*5.2. TCA Cycle Redox-Sensitive Signaling Pathway in Kidney Diseases*

The urinary excretion of the non-diabetic CKD patients shows low levels of TCA cycle metabolites (e.g., citrate, cis-aconitate, isocitrate, alpha-ketoglutarate (α-KG), and succinate) [136]. In addition, kidney biopsies have reduced aconitate, isocitrate, alphaketoglutarate dehydrogenase (α-KGDH), and succinate gene expression. These results show TCA cycle dysfunction [136]. In contrast, in a mouse model of DN, pyruvate, citrate, α-KGDH, and fumarate are upregulated [137]. Moreover, in UUO, succinate levels increase, attributed to TCA cycle dysfunction [138]. Note that the amount of the metabolites is tissueand disease-dependent, so the identification of these metabolites could give advantages in the early detection of mitochondrial damage in these diseases.

TCA cycle dysfunction might be attributed to ROS alterations. In vitro studies have postulated that high glucose oxidation rates lead to the excessive production of electron donors from the TCA cycle. As a consequence, ETS becomes overloaded, promoting O2 •− overproduction [139]. In line with this, podocytes treated with high glucose levels have high ROS levels, and the treatment with mitoTEMPO decreases them [140], suggesting that ROS are specifically delivered from mitochondria. Controversially, the determination of mtROS in the diabetic mouse model shows that it is reduced [141]. Further studies in vivo are needed to elucidate the mtROS overproduction-induced TCA cycle dysfunction in DN.

In the kidney, TCA cycle enzymes can be sulfenylated or S-glutathionylated. For example, Acn can be reversibly inactivated by the oxidation of the sulfhydryl group by O2 •− and H2O2. However, if OS persists, Acn can be irreversibly deactivated by the disruption of the 4Fe-4S group [142]. In AKI induced by folic acid, mitochondrial Acn activity decreases, and the pre-treatment with NAC prevents it [44], suggesting that ROS promote the deactivation of Acn. In addition, the relation between Acn and citrate synthase diminishes, supporting the idea that decreasing in Acn activity is related to OS [44]. Moreover, Mapuskar et al. [143] reported that the persistent increase of O2 •− decreases Acn and citrate synthase activity in cisplatin-induced kidney injury, of which the effects are ameliorated by SOD mimetic avasopasem manganese (GC4419) treatment. The authors reported that in the AKI phase, Acn and citrate synthase activities do not show changes, suggesting that high levels of ROS are required for their inactivation in this model [143]. The latter is demonstrated due to the fact that high levels of ROS are more evident in cisplatin-induced CKD [143].

In kidney pathologies, the levels of mitochondrial isocitrate dehydrogenase isoform 2 (Idh2) are decreased [144,145]. In the cisplatin model, Idh2 function is affected by decreased mitochondrial NADPH and GSH and increased H2O2 production [145]. Furthermore, Han et al. [144] showed that OS generated during I/R reduces Idh2 levels in kidney tubule cells from mice. Since S-glutathionylation deactivates Idh2, this Ox-PTM may be produced during OS under I/R conditions [146]. The deletion of the Idh2 (Idh2−/−) gene in these mice exacerbates kidney tubule injury by increasing plasma creatinine and blood urea nitrogen (BUN) levels. In addition, OS increases the reduction of mitochondrial NADP+ along with GST and GPx activities. In contrast, mitochondrial GSSG/GSH ratio augments. Idh2−/<sup>−</sup> mice show mitochondrial dysfunction and fragmentation, which induces apoptosis in kidney tubule cells [144]. After UUO, Idh2 decreases, and its deletion increases OS markers such as 4-HNE and H2O2 in mitochondrial fractions [147]. Additionally, inflammatory cell filtration was more evident in Idh2−/<sup>−</sup> than wild-type (WT) groups. Together, these results highlighted the importance of Idh2 in managing OS, and its deactivation exacerbates mitochondrial damage.

Note that the fact that ROS-induced TCA cycle dysfunction affects FA β-oxidation has been demonstrated, because TCA cycle impairment is observed early before FA β-oxidation damage in time course studies of the AKI to CKD transition, (Figure 4). In this regard, OXPHOS capacity is also decreased by ROS in early times, suggesting that both events are required to enhancement FA β-oxidation dysfunction [44,108,120].
