Fig. 4. Dynamic metabolic measurements on bIRI kidney PT cells.

a, Representative images of LTL and VCAM1 IF staining on sham and bIRI kidneys (n = 3). b, Left, representative molecular histology of cortical and outer stripe outer medulla (left) areas of bIRI kidney (n = 3), generated from UMAP of recorded lipid profiles. Right, IF staining following MALDI-MSI. c, UMAP plot (left) and representative spatial segmentation (right) showing lipid heterogeneity between PT cells from cortical and OSOM of bIRI kidneys (n = 3). Spatial segmentation image showing the distribution of the UMAP clusters on tissue with same color. d, Representative lipid species distribution in cortical and outer stripe outer medulla areas of bIRI kidney (n = 3). e, Left, Embedding of PT pixels from bIRI kidneys (n = 3), showing the trajectory of PT injury using pseudotime lipidomics analysis (starting point 1). Middle, spatial-trajectory map showing the pseudotime score and UMAP embedding of each pixel on tissue. Right, 3D scatter plot of cell-type-specific trajectories, with the same colors as pixels in the spatial-trajectory map. f, Spearman’s correlation between pseudotime and average ¹³C isotopologue enrichment on all PT pixels. g,h, Dynamic metabolic measurements using [U-¹³C₆]glucose (g) or [U-¹³C₅]glutamine (h), and their trajectory changes on PT cells of bIRI kidneys (n = 3). i,j, Dynamic metabolic comparison of [U-¹³C₆]glucose and total lactate levels (i) or [U-¹³C₅]glutamine measurements (j) between LTL⁺VCAM1^–KIM1^– PT cells (PT-S1/S2) and LTL^–VCAM1⁺ PT cells (FR_PT). A two-tailed paired t-test was performed. k, Direct carbon contribution of different nutrients to glutamate at 2 hours in FR_PT, from the glutamate isotopologues M+2, M+3, and M+5. One-way ANOVA was performed (n = 3). Comparison of dynamic metabolic measurements using [U-¹³C₆]glucose (l), [U-¹³C₁₈]linoleate (m), or [U-¹³C₅]glutamine (n) on PTs from sham kidneys and healthy PTs from IRI kidneys. Two-way ANOVA was performed. Images and graphs show the average ¹³C enrichment (area under curve (AUC) normalized to total time) of isotopologues derived from [U-¹³C₆]glucose or [U-¹³C₅]glutamine, measured at different timepoints (0, 15, 30, 60, and 120 minutes), or [U-¹³C₁₈]linoleate, measured at different timepoints (0, 60, and 120 minutes). Scale bars: (a) 50 µm, (b–e,g,h) 200 µm.

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