**2. Mitochondrial Dysfunction in DKD**

Due to the critical role of mitochondria as the powerhouse of cells, mitochondrial dysfunction traditionally referred to an alteration in the production of adenosine triphos-

**Citation:** Ito, M.; Gurumani, M.Z.; Merscher, S.; Fornoni, A. Glucoseand Non-Glucose-Induced Mitochondrial Dysfunction in Diabetic Kidney Disease. *Biomolecules* **2022**, *12*, 351. https://doi.org/ 10.3390/biom12030351

Academic Editor: Liang-Jun Yan

Received: 25 January 2022 Accepted: 21 February 2022 Published: 23 February 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

59

phate (ATP) by oxidative phosphorylation (OXPHOS). However, as our understanding of the various roles played by mitochondria has expanded, mitochondrial dysfunction now includes any abnormal biological process in mitochondria [7]. In this section, we will discuss different categories of mitochondrial dysfunction occurring in DKD and discuss their potential causative role (Figure 1).

**Figure 1.** Mitochondrial dysfunction in DKD. Whether the level of mitochondrial ROS is increased or decreased is controversial and can vary depending on the stage of DKD. OXPHOS, mitophagy and biogenesis are generally decreased. Increased fission and decreased fusion causes fragmentation of mitochondria. OXPHOS: oxidative phosphorylation, ROS: reactive oxygen species, ↑: increased, ↓: decreased.

#### *2.1. Mitochondrial Oxidative Phosphorylation (OXPHOS)*

As the powerhouse of cells, the central role of mitochondria is the production of adenosine triphosphate (ATP). Metabolites from glucose, lipids and amino acids are transported into the mitochondrial matrix, serving as substrates of the tricarboxylic acid (TCA) cycle. NADH and FADH2 are generated along with the reaction feed electrons into Complexes I and II of the electron transport chain (ETC). As electrons are transported through the ETC, H<sup>+</sup> ions are pumped into the intermembrane space. Complex V or ATP synthase uses this proton gradient to generate ATP (Figure 2). The indices of OXPHOS activity and fitness include oxygen consumption rate (OCR), ATP production, membrane potential and the evaluation of each complex (activity, formation). Generally, it has been observed that OCR in the kidney cortex is increased in early DKD, followed by a decrease as DKD progresses, whereas in glomeruli and podocytes the OCR is decreased in both in early and late phases of the disease [5]. Although some discrepancy exists between studies, ATP production and complex activity have been demonstrated to be decreased at least in the late stage of DKD [8,9]. The contribution of decreased activation of OXPHOS to DKD can be inferred from the observation that some genetic mutations in OXPHOS, such as single-nucleotide polymorphisms (SNPs) in coenzyme Q5 (*COQ5*) and cytochrome *c* oxidase (*COX6A1*), are linked to DKD in humans [10]. *COQ5* encodes methyltransferase located in the mitochondrial matrix and *COX6A1* encodes a subunit of cytochrome c, which is part of the ETC.

**Figure 2.** Electron transport chain (ETC) in mitochondrial inner membrane. NADH and FADH2 from the TCA cycle donate electrons to Complexes I and II. As electrons are transported through the ETC, a proton gradient is generated, which Complex V or ATP synthase couples to ATP synthesis. Electron leakage from the ETC causes the production of ROS. ADP, adenosine diphosphate; ATP, adenosine triphosphate; Cyt C, cytochrome complex; ROS, reactive oxygen species; UQ, ubiquinone; TCA cycle, tricarboxylic acid cycle.

#### *2.2. Mitochondrial Reactive Oxygen Species (mtROS)*

Ever since Brownlee and colleagues proposed that hyperglycemia-induced mitochondrial reactive oxygen species (mtROS) was the unifying mechanism of diabetic microvascular complications in 2000, this paradigm has been prevalent [11,12]. Recently, the source of ROS in DKD and the pathogenic role of ROS have become controversial [13,14]. Although there may be a consensus that ROS-induced damage is increased in DKD, conflicting studies exist regarding the change in mtROS production, which can be attributed to the different methods used to detect mtROS or the varying models or timepoints of DKD. In both live and fixed db/db mouse kidneys, increased mitochondrial ROS was observed using a mitochondrial matrix-localized reduction–oxidation-sensitive green fluorescent protein probe [15]. By contrast, in streptozotocin (STZ)-injected C57BL/6J mice and Ins2- Akita mice (DBA/B6 F1 mice), decreased mitochondrial superoxide was observed upon systemic administration of dihydroethidium (DHE) both in live and fixed kidneys [16]. The latter study does not preclude ROS production in other cell compartments, including the endoplasmic reticulum (ER) or enzyme systems such as nicotinamide adenine dinucleotide phosphate oxidase (Nox). Notably, the restoration of mitochondrial biogenesis and OX-PHOS activity by adenosine monophosphate-activated protein kinase (AMPK) activation increased mtROS and ameliorated the DKD phenotype, arguing against the role of mtROS in inciting DKD.

ROS does not play an exclusively detrimental role in cell biology. Mitochondrial hormesis is the concept that slightly enhanced mitochondrial superoxide at baseline can decrease susceptibility to more severe cell stress [13]. ROS also plays an essential role in certain cell signaling pathways, requiring further elucidation of the intricate characteristics of ROS.

#### *2.3. Biogenesis*

Cells cope with increasing energy demand by increasing mitochondrial biogenesis, in which functional mitochondria are generated by duplication of mitochondrial DNA (mtDNA) and subsequent binary fission. Peroxisome proliferator-activated receptor γ coactivator 1α (PGC1α) plays a central role in mitochondrial biogenesis [17]. PGC1α is a transcriptional regulator of mitochondrial metabolic pathways such as oxidative phosphorylation (OXPHOS), the TCA cycle and fatty acid metabolism. PGC1 peroxisome proliferator-activated receptors (PPARs) and estrogen-related receptors (ERRs) can also

serve as a coactivator of PGC1α. PGC1α dimerizes with transcriptional coactivators to regulate downstream gene transcription, and these partners include cyclic AMP-responsive element-binding protein (CREB) nuclear respiratory factors 1 and 2 (NRF1 and NRF2) and activated PPARs and ERRs [4]. Nutrient-sensing pathways like the mechanistic target of rapamycin (mTOR), AMPK, sirtuin, cyclic AMP (cAMP) and cyclic guanosine monophosphate (cGMP) regulate PGC1α directly or indirectly.

In DKD, although there is some discrepancy between studies possibly due to the analysis at different disease stages, PGC1α activity is considered to be increased in the early stage of diabetes, as demonstrated in 8-week-old db/db mice, followed by a decrease in activity at later stages, as demonstrated in pretransplant patients and mice 24 weeks after diabetes induction with STZ injection [8,16,18,19]. Taurine upregulated gene 1 (*Tug1*), a long noncoding gene, was described as a regulator of PGC1α in podocytes in DKD [20]. It was demonstrated that *Tug1* binds to an element upstream of *Ppargc1a* and interacts with PGC1α binding to its own promoter, subsequently enhancing *Ppargc1a* promoter activity.

#### *2.4. Mitochondrial Fission and Fusion*

Mitochondria are dynamic organelles which undergo tightly controlled processes of fission and fusion. Mitochondrial fission is mediated by dynamin-1-like protein (DRP1) and its receptors such as fission factor 1 (FIS1), mitochondrial fission factor (MFF) and mitochondrial dynamics proteins of 49 and 51 kDa (MID49 and MID51). Mitochondrial fusion is mediated by the long isoforms of optic atrophy protein 1 (OPA1), which plays a role in inner mitochondrial membrane fusion, and the mitofusins (MFN1 and MFN2) which play a role in outer mitochondrial membrane fusion [5,6].

Although the increase in mitochondrial fission and fusion factors such as the long isoforms of OPA1, MFN1, MFN2 and MFF were observed in early DKD, mitochondria were consistently fragmented throughout early and late stage in STZ-injected rats [8]. Human kidney biopsies of patients with DKD also demonstrated fragmented mitochondria in podocytes and proximal tubular cells [21,22]. Consistent with increased fission and decreased fusion, Drp1 and FIS1 expression was increased, while MFN2 expression was shown to be decreased in tubules in the latter study.

#### *2.5. Mitophagy*

Autophagy is a pathway that degrades and recycles damaged organelles and macromolecules, and selective autophagy of mitochondria is termed as mitophagy. Mitophagy has a critical role in the maintenance of mitochondrial quality by removing damaged mitochondria. Mitophagy can be mediated by the phosphatase and tensin homolog-induced putative kinase 1 (PINK1)/parkin-mediated pathway and other outer mitochondrial membrane proteins such as BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3) and NIP3-like protein X (NIX), or the mitophagy receptor FUN14 domain-containing protein 1 (FUNDC1).

The PINK1 and parkin-mediated pathway has been more extensively investigated than the others [23]. PINK1 has a Ser/Thr kinase domain and is found inserted into both the inner and outer mitochondrial membranes. In healthy mitochondria, PINK1 is cleaved at two points by mitochondrial proteases, leading to its dissociation from the mitochondrial membrane and degradation by the ubiquitin-proteasome system. In depolarized mitochondria, PINK1 escapes cleavage and stably resides in the outer membrane. Subsequently, PINK1 homodimerizes and autophosphorylates to recruit E3 ubiquitin ligase parkin and ubiquitin, directing the mitochondria to the mitophagy pathway. In the ubiquitin-independent pathway, outer mitochondrial membrane proteins such as BNIP3, NIX or FUNDC1 recruit microtubule-associated protein 1A/1B light chain 3 (LC3) and induce mitophagy under certain stimuli including hypoxia [24,25]. Cardiolipin, which is located in the inner mitochondrial membrane under normal conditions, is externalized by certain stimuli and detected by LC3, facilitating the engulfment of the mitochondria by autophagosomes [26]. P62 is a marker of autophagy cargo, and its accumulation can

indicate stagnation in degradation via autophagic flux. In general, basal mitophagy levels of podocytes are high, which can be attributed to their terminally differentiated characteristics. In contrast, in tubular cells the mitophagy level is low at baseline but it can be induced as a consequence of stress.

Mitophagy is suppressed in DKD, which was demonstrated by low PINK1/parkinexpression levels in podocytes of STZ-induced diabetic mice and increased p62 expression levels in tubular cells of biopsy obtained from patients with DKD [27–29]. Thioredoxininteracting protein (TXNIP) was implicated in the suppression of tubular autophagy and mitophagy induced by high glucose [27]. High glucose was also shown to inhibit the transcriptional activity of forkhead-box class O1 (FoxO1) via its phosphorylation by Akt (protein kinase B), leading to the downregulation of PINK1 [29]. The protective effect of mitoquinone on DKD was partially attributed to the restoration of PINK1 and parkin protein expression in tubular cells via NRF2 activation [30].
