*2.6. Urinary Mitochondrial Biomarker*

It is of value to detect DKD in the early disease stage in a reliable manner, preferentially before microalbuminuria or a decrease in the estimated glomerular filtration rate (eGFR) becomes evident, to prevent further progression to end stage kidney disease. Since mitochondrial dysfunction is thought to precede overt histological changes in DKD, biomarkers of mitochondrial dysfunction have recently been vigorously investigated. Even though those studies investigated patients with established DKD, they can be useful to identify biomarkers in patients affected by DKD. A study of metabolites in urine found global suppression in mitochondrial respiration in patients with DKD compared to controls without DKD [19]. In concordance with the notion that mtDNA is released from damaged tubular cells, the same study demonstrated increased mtDNA in urine. Although urinary mtDNA had a modest but significant inverse correlation with intra-renal mtDNA and eGFR at baseline, a positive correlation with interstitial fibrosis was found. Urinary mtDNA or mtDNA of biopsy specimens did not significantly correlate with eGFR decline in the 24 months of follow up [31]. Further investigations are warranted to determine if mitochondrial biomarkers can be used clinically.

## **3. Glucose-Induced Mitochondrial Dysfunction in DKD**

In glomerular cells, glucose is taken up by glucose transporters (GLUTs) via facilitated diffusion transport. The expression pattern of each member of the GLUTs is cell-specific. Mesangial cells express GLUT1 and 4, podocytes express GLUT1, 4 and 8 and endothelial cells express GLUT1 [32]. In contrast, tubular cells reabsorb glucose from the glomerular filtrate mainly via sodium-dependent glucose cotransporters (SGLTs). Glucose reabsorbed by tubular cells is dissipated across GLUTs in the basolateral plasma membrane and diffuses into the interstitium. Proximal tubular cells can also produce glucose via gluconeogenesis, which is increased in type 2 diabetes [33]. Hyperglycemia is the major pathogenetic factor and some intermediate metabolites of glucose metabolism have also been implicated in contributing to cell injury in DKD. In the unifying hypothesis offered by Brownlee and colleagues in 2000, it was proposed that the overproduction of mtROS due to increased influx into OXPHOS activated the nuclear DNA-repair enzyme poly(ADP-ribose) polymerase (PARP), leading to the decreased activity of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and the subsequent accumulation of toxic intermediates of glucose metabolism [11]. However, later studies have suggested that mitochondria are dysfunctional and that other mechanisms, in addition to a mere increase in substrates, contribute to increased glycolysis and the subsequent accumulation of toxic metabolites.

#### *3.1. Warburg Effect*

In general, cancer cells display enhanced glycolysis and impaired oxidative phosphorylation. While the short-time and reversible shift of this metabolic process is called the Crabtree effect, the long-term metabolic reprogramming is called the Warburg effect [34]. The Warburg effect is the term originally used to describe a shift from OXPHOS to aerobic glycolysis (in which lactate is produced as a final product from glucose) in cancer [35]. Recent studies have demonstrated that this shift also takes place in diabetic kidneys [9,19]. Mitochondria in diabetic tissues are dysfunctional, as discussed above. Transcriptomic, metabolomic and metabolite flux analysis showed increased glucose metabolism and decreased mitochondrial function in the kidney cortices of db/db mice [9]. A metabolomics analysis of urine samples demonstrated significant decreases of 13 metabolites in patients with DKD compared to healthy controls, 12 of which were associated with mitochondrial metabolism [19]. Whether mitochondrial dysfunction causes the shift to glycolysis or if increased glycolysis causes mitochondrial dysfunction remains to be established [36].

Pyruvate kinase is the enzyme which catalyzes the conversion of phosphoenolpyruvate to pyruvate, the last and irreversible step of glycolysis. Decreased pyruvate kinase M2 activity was identified as the possible mechanism of the Warburg effect in DKD by Qi and colleagues [37]. They first conducted proteomics analysis of glomeruli isolated from patients with type 1 diabetes who did not develop DKD for over 50 years (protected) and those with a histological confirmation of DKD (unprotected). Some enzymes involved in glucose metabolism and antioxidation were found to be increased in protected glomeruli, and in particular, pyruvate kinase M2 (PKM2) expression and activity were upregulated. In mechanistic studies using high-glucose-treated podocytes and STZ-injected mice, it was confirmed that PKM2 activity was decreased in the DKD mouse model, that PKM downregulation contributed to DKD exacerbation and that pharmacological activation of PKM2 reversed the elevation in toxic glucose metabolites and mitochondrial dysfunction. Similarly, decrease in pyruvate kinase activation causes the Warburg effect in cancer [38].

Other factors that are postulated to induce the Warburg effect in DKD include sphingomyelin and fumarate accumulation [39]. Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) revealed significant increases of ATP/AMP ratio and of a specific sphingomyelin species (SM(d18:1/16:0)) in glomeruli of DKD mice [40]. In vitro, addition of SM(d18:1/16:0) to mesangial cells activated glycolysis. Fumarate was identified as a factor that mediated Nox4-induced injury in DKD [41]. Podocyte-specific induction of Nox4 in vivo recapitulated DKD-induced glomerular injury, and metabolomic analysis demonstrated increased fumarate, which was reversed with Nox1/Nox4 inhibition. Fumarate could serve as a hypoxia-inducible factor (HIF) stabilizer, which could cause the activation of glycolysis and suppression of OXPHOS.
