**11. Oxaloacetate**

OAA can be synthesized from malate by the MDH. It can also be derived from pyruvate catalyzed by the pyruvate decarboxylase, or aspartate by the glutamic oxaloacetate transaminase (GOT). OAAs can be condensed with acetyl-CoA to start the cycle again and also can be used for gluconeogenesis.

Currently, there is no information related to OAA levels in kidney diseases, probably by the difficulties in its measurement [150]. However, in kidney injury induced by toxic compounds potassium dichromate [151], gentamicin [152], melamine/cyanuric acid, and in diabetic nephropathy [153], MDH activity reduction has been reported; suggesting a decrease in OAA synthesis. Moreover, GOT serum levels in CKD patients are reduced and correlated with advanced stages of the disease [154].

Contrary, in RCC, MDH and GOT expression are increased [155], suggesting an increase in OAA synthesis. Furthermore, OAA inhibits SDH [156], thus impacting ETS activity and promoting succinate accumulation, which can inhibit 2OGDD as mentioned above (Figure 2i).

#### **12. Clinical Significance of TCA Metabolites**

Clinically, kidney function is evaluated by indirect measurement of glomerular filtration by serum creatinine levels, albuminuria, proteinuria, and eGFR. Recently, the use of mass spectrometry (MS) as a tool with proteomics [157], peptidomics [158], and metabolomics [159] approaches to the discovery of new biomarkers in urine and serum, has increased, showing a large number of molecules with potential use in the clinic. Some examples of molecules identified by mass spectrometry currently useful as biomarkers in clinics include cystatin C [160,161], neutrophil gelatinase-associated lipocalin (NGAL), and kidney injury molecule 1 (KIM1) [162–165]. Hence, the use of new biomarkers in conjunction with the classical method of kidney function evaluation could be helpful in a more accurate diagnosis or prognosis of different kidney diseases.

Due to the involvement of TCA cycle metabolites in kidney physiology and pathophysiology, identifying these in biofluids, such as serum and urine by metabolomics, could give insights into their use as potential biomarkers in different kidney diseases.

**Acetyl-CoA.** Currently, acetyl-CoA has not been identified as a biomarker in kidney diseases, probably by its multiple sources and its implication in diverse biochemical pathways. However, as mentioned above, one of its functions is in the fatty acid metabolism, which seems to be impaired in kidney diseases [11]. Carnitine can react with acetyl-CoA to form acetyl-carnitine during fatty acid metabolism by the carnitine acetyltransferase (CAT).

In CKD, serum levels of acetyl-carnitine increase along with disease progression, whereas in urine are decreased; even more, serum acetyl-carnitine shows a negative correlation with eGRF [166,167]. In AKI patients, serum levels of acetyl-carnitine levels also are increased [168]. In biopsies of renal cell carcinoma, acetyl-carnitine is increased; moreover, there are differences between clear cell, papillary, and chromophobe subtypes, with a more noticeable increase in clear cell RCC subtype [169].

Currently, acetyl-carnitine has been proposed as a biomarker for hepatocellular carcinoma, in which it is increased [170,171]; and in major depressive disorder, in which levels are decreased in serum. In kidney diseases, the use of this metabolite and the eGFR could help evaluate kidney function. However, more in-depth studies are necessary to determine its utility in discriminating against different kidney diseases.

**Citrate.** As mentioned above, urinary excretion of citrate is decreased in patients with CKD [42–44]. Clinically, urinary low citrate excretion is proposed as a marker of acid retention and reduced glomerular filtration in patients with CKD [43]. The meaning of plasma citrate is not clear enough since both negative and positive correlations with estimated glomerular filtration rate (eGFR) have been proposed [51,172]; in addition, the ratio of myo-inositol:citrate in urine seems to predict active renal vasculitis [173]. Similarly, in AKI pediatric patients, urinary citrate levels were found to be reduced [174]. In RCC, citrate levels decreased in urine [175], but these are enriched in tissue [62]. Hence, reduced citrate levels in urine seem to be a promising biomarker of altered kidney function.

**Isocitrate.** There is scarce information related to isocitrate alteration in serum or urine levels in kidney diseases. This metabolite and its derivate cis-aconitate are decreased in the urine of CKD patients [42]; additionally, plasma isocitrate correlates negatively with eGFR [51], being a possible predictor of disease progression. On the other hand, in RCC, low expression of IDH1, the enzyme responsible for isocitrate conversion to AKG, has been associated with a poor prognosis [93]. More in-depth studies are necessary to understand the clinical significance of this metabolite.

**AKG.** In CKD patients, decreased urinary levels of AKG have been reported [42], whereas there are no differences in AKI patients [176]. In RCC, AKG urinary excretion is increased [177], whereas, in biopsies, reduced levels have been reported; even more, tissue levels of this metabolite could be helpful in the prognosis of this neoplasia [81].

**Succinate.** In CKD patients, decreased urinary levels of succinate have been reported [42], and plasma succinate correlates negatively with eGFR [51]. In RCC, succinate levels are increased in tissue [62] and decreased in urine [175]. However, there is no information related to alterations of this metabolite in urine or serum from AKI patients. Currently, increased urinary levels of succinate and AKG have been proposed as biomarkers of major depressive disorder [178], opening a new panorama for the use of these metabolites in the clinic.

**Fumarate.** Increased urinary levels of fumarate in CKD patients have been described [44]; also, plasma fumarate correlates positively with eGFR [51] and has been associated with mortality [179]. In RCC, tissue fumarate levels are decreased [62], and no altered levels are reported in AKI patients. However, in an animal model, FH activity in urine and plasma has been proposed as a biomarker of AKI [147].

**Malate.** As fumarate, increased malate levels in the urine of CKD patients have been reported [44], and plasma malate also correlates negatively with eGFR [51]. In RCC, tissue malate levels are decreased [62]. In AKI patients, there are no reported altered levels of this metabolite.

**OAA**. As mentioned above, OAA is difficult to detect by MS; hence, its potential as biomarker is limited. In CKD, GOT serum levels are reduced and correlated with advanced stages of the disease [154]. In RCC tissue, GOT expression is increased. GOT is currently used for clinical evaluation of liver function; its use in conjunction with other parameters in the diagnosis and prognosis of kidney diseases could be helpful in clinics.

A summary of alterations of some TCA cycle metabolites in kidney diseases in humans and their potential as biomarkers is showed in Table 1.


**Table 1.** TCA cycle metabolites alterations in kidney diseases with potential use as biomarkers.

Summary of tricarboxylic citric acid (TCA) cycle alterations in different kidney diseases in humans. Δ = increased levels, ∇ = decreased levels; CKD, chronic kidney disease; AKI, acute kidney injury; RCC, renal cell carcinoma; AKG, alpha-ketoglutarate.
