*5.1. FA β-Oxidation Dysfunction in Kidney Diseases*

The impairment of β-oxidation has been reported in AKI and CKD. In patients and animal models, mRNAs along with β-oxidation proteins levels are decreased [105–107]. In the folic-acid-induced AKI model, ATP production is reduced, and mitochondria are uncoupling due to β-oxidation dysfunction [108]. Moreover, in maleic-acid (MA)-induced AKI, the FA β-oxidation-linked oxygen consumption rate (OCR) is diminished [109]. On the other hand, the transcriptomic analysis showed that the acyl-CoA dehydrogenase family member 10 (ACAD10) is downregulated in DN [110]. Moreover, in the 5/6 nephrectomy model, medium-chain acetyl dehydrogenase (MCAD) decreases at twenty-eight days [107]. Thus, the decrease in these enzymes is related to FA β-oxidation deregulation in AKI and CKD. Following the latter, FA β-oxidation has been evaluated in a course temporal in 5/6 nephrectomy, which decreases from early times [111]. Consequently, the reduction of FA β-oxidation causes intrarenal lipids accumulation, inducing lipotoxicity and impairing renal function [112]. In line with this, Nishi et al. [113] demonstrated that lipid accumulation is evident in tubular epithelial cells (TECs). Lipid deposition increases according to kidney lesion, suggesting a metabolic reprogramming that shifts β-oxidation to lipid synthesis [114,115]. According to the latter, UUO increases triglycerides synthesis from one day after obstruction [116]. Triglycerides increase is partly due to the overexpression of the transporter of the long-chain FA cluster of differentiation 36 (CD36) in PT [7,117]. CD36 also promotes signaling pathway activation such as epithelial–mesenchymal transition (EMT), inflammation, and others, leading to fibrosis. In this context, CD36−/<sup>−</sup> mice subjected to UUO show less fibrosis than sham groups [118]. However, Kang et al. [106] showed that

mice that overexpress CD36 show the accumulation of lipids but low expression levels of fibrotic markers. Therefore, the authors hypothesized that impaired FA β-oxidation is sufficient to induce the development of fibrosis and lipid accumulation is the only consequence of this dysfunction [106]. Thus, it has been suggested that defective FA β-oxidation is one of the principal mechanisms associated with fibrosis development [103,106].

**Figure 3.** Renal proximal tubule (RPTC) cells use fatty acids (FA) β-oxidation to produce adenosine triphosphate (ATP). In the kidney, the proximal tubules of the nephrons of the renal cortex use fatty acids (FA) as the primary source of energy. FA bound to the fatty acid-binding protein (FABP) enter the RPTC through the cluster of differentiation 36 (CD36). In the cytosol, acyl-coenzyme A (CoA) synthetase (ACS) (attached to the outer mitochondrial membrane (OMM)) activates FA by the addition of acetyl-CoA (CoA-SH). The latter allows FA to enter the OMM through carnitine *O*-palmitoyl transferase I (CPT I). CPTI exchanges acetyl-CoA for L-carnitine. In turn, FA goes to the inner mitochondrial membrane (IMM). In the IMM, carnitine *O*-palmitoyl transferase II (CPT II) removes the carnitine group and adds acetyl-CoA (CoA-SH). The latter allows FA to enter the mitochondrial matrix. Fatty acyl-CoA undergoes β-oxidation, generating nicotinamide adenine dinucleotide phosphate (NADH) and flavin adenine dinucleotide (FADH2). Created with Biorender.com.

The alterations in CPT I levels also contribute to FA β-oxidation impairment. For example, modifications in transporters of plasma acylcarnitine have been reported in CKD [119]. In this context, Prieto-Carrasco et al. [120] showed CPT I levels decreased in a temporal course from 2 to 28 days after nephrectomy, associated with progressive impairment in mitochondrial β-oxidation. The authors concluded that the decrease in CPT I favors mitochondrial β-oxidation impairment and the subsequence fibrosis development. The latter is supported by the fact that patients with CKD show a correlation between low CPT IA levels and fibrosis [121]. In this sense, the transgenic mouse models overexpressing CPT IA are able to restore oxidative metabolism, avoiding fibrosis development in UUO, folic acid nephropathy, and adenine-induced nephrotoxicity [121]. In addition, CPT IA overexpression also reduces fibrosis by decreasing TGFβ1 levels [121].

In summary, defective FA β-oxidation is observed in kidney diseases from early times, promoted through decreased mRNA expression and downregulation in the activity and levels of the proteins involved in this process and ETS activity reduction (discussed below). Later, the overexpression of CD36 contributes to lipid accumulation and the activation of mechanisms that lead to the fibrotic process. However, other factors contribute to the impairment of β-oxidation.

#### Oxidation and OS Production and in Kidney Diseases

Renal pathologies cause a disturbance in mitochondria homeostasis, affecting mitochondrial metabolism, which leads to AKI to CKD transition. OS might cause these alterations in mitochondrial metabolism produced during ETS, β-oxidation, and Krebs cycle activity [103]. For instance, Kowaltowski's group [122,123] demonstrated that H2O2 is produced during the first step of FA β-oxidation, catalyzed by a very long-chain acyl-CoA dehydrogenase (VLCAD) enzyme in liver mitochondria. If H2O2 produced is not degraded, it could induce mitochondria-decoupling electron leakage during OXPHOS. Even β-oxidation may be damaged. The latter has been supported by Aparicio-Trejo et al. [44], showing that folic acid causes damage to mitochondria and decreasing the OXPHOS associated with FA β-oxidation is due to ROS overproduction. The use of the antioxidant N-acetylcysteine (NAC) prevents the reduction in OXPHOS capacity associated with FA β-oxidation impairment from 2 to 28 days of the administration, avoiding CKD transition [108]. In accordance, Briones-Herrera et al. [109] showed that MA, another inductor of OS, decreases β-oxidation and the use of antioxidant sulforaphane (SF) prevents this decrease [109].

On the other hand, Tan et al. [110] showed by transcriptomic analysis in diabetic mice that mitochondrial FA β-oxidation is downregulated. This downregulation is attributed to the overexpression of the C5 substrate of the complement system receptor 1 (C5aR1). Thus, C5aR1 is implicated in lipids metabolism in diabetes [124]. C5aR1 is also upregulated in kidney diseases, producing FA β-oxidation impairment in DN [110]. In addition, C5aR1 upregulation disrupts mitochondrial respiration, generating high levels of ROS. These results showed that C5aR1-induced ROS overproduction alters FA metabolism in DN.

During mitochondria decoupling, electron leakage from ETS occurs, which induces the reduction of oxygen (O2) to the radical O2 •−, a type of ROS that triggers the production of other ROS such as H2O2 [125]. High levels of •OH and ONOO− induce OS and significantly oxidative damage of proteins, lipids, and DNA. The oxidation of lipids produces highly reactive molecules such as MDA and 4-HNE as products of chain lipid peroxidation that also induce mtDNA damage (Figure 4) [68]. Forty-eight hours after cisplatin treatment, induced AKI, 4-HNE, and MDA levels increase along with GPx4 levels decrease in the renal cortex, indicating lipid membrane peroxidation [126]. Furthermore, in MA-induced Fanconi syndrome, 24 h after injection with MA, the mitochondrial levels of 4-HNE are elevated [109]. Additionally, GPx activity decreases, favoring H2O2 accumulation and mitochondrial lipidic peroxidation [109]. In folic-acid-induced AKI, 24 h after folic acid administration, mitochondrial MDA and 4-HNE levels increase [44]. Both OS markers also increase in nephrectomy models [127,128]. Together, these results show that mitochondria suffer lipid peroxidation in AKI and AKI to CKD transition.

In kidney diseases, the uptake of lipids by CD36, along with the dysfunction of FA β-oxidation, causes lipids accumulation in lipid droplets (LDs), inducing ROS overproduction (Figure 4) [129,130]. Since ROS and their products induce severe cell damage, a cellular balance of ROS is needed. This balance is performed by different antioxidants that include enzymatic and non-enzymatic antioxidants [131]. The reduction of the antioxidant system has been widely reported in kidney diseases [11,132]. In renal ischemia and nephrotoxicity, catalase (CAT), SOD, and glutathione S-transferase (GST) levels are depleted [70,133]. Moreover, in cisplatin-induced AKI, mitochondrial GSH and NADPH levels are decreased [134]. In the 5/6 nephrectomy, the activities of CAT, SOD, GPx, GR, and GST fall at 20 h in glomeruli, PT, and DT [128]. Moreover, early after UUO, these enzymes' mRNA and protein levels decrease, while oxidative markers increase [135]. Indeed, the decrease in antioxidant-system-induced OS has been suggested as a factor to induce the AKI to CKD transition.

**Figure 4.** ROS deregulate mitochondrial metabolism in kidney diseases. (1) In renal damage, the cluster of differentiation 36 (CD36) overexpression causes a high fatty acids (FA) uptake. In addition, carnitine *O*-palmitoyl transferase I (CPT I) and carnitine *O*-palmitoyl transferase II (CPT II) are decreased. (2) ROS cause FA β-oxidation decrease, inducing (3) a tricarboxylic acid (TCA) cycle and (4) oxidative phosphorylation (OXPHOS) capacity reduction. The decrease in β-oxidation also (5) induces the accumulation of lipids and a further ROS overproduction. The latter (6) damages mitochondrial membranes by inducing mitochondrial lipid peroxidation, (7) forming the products malondialdehyde (MDA) and 4 hydroxynonenal (4-HNE). These products are highly reactive and damage other lipids, proteins, and mitochondrial DNA (mtDNA). On the other hand, (8) ROS downregulate aconitase (Acn), citrate synthase, and isocitrate dehydrogenase isoform 2 (Idh2), inducing TCA cycle dysfunction. Moreover, (9) TCA cycle impairment induces FA β-oxidation dysfunction. (10) ROS decrease CI and CIII activities, inducing β-oxidation dysfunction [111]. O2 •−, superoxide anion radical; H2O2, hydrogen peroxide; •OH, hydroxyl radical; LOO•, lipid peroxyl radical; LDs, lipid droplets; OA, oxalacetate; α-KG, alpha-ketoglutarate; DNA, deoxyribonucleic acid; NADH, nicotinamide adenine dinucleotide phosphate; FADH2, flavin adenine dinucleotide; CoA, coenzyme A; CII, complex II; CIV, complex IV; CV, complex V. Created with BioRender.com.

The decreases in acetyl-CoA induced by FA β-oxidation impairment reduce the TCA cycle capacity. Interestingly, in AKI and CKD models, the reduction in TCA cycle enzymes is observed, before FA β-oxidation dysfunction occurs, suggesting this point as the start of the vicious cycle, which further increases the mitochondrial damage (Figure 4). In the next section, we will address the impact that ROS have on TCA cycle dysfunction.
