Figure 1.

Comparison of tubule injury after kidney-specific Cullin 3 deletion (KS-Cul3^−/−) and ischemia/reperfusion injury (IRI). (a,b) Western blot of whole kidney lysate showed Cul3 disruption after 6, 9 and 12 days of doxycycline administration. (c,d) Semi-quantitative analysis of periodic acid-Schiff staining revealed mild acute tubule injury upon Cul3 deletion. (e,f,h) Immunofluorescence revealed expression of kidney injury molecule-1 (KIM-1) in lotus tetragonolobus lectin (LTL)⁺ proximal tubules (KIM-1⁺/LTL⁺) which was observed at days 9 and 12. From day 6 on, there were increased numbers of Ki-67⁺ proliferating cells in tubules (e,g), predominantly in proximal tubules, and interstitium, as well as DNA damage in proximal tubules (based on phosphorylated histone H2AX (γ-H2AX) staining (h,i), and this preceded expression of KIM-1. (j–m) In contrast to KS-Cul3^−/− mice, progressive proliferation (based on Ki-67 staining; j,l) or DNA damage (based on γ-H2AX staining; k,m) occurred concomitantly with KIM-1 expression in proximal tubules of WT mice after 18 h of ischemia/reperfusion injury (IRI). In the contralateral control kidney only a few proximal tubules were positive for KIM-1 and Ki-67 (j,l). Scale bars = 100 μm. Mean values are shown ± SEM. In (b, d, f) asterisks show significant differences between control and each KS-Cul3^−/− mice group (n = 3). In (g,i) statistical analysis were performed separately for KIM-1⁺ and KIM-1⁻ tubules and asterisks show significant differences between control (CTRL) and each KS-Cul3^−/− mice group (n = 3). In (l, m) asterisks show significant differences between group “4 h after IRI” and each later time points after IRI (n = 2–3). *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001; Ordinary one-way ANOVA with Dunnett’s multiple comparisons test. Coomassie-stained gels used for Western blot normalization can be found in Supplementary Fig. S4. Western blots were cropped for clarity; uncropped images can be found in Supplementary Fig. S5.