Fig. 3: BCAA-induced metabolic reprogramming in podocyte.

a RNA sequencing of differentiated MPC-5 podocytes treated with HG or HG plus 3 mM BCAAs (HG/BCAA) (leucine: isoleucine: valine =1:1:1) for 24 h. We show the top 7 upregulated pathways (red) and the top 7 downregulated pathways (green) using Gene Ontology (GO) and modified Fisher’s exact test. b The mRNA expression in primary podocytes (upper, n = 6) and isolated glomeruli from mice (lower, n = 5 mice for Tubb2b in all groups; n = 5 mice for Tuba4a in HF and HF/paired groups; n = 6 mice for others). c Representative immunofluorescence images of F-actin expression in MPC-5 podocytes. d Heatmap of the top 33 genes from (a) with cutoff of p < 0.05 and a fold change ≥ 2 (Mann–Whitney U test). The numbers on the right indicate the fold changes. e The mRNA expression in primary podocytes (upper) and isolated glomeruli from mice (lower), n = 6. f Graphical representation of genes involved in de novo serine biosynthesis and one-carbon metabolism. g Representative Western blots of key protein in the serine and folate biosynthetic pathway in both primary podocytes (n = 4) and isolated glomeruli from mice (n = 4 mice per group). h Upper, extracellular acidification rate (ECAR) in cultured primary podocytes; bottom, statistical analyses of post-glucose, post-oligomycin A and reserved capacity in ECAR, n = 5. i Upper, oxygen consumption rate (OCR) of primary podocytes; bottom, statistical analyses of baseline respiratory capacity, ATP-coupled respiratory capacity, maximum respiratory capacity and reserve respiratory capacity in the OCR. 2-DG, 2-deoxyglucose. FCCP, cyanide-4-(trifluoromethoxy) phenylhydrazone. RE, rotenone. Resp, respiration. n = 5. j Heatmap of the relative abundance of the carbon-13 (¹³C)-labeled main metabolites from uniformly carbon-13-labeled glucose ([U-¹³C6] glucose) (25 mM) treated primary podocytes. GAP, glyceraldehyde-3-phosphate; DHAP, dihydroxyacetone phosphate; 3-PG, 3-phosphoglycerate; α-KG, alpha-ketoglutarate. k Incorporation of ¹³C from [U-¹³C6] glucose (25 mM) into the indicated metabolites at 6 h in primary podocytes. n = 3. l Schematic metabolic map of [U-¹³C6]-labeled glucose metabolism. The data were presented as mean ± SD, and analyzed by unpaired two-tailed Student’s t test (a, b upper, d, e upper, h, i, and k) or one-way ANOVA (b bottom, e bottom).