Fig 1: Unbiased metabolomics analysis identifies higher citrate levels and ACLY activity in fibrotic mouse kidneys. (A) Measurement of acetyl‐CoA levels in unilateral ureteral obstruction (UUO) kidneys vs. controls. Acetyl‐CoA levels were increased in the UUO group compared with controls. * p < 0.05, n = 5–6. (B) Schematic of an untargeted metabolomics workflow using LC‐MS/MS to analyze total kidney extracts from healthy and chronic kidney disease (CKD) mice. (C) Volcano plot (log2 fold change vs. –log2(p‐value)) showing altered metabolites in folic acid (FA)‐treated kidneys compared to controls. (D) Citrate levels were quantified by LC–MS/MS. Data are presented as fold change relative to the control group (control = 1.0). *** p < 0.001, n = 5. (E) Citrate levels in UUO and control kidneys were measured using a citrate assay kit according to the manufacturer's instructions (Abcam, ab83396). Data are presented as fold change relative to the control group (normalized to 1.0). **** p < 0.001, n = 7. (F) RNA in situ hybridization showing Acly (magenta) and Lrp2 (blue) colocalization in renal tubules of mice; the down panel shows a magnified region. (G) Single‐cell RNA‐seq analysis showing Acly expression distribution across nephron segments in healthy and CKD mice (including UUO, folic acid nephropathy, adriamycin nephropathy, and diabetic kidney disease models). Expression is enriched in proximal tubule segments and in injured tubules across CKD models. CD_PC: Collecting Duct Principal Cells, CNT: Connecting Tubule Cells, DCT: Distal Convoluted Tubule Cells, DCT2: Distal Convoluted Tubule Segment 2, ICA: Intercalated Cells, Type A, ICB: Intercalated Cells, Type B, Podo: Podocytes, PEC: Parietal Epithelial Cells, PTS1: Proximal Tubule Segment 1, PTS2: Proximal Tubule Segment 2, PTS3: Proximal Tubule Segment 3, injPT: Injured Proximal Tubule Cells, prolif_Tubule: Proliferating Tubular Cells, MD: Macula Densa, TAL: Thick Ascending Limb, DTL_ATL: Descending Thin Limb/Ascending Thin Limb, Immune: Immune Cell Populations, EC: Endothelial Cells, Stromal: Stromal/Fibroblast Cells, injTAL: Injured Thick Ascending Limb, injDCT_CNT: Injured Distal Convoluted Tubule/Connecting Tubule Cells. (H,I) The expression of Acly mRNA (H) and protein levels (I) in FA‐ treated kidneys relative to controls. **** p < 0.0001, n = 6–8. (J,K) The expression of Acly mRNA (J), ACLY and phospho ACLY protein levels (K) in UUO and Sham kidneys. **** p < 0.0001, n = 8. Data represent mean ± SEM. Unpaired two‐tailed Student's t test for A, D, E, H, J.