**4. Crosstalk between NOXs and Mitochondria in Kidney Diseases**

Mitochondria and NOXs are the primary ROS sources in the kidney. The NOXs family consists of seven isoforms, being NOX1, NOX2, NOX4, and NOX5, the most expressed in renal cells [80]. The O2 •− production of NOX1 and NOX2 needs to assemble membrane subunit p22-phox and the cytosolic subunits p47- and p67-phox and ras-related C3 botulinum toxin substrate 1 (Rac1), while NOX4, abundantly expressed in mitochondrial membranes of renal cells, does not require cytosolic subunits and produces H2O2 [81–83].

The levels of NOXs augment in several AKI and CKD models, inducing ROS overproduction [84]. In the folic acid model, NOXs-linked ROS overproduction (in the kidney cortex, proximal tubules (PT), and distal tubules (DT)) is related to mtROS enhancement, because these two sources establish a pathological circle of ROS production, favoring AKI to CKD transition [44]. In the 5/6 nephrectomy model, mtROS also induce NOXs activation, increasing inflammation and fibrosis. Moreover, this mechanism contributes to fibrosis development in UUO [85]. Following the latter, several authors have established that in kidney pathologies, mtROS and NOXs-produced ROS increase mitochondrial damage and mitochondrial membrane potential depolarization (↓ΔΨm) (Figure 2) [86,87]. Ang II with the angiotensin type 1 receptor (ATR-1) also participates in the crosstalk between NOXs (NOX2 and NOX4) and mitochondria [88]. In addition to Ang II, in DN, the interaction of advanced glycation end products (AGE), produced by high glucose levels, activates NOXs by AGE receptor (RAGE) to generate ROS production [89]. Protein kinase C (PKC) epsilon (PKC-ε) also activates NOXs by inducing the phosphorylation of p47-phox, triggering ROS production [90]. NOXs-induced ROS cause the opening of a mitochondrial adenosine triphosphate (ATP)-sensitive potassium (K) channel (mt-KATP), triggering ↓ΔΨm [91]. In AKI and CKD models, the use of the NOXs inhibitor, apocynin, decreases mtROS production [92,93], supporting the idea that crosstalk between NOXs and mitochondria is involved.

**Figure 2.** Crosstalk between nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (NOXs) and mitochondria in the kidney. Angiotensin II (Ang II) binds to the angiotensin type 1 receptor (ATR-1), which activates NOX2 and NOX4. In addition, the binding of advanced glycation end products (AGEs) to the receptor for advanced glycation end products (RAGE) induces the activation of NOX2. Furthermore, protein kinase C (PKC) epsilon (PKC-ε), activated by mitochondrial ROS (mtROS), activates NOX2 through the p47-phox subunit, inducing ROS production. NOXs-induced ROS promote the phosphorylation and opening of an mitochondrial adenosine triphosphate (ATP)-sensitive potassium K channel (mt-KATP), decreasing mitochondrial membrane potential depolarization (↓ΔΨm). ROS and mtROS activate the redox signaling pathways: transforming growth factor-beta 1 (TGFβ1) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). mtROS also affect mitochondrial function by inducing damage to mitochondrial deoxyribonucleic acid (DNA) (mtDNA) and phospholipids that, in the last instance, generates mitochondrial dysfunction. The mtROS also favor the mitochondrial permeability transition pore (MPTP) opening, inducing the release of proapoptotic factors into the cytosol. p67-phox: subunit from NOX2; p22-phox: subunit from NOX2 and NOX4; Rac1: Ras-related C3 botulinum toxin substrate 1; ETS: electron transport system; CI: complex I; CII: complex II; CIII: complex III; CIV: complex IV; CV: complex V. Created with BioRender.com.

The binding of Ang II and AGEs to their receptors activates TGFβ1 and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) redox-sensitive signaling pathways (Figure 2) [94]. Furthermore, mtROS can stimulate TGFβ1 through the upregulation of Smad 2/3, inducing its nuclear translocation. The latter is supported by the fact that the mitochondrial-targeting antioxidants coenzyme Q (mitoQ) and 2- (2,2,6,6-tetramethylpiperidin-1-oxyl-4-ylamino)-2-oxoethyl)triphenylphosphonium chloride (mitoTEMPO) prevents the activation of TGFβ1 along with the transcription of *TGFβ1* genes [95]. mtROS also activate NF-κB by inducing monocyte/macrophage infiltration, increasing interstitial inflammation in UUO kidneys, and treating the antioxidant curcumin to decrease them and preventing interstitial inflammation [96]. mtROS also activate NF-κB in macrophages. In this sense, Herb et al. [97] demonstrated that in macrophages, mtROS, and not ROS produced by NOX2, activate NF-κB by deactivating the regulatory subunit of inhibitor IKK complex (IKKγ) through the disulfide linkage formation. However, in kidney diseases, this mechanism has not been investigated.

TGFβ1 and NF-κB are also localized in mitochondria, regulating mitochondrial proteins [98]. Moreover, the mitochondrial localization of both proteins might indicate that inflammation and fibrosis processes are regulated by mtROS production. Following the latter, in DN, hyperglycemia triggers the Smad4 translocation into mitochondria. This translocation reduces oxidative phosphorylation (OXPHOS), inducing inflammation, fibrosis, and podocyte injury [98].

The production of mtROS and ROS produced by NOXs also damages phospholipids and mitochondrial DNA (mtDNA). mtDNA is particularly susceptible to ROS, because it does not contain histones to protect it, causing DNA integrity loss and resulting in the acquisition of mutations [99]. mtROS also favor the opening of mitochondrial permeability transition pores (MPTPs) into the cytosol [86]. On the other hand, mtROS induce phospholipids oxidation, principally cardiolipin, leading to the ↓ΔΨm, MPTP opening, and ETS activity reduction [100]. Thus, the pathological crosstalk between NOXs and mitochondrial induces ROS, affecting mitochondrial metabolism and biomolecules integrity. The latter induces mitochondrial impairment, favoring the AKI to CKD transition.
