*3.4. The NAD Kinase Pathway*

NAD kinase (NADK) exists both in the cytosol and in the mitochondria [99,100]. This protein is the sole enzyme responsible for conversion of NAD<sup>+</sup> to NADP<sup>+</sup> [101,102] (Figure 3D). Given the key role of NADP+ in maintaining the levels of cellular antioxidant glutathione [103–105], NADK is an indispensable element in the redox metabolic pathways. Although many studies have been conducted on NADK in a variety of experimental systems, the role of this protein in DKD has yet to be explored. Furthermore, as NADK consumes NAD+, how it is involved in maintaining or perturbing NADH/NAD+ redox balance in DKD will also need to be investigated. The major pathways causing increase in NADH and decrease in NAD<sup>+</sup> as well as NAD<sup>+</sup> regeneration by mitochondrial complex I and lactate dehydrogenase (under hypoxia) are summarized in Figure 4.

**Figure 4.** Diagram summarizing the pathways that cause NADH increase and NAD+ decrease in the diabetic kidneys. Regeneration of NAD+ from NADH by either mitochondrial complex I or lactate dehydrogenase (under hypoxic conditions) is also shown.

#### **4. Redox Imbalance-linked Mitochondrial Dysfunction in DKD**

One of the major consequences of NADH/NAD+ redox imbalance is mitochondrial oxidative stress due to oversupply of NADH to the mitochondrial electron transport

chain [47,106,107]. This is caused by electron leakage from the electron transport chain [6,108–112], as it cannot use all of the NADH for ATP production [90,113]. As such, oxygen is partially reduced to form superoxide anion via the electron transport chain, mainly through complexes I, III and IV [114,115]. This mitochondrial superoxide, regardless of the exact site of its generation, is the original source of oxidative stress that can cause oxidative damage to DNA, proteins and lipids [116–119]. Accumulation of these oxidatively damaged macromolecule adducts can eventually lead to cell death and kidney failure [120,121].

While NADH/NAD+ redox imbalance drives the initial event of superoxide production in mitochondria [47,48,122], other abnormalities of mitochondria could also manifest in DKD, culminating in decreased oxygen consumption and ATP production. As mitochondrion is a dynamic organelle, disruption of its fission and fusion processes [123], also knowns as mitochondrial homeostasis [123–125], can also worsen diabetic kidney injury [126]. Indeed, dynamin-related protein 1 (Drp1), well known for its role in regulating mitochondrial fission, has been shown to be upregulated to cause mitochondrial fragmentation in DKD [127–129]. Conversely, mitochondrial fusion regulating proteins such as optic atrophy-1 (opa1) and mitochondrial fusion proteins, in particular, mitochondrial fusion protein 2 (Mfn2), have been shown to be down regulated to impair mitochondrial fusion in DKD [130–132]. This disruption of mitochondrial homeostasis is linked with redox imbalance and oxidative stress accompanied with impairment of mitochondrial membrane potential and release of apoptosis stimulating factors such as cytochrome c and apoptosis inducing factor (AIF) [133–136]. These deranged mitochondrial dynamics, if left unattended or uncorrected, would eventually lead to accumulation of damaged mitochondria, which could overwhelm the mitophagy capacity that is regulated by key proteins such as PINK1 and Parkin [137–140], resulting in cell death and worsened diabetic kidney injury. Therefore, mitochondrial homeostasis and dynamics can also serve as targets for renal therapy in DKD. Figure 5 outlines the potential deleterious mitochondrial consequences of NADH/NAD+ redox imbalance implicated in the pathogenesis of DKD.

**Figure 5.** Mitochondrial dysfunction driven by NADH/NAD<sup>+</sup> redox imbalance and the potential mitochondrial mechanisms underlying pathophysiology of DKD. These mechanisms include increased mitochondrial oxidative damage, decreased ATP production, perturbed mitochondrial membrane potential and deranged mitochondrial homeostasis and impaired sirt3 pathway as well as Nrf2 signaling pathway. The ultimate manifestation of these mitochondrial dysfunctional mechanisms is renal inflammation, fibrosis and diabetic kidney injury.

It is also worth mentioning that a hallmark of the diabetic kidney is hyperfiltration, so that the energy demands of the proximal tubule are greatly increased [141–143]. This may temporarily ameliorate the NADH/NAD<sup>+</sup> redox imbalance as NADH utilization is increased for ATP production. However, as increased NADH consumption means more oxygen consumption and more electron leakage from mitochondria for superoxide production [114,144,145], tubular cells could exhibit increased oxidative stress, which could eventually lead to hyperfiltration linked diabetic nephropathy [53,146]. Nonetheless, whether there is an increased mitochondrial superoxide production linked to hyperfiltration and increased ATP demands remains to be determined.

#### **5. Therapeutic Approaches to Counteracting DKD**

## *5.1. Superoxide Dismutation and Suppression*

There are at least 11 mitochondrial sites that are involved in superoxide generation [147]. Therefore, overall approaches of dismutating superoxide could alleviate DKD [148]. Recently, small molecules that can suppress or inhibit mitochondrial superoxide production have been developed. Typical examples of these small molecules are S1QELs and S3QELs [145,149], which do not interfere with the process of oxidative phosphorylation or ATP production [144]. S1QELs acts at the site IQ of complex I [145,147] while S3QELs acts at the Q site of complex III [150,151]. The usefulness of these suppressors in combating oxidative stress has been tested in certain experimental systems [151–154]. However, studies of these suppressors in alleviation of DKD have yet to be conducted. It is anticipated that these compounds could attenuate the severity of DKD in diabetic subjects.
