*5.3. OXPHOS Redox-Sensitive Signaling Pathway in Kidney Diseases*

As mentioned above, kidney energy demand depends on OXPHOS, which in turn is regulated by Ox-PTMs. However, in the context of kidney diseases, it is poorly studied. OXPHOS capacity is downregulated in renal diseases, inducing ROS overproduction. In this regard, the production of mitochondrial H2O2 has been reported in kidney injury models, associated with ↓ΔΨm, decreasing OXPHOS capacity [8,148]. In remnant kidney from 5/6 nephrectomy, OXPHOS linked to complex I (CI) feeding decrease in a temporal course of nephrectomy from 2 to 28 days [111]. Moreover, male Sprague Dawley rats subjected to nephrectomy showed ATPβ, NDUSF8, and cytochrome c oxidase subunit 1 (Cox I) reduction [8]. Consequently, CI, complex III (CIII) activities, and cyt c diminished, impairing mitochondrial function [8]. Avila-Rojas et al. [148] reported that potassium dichromate

(K2Cr2O7) decreases the CI + CII-linked S3 respiratory state. In addition, ΔΨm in CI + complex II (CII)-linked respiration and respiration associated with OXPHOS are reduced, suggesting that K2Cr2O7 principally affects the synthesis of mitochondrial ATP [148]. Although redox signaling has not been investigated in previous studies, components of OXPHOS might be regulated by Ox-PTMs.

#### **6. ROS Induce Uncoupling Proteins (UCPs) Dysregulation in Kidney Diseases**

UCPs are proton transporters (H+), which move H+ from the IMM into the mitochondrial matrix. These transporters are localized in the IMM and dissipate the proton gradient from the mitochondrial matrix into the IMS [149]. mtROS induce UCP2 activation, decreasing the proton gradient and preventing mtROS overproduction [149].

It has been shown that UCP2 deletion aggravates tubular injury in the I/R model by inducing ROS overproduction, supporting the importance of these transporters in ROS dissipation [150]. Moreover, the UCP2 inhibition worsens the damage caused by lipopolysaccharide (LPS), increasing apoptosis in TECs [151]. In CKD, Jian et al. [152] showed that in renal tubular cells (RTCs), the expression of UCP2 is induced three days after obstruction and continues after seven days, avoiding UUO-induced fibrosis. It suggests that UCP2 is crucial to avert fibrosis development induced by ROS in UUO.

Although UCP1 is commonly found in mitochondria from brown adipose tissue, it is expressed in the kidney. For instance, in AKI models induced by cisplatin or I/R, Jia et al. [153] found that UCP1 is upregulated in renal TECs and its presence is related to OS suppression. Chouchani et al. [154] showed that mtROS alter the redox status of UCP1 by inducing its sulfenylation in Cys 253, promoting UCP1 activity.

#### **7. Redox-Sensitive Signaling Controls Mitochondrial Dynamics, Biogenesis, and Mitophagy**

Mitochondrial dynamic is the balance between mitochondrial fusion and fission, and it is involved in regulating mitochondrial metabolism and cell death. Likewise, the shape and morphology of mitochondria are regulated by the metabolite and ROS concentration concentrations [155]. The integral membrane guanosine triphosphatases (GTPases) perform mitochondrial fusion: mitofusin 1 (Mfn1), mitofusin 2 (Mfn2), and optical atrophy 1 (Opa1) [136]. Meanwhile, mitochondrial fission is mediated by Drp1, the mitochondrial fission factor (Mff), the adaptor protein fission protein 1 (Fis1), and the mitochondrial elongation factor 1 (Mief1) and 2 (Mief2). Chronic OS has been shown to induce mitochondrial fission [155]. However, H2O2 sublethal amounts, and acute OS cause mitochondrial hyperfusion [156]. In HeLa cells, mitochondrial hyperfusion has been associated with the S-glutathionylation of Mfn2 and the formation of Opa1 oligomers. Moreover, mitochondrial hyperfusion increases the resistance to cellular stress and cell death, since it promotes antioxidant defense enzymes activation [156]. In the next section, we analyze the current trends and paradigms of ROS-mediated signaling that form a link between redox-sensing elements and mitochondrial dynamics and their possible role in kidney diseases.

#### *7.1. Redox-Sensitive Proteins Participating in Fission and Fusion*

Redox signaling conveys external and internal signals between redox-sensitive receptors and the downstream effectors of fission machinery. Mitochondrial dynamics require the recruitment of proteins to mitochondria. Indeed, the importation of several proteins to mitochondria depends on proton electrochemical gradient H+ created by ETS at the IMM, which is called the proton motive force (PMF) [157]. In addition, several redox-sensitive proteins are activated to induce proteins translocation from the cytosol. For instance, previous studies in HeLa cells have shown that MAPKs are involved in regulating the fission process in response to OS through Ras [158,159]. In line with this, the redox-sensitive extracellular regulated kinase 2 (ERK2) protein mediates the phosphorylation of Drp1 in Ser 616 to induce mitochondrial fragmentation [158]. In addition to ERK 2, PKC-delta (PKC-δ) promotes Drp1 phosphorylation in Ser 579 under OS [160]. Drp1 is also regulated by ROS. In this context, Kim et al. [161] showed that in vascular diseases related to diabetes and aging models, protein disulfide isomerase A1 (PDIA1) is depleted, inducing the sulfenylation of Drp1 in Cys 644. The latter leads to mitochondrial fragmentation, mtROS increase, and senescence induction. Furthermore, the authors demonstrated that the restoration of the PDIA1/Drp1 axis could be used as a therapeutic strategy to improve vascular diseases [161], suggesting that PDIA1 has a thiol reductase function for Drp1. Drp1 persulfidation was previously reported, inducing its inactivation. Persulfidation is carried out by CARS2, altering mitochondrial dynamics and favoring fusion [66].

In renal pathologies, the upregulation of Drp1 is related to OS conditions [44,147,148]. Likewise, the phosphorylation of ERK 1/2 increases along with Drp1 levels in response to OS in the I/R rat model [162]. Thus, ROS might induce the recruitment of Drp1 through ERK1/2 (Figure 5).

**Figure 5.** Redox-sensitive signaling regulates mitochondrial dynamics in kidney diseases. (1) ROS activate the redoxsensitive extracellular regulated kinase 2 (ERK2) protein and protein kinase C (PKC) isoform δ (PKC-δ). These proteins phosphorylate and activate dynamin-related protein 1 (Drp1), inducing its translocation to the OMM to triggering fission. Likewise, ROS overproduction upregulates Drp1, fission 1 (Fis1), and mitochondrial fission factor (Mff), inducing fission increase. (2) ROS also induce fusion decrease by downregulating optical atrophy 1 (Opa1) and mitofusin 1 (Mfn1) and 2 (Mfn2) proteins. (3) The augment of fission promotes mitophagy activation by inducing the translocation of phosphatase and tensin homolog (PTEN)-induced putative kinase 1 (Pink1) that in turn recruit to parkin in the OMM to induce mitophagosome formation. Mitophagosome fuses with lysosome to form mitophagolysome formation. However, mitophagy flux is impaired, inducing the accumulation of damaged mitochondria. (4) Low ROS levels induce mitochondrial biogenesis by activating the redox-sensitive adenosine monophosphate (AMP)-activated protein kinase (AMPK) and the peroxisome proliferator-activated receptor (PPAR) γ coactivator-1 alpha (PGC-1α). However, high levels of ROS producing oxidative stress (OS) induce mitochondrial biogenesis decrease, triggering mitochondrial mass decrease. Created with BioRender.com.

In kidney diseases, ROS overproduction induces mitochondrial proteins fusion decrease and fission increase. In line with this, in the K2Cr2O7 rat model, Drp1 increase and curcumin treatment decrease it [148], suggesting that ROS induce Drp1 upregulation (Table 1). The latter is supported by the fact that ROS promote Drp-1 translocation to mitochondria. Drp1 is also recruited in RPTC treated with cisplatin, inducing mitochondrial

fragmentation and ATP depletion [163]. Moreover, Drp1 is upregulated in Idh2−/<sup>−</sup> mice due to NADPH and GSH decrease, which increase mtROS. mtROS also downregulate Opa1 (Table 1) [44,144,145]. Thus, mtROS trigger fission increase and fusion decrease. Following the latter, Opa1 levels are diminished under H2O2 treatment, and the diminishment is even higher in Idh2 small interfering RNA (siRNA)-transfected mProx24 cells [144]. Mfn1 is another fusion protein decreased in AKI models (Table 1) [44,164].


**Table 1.** ROS regulate mitochondrial dynamics, biogenesis, and mitophagy in acute kidney injury (AKI) models.

Abbreviations: ↑: increase; ↓: decrease; I/R, ischemia/reperfusion; Drp1, dynamin-related protein 1; Fis1, fission 1; OS, oxidative stress; Opa1, optical atrophy 1; Mf1, mitofusin 1; Pink1, phosphatase and tensin homolog (PTEN)-induced putative kinase 1; Idh2, Isocitrate dehydrogenase isoform 2; K2Cr2O7, potassium dichromate; PGC-1α, peroxisome proliferator-activated receptor (PPAR) γ coactivator-1 alpha; TFAM, transcription factor A; NRF1, nuclear respiratory factor 1; NRF2, nuclear respiratory factor 2; p62, sequestosome; LC3, microtubule-associated protein 1A/1B-light chain 3 phosphatidylethanolamine conjugate; NADPH, nicotinamide adenine dinucleotide phosphate; GSH, glutathione; mtROS, mitochondrial reactive oxygen species; MA, maleic acid; SF, sulforaphane.

> The blockage of mitochondrial fission has been suggested as a strategy to ameliorates mitochondrial damage [166]. For instance, the loss of DRP1, six hours after suffering bilateral I/R, re-establish mitochondrial function due to the reduction of mtROS production associated with fission decrease (Table 1) [165]. In addition, mice Drp1−/<sup>−</sup> does not have

tubulointerstitial fibrosis [165], suggesting that the Drp1 blocking might avoid the AKI to CKD transition. Furthermore, the specific inhibitor of Drp1, the mitochondrial division inhibitor 1 (mdivi-1), ameliorates I/R-mediated AKI (Table 1) [163]. However, in the case of UUO, the usage of mdivi-1 augments fibrosis [167], which correlates with midivi-1-treated human proximal tubular cells (HK2) under hypoxic conditions, showing fibrosis markers increase [167]. Therefore, Drp1 blocking might be employed in the case of AKI, but not in CKD.

The management of redox recovery homeostasis might be utilized as a strategy to improve mitochondrial dynamics. Consistent with this, the use of antioxidants that target mitochondria has been shown to enhance the homeostasis of mitochondrial dynamics [44,148]. Therefore, ROS regulate proteins involved in mitochondrial fission and fusion. According to the latter, it has been hypothesized that the treatment with sublethal amounts of H2O2 induces acute stress, promoting a hyperfused mitochondrial state [24,168]. In folicacid-induced kidney injury, Drp1 and Fis1 increase, and the treatment with NAC decreases them [44]. In response to cisplatin or bilateral I/R, Drp1 is recruited to mitochondria, triggering apoptosis induction by delivering cyt c and decreasing the antiapoptotic protein B cell lymphoma 2 (Bcl-2) [163]. Moreover, the activation of peroxisome proliferator-activated receptor γ (PPARγ) stabilizes mitochondrial potential, reducing ROS. The latter results in decreasing Drp1, augmenting Mfn2, Opa1 and restoring mitochondrial dynamics [8], suggesting that the rescue of mitochondrial biogenesis might recover mitochondrial dynamics due to mtROS decrease. In CKD, the levels of fusion proteins (e.g., Mfn1, Mfn2, and Opa1) are also decreased, while fission proteins are increased (e.g., Drp1 and Mff) (Table 2) [8,167,169,170], attributed to mitochondrial OS increase.


**Table 2.** ROS regulate mitochondrial dynamics, biogenesis, and mitophagy in chronic kidney disease (CKD) models.

Abbreviations: ↑: increase; ↓: decrease; DN, diabetic nephropathy; PPARα, peroxisome proliferator-activated receptor γ coactivator-1α; AMPK, adenosine monophosphate (AMP)-activated protein kinase; PPARγ, peroxisome proliferator-activated receptor γ; OMM, outer mitochondrial membrane; Drp1, dynamin-related protein 1; Fis1, fission 1; OS, oxidative stress; Opa1, optical atrophy 1; Mf1, mitofusin 1; Pink1, phosphatase and tensin homolog (PTEN)-induced putative kinase 1; PGC-1α, peroxisome proliferator-activated receptor (PPAR) γ coactivator-1 alpha; TFAM, transcription factor A; NRF1, nuclear respiratory factor 1; NRF2, nuclear respiratory factor 2; p62, sequestosome; LC3, microtubule-associated protein 1A/1B-light chain 3 phosphatidylethanolamine conjugate.

Ox-PTMs might regulate mitochondrial dynamics proteins; however, they are poorly studied in kidney disease. In line with this, S-nitrosylation in Drp1 Ser 644 induces Drp1 dimerization and augments its GTPase activity. Thus, NO promotes Drp1-induced mitochondrial fission [171]. Furthermore, OS can trigger mitochondrial hyperfusion [156]. Further studies are needed to clarify the role of ROS to induce Ox-PTMs regulation in mitochondrial dynamics and biogenesis in renal disease.

### *7.2. Redox-Sensitive Proteins Participating in Mitochondrial Biogenesis*

Mitochondria biogenesis is triggered to increase the number and size of mitochondria [172]. PPARγ coactivator-1 alpha (PGC-1α) controls the biogenesis process through the transcription of nuclear respiratory factors 1 (NRF-1) and 2 (NRF-2), PPARs, transcription factor A (TFAM), estrogen, and estrogen-related receptors (ERRs), among others [173]. These proteins are redox-sensitive. For instance, the exposure to H2O2 in skeletal muscle cells for 24 h increases the activity of the PGC-1α promoter as well as mRNA expression. These effects are blocked with NAC treatment [174], showing that ROS mediate the activation of PGC-1α. However, the impact of ROS over PGC-1α depends on the concentration and cellular type. For example, low ROS levels lead to the reduced expression of PGC-1α, while high levels induce its transcription through redox-sensitive adenosine monophosphate (AMP)-activated protein kinase (AMPK) [174], which function as a cellular energy sensor by regulating mitochondrial biogenesis and maintaining redox homeostasis [175]. AMPK reduces ROS through PGC1-α, which induces the overexpression of CAT, Mn-SOD, UCP2, and nicotinamide adenine dinucleotide (NAD)-dependent deacetylase sirtuin-3 (SIRT3) [176]. Therefore, the activation of AMPK induces PGC-1α promoter activity and mRNA levels increase. PGC-1α is commonly downregulated in kidney diseases, leading to mitochondrial mass and metabolism decrease [10]. In the folic-acid-induced AKI model, PGC-1α, TFAM, NRF1, and NRF2 levels decrease 24 h after the treatment, and NAC treatment prevents this effect [44]. Moreover, male Wistar rats subject to nephrotoxicity by K2Cr2O7 show a decrease in PGC-1α levels [148]. Both studies showed that the treatment with antioxidants (NAC and curcumin) upregulates biogenesis [47,152,169]. Importantly, NAC and curcumin antioxidants have shown mitochondria protection by promoting bioenergetics preservation and maintaining redox homeostasis to avoid mtROS [16,94]. In 5/6 nephrectomy-induced CKD, the levels of PGC-1α decrease in a temporal course from two days after the nephrectomy, causing reductions in NRF1 and NRF2 [120]. The treatment with pioglitazone, an antidiabetic drug, reduces mtROS, restoring mitochondrial biogenesis. Interestingly, the biogenesis decrease in DN is attributed to the low production of O2 •− [141]. AMPK activity decreases in this model, and AMPK activation induces O2 •− production, activating PGC-1α [141]. Therefore, in the DN mice model, low ROS levels are essential factors to trigger mitochondrial biogenesis.
