Figure 3. Knockdown of CNN2 attenuates tubular cell death and kidney inflammation in AKI.

(A and B) Representative micrographs (A) and quantitative data (B) of TUNEL staining in ShNC and ShCNN2 mouse kidneys 1 day after IRI or 3 days after cisplatin injection. Arrows indicate apoptotic cells. DAPI is a nuclear counterstain. Scale bar, 50 µm. *P < 0.05 (n = 5). (C) Western blot assay demonstrated the expression of AIF, FADD, Bax, and ARC in ShNC and ShCNN2 mouse kidneys at 1 day after IRI (n = 6). (D) Western blot assay demonstrated p-MLKL, MLKL, and GPX4 expression in ShNC and ShCNN2 mouse kidneys at 1 day after IRI. (E) Representative immunohistochemical staining micrographs for p-MLKL, GPX4, CD45, Ki67, and PCNA in ShNC and ShCNN2 mouse kidneys 1 day after IRI or 3 days after cisplatin injection. Scale bar, 50 µm. Arrows indicate positive staining. (F) Quantitative real-time PCR analyses revealed the mRNA abundance of Rantes, IL-6, and TNF-α in ShNC and ShCNN2 mouse kidneys 1 day after IRI or 3 days after cisplatin injection. *P < 0.05 (n = 6). (G) Immunofluorescence staining showed F4/80⁺ macrophages in ShNC and ShCNN2 mouse kidneys 1 day after IRI. Scale bar, 50 µm. Arrows indicate positive staining. (H) Western blot assays demonstrated PCNA expression in ShNC and ShCNN2 mouse kidneys at 1 day after IRI or 3 days after cisplatin injection. (I) Western blot assays demonstrated PDGFR-β and α-SMA expression in the kidneys from ShNC and ShCNN2 mice 1 day after IRI. For all Western blot panels, numbers indicate individual animals within each group. Graphs are presented as means ± SEM. Differences between groups were analyzed using unpaired t tests or ANOVA followed by the Student-Newman-Keuls test. AIF, apoptosis inducing factor; ARC, apoptosis repressor with caspase recruitment domain; FADD, Fas-associated protein with death domain; p-MLKL, phosphorylated mixed lineage kinase domain-like protein; GPX4, glutathione peroxidase 4; α-SMA, α–smooth muscle actin.