Progressive kidney disease, including diabetic nephropathy, continues to rise each year and is a worldwide problem, resulting in an increased number of people with end-stage renal disease . Chronic kidney disease includes both glomerulosclerosis and tubulointerstitial scarring, often resulting in renal failure. Hyperglycaemia is a known cause and accelerator of kidney disease, as seen in diabetic nephropathy. A number of mechanisms have been implicated in the tissue-damaging effects of hyperglycaemia, such as the polyol pathway , increased production of advanced glycation endproducts (AGEs) , activation of protein kinase C (PKC) isoforms  and enhancement of oxidative stress [5–9]. A unifying hypothesis has been postulated to connect all these mechanisms by Brownlee , suggesting that excess generation of mitochondrial superoxide by hyperglycaemia is the primary initiating factor linking these pathways to tissue damage. Mitochondria are the major source of reactive oxygen species (ROS) in cells as they produce superoxide as a byproduct of their normal function, namely electron transport for the production of energy as ATP .
More recent studies have postulated that NADPH oxidases, a family of enzymes known as Nox, are also a main source of ROS production in diabetes . NADPH oxidase is composed of two membrane-associated components, p22phox and gp91phox, and four major cytosolic components, p47phox, p40phox, p67phox and rac-1/2. The gp91phox component has a number of other homologues that are present within the kidney, namely Nox-1, Nox-2 and Nox-4, and all these isoforms have been examined in diabetic nephropathy .
In this review, the role of Nox-4 will be explored as it pertains to kidney disease, with a particular emphasis on diabetic nephropathy. Nox-4 has been shown to be a major source of ROS in the renal cells of diabetic animals [12,14–16]. Furthermore, a study by Block et al. found that Nox-4 localizes to membranes and mitochondria, and is upregulated in the kidney cortex in diabetes. Such studies, and the failure of a range of relatively nonspecific antioxidants in the treatment of kidney disease [18,19], have stimulated further exploration of Nox-4 as an attractive target for developing new renoprotective strategies.
NADPH OXIDASE AS A SOURCE OF CELLULAR REACTIVE OXYGEN SPECIES
ROS are important signalling molecules but are also important in producing an oxidative burst critical for various immune processes. However, excess production or reduced clearance of ROS resulting in oxidative stress can often be deleterious and is involved in the pathogenesis of chronic diseases such as progressive kidney disease, including diabetic nephropathy [7,10,20]. One important source of ROS production is the enzyme family of NADPH oxidases (Nox). They are membrane-associated proteins that can transfer electrons across the biological membranes using NADPH as an electron donor, resulting in the generation of ROS. Nox generates superoxide (O2-) during the respiratory burst in phagocytes but is also responsible for the production of superoxide in nonphagocytic cells. Classically, Nox is composed of two membrane-associated components, p22phox and gp91phox, and four major cytosolic components, p47phox, p40phox, p67phox and rac-1/2, that, upon activation, form a functional enzyme complex. Furthermore, it has been shown that gp91phox is only one member of a homologous group of proteins termed ‘Nox’ [21–23] and to date seven isoforms (Nox1–Nox7) have been identified , with many cells expressing multiple Nox proteins . This Nox family of enzymes has been implicated as playing a role in many cell functions including regulation of various transcription factors involved in cell proliferation, differentiation and apoptosis, host defence, growth factor receptor signalling, senescence, gene expression, oxygen sensing and angiogenesis [26–30].
RENAL Nox-4 AND OTHER Nox ISOFORMS
Nox-4 is predominantly expressed in the kidney cortex and has been previously described as ‘Renox’ [31,32]. A study by Martyn et al. reported that Nox-4 does not require cytosolic subunits for activation; however, this Nox isoform does co-localize with the p22phox subunit at the site where superoxide generation occurs at the internal membranes. This constitutive activity of Nox-4 results in the release of hydrogen peroxide to the extracellular space .
A number of Nox proteins have been demonstrated in the various compartments of the kidney. In human podocytes, expression of Nox-2, p22phox, p47phox and p67phox has been demonstrated, and ATP treatment results in p67phox being upregulated . In the adult rat kidney, a number of Nox subunits are localized to the nephron including Nox-1, Nox-2, Nox-4, p22phox, p47phox and p67phox. In a previous study, our group identified upregulation of certain Nox enzyme subunits including p47phox in the diabetic kidney .
Nox enzymes have been shown to contribute to the pathophysiology of progressive kidney disease. Multiple factors implicated in the progression of kidney disease, including hyperglycaemia, the renin–angiotensin system (RAS), including specifically angiotensin-II (AngII), AGEs, transforming growth factor-β (TFG-β) and PKC have all been implicated in altering the expression of Nox proteins and their regulatory units, as well as influencing the amount of ROS ultimately produced (see Fig. 1) [13,17,26,36–46]. In-vitro studies in renal cells cultured in high glucose and in-vivo studies in the experimental models of diabetes have demonstrated upregulation of Nox-4 in the kidney with associated increases in ROS, including both superoxide and hydrogen peroxide [13,17,36,37,41–43,47].
Nox-4 AND KIDNEY INJURY
The importance of Nox-4 in progressive kidney disease is supported by a number of previous studies demonstrating increased renal Nox-4-derived ROS [41–43,48–52]. A number of studies have shown a link between certain Nox isoforms and hypertension. It was demonstrated that there is upregulation of the Nox isoforms p47phox and p67phox in the spontaneously hypertensive rat kidney, a model of essential hypertension . In mesangial cells, the activation of Nox-4 and subsequent generation of ROS contributed to mediate AngII-induced activation of Akt/protein kinase B (PKB)  as well as mesangial cell hypertrophy and fibronectin accumulation [37,53,54]. In a more recent study, AngII induced a chronic increase in Nox-4 protein and mRNA expression in association with increased ROS generation in mesangial cells . In-vivo studies by Zhang et al.[56▪] demonstrated enhanced activity and expression of Nox-4 in the macula densa in AngII-induced hypertensive mice. Furthermore, Gorin et al. demonstrated that Nox-4 was the major source of ROS in the kidney in early diabetes and that administration of an antisense Nox-4 cDNA prevented the development of diabetic nephropathy in rats, as reflected by reduced kidney and glomerular hypertrophy, as well as attenuated expression of fibronectin in the renal cortex.
Oxidative stress is implicated in TGF-β-mediated tubular cell injury, with TGF-β having been shown to promote the upregulation of NADPH oxidase subunits in rat renal tubular cells . Furthermore, in response to TGF-β, Nox-4 is the predominant isoform implicated in kidney myofibroblast differentiation and expression of fibronectin [38,58]. Plumbagin, which has multiple actions including Nox-4 inhibition, was shown to reduce the TGF-β-induced fibrosis in diabetic C57BL/6J mice as well as immortalized human proximal tubule cells (HK2) [59▪]. In a recent report, albeit in a nonrenal fibroblast cell line, the effects of TGF-β on Nox-4 regulation were examined. This study expanded the possibility of posttranslational regulation of Nox-4 .
Activation or signalling via PKC has also been reported to modulate Nox-4 expression. Specifically, ROS generation in mesangial cells cultured in high glucose and subsequent collagen IV expression resulting from the activation of NADPH oxidase were shown to be dependent on the activation of conventional PKC isoforms, particularly PKC-α and PKC-β, thus contributing to early diabetic nephropathy . Another study suggests a positive feedback loop between ROS generation and the activation of PKC-ζ in the mesangial cells cultured in high glucose . Indeed, we have previously shown that activation of Nox and increased superoxide and p47phox expression were via phosphorylation of PKC-α in a streptozotocin model of type 1 diabetes .
The role of Nox-4 in podocytes, which are increasingly considered to play a key role in the pathogenesis of chronic renal diseases that are characterized by albuminuria, has been explored in a number of studies which demonstrated that Nox-4 was increased in certain renal diseases, particularly glomeruli, including podocytes with an associated increase in ROS and albuminuria [41,42,62,63]. In mouse podocytes, Das et al. showed that Nox-4 was predominately localized to the mitochondria and that Nox-4 upregulation by TGF-β resulted in increased ROS production, mitochondrial dysfunction and apoptosis, all of these pathological processes considered to contribute to diabetic nephropathy. Furthermore, we have recently shown in vitro that deletion of Nox-4 in podocytes leads to decreased ROS production, as well as a decrease at the gene level of collagen IV, fibronectin and vascular endothelial growth factor expression [65▪▪].
Our group has previously shown an interaction between cytosolic and mitochondrial sources of ROS to amplify kidney disease in diabetes . More recently, high glucose elicited upregulation of Nox-4 protein expression in the mitochondria of cultured mesangial cells [17,67]. These in-vitro findings were confirmed in vivo, in which in the kidney cortex from streptozotocin-induced type 1 diabetic rats including the mitochondrial fraction showed an increase in Nox-4 . We have previously shown an interaction between cytosolic and mitochondrial sources of ROS to amplify kidney disease in diabetes . More recently, total cellular and mitochondrial ROS was associated with increased NADPH oxidase activity and Nox-4 protein expression in mesangial cells in response to high glucose . This finding is potentially important as previous seminal studies by Brownlee and colleagues , albeit in endothelial cells, had not considered Nox-4 but rather the electron transport chain (ETC) as the major source of mitochondrial ROS in diabetes as it pertains to the vascular complications. On the basis of these and other findings, it is now viewed as likely that Nox-4 activation is a major driving force for excess ROS generation in the diabetic kidney and that this represents a potentially important target for therapeutic intervention.
PROXIMAL TUBULAR Nox-4
Proximal tubular epithelial cells (PTCs) synthesize matrix proteins, which are responsible for extracellular matrix (ECM) accumulation resulting in the progression of chronic kidney disease. Hyperglycaemia-induced ROS generation in particular by the Nox-4 isoform has been associated with tubulointerstitial fibrosis. Furthermore, PTCs are particularly rich in mitochondria and it has been reported that Nox-4 is expressed in the mitochondria of these cells . In cultured renal PTCs exposed to high glucose, there was increased expression of Nox-4 but not Nox-1 or Nox-2 proteins. This NADPH oxidase-derived ROS generation led to increased TGF-β and fibronectin, which was attenuated by a Nox inhibitor, GKT-136901 . In addition, overexpression of Nox-4 in PTCs resulted in increased fibronectin expression . Chronic exposure of PTCs to AngII caused upregulation of Nox-4-dependent ROS production, resulting in the increased expression of epithelial-to-mesenchymal transition (EMT) markers, these findings interpreted as representing a potential Nox-4-dependent mechanism for progressive renal injury . Furthermore, a study by Lee et al. in tubular epithelial cells showed that 5’ adenosine monophosphate-activated protein kinase activation was able to attenuate the TGF-β, AngII and high glucose induced increases in ROS and Nox-4 expression. This occurred in association with the suppression of the induction of EMT. Further studies confirm that Nox-4-derived ROS are involved in TGF-β1-induced rat kidney myofibroblast differentiation associated with renal fibrosis . In human renal tubular cells, accumulation of p-cresyl sulphate (PCS), a uraemic toxin associated with mortality in chronic kidney disease patients, leads to increased NADPH oxidase activity and ROS production which then triggers inflammatory cytokines in renal fibrosis. To further define this pathway, knockdown of Nox-4 expression was able to suppress the effects of the uraemic toxin, PCS . In a sepsis-induced in-vitro model using PTCs, the bacterial endotoxin lipopolysaccharide increased the cytosolic expression of inducible nitric oxide synthase and Nox-4, resulting in the interruption of mitochondrial oxidative phosphorylation by reducing cytochrome c oxidase activity. This targeting of mitochondria by ROS resulted in further ROS production from mitochondria, thereby contributing to mitochondrial dysfunction in this model .
The study of the role of Nox-4 in progressive kidney injury has been greatly facilitated by the advent of a number of knockout mice, although the findings have not been uniform. Babelova et al. examined three experimental animal models of renal injury using Nox-4-knockout animals (streptozotocin diabetes I, unilateral ureteral ligation and 5/6 nephrectomy). The results showed data suggesting that Nox-4 does not promote renal disease but could have a partial protective effect. These authors suggested that these renal data do not support the view that Nox-4 is a major driver of renal disease. However, recent studies by our group in another model of diabetic nephropathy showed that in ApoE/Nox-4 double knockout mice there was not only reduced albuminuria but also decreased renal morphological injury [65▪▪]. These conflicting results could be explained, at least in part, by the use of different models of experimental diabetes as well as the different mouse backgrounds [77,78]. Furthermore, the type of genetic manipulation used to generate these global or cell specific Nox-4-deleted mice could be another contributing factor for the differences in results amongst the various research groups. However, it should also be noted that genetic mouse models cannot be considered to accurately represent the human condition; therefore, use of Nox-4 inhibitors may be a preferred approach to define the role of this isoform in progressive kidney disease as well as providing a superior approach for ultimate clinical translation.
CURRENT INTERVENTIONS AND REACTIVE OXYGEN SPECIES GENERATION
In the previous studies, we have demonstrated that excess mitochondrial superoxide as observed in the diabetic kidney is not affected by a number of current clinically used therapies, including angiotensin-converting enzyme (ACE) inhibitors . By contrast, an inhibitor of AGE accumulation, deletion of the AGE receptor for advanced glycation end-products and a nonspecific antioxidant apocynin were shown to inhibit ROS of both mitochondrial and cytosolic origin [36,80,81] in association with improved renal injury. These disparate effects of apocynin and ACE inhibitors on mitochondrial ROS appear to be therapeutically relevant as a combination of apocynin and ramipril was more effective than either monotherapy . However, the use of some of these Nox inhibitors, such as apocynin and diphenylene iodonium (DPI), as have been employed by various researchers to elucidate the role of these enzymes in progressive kidney disease, may have limited utility because of their nonspecificity [83–85].
Recently, more specific Nox inhibitors have been developed by GenKyoTex (Geneva, Switzerland) and have been utilized in a number of in-vitro and in-vivo studies. In mouse proximal tubular cells, a Nox1/4 inhibitor, GKT136901, was able to ameliorate the high-glucose-induced activation of Nox-4, and thus this agent was associated with reduced oxidative stress and less profibrotic signalling . A more recent study by this group , in the db/db model of diabetes, demonstrated some renoprotective effects of GKT136901, resulting in reduced oxidative stress, decreased albuminuria and preservation of renal structure. We have recently shown in vitro that deletion of Nox-4 in podocytes leads to decreased ROS production, and that pretreatment of human podocytes with another Nox inhibitor GKT137831 which had been grown in high glucose or TGF-β also resulted in decreases in ECM proteins and growth factors [65▪▪]. Furthermore, we have shown in streptozotocin-induced diabetic ApoE(-/-) mice that deletion of the Nox-4 but not the Nox-1 isoform was renoprotective as there was reduced glomerular injury. Indeed, administration of GKT137831 replicated the renoprotective effects of Nox-4 deletion [65▪▪]. In the context of a recent publication showing that GKT137831 was also able to reverse lung fibrosis associated with aging in a model of idiopathic pulmonary fibrosis [86▪▪], this first-in-class drug, GKT137831, is now beginning phase II clinical trials in patients with diabetic nephropathy (GSN000200). Other potential clinical indicators of this drug, although beyond the scope of this report, include osteoporosis [87▪].
A large body of evidence is available on Nox-4 and its role in progressive kidney disease. However, given that some of the data are conflicting with respect to Nox-4 as preventing or inhibiting renal disease, further studies are needed to elucidate the physiological functions of Nox-4. The recent targeted inhibition of Nox-4 by GKT137831 will hopefully lead to a promising therapeutic strategy for the treatment of progressive kidney disease. However, other adjunct therapies that inhibit PKC, AGE or the RAS (see Fig. 1) that target signalling molecules, whether upstream or downstream of Nox expression, thereby influencing the subsequent production of ROS should also be considered.
Financial support and sponsorship
Conflicts of interest
V.T-B. is a recipient of an Advanced Postdoctoral JDRF Fellowship (10-2012-227).
M.E.C. and K.A.M.J-D. are recipients of a JDRF Grant (4-2010-52). K.A.M.J-D. has received a small research grant from Genkyotex Inc.
REFERENCES AND RECOMMENDED READING
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