUCTION

Oxidative stress plays a critical role in the initiation and development of diabetic nephropathy (DN) (Baynes, 1991; Forbes et al., 2008; Giacco and Brownlee, 2010; Hinokio et al., 1999; Kashihara et al., 2010; Schnackenberg, 2002; Singh et al., 2011; Son et al., 2004; Stanton, 2011). Diabetes is associated with an increase in generation of reactive oxygen species (ROS) in the glomerular and tubulointerstitial compartments of the kidney (Forbes et al., 2008; Kanwar et al., 2008; 2011; Kashihara et al., 2010; Koya et al., 2003; Singh et al., 2011; Stanton, 2011; Vasavada and Agarwal, 2005). Although chronic hyperglycemia is sufficient to promote renal cell injury, data from experimental animal models as well as cultured cells indicate that a combination of growth factors, hormones and cytokines, in addition to high glucose concentrations, alter the redox status of the cells and contribute to cell damages in the diabetic kidney (Abboud, 1997; Campbell et al., 2011; Gorin and Block, 2013a; 2013b; Kanwar et al., 2008; 2011; Kashihara et al., 2010; Rincon-Choles et al., 2002; Singh et al., 2011). While multiple pathways lead to ROS generation, i.e., mitochondrial electron transport chain, xanthine oxidase or uncoupled nitric oxide synthase, recent evidence demonstrates that the Nox family of NADPH oxidases constitutes a major source of ROS in nonphagocytic cells, including renal cells (Ago et al., 2010; Barnes and Gorin, 2011; Bedard and Krause, 2007; Block et al., 2009; Brown and Griendling, 2009; Brownlee, 2005; Coughlan et al., 2009; Craven et al., 2001; Dikalov, 2011; Gill and Wilcox, 2006; Gorin and Block, 2013a; 2013b; Gorin et al., 2005; Griendling and FitzGerald, 2003; Hwang et al., 2012; Kiritoshi et al., 2003; Kitada et al., 2011; Lambeth, 2007; Lambeth et al., 2007; Lassegue and Clempus, 2003; Lassegue and Griendling, 2010; Lassegue et al., 2012; Nishikawa et al., 2000; Octavia et al., 2012; Paravicini and Touyz, 2008; Rivera et al., 2010; Sedeek et al., 2012). The present review will focus on the upstream regulators controlling the expression and activity of the Nox enzymes as well as the downstream effectors targeted by Nox-derived ROS to initiate and propagate diabetes-induced glomerular and tubulointerstitial cell injury.

NADPH OXIDASES OF THE NOX FAMILY PREDOMINANTLY EXPRESSED IN THE KIDNEY

To date, the Nox family comprises seven members: Nox1-5, and the dual oxidases (Duox) Duox1 and -2 (Bedard and Krause, 2007; Geiszt, 2006; Lambeth et al., 2007; Selemidis et al., 2008). Nox4, Nox1, Nox2 (a.k.a. gp91phox), and Nox5 are the Nox homologues that are predominantly expressed in glomerular cells (mesangial cells, glomerular epithelial cells or podocytes), glomerular endothelial cells and tubulointerstitial cells (tubular epithelial cells and interstitial fibroblasts) (Gorin and Block, 2013a; 2013b). These Nox homologues consist of six membrane-spanning regions with binding sites for NADPH, flavin adenine dinucleotide (FAD), and heme. The latter comprise electron transfer centers that pass electrons from NADPH to oxygen forming superoxide (O2?) and hydrogen peroxide (H2O2) (Bedard and Krause, 2007; Brown and Griendling, 2009; Geiszt, 2006; Geiszt et al., 2000; Gorin and Block, 2013a; Lassegue and Griendling, 2010; Lassegue et al., 2012; Selemidis et al., 2008; Shiose et al., 2001). The calcium-dependent homolog Nox5 is found in the human kidney tissue and cells, but is not present in mice and rats (Brandes et al., 2010; Lassegue and Griendling, 2010).

The isoforms Nox2 and Nox1 require p22phox as an activating, stabilizing and/or regulatory subunit for binding to p47phox or NoxO1 (Bedard and Krause, 2007; Geiszt, 2006; Selemidis et al., 2008). Activation mechanisms for Nox1 are similar to those of Nox2, and involve complex formation with regulatory subunits upon agonist stimulation. Although Nox1 seems to primarily interact with the p47phox homolog NoxO1 (Nox organizer 1), the p67phox homolog NoxA1 (Nox activator 1), and small GTPase Rac upon activation, it was reported that p47phox and p67phox can partially replace NoxO1 and NoxA1, respectively (Bedard and Krause, 2007; Brandes and Schroder, 2008; Brandes et al., 2010; Geiszt, 2006; Lambeth, 2007; Lassegue and Griendling, 2010; Selemidis et al., 2008;). Nox4 heterodimerization with p22phox enhances the enzyme activity but the oxidase does not required the regulatory subunits essential to other Nox isoforms and is a constitutively active enzyme regulated primarily at the level of its expression in response to various stimuli, including mediators of DN such as high glucose (HG) (Block et al., 2009; Eid et al., 2009; 2013b) transforming growth factor-β (TGF-β) (Bondi et al., 2010; Cucoranu et al., 2005), angiotensin II (Ang II) (Block et al., 2008), insulin, insulin-like growth factor (IGF-I), and advanced glycation end products (AGEs) or advanced oxidation protein products (AOPPs) (Bedard and Krause, 2007; Lassegue and Clempus, 2003; Lassegue and Griendling, 2010; Lassegue et al., 2012; Lee et al., 2013a; Mahadev et al., 2004; Meng et al., 2008; Menini et al., 2007; New et al., 2012; Selemidis et al., 2008; Thallas-Bonke et al., 2008). As a consequence, the overall ROS output of Nox4 is directly governed by its expression level. Importantly, it is suggested that transcriptional events are implicated in the chronic control of Nox4 protein expression (Chai et al., 2008; Cucoranu et al., 2005; Eid et al., 2009; 2010; 2013a; Hecker et al., 2009; Moe et al., 2006; Mittal et al., 2007; Pedruzzi et al., 2004; Serrander et al., 2007; Wingler et al., 2001; Yamagishi et al., 2005), whereas acute regulation of Nox4 occurs via translational mechanisms without change in its mRNA levels (Block et al., 2008; 2009; Bondi et al., 2010; Meng et al., 2008; New et al., 2012; Peshavariya et al., 2009). Regulatory proteins that enhance Nox4 activity like polymerase delta-interacting protein 2 (Poldip2) and the p47phox-related adaptor protein, Tks (tyrosine kinase substrate with five SH3 domains) 5, have recently been identified (Diaz et al., 2009; Lyle et al., 2009; Sedeek et al., 2009). A potential implication of Rac in the control of Nox4 function was suggested in endothelial and mesangial cells (Chai et al., 2008; Gorin et al., 2003; Wu et al., 2007); however, it remains a controversial topic because, unlike Nox2 or Nox1, Rac1 does not activate Nox4 in transfected cells (Bedard and Krause, 2007). The activity of Nox5 is not controlled by any other subunits and is strictly dependent on calcium binding to the enzyme through its EF hand domains (Bedard and Krause, 2007).

It is important to mention that Nox4 seems to produce a higher hydrogen peroxide to superoxide ratio than Nox1, Nox2 and Nox5. Recent studies strongly suggest that hydrogen peroxide formation occurs through Nox4 third extracytosolic loop (E-loop) and that the structure of the E-loop may obstruct superoxide release as well as provide a source for protons, thus permitting rapid dismutation of superoxide to generate hydrogen peroxide (Takac et al., 2011). Although Nox4 predominantly produces hydrogen peroxide, numerous studies in vascular or renal cells and tissue detected Nox4-dependent superoxide production (Ago et al., 2010; Block et al., 2009; Clempus et al., 2007; Cucoranu et al., 2005; Eid et al., 2009; 2010; Gorin et al., 2005; Kuroda et al., 2005; 2010; Liu et al., 2010; Maalouf et al., 2012; Peshavariya et al., 2007; Shiose et al., 2001). Figure 1 shows the structure and molecular organization of the renal Nox NADPH oxidases.

UPSTREAM REGULATORS AND DOWNSTREAM EFFECTORS OF NOX NADPH OXIDASES IN DIABETES-INDUCED GLOMERULAR MESANGIAL CELL INJURY

The importance of Nox4 in mesangial cell injury is supported by studies in experimental rodent models of diabetes as well as in vitro work in cultured cells exposed to HG. Nox4 protein expression increases in the glomeruli, including the mesangium, and Nox4-derived ROS contribute to oxidative stress during the initial and chronic stages of diabetes (Eid et al., 2009; 2010; Etoh et al., 2003; Fujii et al., 2007; 2010; Gorin et al., 2005; Maeda et al., 2010; Sonta et al., 2005). The elevation in Nox4 protein and ROS generation are reversed by insulin treatment, confirming that hyperglycemia and hyperglycemia-induced mediators are responsible for these effects (Etoh et al., 2003; Gorin et al., 2005). Our group provided the initial evidence that Nox4-dependent ROS generation mediates glomerular hypertrophy and mesangial matrix accumulation (Gorin et al., 2005). We showed that inhibition of Nox4 oxidase by administration of antisense oligonucleotides for Nox4 significantly reduced glomerular enlargement as well as fibronectin accumulation in glomeruli from type 1 diabetic rats (Gorin et al., 2005). Recent studies using ApoE/Nox4 double knockout mouse or Nox4 knockout mice on C57BL6/J background made type 1 diabetic with streptozotocin showed that genetic deletion of Nox4 markedly attenuated diabetes-induced oxidative stress, mesangial matrix expansion as well as extracellular matrix protein fibronectin and collagen IV accumulation in the glomeruli (Jha et al., 2014; Thallas-Bonke et al., 2014). It should be mentioned that both Nox4 and Nox5 expression are increased in human diabetic glomeruli (Holterman et al., 2014).

In cultured mesangial cells, glucose elicits a rapid upregulation in Nox4 protein levels, including in the mitochondrial fraction, which is associated with an increase in cellular and mitochondrial ROS production (Block et al., 2009; Eid et al., 2013b; Papadimitriou et al., 2014; Shah et al., 2013). Moreover, prolonged exposure of mesangial cells to HG has also been described to augment Nox4 mRNA and protein expression (Etoh et al., 2003; Fu et al., 2010; Jeong et al., 2012). Nox4 is required for HG-induced (acute or chronic) increase in ROS production and accumulation of fibronectin in these cells (Gorin et al., 2005). Furthermore, Nox4 participates to HG-mediated mitochondrial ROS generation in mesangial cells (Block et al., 2009), suggesting that Nox4-derived ROS may affect mitochondrial function. This contention is supported by the recent observation that ROS generated by overexpression of Nox4 are able to oxidize and affect the activity of mitochondrial proteins in cardiac myocytes (Ago et al., 2010). Moreover, Nox4-derived ROS have been reported to decrease mitochondrial function via disruption of complex I in endothelial cells (Koziel et al., 2013). These findings suggest that mitochondrial electron transport chain may be a downstream effector of Nox4. A short paracrine loop may exist, by which ROS production by mitochondrial Nox4 alters mitochondrial respiratory chain activity, thereby leading to more ROS generation by the dysfunctional mitochondrial electron transport chain and alteration of mitochondrial function.

Recent work from our group identified important downstream targets of Nox4-derived ROS in the pathway linking HG to mesangial cell fibrotic injury (Eid et al., 2013b). The study revealed the role of Nox4 as a critical mediator of endothelial nitric oxide synthase (eNOS) uncoupling and decrease in nitric oxide (NO) bioavailability induced by HG in cultured mesangial cells and in diabetes in vivo (Eid et al., 2013b). We demonstrate that ROS derived from dysfunctional eNOS contribute to fibronectin expression in mesangial cells exposed to HG. The molecular mechanisms underlying this process involve the reaction of Nox4-derived superoxide with NO generated constitutively by functional eNOS resulting in the formation of peroxynitrite that subsequently uncouples eNOS, further promoting superoxide generation (Eid et al., 2013b). In the diabetic milieu, Nox4-dependent eNOS uncoupling, not only eliminates the protective effect of eNOS-derived NO, but also converts the enzyme to a phlogistic mediator that further enhances ROS generation and mesangial cell fibrotic response. A role for Nox4 in peroxynitrite production and NOS (eNOS or neuronal NOS) uncoupling have been confirmed in other systems (de Mochel et al., 2010; Ito et al., 2013; Siu et al., 2015) as well as in the diabetic kidney where Nox4 deletion reduced peroxynitrite production in the glomeruli (Jha et al., 2014; Thallas-Bonke et al., 2014). Protein kinase C (PKC) is also a downstream target of Nox4 in diabetic glomeruli. This is supported by the findings that the major isoforms of PKC that are increased with diabetes in the glomeruli can be normalized in Nox4 knockout mice (Thallas-Bonke et al., 2014).

Novel upstream regulators of Nox4-dependent ROS generation were also recently characterized. We demonstrated that Sestrin 2 attenuates HG-induced ROS generation and MC mesangial cell fibrotic injury through blockade of Nox4-dependent eNOS dysfunction/decline in NO levels (Eid et al., 2013b). Specifically, Sestrin 2 counteracts Nox4-mediated ROS production via impeding the rapid upregulation of Nox4 protein elicited by HG. The stress-inducible protein Sestrin 2 is known to suppress ROS and protect cells from oxidative stress but the mechanisms by which Sestrin 2 exerts its antioxidant properties remain unclear. The characterization of Sestrin 2 as an upstream negative regulator of Nox4 unveiled a novel molecular mechanism by which Sestrin 2 is able to inhibit oxidative stress. Importantly, AMP-activated protein kinase (AMPK) functions as a mediator of Sestrin 2 inhibitory effects on HG-stimulated and Nox4-dependent eNOS dysfunction and extracellular matrix protein accumulation in mesangial cells (Eid et al., 2013b). The function of AMPK as a negative modulator of HG-mediated increase in Nox4 expression in mesangial cells is further supported by a recent report (Papadimitriou et al., 2014). It should be mentioned that the protective effects of Sestrin 2/AMPK is reciprocally blunted by HG. Hence, HG promotes AMPK inactivation via downregulation of Sestrin 2, which in turn results in increased Nox4 and Nox4-dependent ROS production followed by eNOS uncoupling and decrease in NO bioavailability and enhancement of mesangial cell fibrotic response (Eid et al., 2013b).

Other studies have demonstrated that statins and Rho kinase inhibitors prevent increased Nox4 expression and oxidative stress in diabetic kidney, including glomeruli, concomitantly to the amelioration of mesangial matrix expansion and renal function, indicate that small GTPases Rho and Rac pathways (known targets of statins) may act as upstream modulators of Nox4 expression and Nox4-dependent ROS release (Fujii et al., 2007; Gojo et al., 2007). microRNAs also play a role in Nox4 expression and serves as an endogenous silencer for Nox4 gene in mesangial cells (Fu et al., 2010). miR-25 seems to negatively regulate Nox4 expression by directly targeting its 3’ untranslated region (3′-UTR) (Fu et al., 2010). The downregulation of microRNA-25 (miR-25) by HG in mesangial cells or by hyperglycemia in diabetic kidney results in the relief of Nox4 gene silencing that lead to increased Nox4 expression and ROS production (Fu et al., 2010). Similar functional relationship between miR-25 and Nox4 was reported in the heart where downregulation of miR-25 results in increased Nox4 expression and subsequent oxidative and nitrative stress (Varga et al., 2013). Thioredoxin-interacting protein (TxNIP) has been recently identified as an upstream regulator of Nox4 protein expression in mesangial cells exposed to HG. TxNIP mediates HG-induced ROS generation by Nox4 in mesangial cells (Shah et al., 2013).

The mRNA and protein expression of p22phox, the subunit required for full activation of Nox4, are upregulated by glucose in mesangial cells as well as in glomeruli from diabetic animals (Maeda et al., 2010; Whiteside et al., 2009; Xia et al., 2006; Zhang et al., 2012). Interestingly, Nox4 and p22phox both contribute to HG-dependent oxidative stress and fibronectin or collagen IV accumulation as well as the expression of other markers of fibrotic injury in mesangial cells (Xia et al., 2006; 2008; Zhang et al., 2012). Equivalent to what is seen for Nox4, AMPK activation is also able to counteract the induction of ROS production by HG via blockade of a p22phox upregulation in mesangial cells (Whiteside et al., 2009). These observations are consistent with the concept proposing that AMPK act as a suppressor of oxidative stress via inhibition of NADPH oxidase subunits expression in various biological systems including vascular tissues (McCarty et al., 2009; Schuhmacher et al., 2011; Wang et al., 2010). Because it is known that p22phox interacts with Nox4 and enhances its activity, it is reasonable to think that in these cells, Nox4 and p22phox may form a complex that accounts for HG-induced ROS generation and the subsequent fibrotic response.

Nox4 also confers Ang II-mediated harmful effects in mesangial cells. Ang II elicits an acute increase in Nox4 protein expression as well as a chronic prolonged upregulation of Nox4 mRNA and protein levels associated with enhanced ROS production in these cells (Block et al., 2008; Fujii et al., 2010). Ang II also elicits an increase in mitochondrial abundance of Nox4 protein and the oxidase contributes to ROS production in mitochondria (Lee et al., 2013a). Ang II-induced ROS generation in mesangial cells acts principally through Nox4 as an upstream activator of extracellular signal-regulated kinase 1/2 (ERK1/ERK2), proline-rich tyrosine kinase-2 (Pyk-2)/Src/3-phosphoinositide-dependent protein kinase-1 (PDK-1), Akt/protein kinase B (Akt/PKB), and/or p70 S6 kinase (p70S6K) pathways that lead to cell hypertrophy and increased protein synthesis and/or fibronectin expression (Barnes and Gorin, 2011; Block et al., 2008; Gorin et al., 2003; 2004). More specifically, Pyk-2 appears to act as a molecular scaffold binding to both PDK-1 and Src, thereby allowing Src to tyrosine phosphorylate and activate PDK-1, which in turn activate its downstream effectors, Akt/PKB and p70S6K (Block et al., 2008). Interestingly, PDK-1/Src/Pyk-2 complex may favor the formation of signaling platforms bringing key intermediates into proximity, thereby facilitating the contact between Nox4-derived ROS and downstream effectors (Block et al., 2008; Ushio-Fukai, 2006). Importantly, the most proximal event physically tethering Nox4 activation by Ang II to the rest of the signaling cascade is a redox-dependent posttranslational modification of Src on cysteine residues induced by Nox4-derived ROS (Block et al., 2008). It was reported that activation of Rac1 by phospholipase A2 (PLA2)-mediated arachidonic acid (AA) release appears to be implicated in Nox4-dependent ROS production and subsequent Akt/PKB-mediated cellular hypertrophy in mesangial cell treated with Ang II (Gorin et al., 2003; 2004). Our recent work also placed Nox4 as central mediator that control Ang II-induced redox signaling that lead to peroxynitrite-dependent eNOS dysfunction, decline in NO bioavailability and MC fibrotic injury (Lee et al., 2013a). The p22phox subunit is also required for the hypertrophic and fibrotic actions of Ang II in mesangial cells (Block et al., 2006).

Comparable to what was observed in a myriad of cell types (Barnes and Gorin, 2011; Bondi et al., 2010; Cucoranu et al., 2005), TGF-β specifically increases the expression of Nox4 and ROS production in mesangial cells. Moreover, a role for Nox4 in TGF-β-mediated ROS generation and mesangial cell fibrotic injury was established (Jha et al., 2014; Papadimitriou et al., 2014). p22phox is required for TGF-β-induced ROS production in mesangial cells (Xia et al., 2008). As described above for HG, AMPK also acts as a negative regulator of TGF-β-dependent Nox4 upregulation and subsequent ROS production in these cells.

PKC isozymes are positioned upstream and downstream of p22phox-containing NADPH oxidases and ROS production in mesangial cells exposed to HG or TGF-β (Kwan et al., 2005; Xia et al., 2006). Furthermore, in mesangial cells, HG-induced oxidative stress involves autocrine TGF-β stimulation of PKC and resultant generation of ROS by p22phox-based Nox oxidase via p22phox protein upregulation (Xia et al., 2008). Other studies with mesangial cells exposed to AOPPs place an NADPH oxidase, distal to PKC activation, resulting in extracellular matrix protein overproduction and upregulation of TGF-β (Wei et al., 2009).

The role of other Nox catalytic isoforms or regulatory subunits in mesangial cell injury in the diabetic kidney has been less studied. A role for Nox2 or Nox1 is indirectly suggested by the observation that administration of apocynin significantly reduced glomerular fibronectin and collagen accumulation in type 1 diabetic rats (Asaba et al., 2005). However, The involvement of Nox2 in the mesangium is not clear as some studies indicate that Nox2 is not detected in cultured human and rat mesangial cells whereas Nox2 is detected in isolated mouse mesangial cells and in diabetic glomeruli (Jones et al., 1995; Liu et al., 2012; Miyata et al., 2005). Nox2 regulatory subunits p47phox and p67phox are systematically found in mesangial cells (Jones et al., 1995; Pleskova et al., 2006) and the expression and translocation of p47phox and p67phox to the membrane is increased in the diabetic glomeruli (Kitada et al., 2003). HG causes an increase in p47phox protein expression in mesangial cells and downregulation of p47phox blunts high glucose-induced ROS and extracellular matrix accumulation (Hua et al., 2003; Kwan et al., 2005; Xia et al., 2006). Importantly, diabetes-induced oxidative stress and glomerular injury are attenuated in p47phox-deficient type 1 diabetic Akita mice (Liu et al., 2012). Moreover, deletion of p47phox attenuates diabetes-induced and HG-induced Nox2 expression in glomeruli and mesangial cells, respectively (Liu, et al., 2012). This indicates that p47phox-dependent activation of Nox oxidase, likely Nox2, is determinant for the promotion of DN. However, a recent study showed that glomerular mesangial matrix expansion and albuminuria are not attenuated in type 1 diabetic Nox2 knockout mice (You et al., 2013). Interestingly, an upregulation of Nox4 is observed in these mice (You et al., 2013). Together, these findings warrant a reassessment of the role of Nox2 in DN. While Nox1 is thought to promote the deleterious effects of glucose or Ang II in the vasculature (Lassegue et al., 2001; Lavrentyev and Malik, 2009), its function in mesangial cell injury in the diabetic milieu has not been reported. Indeed, a role for Nox1 as a source of renal ROS and mediator of DN has been challenged by the recent report showing that deletion of Nox4, but not Nox1, in a mice model of type 1 diabetes in ApoE knockout mice resulted in renal protection from glomerular injury (Jha et al., 2014). However, p47phox and p67phox recruitment to the membrane contributes to Ang II-mediated oxidative stress and mesangial cell growth (Ding et al., 2007) and TGF-β was reported to upregulate the expression of Nox2, p22phox, p47phox and p67phox in mesangial cells (Lee et al., 2003). AOPPs also increase p22phox, p47phox and Nox4 protein expression (Wei et al., 2009). AOPPs promote an increase in p47phox expression and translocation as well as its interaction with p22phox expression (Wei et al., 2009). These events are associated with enhanced extracellular matrix protein synthesis. Although PKC-dependent NADPH oxidase activation mediates AGEs-induced mesangial cell fibrotic injury (Thallas-Bonke et al., 2008), the Nox subunits implicated in these processes remain to be identified. In vivo studies suggested that induction of glomerular injury by AGEs is due to Src homology 2 domain-containing transforming protein C1 (Shc1) isoform p66Shc-mediated Nox4 expression (Menini et al., 2007).

Figure 2 is an overview of the major redox pathways and signaling intermediates positioned upstream and downstream of Nox enzymes that are stimulated by diabetic stimuli to promote glomerular mesangial cell injury.

UPSTREAM REGULATORS AND DOWNSTREAM EFFECTORS OF NOX NADPH OXIDASES IN DIABETES-INDUCED GLOMERULAR EPITHELIAL CELL/PODOCYTE INJURY

Upregulation of Nox4 protein expression in diabetic glomeruli including podocytes is accompanied by increased oxidative stress, loss of podocyte or foot process effacement as well as albuminuria in OVE26 type 1 diabetic mice (Eid et al., 2009; 2010; 2013a; Khazim et al., 2013; Sharma et al., 2008). In cultured podocytes exposed to HG for prolonged time period, Nox4 mRNA and protein are augmented and Nox4-derived ROS play a key role in apoptotic cell death (Eid et al., 2009; 2010; 2013a; Jha et al., 2014; Khazim et al., 2013; Piwkowska et al., 2011; Sharma et al., 2008). In human podocytes where both Nox4 and Nox5 are present, HG had no effect on Nox5 mRNA expression but significantly increased Nox4 mRNA levels (Jha et al., 2014). Transgenic mice overexpressing human Nox5 in a podocyte-specific manner exhibited early onset of albuminuria and podocyte foot process effacement (Holterman et al., 2014). Subjecting the mice to streptozotocin-induced type 1 diabetes further exacerbated these changes (Holterman et al., 2014).

A sequential regulation of Nox oxidases by cytochrome P450 of the 4A family (CYP4A) was recently identified in podocytes in which 20-hydroxyeicosatetraenoic acid (20-HETE) generation by CYP4A mediates the stimulatory effect of HG on Nox4 and Nox1 expression and the resultant ROS production at later time points (Eid et al., 2009). In the presence of HG concentrations, Nox4 promotes podocyte cell death via activation of p53- and PUMA-dependent apoptotic pathway (Eid et al., 2010). The oxidative stress triggered by HG appears to be exacerbated by the fact that the ROS generated by Nox4 affects the balance between oxidants and antioxidants by decreasing the activity of key antioxidant enzymes such as glutathione peroxidase (GPx) and catalase (Piwkowska et al., 2011). Importantly, inactivation of AMPK-activated protein kinase (AMPK) by HG accounts for the increase in Nox4 mRNA and protein expression as well as subsequent ROS production and podocyte apoptosis (Eid et al., 2010). AMPK activators, e.g. AICAR (5-aminoimidazole-4-carboxamide-1-riboside) or adiponectin, significantly reduced Nox4 expression, oxidative stress and podocyte injury in vitro or in vivo (Eid et al., 2010; Piwkowska et al., 2010; Sharma et al., 2008). A recent report indicates that mammalian target of rapamycin complex 1 (mTORC1) is an upstream positive regulator of Nox4 expression and subsequent ROS production and injury in podocytes exposed to HG or in vivo in glomeruli from OVE26 type 1 diabetic mice (Eid et al., 2013a). The same report showed that AMPK exerts its protective actions by inhibiting mTORC1 pathway via tuberin activation (Eid et al., 2013a).

Although podocytes also express Nox2, p22phox, p47phox and p67phox (Greiber et al., 1998; Nistala et al., 2008), there is no evidence of regulation of these subunits by HG. Similar to Nox4, Nox1 protein levels are increased in glomeruli from OVE26 mice (Eid et al., 2009; 2010). Nox1 protein is upregulated by HG in cultured podocytes and, as for Nox4, mTORC1 positively regulates Nox1 expression (Eid et al., 2013a). While Ang II- or TGF-β-induced oxidative stress mediates podocyte injury (Campbell et al., 2011; Nistala et al., 2008; Ziyadeh and Wolf, 2008), very little is known regarding the role of the Nox oxidases in the podocyte dysfunction promoted by these agonists or the other major mediators of DN. Similar to what is observed in MCs, Ang II-dependent increase in NADPH oxidase activity is associated with the upregulation of Nox4, Nox2, Rac and p22phox expression in podocytes (Nistala et al., 2008; Whaley-Connell et al., 2008). Stimulation with Ang II upregulates Nox5 expression in human podocytes and Nox5 is required for Ang II-induced ROS production as well as altered podocyte cytoskeletal dynamics (Holterman et al., 2014). Nox4 was detected within the mitochondria in podocytes and a recent report suggested that TGF-β-induced mitochondrial Nox4 upregulation via the Sma and Mad homologue (Smad) 2/3 pathway is responsible for ROS production, mitochondrial dysfunction, and apoptosis in podocytes (Block et al., 2009; Das et al., 2014; Yu et al., 2014a). Note that in human podocytes, Nox5 mRNA expression is enhanced following treatment with TGF-β (Jha et al., 2014).

Figure 3 is an overview of the major redox pathways and signaling intermediates positioned upstream and downstream of Nox enzymes that are stimulated by diabetic stimuli to promote glomerular mesangial cell and podocyte injury.

UPSTREAM REGULATORS AND DOWNSTREAM EFFECTORS OF NOX NADPH OXIDASES IN DIABETES-INDUCED TUBULAR CELL INJURY

Similar to glomeruli, tubules from type 1 diabetic rats show an increase in Nox4 mRNA and protein expression and downregulation of tubular Nox4 levels with in vivo administration of antisense oligonucleotides reduces diabetes-mediated ROS production and extracellular matrix protein synthesis in the renal cortex that is mainly composed of tubular epithelial cells (Etoh et al., 2003; Fujii et al., 2010; Gorin et al., 2005). The importance of Nox4 as a major source of ROS in the tubular compartment was further supported by a recent study in type 1 diabetic ApoE/Nox4 double knockout mice or Nox4 knockout mice on C57BL6/J background showing that Nox4 genetic deletion markedly reduced ROS production in renal cortex (Jha et al., 2014; Thallas-Bonke et al., 2014). Noteworthy, Nox4 was also shown to be responsible for diabetes-induced ROS production in mitochondria isolated from cortex (Jha et al., 2014). Importantly, the function of Nox1 as a generator of ROS in tubules is weakened by the observations that ROS production is not affected in the renal cortex of type 1 diabetic ApoE/Nox1 knockout mice (Jha et al., 2014). Interestingly, Nox4 protein expression is increased in renal cortex but is unchanged in medulla from type 2 diabetic mice (Sedeek et al., 2010). Increased Nox4 expression in diabetic tubules correlates with an augmentation in p22phox levels (Etoh et al., 2003; Sedeek et al., 2010). Whilst the levels of Nox2 and p47phox are not affected in renal cortex from type 2 diabetic mice (Sedeek et al., 2010), Nox2 is increased in the cortex from type 1 diabetic rats (Gorin et al., 2005). A role for Nox2 is challenged by a recent report showing that tubulointerstitial injury is not ameliorated in type 1 diabetic Nox2 knockout mice (You et al., 2013). Exposure of cultured renal proximal tubular epithelial cells to HG leads to the upregulation of Nox4 protein expression but seems to have no effect on Nox2, Nox1, p22phox or p47phox expression (Ford et al., 2013; Sedeek et al., 2010). Furthermore, Nox4-dependent ROS production is required for glucose-induced increase in fibronectin accumulation and TGF-β expression in these cells (Ford et al., 2013; Sedeek et al., 2010). The profibrotic action of the oxidase is corroborated by the finding that overexpression of Nox4 in tubular cells causes a robust increase in fibronectin synthesis (New et al., 2012). In human tubular epithelial cells, Nox5 has been shown to be express together with Nox1, Nox4 and Nox2 but its role in HG-induced ROS production and cell injury was not studied (Yu et al., 2014b).

A potential upstream regulator of Nox4 expression in the tubules is protein kinase C-β since diabetes-induced Nox4 expression and oxidative stress are attenuated in renal cortex from PKC-β-null mice kidney (Ohshiro et al., 2006). Interestingly, PKC seems to be positioned both upstream and downstream of Nox4 since it was recently reported that the increase in the major isoforms of PKC in tubules was blunted in the renal cortex from diabetic Nox4 knockout mice (Thallas-Bonke et al., 2014). A role for matrix metalloprotease a disintegrin and metalloprotease1 7 (ADAM17) as upstream regulator of Nox4 was recently established in vivo and in vitro. Pharmacological inhibition of ADAM17 attenuated diabetes-mediated Nox4 upregulation, oxidative stress and matrix protein accumulation in OVE26 mice kidney cortex (Ford et al., 2013). In cultured tubular epithelial cells, ADAM17 activity is required for HG-induced increase in Nox4 protein expression, ROS generation and fibrotic injury (Ford et al., 2013). Similar to podocytes, AMPK activation attenuates HG-mediated Nox4 protein upregulation in tubular epithelial cells, supporting the concept that the kinase acts as a suppressor of oxidative stress (Lee et al., 2013b). p38MAPK has been also identified as a critical downstream effector of Nox4 in the signaling pathway linking HG and tubular cell injury (Sedeek et al., 2010).

Ang II has been shown to utilize Nox4 as a mediator of its injurious effects in tubular epithelial cells. Chronic angiotensin II treatment upregulates Nox4 expression and induces epithelial-to-mesenchymal transition in renal epithelial cells through Nox4-dependent ROS production and resultant Src/caveolin-mediated activation of epidermal growth factor receptor/ERK signaling pathway (Chen et al., 2012; Peng et al., 2009; Xu et al., 2010). Furthermore, Ang II upregulates Nox4 expression in the mitochondrial fraction of renal tubular cells and Nox4 is required for Ang II-mediated ROS mitochondrial production (Kim et al., 2012). ROS derived from Nox4 contribute to Ang II-dependent apoptosis in these cells (Kim et al., 2012). Ang II enhances the expression of Nox2, p22phox and p47phox subunits along with Nox4 in tubular cells (Hannken et al., 1998; Xu et al., 2010). The effects on p22phox, p47phox and Nox4 appear to be mediated by lectin-like oxidized low-density lipoprotein receptor-1 (Hannken et al., 1998; Xu et al., 2010). Another report shows that HG induces p22phox expression and oxidative stress via angiotensin II type 1 (AT1) receptor activation, further underlining the important role of Ang II as a regulator of Nox signaling in tubular cells (Takao et al., 2011). Furthermore, a p22phox-containing NADPH oxidase is critical for Ang II-mediated tubular cell hypertrophy through induction of p27Kip1, a factor promoting cell cycle arrest (Hannken et al., 1998). As in mesangial cells, Src and ERK pathways appears to be a primary target of Nox4-derived ROS and plays a key role in the resultant fibrotic or hypertrophic response in tubular cells. AMPK also inhibits Ang II-induced increase in Nox4 expression and tubular epithelial-to-mesenchymal transition (EMT) (Lee et al., 2013b).

Although it is clear that oxidative stress is implicated in TGF-β-mediated tubular cell injury (Rhyu et al., 2005), there is a deficit of causal evidence supporting the role of the oxidase or other Nox enzymes in TGF-β signaling in tubular cells. Stimulation of tubular cells by TGF-β results in an increase in Nox4 protein expression, an effect prevented by AMPK activation (Lee et al., 2013b). Conflicting results exist concerning the regulation of other Nox subunits by TGF-β in these cells. On one hand, TGF-β has no effect on Nox2, p22phox and p47phox expression and that only p67phox seems to be required for the deleterious actions of the cytokine in renal tubular cells (Zhang et al., 2009). On the other hand, TGF-β was shown to upregulate p22phox (Lee et al., 2003). Nox4-derived ROS also serve as signal transducer for the fibrotic effects of IGF-I in renal tubular epithelial cells via Akt/PKB and mTORC1/p70S6K signaling pathways activation (New et al., 2012).

Figure 4 is an overview of the major redox pathways and signaling intermediates positioned upstream and downstream of Nox enzymes that are stimulated by diabetic stimuli to promote tubulointerstitial cell injury.

UPSTREAM REGULATORS AND DOWNSTREAM EFFECTORS OF NOX NADPH OXIDASES IN DIABETES-INDUCED INTERSTITIAL CELL INJURY

The information regarding the roles of hyperglycemia-mediated oxidative stress or Nox-derived ROS in interstitial cell injury, particularly in the activation of renal fibroblast into myofibroblasts, remains very limited. There is indication that exposure of kidney fibroblasts to HG upregulates Nox4 and Nox2 mRNA and protein expression and that this is associated with an increase in ROS generation and hypertrophy (Williams and Gooch, 2014). Calcineurin A-β (CnAβ) maintains basal Nox4 and Nox2 expression and is required for HG-induced increase in Nox4 and Nox2 expression in these cells (Williams and Gooch, 2014). It seems that CnAβ promotes Nox4 and Nox2 transcription via activation of the nuclear factor of activated T cells (NFAT) (Williams and Gooch, 2014). A functional link between Nox4 or Nox2 and ROS generation and hypertrophy is suggested by the fact that expression of Nox4 or Nox2 in CnAβ deficient kidney fibroblasts (where Nox4 and Nox2 are downregulated) is able to restore the stimulatory effects of HG on these biological outcomes (Williams and Gooch, 2014).

Nox4 is the predominant Nox homolog implicated in kidney myofibroblast differentiation and expression of fibronectin and other fibrotic markers in response to TGF-β (Barnes and Gorin, 2011; Bondi et al., 2010). However, unlike cardiac and lung myofibroblasts where Nox4 acts upstream of transcription factor Smad2/3, Nox4 is positioned downstream of Smad3 and proximal to ERK in the pathway linking TGF-β to the fibrotic response in renal fibroblasts (Barnes and Gorin, 2011; Cucoranu et al., 2005; Hecker et al., 2009). These results are in agreement with what is reported in pulmonary vascular smooth muscle cells, where TGF-β-induced proliferation occurs through a Nox4-dependent pathway downstream of Smad3 (Sturrock et al., 2006). Small GTPase RhoA and its downstream target Rho kinase (ROCK) have been identified as upstream positive regulators of Nox4 expression and activity in the pathway linking TGF-β to kidney myofibroblast differentiation (Manickam et al., 2014). Interestingly, RhoA/ROCK seems to act via the increasing the expression of Poldip2, a newly discovered Nox4 enhancer protein (Manickam et al., 2014). Moreover, the data suggest that besides enhancing Nox4 activity Poldip2 is also able to regulate the expression of the oxidase.

CONCLUSION

It is apparent from this review that the in vivo and in vitro experimental evidence support a fundamental role for NADPH oxidases of the Nox family, and especially the homologue Nox4, in the pathogenesis and pathophysiology of DN. The consequence of these observations is the consideration of the Nox homologs and their associated subunits as relevant therapeutic targets for the treatment of DN. There has been recently a considerable effort for the generation and development of agents able to inhibit the Nox enzymes in a homolog-specific manner (Altenhofer et al., 2012; Drummond et al., 2011; Gaggini et al., 2011; Jaquet et al., 2009; Kim et al., 2011; Laleu et al., 2010; Lambeth et al., 2008). Among these inhibitors, the small-molecule dual Nox4 and Nox1 inhibitors from the Pyrazolo pyridine chemical series, referred as to GKT136901 and GKT-137831, have drawn considerable attention. Preclinical studies performed with these inhibitors in experimental animal models indicate that they effectively attenuate the pathological changes observed in renal complication of type 1 and type 2 diabetes, atherosclerosis, ischemic retinopathy, liver fibrosis and idiopathic pulmonary fibrosis (Aoyama et al., 2012; Carnesecchi et al., 2011; Di Marco et al., 2014; Gorin and Block, 2013a; 2013b; Gray et al., 2013; Hecker et al., 2014; Jha et al., 2014; Jiang et al., 2012; 2014; Laleu et al., 2010; Sedeek et al., 2013; Vendrov et al., 2010; Wilkinson-Berka et al., 2014). GKT137831 have recently been successfully used in a phase 1 clinical trial and is currently being evaluated in a phase 2 clinical trial in patients with type 2 diabetes and albuminuria (Clinical-Trials.gov reference number NCT02010242).

Beside the direct inhibition of Nox4 and others Nox oxidases, the present review suggests that adjunct therapies targeting the agonists or signaling intermediates that regulate the expression or function of Nox subunits and subsequent ROS production as well as the downstream targets of Nox oxidases implicated in the pathological processes should be considered for the treatment of diabetic complications. In regards to the observation reported above, such strategies could involve the use of PKC inhibitors, agents that disrupt the AGE signaling, inhibitors of the renin-angiotensin system (angiotensin-converting enzyme inhibitors and Ang II receptor blockers), statins (that inhibit Rac1 and Rho), Rho/Rho kinase inhibitors, AMPK activators (i.e. metformin), Sestrin-mimicking small molecules or peptides Sestrin analogues/agents that stimulate Sestrin activity or expression. Importantly, the small molecules Nox inhibitors that are currently available affect Nox activity without altering the expression of the oxidases. Therefore, the utilization of agents that modulate Nox expression or that target downstream effectors of the oxidases as adjunct therapy to Nox allosteric inhibitors may allow a more effective neutralization of the enzyme due to the blockade of the sustained increase in Nox protein levels promoted by chronic hyperglycemia or other mediators of DN.

Fig 1.

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Figure 1.Structure and molecular organization of the renal Nox NADPH oxidases. The top right left panel illustrates the topology of and the enzymatic reaction catalyzed by the Nox enzymes. The other panels represent the molecular structure of the Nox oxidase homologues predominantly expressed in the kidney, Nox2 (a.k.a. gp91phox), Nox1, Nox4, and Nox5. The regulatory subunits differ from a Nox homologue to another.
Molecules and Cells 2015; 38: 285-296https://doi.org/10.14348/molcells.2015.0010

Fig 2.

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Figure 2.Upstream and downstream effectors of Nox oxidases implicated in glomerular mesangial cell injury triggered by diabetic stimuli. See text for detail.
Molecules and Cells 2015; 38: 285-296https://doi.org/10.14348/molcells.2015.0010

Fig 3.

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Figure 3.Upstream and downstream effectors of Nox oxidases implicated in podocyte injury triggered by diabetic stimuli. See text for detail.
Molecules and Cells 2015; 38: 285-296https://doi.org/10.14348/molcells.2015.0010

Fig 4.

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Figure 4.Upstream and downstream effectors of Nox oxidases implicated in tubular cell injury triggered by diabetic stimuli. See text for detail.
Molecules and Cells 2015; 38: 285-296https://doi.org/10.14348/molcells.2015.0010

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Mol. Cells
May 31, 2023 Vol.46 No.5, pp. 259~328
COVER PICTURE
The alpha-helices in the lamin filaments are depicted as coils, with different subdomains distinguished by various colors. Coil 1a is represented by magenta, coil 1b by yellow, L2 by green, coil 2a by white, coil 2b by brown, stutter by cyan, coil 2c by dark blue, and the lamin Ig-like domain by grey. In the background, cells are displayed, with the cytosol depicted in green and the nucleus in blue (Ahn et al., pp. 309-318).
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