Fig. 2: Necrotic cell death distribution along the nephron in post-ischemic tissue.
From: Longitudinal tracking of acute kidney injury reveals injury propagation along the nephron
a In vivo 2-photon image of a healthy mouse glomerulus (g) and surrounding tubules allows a clear distinction between proximal S1 (PT-S1), proximal S2 (PT-S2), and distal convoluted/collecting duct tubules (DCT/CD) based on distinct morphologic and autofluorescence patterns. Scale bar: 50 μm. b Tubule segment identity can be assigned based on quantitative assessment of green (λEm = 500–550 nm) and blue (λEm = 435–485 nm) tubular epithelial autofluorescence recorded at λEx = 750 nm (n = 152, 190, and 38 segments for PT-S1, PT-S2, and DCT/CD, from 4 mice 24h after partial IRI, respectively); boxplot, line at median, edges at 25th and 75th percentiles, whiskers from min to max, individual points considered outliers. Statistical test: linear mixed-effect model, p-values from two-sided tests (Supplementary Extended Statistics.f). c Upper panel: Representative in vivo 2-photon images acquired during bolus injection of FITC-4kDa-dextran reveal the arrival of filtered FITC-4kDa-dextran first in as PT-S1 and later in as PT-S2 classified tubules. Scale bar: 50 μm. Lower panel: Luminal FITC-4kDa-dextran fluorescence measured in PT-S1 and PT-S2 segments over time (n = 15 and 14 segments from 4 mice, respectively); mean ± SEM. d In vivo 2-photon image of a partial IRI kidney (ischemic region) at 2 h after reperfusion shows the distribution of propidium iodide (PI)+ nuclei across different tubule segments. Scale bar: 50 μm. e Necrotic injury distribution in IR regions of partial IRI kidneys across different nephron segments clustered by percentage of segments with indicated threshold of PI+ nuclei (% of total nuclei/ segment). f, g Volumetric quantification of PI+ nuclei (% of total nuclei/ segment) along different tubule segments in IR regions (n = 195, 90, 105 segments for PTs, PT-S1, PT-S2 from 6 mice, and 14 DCT/CD from 4 mice, respectively); mean ± 95% CI with scatterplot. Statistical test: linear mixed-effect model, p-values from two-sided tests (Supplementary Extended Statistics.g). *: p < 0.05; **: p < 0.01; ***: p < 0.001.
