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. 2024 Oct 8;9(19):e179871. doi: 10.1172/jci.insight.179871

Figure 6. Acrolein-modified PKM2 shifts mitochondrial oxidative phosphorylation to aerobic glycolysis in NRK-52E cells and primary mouse renal tubular epithelial cells.

Figure 6

(A–C) NRK-52E cells were exposed to acrolein (0–30 μM) for 24 hours. (A) Western blot analysis of Acr-PCs is presented. (B) Tandem mass spectrometry illustrating the acrolein-modified peptide in acrolein-treated NRK-52E cells. (C) Pyruvate kinase (PK) activity was determined. (D) Immunofluorescence staining of PKM2 in acrolein-treated NRK-52E cells. Scale bar: 10 μm. (E) Subcellular localization of PKM2 in acrolein-treated NRK-52E cells. Cells treated with acrolein (20 μM, 24 hours) were subjected to the Cell Fractionation Kit, followed by Western blot analysis. (F) Co-immunoprecipitation analysis of nuclear fractions prepared from acrolein-treated NRK-52E cells using an anti-PKM2 antibody or IgG antibody, followed by Western blot analysis. (G) Cells treated with acrolein (20 μM, 24 hours) were subjected to chromatin immunoprecipitation (ChIP) assays with antibodies against HIF-1α (the left panel), PKM2 (the right panel), or IgG, followed by real-time quantitative PCR for PDK1 and HXK2. (H) NRK-52E cells treated with acrolein (0–30 μM) for 24 hours were subjected to Western blot analysis with quantification. (I) Primary renal tubular epithelial cells isolated from Aldh2 WT or Aldh2*2/*2 mice treated with acrolein (0–30 μM) for 24 hours were subjected to Western blot analysis with quantification. (J) Oxygen consumption rate (OCR) was analyzed using the Seahorse XFe24 Metabolic Flux Analyzer. (K and L) ALDH2 overexpression in NRK-52E cells using AAV8-ALDH2-EGFP transient transfection for 24 hours followed by acrolein treatment (20 μM, 24 hours). Western blot analysis was performed. Data are presented as mean ± SD. Statistical significance was determined using Kruskal-Wallis tests, with 2-tailed P values indicated. *P < 0.05, **P < 0.01, ***P < 0.001 compared with vehicle treatment. Acr-PCs, acrolein-protein conjugates; PKM2, pyruvate kinase M2; HXK2, hexokinase 2; PDK1, pyruvate dehydrogenase kinase 1.