Fig. 1

High fructose drives mitochondrial metabolic reprogramming in podocytes via Hmgcs2-stimulated fatty acid degradation. a Quantitative proteomics revealed a continuously upregulated protein cluster causing mitochondria-related events in rat glomeruli during 16-week fructose modeling. b–d The mitochondria ultrastructure was severely disrupted in glomerular podocytes of high fructose-fed rats. b Representative images of mitochondria ultrastructure by transmission electron microscopy. The size and total number of mitochondria in glomerular podocytes of normal and model groups are shown in c, d, respectively (n = 5 per group). e The mitochondria depolarization and ΔΨm reduction were detected in 5 mM fructose-exposed differentiated podocytes (n = 5 per group). f Mito stress test was performed to measure the key parameters of mitochondrial function in differentiated podocytes with or without 5 mM fructose exposure (n = 10 per group). The quantification of OCR data in f, w, ab have been normalized by mitochondrial protein. g, h Ketogenesis was significantly upregulated in rat glomeruli starting from the eighth week of fructose modeling. Upregulation of ketogenesis was shown by both heatmap (g) and volcano plot (h). i Fatty acid degradation was significantly upregulated in rat glomeruli starting from the 12th week of fructose modeling. j Time-dependent increase of ketone body 3-HB in rat renal cortex during fructose modeling (n = 4 per group). k HDACs activity was time-dependently inhibited in renal cortex of high fructose-fed rats (n = 4 per group). l, m Knockdown of Hmgcs2 reversed upregulation of 3-HB induced by high fructose in differentiated podocytes (l) and in mouse glomeruli (m) (n = 4 per group). n, o Knockdown of Hmgcs2 partially rescued the inhibited HDACs activity induced by high fructose in differentiated podocytes (n) and in mouse glomeruli (o) (n = 4 per group). p, q Knockdown of Hmgcs2 reversed upregulation of H3ac and H4ac induced by high fructose in differentiated podocytes (p) and in mouse glomeruli (q) (n = 6 per group). r, s The increasing levels of H3K9ac and H4K12ac at Eci1 or Eci2 promoter induced by high fructose were significantly reversed by knockdown of Hmgcs2 in differentiated podocytes (r) and mouse glomeruli (s). Enrichment of H3K9ac and H4K12ac at Eci1 or Eci2 promoter were measured by real-time qPCR and quantitated in differentiated podocytes or mouse glomeruli under indicated conditions. Gapdh was used as negative control (n = 6 per group). t, u Knockdown of Hmgcs2 reversed the increase of Eci1 induced by high fructose in differentiated podocytes (t) and mouse glomeruli (u) as measured by real-time qPCR and Western blot (n = 6 per group). Relative mRNA or protein levels of Eci1 were normalized to β-Actin, respectively. v Knockdown of Hmgcs2 efficiently rescued the decrease of mitochondrial membrane potential induced by high fructose in differentiated podocytes. Flow cytometric analysis of ΔΨm was performed in differentiated podocytes transfected with Hmgcs2 siRNA as well as siNC and then cultured with or without 5 mM fructose (n = 5 per group). w Knockdown of Hmgcs2 reversed the inhibitory effects of high fructose on basal rate, ATP generation, and maximal respiration in differentiated podocytes. Bioenergetics profile was measured by OCR with a Seahorse ×96 Extracellular Flux Analyzer (Seahorse Bioscience) in differentiated podocytes transfected with Hmgcs2 siRNA as well as the respective negative control (siNC) and then cultured with or without 5 mM fructose (n = 6 per group). x Knockdown of Hmgcs2 reversed the disruption of mitochondria ultrastructure observed by transmission electron microscopy in glomerular podocytes of high fructose-fed mice (n = 4 per group). y, z Knockdown of Eci1 reversed the increase of Hmgcs2 induced by high fructose in differentiated podocytes. mRNA (y) and protein (z) levels of Hmgcs2 in differentiated podocytes transfected with Eci1 siRNA as well as siNC and then cultured with or without 5 mM fructose (n = 6 per group). Relative mRNA or protein levels of Hmgcs2 were normalized to β-Actin, respectively. aa Knockdown of Eci1 efficiently rescued the decrease of mitochondrial membrane potential induced by high fructose in differentiated podocytes. Flow cytometric analysis of ΔΨm was performed in differentiated podocytes transfected with Eci1 siRNA as well as siNC and then cultured with or without 5 mM fructose (n = 5 per group). ab Knockdown of Eci1 reversed the inhibitory effects of high fructose on basal rate, ATP generation, and maximal respiration in differentiated podocytes. Bioenergetics profile was measured by OCR with a Seahorse ×96 Extracellular Flux Analyzer (Seahorse Bioscience) in differentiated podocytes transfected with Eci1 siRNA as well as the respective negative control (siNC) and then cultured with or without 5 mM fructose (n = 6 per group). ac Working model: an epigenetic modification mediated-positive feedback consisting of ketogenesis and fatty acid degradation drives mitochondrial metabolic reprogramming during fructose-induced podocyte injury. Mean values ± SD are shown. Student’s two-tailed paired t test was used for comparing two groups. One-way ANOVA with Tukey’s post hoc test was used for multi-group comparisons. *p < 0.05, **p < 0.01, and ***p < 0.001 denote the significant difference as compared to the corresponding normal animal group or normal cell group