**4. Non-Glucose-Induced Mitochondrial Dysfunction in DKD**

Although hyperglycemia is a key factor in the development of DKD, other factors involved in DKD can also contribute to mitochondrial dysfunction. Dyslipidemia and lipid overload is a common complication in DKD, and hypoxia in the tubulointerstium occurs regardless of the cause of chronic kidney disease (CKD) [48]. Endothelin-1 (Edn1) was first identified as a downstream factor of transforming growth factor-β (TGF-β) in a model of focal segmental glomerulosclerosis (FSGS) and shown to induce albuminuria via mtROS in glomerular endothelial cells. Later, the same signaling pathway was also found to be upregulated in DKD [49,50]. In this section, we discuss the roles of these factors in the development and progression of DKD in relation to mitochondrial dysfunction.

#### *4.1. Lipotoxicity*

In kidney biopsies of patients with DKD, extensive lipid droplet accumulation was observed by electron microscopy in glomerular endothelial cells, podocytes and tubular cells compared to healthy counterparts [51,52]. Genetic analysis of these samples revealed downregulation of fatty acid oxidation (FAO)-related genes including peroxisome proliferator-activated receptor (PPAR)-α, carnitine palmitoyltransferase 1 (CPT1), acyl-CoA oxidase, and L-FABP; upregulation of cholesterol receptors including low-density lipoprotein (LDL) receptors, oxidized LDL receptors, and acetylated LDL receptors; and downregulation of cholesterol-efflux-related genes including ATP-binding cassette transporter A1 (ABCA1), ATP-binding cassette transporter G1 (ABCG1), and apolipoprotein (APOE) [51]. While downregulation of FAO-related genes suggests a decrease in mitochondrial lipid metabolism as the cause of lipid accumulation, lipid accumulation itself can induce mitochondrial dysfunction. We previously showed that human podocytes treated with the serum of patients with DKD show increased tumor necrosis factor (TNF) expression and that local rather than systemic TNF causes free cholesterol accumulation and injury via the suppression of ABCA1 in podocytes [53,54]. Notably, ABCA1 suppression induced cardiolipin accumulation and peroxidation in mitochondria, sensitizing

podocytes to injury [55]. ABCA1 overexpression or inhibition of cardiolipin peroxidation by elamipretide rescued podocyte injury in experimental DKD.

Tubular cells require large amounts of ATP for solute reabsorption and depend on FAO because fatty acids yield more ATP per gram than other energy sources [56]. In the early stage of diabetes, FAO is increased in accordance with increased FA flux, and ROS production is attributed to FAO, especially to electron leakage at the electron transfer flavoprotein that shuttles electrons from acyl-CoA dehydrogenases to coenzyme Q [57]. Nevertheless, FAO is eventually decreased in established diabetes as described earlier [51,58].

Tubular cells in patients with DKD are likely exposed to fatty acid-bound albumin, since dyslipidemia and proteinuria often accompany diabetes mellitus. Whereas albumin itself can cause tubular cell damage on its reabsorption, FA-bound albumin was shown to induce more severe tubular damage [59,60]. Cytotoxicity caused by FA or glycated albumin was shown to be mediated by the uptake via the protein cluster of differentiation 36/FA translocase (CD36/FAT) in the brush border in humans, in contrast to usual albumin reabsorption via a complex of megalin, cubilin and amnionless [61]. In addition to reabsorption, synthesis of FA is also upregulated [62,63]. FAs are esterified by long-chain acyl-CoA (LC-CoA) synthetase (ACSL), which is upregulated in both mouse db/db model and human DKD kidney samples [64,65]. LC-CoA is transferred to mitochondria via CPT1 and CPT2 to produce ATP via FAO and unmetabolized LC-CoA is cleaved or stored in lipid droplets to prevent lipotoxicity. When buffering ability is saturated, LC-CoA serves as an inhibitor of the Na+/H+ exchanger 1 (NHE1) and phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] binding to cause apoptosis in proximal tubular cells [66].

Genetic and pharmacological enhancement of FAO could represent a new therapeutic treatment strategy for patients with DKD. Transgenic expression of PCG1α and fenofibrate treatment ameliorated tubular cell apoptosis via restoration of CPTs and/or acyl-CoA oxidases [58,67].

#### *4.2. Hypoxia*

Tubulointerstitial hypoxia is known to be a final common pathway of CKD progression [48]. Regardless of the cause of the disease, hypoxia can occur due to decreased oxygen supply via blood flow because of impaired vasodilation, loss of vasculature, fibrosis, anemia, increased oxygen demand due to increased solute reabsorption, and inefficient ATP production because of mitochondrial uncoupling, which are the shared mechanisms in CKD.

Recently, the mechanism by which acute hypoxia causes mtROS production in the ETC has been revealed [68]. Acute hypoxia prompts a conformational shift in Complex I, leading to the activation of the Na+/Ca2+ exchanger in the inner mitochondrial membrane. Na<sup>+</sup> imported into the matrix then interacts with phospholipids to reduce membrane fluidity, resulting in the inability of free ubiquinone to move between Complex II and Complex III. Thus, Complex III produces ROS.

Hypoxia had long been deemed a direct suppressor of OXPHOS because oxygen is indispensable as a receiver of electrons in the ETC. However, to decelerate OXPHOS acutely and directly, the oxygen concentration has to be as low as 0.3% [69]. In milder and prolonged hypoxia, HIF1α acts as a mediator to suppress OXPHOS and to increase glycolysis in order to prevent ROS production. HIF1α modifies ETC complexes and promotes metabolic shifts from aerobic OXPHOS to anaerobic glycolysis [69]. Nevertheless, in chronic hypoxia, these changes limit the mitochondrial ability to produce ATP and could induce ATP deficiency, possibly leading to cytotoxicity.

### *4.3. Endothelin-1 (Edn1)/Edn1 Receptor Type A (Endra) Signaling*

Endothelin-1 (Edn1) was first characterized as a signaling molecule released by podocytes in TGF-β-induced FSGS [49]. Surprisingly, Edn1 causes mtROS, decreases reserve respiratory capacity and mtDNA damage in endothelial cells via Edn1 receptor type A (Ednra) activation, but this is not seen in podocytes. This pathway induces

glycosaminoglycan degradation in the endothelial surface layer, leading to the loss of fenestration [70]. Interestingly, EDNRA activation in endothelial cells is also required for podocyte foot process effacement and apoptosis [49].

The similar role of mtROS and EDNRA in endothelial cells and Edn1 is also described in DKD [50]. Circulating Edn1 was increased in diabetic humans and mice [50,71]. Notably, Ednra was not detectable in the glomeruli of healthy human kidneys or DKD-resistant C57BL/6J mice, but was present in those of human DKD kidneys and diabetic DBA/2J mice. Ednra could be induced in high-glucose-treated podocytes, but these podocytes did not express Edn1, implicating other cell types or stimuli in this signaling cascade [50]. Thus, Edn1/Ednra signaling has a critical role in the reciprocal crosstalk between podocytes and endothelial cells via mitochondrial dysfunction both in FSGS and DKD.

Another vasoactive pathway, the renin–angiotensin–aldosterone system (RAAS), is also known to participate in CKD progression, including in DKD. Aside from the detrimental effects of increasing systemic and intraglomerular blood pressure, angiotensin II treatment was found to exacerbate mtROS production and mitochondrial fragmentation in podocytes both in vivo and in vitro, which can be reversed by mitoquinone, a mitochondria-targeted antioxidant [72].

#### **5. Conclusions**

Mitochondrial dysfunction plays a central role in the development and progression of DKD. Thus, targeting mitochondrial dysfunction in DKD could represent a novel therapeutic strategy for patients with DKD. However, as can be learned from studies investigating the beneficial effect of systemic antioxidant administration as a treatment for DKD, it seems that intervention in mitochondrial dysfunction has to be cell type- and context-specific. Another intriguing perspective is age- and sex-related difference in mitochondrial dysfunction in DKD. Although age is an important factor that affects mitochondrial function and the development and progression of DKD, not much is known about the exact mechanisms. In terms of sex, much difference is observed between males and females in the mitochondria of certain tissues, and estrogen and possibly testosterone can mediate renal mitochondrial bioenergetics [73]. Further investigations are needed to elucidate the exact mechanisms leading to mitochondrial dysfunction in DKD, to the extent that findings can be clinically applied to patients and change their prognoses.

**Author Contributions:** Conceptualization, M.I., M.Z.G. and A.F.; writing—original draft preparation, M.I.; writing—review and editing, M.I., S.M. and A.F. All authors have read and agreed to the published version of the manuscript.

**Funding:** M.I. is supported by Manpei Suzuki Diabetes Foundation. A.F. and S.M. are supported by National Institutes of Health grants R01DK117599, R01DK104753 and R01CA227493. A.F. is also supported by U54DK083912, UM1DK100846, U01DK116101 and UL1TR000460 (Miami Clinical Translational Science Institute).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** A.F. and S.M. are inventors of pending and issued patents (US10183038; US10052345; PCT/US2019/032215; PCT/US2019/041730; PCT/US2013/036484; US17/057247; US17/ 259883; Japan no. 501309/2021; Europe no. 19834217.2; China no. 201980060078.3; Canada no. 2852904; 2930119; 3012773) aimed at preventing and treating renal disease. They stand to gain royalties from the future commercialization of these patents. S.M. and A.F. hold equity interest in L&F Research and ZyVersa Therapeutics, Inc. which has licensed worldwide rights to develop and commercialize hydroxypropyl-beta-cyclodextrin from L&F Research for the treatment of kidney disease. A.F. also holds equities in Renal 3 River Corporation. A.F. and S.M. are supported by Aurinia Pharmaceuticals and Boehringer Ingelheim.
