For the kidney, reactive oxygen species (ROS) are a double-edged sword. Although ROS sustain physiological processes, disturbances of synthesis or degradation offset the delicate balance required for renal health. ROS are comprised of superoxide anion (O2–), hydroxyl anion (OH–), peroxynitrite (ONOO–), hydrogen peroxide (H2O2), hypochlorite anion (HOCl–), and hypochlorite (ClO–), which are rapidly metabolized by scavenging antioxidant enzymes that include superoxide dismutase (convert O2– to H2O2), glutathione peroxidase (breakdown H2O2 to H2O and O2), catalase (breakdown H2O2 to H2O and O2), and myeloperoxidase (convert H2O2 to HOCl–). Accordingly, ROS exist transiently but are nevertheless critical players that can alter the structure and function of lipids, proteins, and DNA. These ROS affect numerous downstream signaling pathways, including those involving the activity and expression of kinases, phosphatases, cytoskeletal proteins, transcription factors, cell-surface receptors, and ion/water channels, among others. Although there are several intrarenal sources of ROS, including xanthine oxidases, lipoxygenases, cyclooxygenases, P450 monooxygenase, and mitochondrial respiratory chain oxidation, NADPH oxidases (Nox) have evolved as key players in the renal pathophysiology as these produce the majority of renal superoxide . Noxs comprise seven members (Noxs 1–5, Duox1,2). All Noxs are transmembrane proteins with conserved structural properties that transport electrons across the membranes to reduce O2 to •O2–, whereas the Duoxs, which contain an N-terminal extracellular peroxidase region, generate H2O2. Indeed, Nox-derived oxidative stress has been implicated in the progression of several renal diseases. Whereas the predominant Nox in the kidney, NADPH oxidase 4 (Nox4; originally RENOX), along with NADPH oxidase 1 (Nox1) and NADPH oxidase 2 (Nox2), plays a role in diabetic nephropathy (DN) and hypertension-associated chronic kidney disease, the contribution of NADPH oxidase 5 (Nox5) is relatively unknown.
Nox5 is the newest Nox family member to be discovered [2,3]. Although Nox5 was only recently cloned, its activity may have been assayed as far back as 1943 by MacLeod  with his work on human spermatozoa. The human NOX5 gene spans 18 exons over a region of 126 kb on chromosome 15q23, encodes six splice variants (v1–6), shares a number of structural features common to all Noxs, including six transmembrane spanning domains, two groups of heme-spanning histidines in transmembrane domains 3 and 5, an NADPH-binding motif in the distal region of its C-terminus, and a flavin adenine dinucleotide-binding domain. However, Nox5 differs from other Nox family members as it does not require cytosolic subunits for activation and by several regulatory domains including four N-terminal Ca2+-binding EF-hands, two polybasic regions (binding of phosphotidylinositol 4,5 bisphosphate), a calmodulin-binding consensus sequence, and serine/threonine phosphorylation sites (Ser475, Ser502, Thr494, Ser498, and Ser659) . Importantly, insights into the role of Nox5 have been limited as it is not expressed in most rodents, including model species such as rat and mouse.
This review will focus on the recent findings that suggest a role for Nox5 in renal pathophysiology and will map out key avenues that could allow for future work to fill in the critical gaps in our knowledge.
NADPH OXIDASE 5 ORIGINS: NEW FACE OR OLD FRIEND?
Nox5 is the most recently identified member of the mammalian Nox family and is perhaps the least understood of these enzymes, owing partly to its absence from several model organisms including Caenorhabditis elegans, Mus musculus, and Rattus norvegicus. However, the origins of Nox5 may be as old as any of the currently recognized mammalian Noxs. Comparison of Nox and Duox orthologs across a wide array of species suggests that the catalytic domains of the primordial NoxD, found in algae, is most closely related to the catalytic domain of Nox5-like enzymes [5,6]. However, NoxD lacks EF-hand domains and therefore, unlike other Nox5-like family members, cannot bind Ca2+. Further examination also revealed the presence of a Nox5-like member in slime mold, NoxC, which has an EF-hand domain and is regulated by calcium. Thus, Nox5-like enzymes appear to have arisen early on in evolution (Fig. 1) [6–8]. Indeed, it has recently been suggested that the primordial EF-hand-containing Nox may be the original family member from which all other Noxs diverged and that the EF-hand domain was lost in an ancestral gene, rather than gained, giving rise to the current family members [9▪▪]. Interestingly, the majority of Nox5-like enzymes, such as those found in plants and fungi, as well as the Ca2+-binding Duox family, contain two EF-hand domains, whereas the mammalian Nox5 gene is unique in encoding four EF-hand domains, suggesting a recent evolutionary divergence. A dichotomy emerges only when one compares the genomes of higher organisms such as plants, fungi, and animals. A large number of Nox enzymes have been identified in the plant kingdom, all of which contain EF-hands and are therefore considered to be Nox5-like. Although this suggests that Nox5 is sufficient for viability, at least in plants, its absence from the genome of select animal species and its deletion from fungi yielding no apparent phenotype suggest it is not absolutely required for survival. Accordingly, there may be redundancy within the Nox family and that absence of a specific Nox family member may be compensated for by other family members. Indeed, in the renal proximal tubule cells (PTCs), Nox4 and Nox2 are highly expressed in mouse [10,11], whereas recent evidence suggests that in hypertensive humans, Nox5 predominates [12▪▪]. Thus, there may be compensation in Nox family member expression in the presence or absence of specific family members between species.
Currently, little is known regarding the tissue distribution pattern of Nox5 as its promoter and enhancer regions are not well characterized. The absence of NOX5 from rodent genomes has also contributed to our lack of expression data, preventing traditional β-gal knock-in tracing studies. Original expression studies using northern blots demonstrated Nox5 mRNA transcripts confined to a limited number of adult tissues (i.e. spleen, thymus, and testis) [2,3]. More thorough examination revealed the presence of six splice variants of the NOX5 gene in humans, with Nox5v1 (Nox5a) and Nox5v2 (Nox5b) being the predominant splice variants in most cell lines assayed, whereas Nox5v3 and Noxv4 are expressed sparingly . The truncated Nox5v5 (Nox5s), which encodes a protein-lacking EF-hands, is detected in esophageal cancer cell lines, but few data regarding its expression in other tissues are available [13,14]. Although the data are limited, Nox5 is expressed in the fetal tissue  and also appears to be upregulated in several disease contexts [12▪▪,15,16,17▪▪]. A number of genetic pathways activated during embryonic development are switched off in adulthood. However, these developmentally controlled systems are often reactivated in response to damage and act as part of repair mechanisms . Accordingly, Nox5 induction in adult tissues may be a regenerative response. In-silico analysis of the human Nox5 locus indicates the presence of two distinct promoter regions thought to be responsible for driving Nox5 splice variant expression . More recently, luciferase reporter assays and chromatin immunoprecipitation approaches revealed several potential transcription-factor-binding sites capable of regulating transcription within one of these proximal promoter regions [19,20▪]. The in-vivo role of this region has yet to be verified. Furthermore, there are no data regarding possible distal promoter or enhancer regions. The development of a humanized mouse model, engineered to express the full human Nox5 gene within the analogous (i.e., syntenic) region of the mouse genome, would provide significant insight into the expression patterns and the regulatory regions governing Nox5 expression.
RENAL NADPH OXIDASE 5 LOCALIZATION
Given its absence from the rodent genome, our ability to evaluate the role of Nox5 in renal development and disease is limited. Data from our laboratory show immunodetectable glomerular Nox5 expression in renal biopsies from patients with DN. In these tissues, Nox5 expression overlaps with nephrin, consistent with a podocyte-specific localization [17▪▪]. Furthermore, Nox5v2 expression is the predominant form in a human-podocyte cell line and is induced at both mRNA and protein levels by angiotensin II (AngII). Nox5 activity was confirmed through its ability to generate superoxide following AngII stimulation [17▪▪]. A recent study by Pandey et al. was aimed at assessing Nox5 splice variant expression and function in human vasculature. The authors detected mRNA and protein of Nox5v1 and Nox5v2 in cultured vascular smooth muscle cells (VSMCs) and endothelial cells. In addition, both isoforms were shown to induce ROS generation. Stimulation with the known Nox5-activators (AngII, ET-1, and TNF-α) increased Nox5 mRNA in VSMCs, whereas adenoviral-mediated Nox5 overexpression in endothelial cells activated various mitogen-associated protein kinase pathways via increased phosphorylation . These findings are consistent with the previous data proposing the presence of functionally active Nox5 variants [v1 and Nox5S(v5)] in human microvascular endothelial cells and raise the possibility that Nox5 might be expressed in the glomerular arterioles, in which ROS are known to modulate the vascular tone and thereby affect glomerular filtration rate and renal blood flow . The presence of Nox5 was also validated in an elegant manner downstream of the glomerulus. Nox5 expression levels and localization were compared in human PTCs from normotensive and hypertensive patients. This study found that mRNA and protein Nox5 expression levels, as well as its apical membrane localization, were significantly enhanced in hypertensive versus normotensive PTCs. Functionally, hypertensive-derived PTC Nox5 harbored a five-fold increase in total NADPH oxidase activity, which could be blocked using Nox5-specific small interfering RNA [12▪▪]. Taken together, it seems that Nox5-mediated generation of superoxide in renal disease could originate from some or all of the aforementioned cell types, leading to oxidative stress in various renal locales.
NADPH OXIDASE 5 IN DIABETIC NEPHROPATHY
The role of Nox-derived ROS, in particular Nox4, in driving DN has garnered much attention over the last decade [23–25]. Given that most models of DN rely significantly on the use of rodents, a role for Nox5 remained unexplored. Our studies demonstrate that Nox5 is expressed in immortalized human podocytes in vitro and is upregulated in the glomeruli of individuals with diabetes in vivo[17▪▪]. Co-staining with the podocyte-specific marker nephrin (Fig. 2) suggests that both podocyte and nonpodocyte cells, potentially mesangial cells, express Nox5 in this context [17▪▪]. Transgenic mice expressing Nox5 in a podocyte-specific manner (Nox5pod+) developed foot process effacement, albuminuria, and elevations in SBP [17▪▪]. When Nox5pod+ animals were rendered diabetic via streptozotocin injection, the severity of nephropathy was exacerbated, with the animals showing more severe albuminuria, greater glomerular basement membrane thickening, and more widespread foot process effacement than diabetic nontransgenic mice. Nox5pod+ animals also showed significant increases in SBP that were not observed in diabetic controls, a pathological change associated with diabetes that is absent from most rodent models [17▪▪]. Interestingly, a novel inducible transgenic mouse line with podocyte-specific expression of Nox5 appears to be more sensitive to AngII infusion with exacerbated increases in SBP compared with control animals treated with AngII further, confirming that Nox5 is regulated by the renin–angiotensin system in vivo and suggesting that Nox5 may play a role in the renal damage induced by hypertension (Kennedy, unpublished observation).
Genetic PKCα deletion or pharmacological inhibition ameliorates the albuminuria in the murine models of DN . Although rodents lack Nox5, an interaction with PKCα should not be ignored. The PKCα isoform was recently shown to activate Nox5 in vitro through phosphorylation of Ser490, Thr494, and Ser498 [27▪]. High-glucose stimulation of human endothelial cells promoted PKCα activity and increased Nox5-generated reactive oxygen species that could be abrogated by a PKCα inhibitor (Go 6976). Such PKCα-induced Nox5 activity could restrict endothelial-dependent NO bioavailability via enhanced superoxide production to alter renal blood flow during diabetes [27▪]. Moreover, PKCα expression is upregulated in the podocytes of humans with DN and is associated with endocytosis of the slit diaphragm protein, nephrin . Therefore, future work could be aimed at testing whether Nox5-derived ROS generation is associated with diabetes-associated PKCα activity and nephrin cell-surface expression. It should also be noted that based on the studies of the vasculature demonstrating Nox5 expression in the endothelial cells and VSMCs, one might expect Nox5 to be expressed in the glomerular capillary beds; however, additional work will be required to address this question.
NADPH OXIDASE 5 IN RENAL DISEASE
Given that Nox5 activity is governed partly by intracellular calcium, it is interesting to speculate that Nox5 may play an important role in the pathogenesis of focal segmental glomerular sclerosis (FSGS). Familial cases of FSGS have been linked to mutations in the TRPC6 Ca2+ channel . TRPC6 gene mutations which enhance its cell-surface expression and activity, or TRPC6 overexpression itself trigger podocyte cell stress, effacement, and apoptosis through increased intracellular calcium levels . It is tempting to ask whether such elevations in calcium drive damage-inducing ROS production via Nox5 in humans. It is also interesting to note that Nox-derived ROS production has been linked to increased cell-surface localization and activity of TRPC6 in podocytes . Thus, increased podocyte oxidative stress through activation of other Nox family members may induce TRPC6-mediated Ca2+ flux, leading to a feed-forward mechanisms resulting in Nox5 activation. Although much remains to be learned regarding the regulation of expression and activity of Nox5 in the various cell types within the glomerulus, these preliminary studies warrant further investigation into the role of Nox5 in glomerulopathies.
Nox-induced ROS following ischemia–reperfusion injury (IRI), along with the downregulation of antioxidant enzymes, appears to play a significant role in acute kidney injury (AKI). It would appear that persistent Nox-mediated ROS production and oxidative stress after AKI predispose the animal models to chronic kidney disease [32,33]. Interestingly, a subgroup of patients with acute renal failure have polymorphisms in the p22phox gene and show increased susceptibility to oxidative stress, leading to higher requirement for dialysis and increased rates of hospital deaths . Although Nox5 has yet to be implicated in this process, it is interesting to note that after AKI, the recovering kidney displays an increased systemic pressor response to AngII despite ameliorations in glomerular filtration rate and tubular remodeling [32,35,36]. Increased calcium flux through enhanced sensitivity to AngII may result in overt activation of Nox5 and increased tubular oxidative stress. Indeed, it has recently been demonstrated that Nox5 protein expression and ROS production are upregulated in the proximal tubules (Fig. 2) of individuals with essential hypertension, implicating Nox5 in the regulation of renal hemodynamics [12▪▪]. Unfortunately, pathological changes to tubular structure were not assessed in this study, though one would expect increased fibrosis in individuals with essential hypertension. Although the mechanism through which Nox5 is activated may differ between individuals with AKI versus essential hypertension, the end result would appear to be the same. Thus, targeted inactivation of Nox5 may prove to be protective in the context of tubular damage.
NADPH OXIDASE 5 POLYMORPHISMS
At a lengthy 126 kb, the human NOX5 gene shows a high frequency of single-nucleotide polymorphisms (SNPs). A recent review identified 100 SNPs encoding missense, nonsense, and synonymous mutations in the intronic and exonic sequences of the NOX5 gene . Using the National Center for Biotechnology Information sequence analysis software, some missense SNPs were found in regions encoding the EF-hands, as well as for transmembrane and C-terminal NADPH-binding domains. The localization of these mutations could in theory affect Nox5 activity. The latest evidence regarding the relationship of NOX5 SNPs on its activity was presented by Wang et al.[37▪▪], in which 15 mutants of the Nox5v1(β) splice variant were studied. Although the majority of Nox5v1(β) mutants were unaffected by these SNPs, seven of these did, however, lead to a decrease in the basal Nox5-mediated superoxide production in transfected COS-7 cells, independently of Nox5 protein levels or localization. However, following stimulation with either ionomycin to raise intracellular calcium levels or phorbol ester to phosphorylate Nox5, superoxide production was attenuated in three mutants (S236R, G542R, and V689A) compared with wildtype Nox5. No Nox5 gain-of-function mutations have been identified to date. Furthermore, a unique Nox5 mutant in which a lysine residue was introduced replacing a methionine at position 77 (M77K) was of particular interest in this study, in that it led to a diminished superoxide-generating capacity under basal and stimulated conditions. It was postulated that the M77K mutation may hinder phorbol myristate acetate-induced phosphorylation at Ser490, Thr494, and Ser498, which can lower the Ca+-dependent Nox5 activation threshold. This was, however, not the case as phospho-specific antibodies revealed similar phosphorylation levels in M77K mutant versus wildtype Nox5. In addition, as the M77K mutation is predicted to be found between the sequence encoding two calcium-binding EF hands, its impact on Ca2+ binding was evaluated. Indeed, ionomycin stimulation led to a 50% decrease in Ca2+-dependent Nox5 activation compared with the wildtype enzyme. The frequency at which these loss-of-function mutations occur in humans depends on ethnicity, as the R530H SNP is more commonly associated with Asian and African populations compared with European populations, whereas the W254Ter SNP is predominant in Africans but is essentially absent in other populations [37▪▪]. Also, data suggest that a validated nonsense SNP at position 300 of the Nox5v3 (gamma) isoform, yielding in theory a truncated enzyme, appears more frequently in the sub-Saharan population (∼4%) than in North Americans  (0.01%) and is absent in Europeans. Given the proposed role of Nox5-derived oxidative stress in driving renal diseases, one could postulate that polymorphisms that decrease Nox5 activity may be protective in these settings. On the other hand, if Nox5 is eventually shown to play a role in renal development, such activity reducing polymorphisms might prove detrimental. As it stands, we are unaware of any studies linking the various Nox5 polymorphisms to susceptibility or resistance to renal dysfunction at this time. Furthermore, thorough characterization of the Nox5 promoter and enhancer regions may identify further polymorphisms responsible for modified Nox5 activity and expression that may also be linked to disease progression.
The accumulated evidence points to a role for ROS as both protective and detrimental during renal disease progression. The major ROS generators in the kidney are the Noxs. Although most studies using rodent models have focused upon Noxs 1, 2, and 4, the more recently identified family member, Nox5 is beginning to emerge as a potential player. Whereas future work should comprehensively map the intrarenal distribution of Nox5 in health and disease, initial findings suggest that glomerular and tubular expression of this Nox isoform is induced during diabetes and hypertension, respectively. Furthermore, identification of SNPs in distinct human populations suggests a role for either good or ill for this understudied ROS-generating enzyme. Clearly, additional studies are warranted involving both human samples supported by novel gene-targeted animal models to properly define its role in renal disease. If proven to be a significant contributor to renal ROS production, Nox5 might emerge as an attractive target for pharmacological intervention.
Financial support and sponsorship
C.R.J.K. is supported by a grant from the Canadian Institutes of Health Research (CIHR). C.H. is a recipient of the Kidney Foundation of Canada (KFOC) Post-Doctoral Fellowship Award – Agostino Monteduro Endowment Fund. J.F.T. is an Ontario Graduate Scholarship (OGS) Doctoral Award recipient.
Conflicts of interest
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