Figure 2.
Gsdmd deficiency attenuates focal crystalline TMA, AKI, and ischemic infarction. (A) Illustration of the experimental design. CC (20 mg/kg) was injected into the left renal artery to induce focal TMA in WT and GSDMD knockout (Gsdmd−/−) mice. The mice were euthanized and analyzed after 24 hours. (B) Representative immunohistochemical images of αSMA and fibrin staining of interlobar, arcuate, and interlobular arteries in the kidneys. (C) Quantification of arterial obstruction in sham (n = 13) and TMA kidneys from WT (n = 10) and Gsdmd−/− (n = 13) mice. (D) Representative immunohistochemical images of αSMA and fibrin staining within the glomerular capillaries. (E) Representative images of periodic acid-Schiff (PAS) staining within glomeruli showing characteristics indicative of TMA, including glomerular capillary thrombi and increased capillary wall thickness. (F) Quantification of glomerular fibrin thrombi in sham (n = 4) and TMA kidneys from WT (n = 6) and Gsdmd−/− (n = 6) mice. (G) Glomerular filtration rate (GFR) at baseline and 24 hours after focal TMA induction in WT mice (n = 16) and Gsdmd−/− mice (n = 18). (H) Representative images of 2,3,5-Triphenyltetrazolium chloride (TTC) staining of TMA (left) and sham (contralateral right) kidneys of WT and Gsdmd−/− mice. Red areas indicate living kidney tissue, whereas white areas indicate infarcted kidney tissue. (I) Quantification of infarct size in sham (n = 25) and TMA kidneys from WT (n = 12) and Gsdmd−/− (n = 13) mice. (J) Representative images of PAS staining in sham and TMA kidneys from WT and Gsdmd−/− mice. (K) Quantification of tubular injury in sham (n = 13) and TMA kidneys from WT (n = 10) and Gsdmd−/− (n = 13) mice. (L) Immunoblot analysis of mature IL-1β (p17) in the kidneys of 3 sets of sham, WT, and Gsdmd−/− mice with focal TMA. β-actin was used as a loading control. (M) Plasma levels of IL-1β in healthy mice (n = 6) and focal TMA mice (WT: n = 9, Gsdmd−/−: n = 9) were quantified using enzyme-linked immunosorbent assay (ELISA). Scale bars: for panels B,D-E,J, 20 μm, and for panel H, 4 mm. The data represent mean ± SD. ∗P < .05, ∗∗P < .01, and ∗∗∗P < .001 using 2-way ANOVA with Bonferroni multiple comparisons test for panels C,G, or 1-way ANOVA with Tukey post hoc test for panels F,I,K,M.

Gsdmd deficiency attenuates focal crystalline TMA, AKI, and ischemic infarction. (A) Illustration of the experimental design. CC (20 mg/kg) was injected into the left renal artery to induce focal TMA in WT and GSDMD knockout (Gsdmd−/−) mice. The mice were euthanized and analyzed after 24 hours. (B) Representative immunohistochemical images of αSMA and fibrin staining of interlobar, arcuate, and interlobular arteries in the kidneys. (C) Quantification of arterial obstruction in sham (n = 13) and TMA kidneys from WT (n = 10) and Gsdmd−/− (n = 13) mice. (D) Representative immunohistochemical images of αSMA and fibrin staining within the glomerular capillaries. (E) Representative images of periodic acid-Schiff (PAS) staining within glomeruli showing characteristics indicative of TMA, including glomerular capillary thrombi and increased capillary wall thickness. (F) Quantification of glomerular fibrin thrombi in sham (n = 4) and TMA kidneys from WT (n = 6) and Gsdmd−/− (n = 6) mice. (G) Glomerular filtration rate (GFR) at baseline and 24 hours after focal TMA induction in WT mice (n = 16) and Gsdmd−/− mice (n = 18). (H) Representative images of 2,3,5-Triphenyltetrazolium chloride (TTC) staining of TMA (left) and sham (contralateral right) kidneys of WT and Gsdmd−/− mice. Red areas indicate living kidney tissue, whereas white areas indicate infarcted kidney tissue. (I) Quantification of infarct size in sham (n = 25) and TMA kidneys from WT (n = 12) and Gsdmd−/− (n = 13) mice. (J) Representative images of PAS staining in sham and TMA kidneys from WT and Gsdmd−/− mice. (K) Quantification of tubular injury in sham (n = 13) and TMA kidneys from WT (n = 10) and Gsdmd−/− (n = 13) mice. (L) Immunoblot analysis of mature IL-1β (p17) in the kidneys of 3 sets of sham, WT, and Gsdmd−/− mice with focal TMA. β-actin was used as a loading control. (M) Plasma levels of IL-1β in healthy mice (n = 6) and focal TMA mice (WT: n = 9, Gsdmd−/−: n = 9) were quantified using enzyme-linked immunosorbent assay (ELISA). Scale bars: for panels B,D-E,J, 20 μm, and for panel H, 4 mm. The data represent mean ± SD. ∗P < .05, ∗∗P < .01, and ∗∗∗P < .001 using 2-way ANOVA with Bonferroni multiple comparisons test for panels C,G, or 1-way ANOVA with Tukey post hoc test for panels F,I,K,M.

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