Fig 2: Protein expression of glycolytic regulators in hACs on different treatments. Representative Western blots and quantifications of glycolytic proteins in cell cultures treated with vehicle (control), TGF-ß1 (5 ng/ml) or BMP2 (100 ng/ml) for 72 h. TGF-ß1–treated cells have increased protein expression of Glut1, HKI and HKII compared with the control. BMP2-treated cells have slightly increased protein expression of Glut1 and HKI, with HKII unchanged, compared with the control. Data are means ± SD of four independent experiments (*p < 0.05 by two-tailed t test).

Fig 3: FH null cells have a higher protein expression of Glucose 6-Phosphate Dehydrogenase (G6PDH) than FH restored and normal renal cells. A - Western blots for HK1, LDHA, G6PDH, FASN and ß-Actin were performed for UOK262, UOK262EV, UOK268 (FH null), UOK262WT (FH restored), and HRCE (human renal cortical epithelial) cells (a representative normal kidney cell), as described in Methods. B - The relative levels of two fatty acids (palmitate and oleate) were determined by Gas chromatography/mass spectrometry (GC/MS) of UOK262, UOK262EV, UOK268 and UOK262WT. The bar graph shows the mean values of three replicates along with the standard deviation as error bars. * indicates a statistically significant result (p<0.05) from a t-test.

Fig 4: Patients with chronic kidney disease (CKD) show an increased level of HK2, and CKD mice-derived mesenchymal stem/stromal cells (MSCs) show impaired mitochondrial functionality. (A) Serum samples from patients with CKD (n=30) and healthy controls (n=30) were subjected to enzyme-linked immunosorbent assay (ELISA) for HK2. (B) Methylglyoxal (MG) content in human serum samples from the same experimental groups (CKD patients vs. healthy control group). (C) Expression of HK1 and HK2 in mitochondrial fractions of MSCs derived from the CKD mouse model (CKD-mMSCs) and normal control mice (Nor-mMSCs), as detected by immunoblotting (n=5). Expression levels of HK1 and HK2 were normalized relative to levels of the mitochondrial marker VDAC1. (D) Normal-mMSCs and CKD-mMSCs were assayed using MitoSOX to assess mitochondrial oxidative stress. MitoSOX signals were quantified using flow cytometry (n=5). (E) Normal-mMSCs and CKD-mMSCs were assayed using TMRE to assess the mitochondrial membrane potential. TMRE signals were quantified using flow cytometry (n=5). Values represent the mean ± standard error of the mean (SEM). *p<0.05 or **p<0.01 vs. control.

Fig 5: Melatonin improved oxidative stress and mitochondrial membrane potential by lowering mitochondrial HK2 expression in chronic kidney disease mouse model-derived mesenchymal stem cells (CKD-mMSCs). (A) Immunoblotting results showing the expression of HK1 and HK2 in each experimental mMSC group: untreated CKD-mMSCs, CKD-mMSCs treated with melatonin, and melatonin-treated CKD-mMSCs pre-treated with luzindole. Expression levels of HK1 and HK2 were normalized relative to VDAC1 levels. Values represent the mean ± SEM (n=3). *p<0.05 or **p<0.01 vs. control; ##p<0.01 vs. melatonin-treated CKD-mMSCs. (B) CKD-mMSCs from each experimental group assayed using MitoSOX to assess mitochondrial oxidative stress. MitoSOX signals were quantified using flow cytometry. Values represent the mean ± SEM (n=5). **p<0.01 vs. control, ##p<0.01 vs. melatonin-treated CKD-mMSCs. (C) CKD-mMSCs from each experimental group assayed using TMRE to assess mitochondrial membrane potential. TMRE signals were quantified using flow cytometry. Values represent the mean ± SEM (n=5-6). **p<0.01 vs. control, ##p<0.01 vs. melatonin-treated CKD-mMSCs.