*5.2. NADPH Oxidase*

NADPH oxidase (NOX) can generate superoxide anion using NADPH as its reducing agent [99]. So far, seven NOXs have been identified (NOX 1-5, Duox1 and DuoX2) [99]. These isoforms differ in many aspects including catalytic oxidase subunit, tissue distribution, intra-cellular location, and mechanisms of regulation [100,101]. All NOXs are composed of multiple subunits. Upon stimulation, these subunits will come together and assemble to form a membrane-associated complex to generate superoxide at the expense of NADPH [102]. Figure 4 shows a representative diagram of NOX assembly upon stimulation whereby the major site of ROS production is the gp91phox subunit with other proteins being the ancillary units required for the regulation and functioning of the whole enzyme complex. It should be noted that Figure 4 only shows the assembly of NOX2. The structural and compositional variations of other NOX isoforms [103] and their potential interaction with cadmium may also play a role in cadmium induced renal toxicity. Under normal conditions, these NOXs function in a beneficial way by regulating kidney metabolism and homeostasis including glucose transport, gluconeogenesis, renal hemodynamics, and electrolyte transport and balance [99]. Under pathophysiological conditions, these NOXs, in particular NOX2 and NOX4 in the kidney, can overgenerate ROS that are damaging to cellular components including DNA, proteins, and lipids, causing cell death and kidney injury [99,104–106]. It has been reported that cadmium exposure can increase the expression of NOX1 subunits, leading to increased ROS production from the enzyme [107]. Nevertheless, it is not known exactly which subunit in the NADPH oxidase physically interacts with cadmium at the present time. It should be noted that xanthine oxidase [108,109] and nitric oxide synthase [110–112], although not a major source of ROS in the kidney, may also contribute to renal oxidative stress under a variety of pathological and experimental conditions including cadmium exposure. It should also be pointed out that comprehensive

evaluations of the roles of NADPH oxidases, xanthine oxidase, and nitric oxide synthase in cadmium-induced kidney injury remain to be conducted.

**Figure 4.** NADPH oxidase assembly and superoxide production at the expense of NADPH. Upon stimulation, each individual subunit of the enzyme is recruited to the membrane and form a membrane-associated complex. Only one subunit GP91 catalyzes partial reduction of oxygen. This figure is adapted from reference [102]. Please not that shown here is NOX2 assembly. For structures and components of other NXO isoforms, please refer reference [103].

#### **6. Effects of Cadmium on Mitochondrial Function**

Cadmium can enter mitochondria and accumulate therein [113,114]. This is likely facilitated by mitochondrial membrane channels, and solute molecule carriers and receptors [114]. Once inside the mitochondria, cadmium can bind thiol-containing proteins and impair the corresponding protein function [80]. Studies have demonstrated that upon exposure to cadmium, kidney mitochondria displayed deformation, swelling, and vaculation, concurrent with increased SOD1 expression and decreased SOD2 and catalase expression [115]. Additionally, the anti-apoptotic protein BCL-2 was also found decreased by cadmium exposure [115]; so was the ratio between reduced glutathione and oxidized glutathione [60]. All these could be a generalized cadmium mitochondrial toxicity and the ultimate outcome would be reflected by overproduction of mitochondrial ROS, disruption of mitochondrial metabolic pathways, and impairment of mitochondrial pores, membrane channels and transporters [80]. It has been reported that complex II and complex III may be the major sites impaired by cadmium in the nephrons [98] while the effects of cadmium on proximal tubular mitochondrial complex I (NADH-ubiquinone oxidoreductase) remain unclear. Disruption of all these processes by cadmium would increase mitochondrial ROS production and eventually lead to cell death and kidney injury [80,114,116–119]. An outline of cadmium induced ROS production, oxidative damage to macromolecules, cell death, and kidney injury is shown in Figure 5, highlighting the concept that oxidative damage is a unifying mechanism of cadmium-induced kidney injury.

**Figure 5.** ROS can damage DNA, proteins, and lipids. Damage of the molecules impairs the biological function of each molecule, leading to cell death and kidney injury. Cell death may include both necrosis and apoptosis.
