Secondary Logo

Journal Logo

Nox-4 and progressive kidney disease

Thallas-Bonke, Vickia,b; Jandeleit-Dahm, Karin A.M.a,c; Cooper, Mark E.a,c

Current Opinion in Nephrology and Hypertension: January 2015 - Volume 24 - Issue 1 - p 74–80
doi: 10.1097/MNH.0000000000000082

Purpose of review Nox-4 is a member of the NADPH oxidase (Nox) family of enzymes implicated in reactive oxygen species generation. Nox-4 is distributed in many tissues; however, its physiological functions remain poorly understood. In contrast to other Nox isoforms, it is unique in that it produces large amounts of hydrogen peroxide constitutively and does not require other cytosolic oxidase components for its activation. This review highlights the recent developments in Nox-4 research and progressive kidney disease as well as the potential of new Nox-4 inhibitors to reduce renal damage.

Recent findings Recently, Nox-4 was shown to be implicated in kidney diseases such as diabetic nephropathy. Nox-4 has been identified as playing a role in damage to the kidney induced by hyperglycaemia and other major pathways of renal damage, including advanced glycation end-products, the renin–angiotensin system, TGF-β and protein kinase C.

Summary The role of Nox-4 as a target for renoprotection remains controversial, although recent positive preclinical data have stimulated increased interest in inhibiting the enzyme in clinical trials of renal disease.

aDiabetes Complications Division, Baker IDI Heart & Diabetes Institute, JDRF Danielle Alberti Memorial Centre for Diabetic Complications

bDepartment of Medicine, Austin and Northern Clinical Schools, University of Melbourne

cDepartment of Medicine, Central Clinical School, Monash University, AMREP Precinct, Melbourne, Victoria, Australia

Correspondence to Dr Vicki Thallas-Bonke, Baker IDI Heart & Diabetes Institute, P.O Box 6492, St Kilda Road Central, Melbourne, VIC 3004, Australia. Tel: +61 3 8532 1451; fax: +61 3 8532 1288; e-mail:

Back to Top | Article Outline


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 [1]. 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 [2], increased production of advanced glycation endproducts (AGEs) [3], activation of protein kinase C (PKC) isoforms [4] and enhancement of oxidative stress [5–9]. A unifying hypothesis has been postulated to connect all these mechanisms by Brownlee [10], 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 [11].

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 [12]. 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 [13].

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.[17] 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.

Box 1

Box 1

Back to Top | Article Outline


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 [24], with many cells expressing multiple Nox proteins [25]. 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].

Back to Top | Article Outline


Nox-4 is predominantly expressed in the kidney cortex and has been previously described as ‘Renox’ [31,32]. A study by Martyn et al.[33] 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 [33].

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 [34]. 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[35]. In a previous study, our group identified upregulation of certain Nox enzyme subunits including p47phox in the diabetic kidney [36].

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].



Back to Top | Article Outline


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 [35]. 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) [53] 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 [55]. 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.[43] 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 [57]. 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 [60].

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 [16]. Another study suggests a positive feedback loop between ROS generation and the activation of PKC-ζ in the mesangial cells cultured in high glucose [61]. 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 [36].

Back to Top | Article Outline


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.[64] 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▪▪].

Back to Top | Article Outline


Our group has previously shown an interaction between cytosolic and mitochondrial sources of ROS to amplify kidney disease in diabetes [66]. 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 [17]. We have previously shown an interaction between cytosolic and mitochondrial sources of ROS to amplify kidney disease in diabetes [66]. 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 [67]. This finding is potentially important as previous seminal studies by Brownlee and colleagues [68], 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.

Back to Top | Article Outline


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 [17]. 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 [69]. In addition, overexpression of Nox-4 in PTCs resulted in increased fibronectin expression [70]. 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 [71]. Furthermore, a study by Lee et al.[72] 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 [73]. 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 [74]. 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 [75].

Back to Top | Article Outline


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.[76] 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.

Back to Top | Article Outline


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 [79]. 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 [82]. 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 [69]. A more recent study by this group [69], 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▪].

Back to Top | Article Outline


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.

Back to Top | Article Outline



Back to Top | Article Outline

Financial support and sponsorship


Back to Top | Article Outline

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.

Back to Top | Article Outline


Papers of particular interest, published within the annual period of review, have been highlighted as:

  • ▪ of special interest
  • ▪▪ of outstanding interest
Back to Top | Article Outline


1. Shaw JE, Sicree RA, Zimmet PZ. Global estimates of the prevalence of diabetes for 2010 and 2030. Diabetes Res Clin Pract 2010; 87:4–14.
2. Yabe-Nishimura C. Aldose reductase in glucose toxicity: a potential target for the prevention of diabetic complications. Pharmacol Rev 1998; 50:21–33.
3. Brownlee M, Cerami A, Vlassara H. Advanced products of nonenzymatic glycosylation and the pathogenesis of diabetic vascular disease. Diabetes Metab Rev 1988; 4:437–451.
4. Lee AT, Cerami A. Nonenzymatic glycosylation of DNA by reducing sugars. Prog Clin Biol Res 1989; 304:291–299.
5. Baynes JW, Thorpe SR. Role of oxidative stress in diabetic complications: a new perspective on an old paradigm. Diabetes 1999; 48:1–9.
6. Choi SW, Benzie IF, Ma SW, et al. Acute hyperglycemia and oxidative stress: direct cause and effect? Free Radic Biol Med 2008; 44:1217–1231.
7. Forbes JM, Coughlan MT, Cooper ME. Oxidative stress as a major culprit in kidney disease in diabetes. Diabetes 2008; 57:1446–1454.
8. Giacco F, Brownlee M. Oxidative stress and diabetic complications. Circ Res 2010; 107:1058–1070.
9. Patel H, Chen J, Das KC, Kavdia M. Hyperglycemia induces differential change in oxidative stress at gene expression and functional levels in HUVEC and HMVEC. Cardiovasc Diabetol 2013; 12:142.
10. Brownlee M. The pathobiology of diabetic complications: a unifying mechanism. Diabetes 2005; 54:1615–1625.
11. Chance B, Sies H, Boveris A. Hydroperoxide metabolism in mammalian organs. Physiol Rev 1979; 59:527–605.
12. Zou AP, Li N, Cowley AW Jr. Production and actions of superoxide in the renal medulla. Hypertension 2001; 37 (2 Part 2):547–553.
13. Gill PS, Wilcox CS. NADPH oxidases in the kidney. Antioxid Redox Signal 2006; 8:1597–1607.
14. Asaba K, Tojo A, Onozato ML, et al. Effects of NADPH oxidase inhibitor in diabetic nephropathy. Kidney Int 2005; 67:1890–1898.
15. Wendt MC, Daiber A, Kleschyov AL, et al. Differential effects of diabetes on the expression of the gp91phox homologues nox1 and nox4. Free Radic Biol Med 2005; 39:381–391.
16. Xia L, Wang H, Goldberg HJ, et al. Mesangial cell NADPH oxidase upregulation in high glucose is protein kinase C dependent and required for collagen IV expression. Am J Physiol Renal Physiol 2006; 290:F345–F356.
17. Block K, Gorin Y, Abboud HE. Subcellular localization of Nox4 and regulation in diabetes. Proc Natl Acad Sci USA 2009; 106:14385–14390.
18. Cooper ME, Jandeleit-Dahm K, Thomas MC. Targets to retard the progression of diabetic nephropathy. Kidney Int 2005; 68:1439–1445.
19. Drummond GR, Selemidis S, Griendling KK, Sobey CG. Combating oxidative stress in vascular disease: NADPH oxidases as therapeutic targets. Nat Rev Drug Discov 2011; 10:453–471.
20. Duchen MR. Roles of mitochondria in health and disease. Diabetes 2004; 53 (Suppl. 1):S96–S102.
21. Banfi B, Maturana A, Jaconi S, et al. A mammalian H+ channel generated through alternative splicing of the NADPH oxidase homolog NOH-1. Science 2000; 287:138–142.
22. Lambeth JD. Nox/Duox family of nicotinamide adenine dinucleotide (phosphate) oxidases. Curr Opin Hematol 2002; 9:11–17.
23. Lambeth JD, Cheng G, Arnold RS, Edens WA. Novel homologs of gp91phox. Trends Biochem Sci 2000; 25:459–461.
24. Briones AM, Touyz RM. Oxidative stress and hypertension: current concepts. Curr Hypertens Rep 2010; 12:135–142.
25. Cheng G, Cao Z, Xu X, et al. Homologs of gp91phox: cloning and tissue expression of Nox3, Nox4, and Nox5. Gene 2001; 269:131–140.
26. Bedard K, Krause KH. The NOX family of ROS-generating NADPH oxidases: physiology and pathophysiology. Physiol Rev 2007; 87:245–313.
27. Chen K, Thomas SR, Keaney JF Jr. Beyond LDL oxidation: ROS in vascular signal transduction. Free Radic Biol Med 2003; 35:117–132.
28. Nakashima I, Kato M, Akhand AA, et al. Redox-linked signal transduction pathways for protein tyrosine kinase activation. Antioxid Redox Signal 2002; 4:517–531.
29. Nimnual AS, Taylor LJ, Bar-Sagi D. Redox-dependent downregulation of Rho by Rac. Nat Cell Biol 2003; 5:236–241.
30. Sauer H, Wartenberg M, Hescheler J. Reactive oxygen species as intracellular messengers during cell growth and differentiation. Cell Physiol Biochem 2001; 11:173–186.
31. Geiszt M, Kopp JB, Varnai P, Leto TL. Identification of renox, an NAD(P)H oxidase in kidney. Proc Natl Acad Sci USA 2000; 97:8010–8014.
32. Shiose A, Kuroda J, Tsuruya K, et al. A novel superoxide-producing NAD(P)H oxidase in kidney. J Biol Chem 2001; 276:1417–1423.
33. Martyn KD, Frederick LM, von Loehneysen K, et al. Functional analysis of Nox4 reveals unique characteristics compared to other NADPH oxidases. Cell Signal 2006; 18:69–82.
34. Greiber S, Munzel T, Kastner S, et al. NAD(P)H oxidase activity in cultured human podocytes: effects of adenosine triphosphate. Kidney Int 1998; 53:654–663.
35. Chabrashvili T, Tojo A, Onozato ML, et al. Expression and cellular localization of classic NADPH oxidase subunits in the spontaneously hypertensive rat kidney. Hypertension 2002; 39:269–274.
36. Thallas-Bonke V, Thorpe SR, Coughlan MT, et al. Inhibition of NADPH oxidase prevents advanced glycation end product-mediated damage in diabetic nephropathy through a protein kinase C-alpha-dependent pathway. Diabetes 2008; 57:460–469.
37. Block K, Eid A, Griendling KK, et al. Nox4 NAD(P)H oxidase mediates Src-dependent tyrosine phosphorylation of PDK-1 in response to angiotensin II: role in mesangial cell hypertrophy and fibronectin expression. J Biol Chem 2008; 283:24061–24076.
38. Bondi CD, Manickam N, Lee DY, et al. NAD(P)H oxidase mediates TGF-beta1-induced activation of kidney myofibroblasts. J Am Soc Nephrol 2010; 21:93–102.
39. Chai D, Wang B, Shen L, et al. RXR agonists inhibit high-glucose-induced oxidative stress by repressing PKC activity in human endothelial cells. Free Radic Biol Med 2008; 44:1334–1347.
40. Cucoranu I, Clempus R, Dikalova A, et al. NAD(P)H oxidase 4 mediates transforming growth factor-beta1-induced differentiation of cardiac fibroblasts into myofibroblasts. Circ Res 2005; 97:900–907.
41. Eid AA, Ford BM, Block K, et al. AMP-activated protein kinase (AMPK) negatively regulates Nox4-dependent activation of p53 and epithelial cell apoptosis in diabetes. J Biol Chem 2010; 285:37503–37512.
42. Eid AA, Gorin Y, Fagg BM, et al. Mechanisms of podocyte injury in diabetes: role of cytochrome P450 and NADPH oxidases. Diabetes 2009; 58:1201–1211.
43. Gorin Y, Block K, Hernandez J, et al. Nox4 NAD(P)H oxidase mediates hypertrophy and fibronectin expression in the diabetic kidney. J Biol Chem 2005; 280:39616–39626.
44. Lambeth JD, Kawahara T, Diebold B. Regulation of Nox and Duox enzymatic activity and expression. Free Radic Biol Med 2007; 43:319–331.
45. Lee HS, Song CY. Oxidized low-density lipoprotein and oxidative stress in the development of glomerulosclerosis. Am J Nephrol 2009; 29:62–70.
46. Miyata K, Rahman M, Shokoji T, et al. Aldosterone stimulates reactive oxygen species production through activation of NADPH oxidase in rat mesangial cells. J Am Soc Nephrol 2005; 16:2906–2912.
47. Gorin Y, Block K. Nox as a target for diabetic complications. Clin Sci 2013; 125:361–382.
48. Etoh T, Inoguchi T, Kakimoto M, et al. Increased expression of NAD(P)H oxidase subunits, NOX4 and p22phox, in the kidney of streptozotocin-induced diabetic rats and its reversibity by interventive insulin treatment. Diabetologia 2003; 46:1428–1437.
49. Fu Y, Zhang Y, Wang Z, et al. Regulation of NADPH oxidase activity is associated with miRNA-25-mediated NOX4 expression in experimental diabetic nephropathy. Am J Nephrol 2010; 32:581–589.
50. Fujii H, Kono K, Nakai K, et al. Oxidative and nitrosative stress and progression of diabetic nephropathy in type 2 diabetes. Am J Nephrol 2010; 31:342–352.
51. Fujii M, Inoguchi T, Maeda Y, et al. Pitavastatin ameliorates albuminuria and renal mesangial expansion by downregulating NOX4 in db/db mice. Kidney Int 2007; 72:473–480.
52. Sonta T, Inoguchi T, Matsumoto S, et al. In vivo imaging of oxidative stress in the kidney of diabetic mice and its normalization by angiotensin II type 1 receptor blocker. Biochem Biophys Res Commun 2005; 330:415–422.
53. Gorin Y, Ricono JM, Kim NH, et al. Nox4 mediates angiotensin II-induced activation of Akt/protein kinase B in mesangial cells. Am J Physiol Renal Physiol 2003; 285:F219–F229.
54. Gorin Y, Ricono JM, Wagner B, et al. Angiotensin II-induced ERK1/ERK2 activation and protein synthesis are redox-dependent in glomerular mesangial cells. Biochem J 2004; 381 (Pt 1):231–239.
55. Fujii M, Inoguchi T, Sasaki S, et al. Bilirubin and biliverdin protect rodents against diabetic nephropathy by downregulating NAD(P)H oxidase. Kidney Int 2010; 78:905–919.
56▪. Zhang J, Chandrashekar K, Lu Y, et al. Enhanced expression and activity of Nox2 and Nox4 in the macula densa in ANG II-induced hypertensive mice. Am J Physiol Renal Physiol 2014; 306:F344–F350.

The status of Nox isoforms in renal injury in this study in the setting of AngII-induced hypertension is further explored. Indeed, Nox-4 expression and activity is upregulated in the macula densa in this hypertension model.

57. Zhang H, Jiang Z, Chang J, et al. Role of NAD(P)H oxidase in transforming growth factor-beta1-induced monocyte chemoattractant protein-1 and interleukin-6 expression in rat renal tubular epithelial cells. Nephrology 2009; 14:302–310.
58. Barnes JL, Gorin Y. Myofibroblast differentiation during fibrosis: role of NAD(P)H oxidases. Kidney Int 2011; 79:944–956.
59▪. Yong R, Chen XM, Shen S, et al. Plumbagin ameliorates diabetic nephropathy via interruption of pathways that include NOX4 signalling. PLoS One 2013; 8:e73428.

This interesting study explored a novel agent, plumbagin, which appears to be renoprotective. The authors suggested that at least in part this drug acts via effects on Nox-4 signalling pathways.

60. Desai LP, Zhou Y, Estrada AV, et al. Negative regulation of NADPH oxidase 4 by hydrogen peroxide-inducible Clone 5 (Hic-5) protein. J Biol Chem 2014; 289:18270–18278.
61. Kwan J, Wang H, Munk S, et al. In high glucose protein kinase C-zeta activation is required for mesangial cell generation of reactive oxygen species. Kidney Int 2005; 68:2526–2541.
62. Eid AA, Ford BM, Bhandary B, et al. Mammalian target of rapamycin regulates Nox4-mediated podocyte depletion in diabetic renal injury. Diabetes 2013; 62:2935–2947.
63. Sharma K, Ramachandrarao S, Qiu G, et al. Adiponectin regulates albuminuria and podocyte function in mice. J Clin Invest 2008; 118:1645–1656.
64. Das R, Xu S, Quan X, et al. Upregulation of mitochondrial Nox4 mediates TGF-beta-induced apoptosis in cultured mouse podocytes. Am J Physiol Renal Physiol 2014; 306:F155–F167.
65▪▪. Jha JC, Gray SP, Barit D, et al. Genetic targeting or pharmacologic inhibition of NADPH oxidase nox4 provides renoprotection in long-term diabetic nephropathy. J Am Soc Nephrol 2014; 25:1237–1254.

Global deletion of Nox-4 resulted in protection against glomerular injury and GKT137831, a novel Nox1/4 inhibitor, was able to replicate these renal benefits.

66. Coughlan MT, Thorburn DR, Penfold SA, et al. RAGE-induced cytosolic ROS promote mitochondrial superoxide generation in diabetes. J Am Soc Nephrol 2009; 20:742–752.
67. Shah A, Xia L, Goldberg H, et al. Thioredoxin-interacting protein mediates high glucose-induced reactive oxygen species generation by mitochondria and the NADPH oxidase, Nox4, in mesangial cells. J Biol Chem 2013; 288:6835–6848.
68. Nishikawa T, Edelstein D, Brownlee M. The missing link: a single unifying mechanism for diabetic complications. Kidney Int Suppl 2000; 77:S26–S30.
69. Sedeek M, Callera G, Montezano A, et al. Critical role of Nox4-based NADPH oxidase in glucose-induced oxidative stress in the kidney: implications in type 2 diabetic nephropathy. Am J Physiol Renal Physiol 2010; 299:F1348–F1358.
70. New DD, Block K, Bhandhari B, et al. IGF-I increases the expression of fibronectin by Nox4-dependent Akt phosphorylation in renal tubular epithelial cells. Am J Physiol Cell Physiol 2012; 302:C122–C130.
71. Chen J, Chen JK, Harris RC. Angiotensin II induces epithelial-to-mesenchymal transition in renal epithelial cells through reactive oxygen species/Src/caveolin-mediated activation of an epidermal growth factor receptor–extracellular signal-regulated kinase signaling pathway. Mol Cell Biol 2012; 32:981–991.
72. Lee JH, Kim JH, Kim JS, et al. AMP-activated protein kinase inhibits TGF-beta-, angiotensin II-, aldosterone-, high glucose-, and albumin-induced epithelial–mesenchymal transition. Am J Physiol Renal Physiol 2013; 304:F686–F697.
73. Manickam N, Patel M, Griendling KK, et al. RhoA/Rho kinase mediates TGF-beta1-induced kidney myofibroblast activation through Poldip2/Nox4-derived reactive oxygen species. Am J Physiol Renal Physiol 2014; 307:F159–F171.
74. Watanabe H, Miyamoto Y, Honda D, et al. p-Cresyl sulfate causes renal tubular cell damage by inducing oxidative stress by activation of NADPH oxidase. Kidney Int 2013; 83:582–592.
75. Quoilin C, Mouithys-Mickalad A, Lecart S, et al. Evidence of oxidative stress and mitochondrial respiratory chain dysfunction in an in vitro model of sepsis-induced kidney injury. Biochim Biophys Acta 2014; 1837:1790–1800.
76. Babelova A, Avaniadi D, Jung O, et al. Role of Nox4 in murine models of kidney disease. Free Radic Biol Med 2012; 53:842–853.
77. Alpers CE, Hudkins KL. Mouse models of diabetic nephropathy. Curr Opin Nephrol Hypertens 2011; 20:278–284.
78. Brosius FC 3rd, Alpers CE, Bottinger EP, et al. Mouse models of diabetic nephropathy. J Am Soc Nephrol 2009; 20:2503–2512.
79. Coughlan MT, Thallas-Bonke V, Pete J, et al. Combination therapy with the advanced glycation end product cross-link breaker, alagebrium, and angiotensin converting enzyme inhibitors in diabetes: synergy or redundancy? Endocrinology 2007; 148:886–895.
80. Tan AL, Sourris KC, Harcourt BE, et al. Disparate effects on renal and oxidative parameters following RAGE deletion, AGE accumulation inhibition, or dietary AGE control in experimental diabetic nephropathy. Am J Physiol Renal Physiol 2009; 298:F763–F770.
81. Thallas-Bonke V, Coughlan MT, Tan AL, et al. Targeting the AGE–RAGE axis improves renal function in the context of a healthy diet low in advanced glycation end-product content. Nephrology 2013; 18:47–56.
82. Thallas-Bonke V, Coughlan MT, Bach LA, et al. Preservation of kidney function with combined inhibition of NADPH oxidase and angiotensin-converting enzyme in diabetic nephropathy. Am J Nephrol 2010; 32:73–82.
83. Aldieri E, Riganti C, Polimeni M, et al. Classical inhibitors of NOX NAD(P)H oxidases are not specific. Curr Drug Metab 2008; 9:686–696.
84. Jaquet V, Scapozza L, Clark RA, et al. Small-molecule NOX inhibitors: ROS-generating NADPH oxidases as therapeutic targets. Antioxid Redox Signal 2009; 11:2535–2552.
85. Selemidis S, Sobey CG, Wingler K, et al. NADPH oxidases in the vasculature: molecular features, roles in disease and pharmacological inhibition. Pharmacol Ther 2008; 120:254–291.
86▪▪. Hecker L, Logsdon NJ, Kurundkar D, et al. Reversal of persistent fibrosis in aging by targeting Nox4–Nrf2 redox imbalance. Sci Transl Med 2014; 6:231ra47.

Targeting of Nox4 in a mouse model of idiopathic pulmonary fibrosis resulted in the reversal of persistent fibrosis and produces further evidence for a Nox-4 inhibitor GKT137831, as an antifibrotic agent.

87▪. Goettsch C, Babelova A, Trummer O, et al. NADPH oxidase 4 limits bone mass by promoting osteoclastogenesis. J Clin Invest 2013; 123:4731–4738.

diabetic nephropathy; NADPH oxidase; protein kinase C; reactive oxygen species

Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.