Fig 2: PV drives disruption of Warburg-like aerobic glycolysis. Solid in vitro LDHA enzymatic activity was estimated using recombinant human LDHA with the indicated concentrations of PV (0, 10, 50, 100, 500 and 1000 µg/ml). Oxamate (50 mM) was used as a positive control. To evaluate intracellular LDHA activity, 12Z cells were treated with various concentrations of PV (0–250 µg/ml) for 4 h. LDHA activity was measured using two independent methods of colorimetric observance at 340/460 nm (A,B). Total forms of LDHA, PDHA, PDK1, PDK3, and phosphorylated PDHA (Ser293) were detected using western blotting. Hsp90 and GAPDH were used as normalization controls (C,D). The lactate production and oxygen consumption rate in 12Z cells was measured after increasing doses of PV treatment. Oxygen consumption rate was measured in black 96-well microplates and lactate was quantified in phenol red and serum free DMEM by fluorescence at 535/590 nm (E,F). Data are presented as mean ± SEM. Statistical analysis was conducted using a Dunnet one-way ANOVA (*; p < 0.05, **; p < 0.01, ***; p < 0.001).

Fig 3: Paricalcitol alleviated LPS-induced injury through phosphorylation of PDHA1. (A) Oxygen consumption rate (OCR) (top) measured by Seahorse metabolic analyzer and quantitative analysis (bottom) of mitochondrial function parameters (basal respiration, maximal respiration). (B) lactate content in HK-2 cells treated with LPS, LPS + P, LPS + D (D: DCA, 5mM, pretreated 2 h), LPS + P + D for 24 h. (C) Western blot analysis (top) and densitometric quantitation (bottom) of PDHA1, p-PDHA1, cleaved caspase3 and bcl2 was performed in the five groups of HK-2 cells. (D) Real-time RT-PCR quantification of IL-6 and MCP1. *p < 0.05; **p < 0.01; ***p < 0.001. P, paricalcitol; D: DCA, dichloroacetic acid solution. Oligo, oligomycin; Rote, rotenone; Anti, antimycin A.

Fig 4: AG inhibits the PDK protein and mRNA expression. (A) H292 cells were treated with AG at the indicated doses for 1 h. (A) AG reduced lactate generation in a dose-dependent manner. (B) AG significantly inhibited the expression of p-PDHA1, which is phosphorylated by PDKs. Total amount of PDHA1 was not changed by AG treatment. Expression of LDHA also was not affected by AG treatment. HSP90 represents the normalized control of each well. (C) After H292 cells were treated with various concentrations of AG (20, 50 and 75 µM) for 6 h, the protein expression levels of mitochondrial PDK1, PDK2, and PDK3 were determined via western blotting. GAPDH represents the normalized control of each well. Protein expression levels of PDKs decreased in a dose-dependent manner after AG treatment. Notably, PDK1 expression levels significantly decreased after AG treatment. (D) In vitro PDK activity assay was performed to determine whether PDK1 activity was regulated by AG. AG did not directly regulate the activity of PDK1. (E-G) H292 cells were treated with various concentrations of AG (20, 50 and 75 µM) for 6 h, and the mRNA levels of PDK1, PDK2 and PDK3 were determined via reverse transcription-quantitative polymerase chain reaction. AG significantly decreased the mRNA levels of PDKs in H292 cells. Data are represented as the mean ± standard deviation compared with the control from three independent experiments. ***P<0.001 vs. control. AG, andrographolide; PDK, pyruvate dehydrogenase kinase; PDHA1, pyruvate dehydrogenase E1 subunit alpha 1; LDHA, lactate dehydrogenase A; HSP90, heat shock protein 90; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; p-, phosphorylated.

Fig 5: Paricalcitol alleviated glucose metabolism reprogramming of LPS-induced AKI mice. (A) Renal lactate content and hexokinase activity of the four groups. (B) Western blot analysis (left) and densitometric quantitation (right) of PDHA1 and p-PDHA1 and was performed in the four groups of mice. (C) Immunofluorescence analysis and its quantitative analysis of p-PDHA1 (green) and PDHA1 (red) of kidney sections. White arrow: glomerulus; yellow arrow: renal tubules. (D) Images of mitochondrial injury of proximal tubule epithelial cells from the four groups of mice by TEM. *p < 0.05; **p < 0.01; ***p < 0.001.